Why is the Development of Disease-Modifying Osteoarthritis Drugs (DMOADs) Required?Disease Burden

Osteoarthritis (OA) is the most prevalent arthritis globally and represents a major challenge for twenty-first century health care systems.1,2 The Global Burden of Disease 2020 report showed an increase of 9.3% and 8.2% in the age-standardized OA point prevalence and annual incidence rate from 1990 to 2017.3 The prevalence rises with increasing age; in the USA (United States of America), OA was found in 13.9% of adults aged 25 years and 33.6% for those aged 65 years respectively in 2005.4 The lifetime risk of having symptomatic knee OA is about 40% in men and 47% in women, and the risk increases to 60.5% among obese persons.5 By the year 2040, an estimated 25.9% of the total adult population will have doctor-diagnosed arthritis in the USA.6

Globally, 80% of patients with OA suffer from limitations in movement, and 25% from difficulty in performing their major daily activities of life; representing a significant impact of OA on functional impairment and disability.7 In terms of economic burden, mean per-person earnings losses caused by OA were, on average, 7548 US$ per year from 2008 to 2011.8 The mean all-cause health care utilization of working-age patients with OA is $14,521 US$ per year.9 The socio-economic costs of OA were reported to range between 0.25% and 0.50% of a countrys GDP.10 In an individual patient data meta-analysis, the pooled estimate for premature mortality revealed a 23% increased risk (95% CI 1.07, 1.42) in patients with knee OA and a 20% increased risk (95% CI 1.04, 1.37) in hip OA.11

Current OA treatment options are focused on symptomatic improvement in pain and joint function and include paracetamol, nonsteroidal anti-inflammatory drugs (NSAIDs), opioid analgesics, and intra-articular medications such as steroids and hyaluronic acids.14 Surgical treatments are typically indicated only for patients with end-stage OA, as a last resort. Recently, paracetamol and opioids are only conditionally or not recommended by several scientific organisations,12,13 highlighting the importance of finding new effective treatments for OA. In addition, outcomes for patients with OA are usually suboptimal and patients remain vulnerable to the clinical consequences of the disease on pain and physical function.14

OA was previously regarded as a degenerative disorder resulting from cartilage damage;15 however, the development and utilization of modern imaging methods revealed that it results from the failure of the joint organ with a heterogeneous involvement of the whole joint structures, including cartilage damage, subchondral bone remodeling, synovial inflammation and osteophyte development.16 Therefore, OA can be defined as a complex heterogeneous syndrome with multiple joint tissue involvement of varying severity. In part as a consequence, it is a huge challenge to develop a single one size fits all therapy that may be suitable and effective for all patients with OA.17

The central hallmark in the pathologic process of OA disease is the progressive deterioration in the biological, structural and mechanical properties and function of the joint tissues, and an effective medical treatment should possess the ability to delay these processes or ideally even halt them completely. Such pharmaceutical agents that will alter the natural history of disease progression by arresting joint structural change and ameliorating symptoms, either by reducing pain or improving physical function are termed as DMOADs.18

Currently, regulatory bodies such as US Food and Drug Administration (FDA)19 and the European Medicines Agency (EMA)20 have not approved any drug as an effective DMOAD, as the approval guide requires a potential DMOAD to demonstrate a slowing in the loss of knee or hip joint space width (JSW) on x-ray with associated symptomatic improvement.17 Therefore, current OA trials for DMOAD development pipeline need to meet both clinically meaningful symptom improvement with concomitant structural benefits according to US FDAs published draft industry guidance on structural endpoints for OA published in 2018.18

Because OA is characterised by its extraordinary inter-patient variability in clinical and structural manifestations, identification of patient/disease subtypes appropriate for targeted therapy is probably one of the promising ways forward in drug development research.21,22 In addition, structural changes in OA result from complex interactions among different pathobiological pathways, which implicate a variety of catabolic factors and cytokines in the different joint tissues (molecular cross-talk).23 Therefore, a new model of classifying OA based on pathophysiological disease subtypes is needed.

These subtypes can be clinical phenotypes or molecular/mechanistic endotypes.24 A clinical phenotype can be defined as a group of observable traits (ie aetiologic factors, risk factors) that can identify and characterize a subtype in a defined population.25,26 In other words, these subgroups of patients have similar clinically observable characteristics for better identifying individuals who are at higher risk of progression (prognostic) or who are more likely to respond to a specific intervention (prescriptive).27,28

An endotype is a disease subtype defined by distinct pathophysiologic mechanisms, including cellular, molecular and biomechanical signalling pathways.29 Therefore, the endotype is distinct from a phenotype, and indicates the presence of a well-defined molecular mechanism. A given clinical phenotype of OA may comprise overlapping molecular endotypes (ie, different mechanisms giving rise to the same manifestation at varying degrees during different phases of the disease).24

From the point of view of targeted drug discovery, where identifying and directing the right pathobiological mechanism and structural manifestations of disease is key for success, drug development in OA should be based on the endotypes as the basis of the main drivers of OA disease.30 In this review, we will, therefore, focus on currently ongoing phase 2 and 3 clinical trials of active drug development (Figure 1) related to three main molecular/mechanistic endotypes: 1) Cartilage-driven endotype, 2) Bone-driven endotype, 3) Inflammation-driven endotype. While each drug has been assigned to and is discussed under one endotype based on its predominant activity, a particular therapeutic may have broader endotype-effects and where present, these are duly noted.

Figure 1 Active drugs related to the three main molecular or mechanistic OA endotypes (phase 2 and 3).

One author (WMO) conducted electronic and manual searches on the https://clinicaltrials.gov/ for identifying ongoing phase 2/3 clinical trials in active drug development pipelines, as well as electronic database searches in the PubMed and Embase via Ovid for published reports of phase-2/3 clinical trials results from the inception of these databases to 31st March 2021 using the following MESH or keywords: osteoarthritis OR osteoarthrosis AND DMOAD/ OR structure modification OR disease-modifying osteoarthritis drugs/.

Cartilage damage is considered as a central part of OA disease process, which involves a variety of catabolic and reparative mechanisms at the molecular level. The pharmaceutical drugs in phase 2 and 3 stages of development for cartilage-driven endotype are summarized in Table 1.

Matrix-degrading enzymes in the joint such as collagenases and aggrecanases are responsible for proteolysis of extracellular matrix components such as type II collagen and aggrecan, which is the most abundant proteoglycan in cartilage.31 Proteinases such as matrix metalloproteinase 13 (MMP13) and ADAMTS5 (a Disintegrin And Metalloproteinase with ThromboSpondin-motif-5) are involved in cartilage destruction and progression of cartilage damage in OA pre-clinical models.32,33 The potential benefits of MMP inhibitors in preserving the OA joint have been investigated. However, in patients with knee OA, broad-spectrum MMP inhibitors such as PG-116800 showed reversible musculoskeletal toxicities in a dose-dependent manner without clinical benefits, leading to the termination of further development of this drug.34

S201086/GLPG1972 is a potent and highly selective active site inhibitor of ADAMTS5. It possesses an excellent selectivity profile in animal models and high stability in dog and human liver microsomes and hepatocytes.35 Phase-1 clinical studies revealed favorable pharmacokinetics as well as a strong and consistent target engagement in both healthy subjects and OA patients (n=171).36 In a phase-2 study (Roccella study) which investigated the efficacy and safety profile of three different once-daily oral doses of GLPG1972/S201086 (n=932), the change in cartilage thickness [in mm (SD)] of central medial tibiofemoral compartment of the target knee via quantitative MRI was 0.116 (0.27) for the placebo group and 0.068 (0.20), 0.097 (0.27) and 0.085 (0.22), for the low, medium and high dose, respectively. There was no statistically significant difference versus placebo in both MRI and clinical outcome measures.37 Another ADAMTS5-targeting agent, M6495 an anti-ADAMTS5 Nanobody (Ablynx), showed an acceptable safety profile and dose-dependent effects in a phase-1 study.38

Sprifermin is a recombinant human fibroblast growth factor 18 (FGF18) which binds to fibroblast growth factor receptor-3 (FGFR-3) in cartilage.39 It stimulates the proliferation of articular chondrocytes and induces hyaline extracellular matrix synthesis in rat OA models.40 At the cellular level, intermittent administration may transiently promote an anabolic effect, while continuous administration may stimulate other signalling pathways, leading to a weaker effect.41

Lohmander et al reported in 2014 that intra-articular (IA) sprifermin administration did not improve medial tibiofemoral cartilage-thickness over 12 months quantified by MRI (n=168) possibly as follow-ups were too short for detection of the full disease-modifying effect of treatment.39 However, a significant dose-dependent response was detected in total and lateral tibiofemoral cartilage-thickness and radiographic JSW over 12 months. The authors speculated that the dynamic loading implicated in predominantly medial tibiofemoral involvement seems to impede attempts to prevent cartilage loss or regenerate cartilage tissue. Sprifermin had no major local or systemic adverse events compared with placebo. Conference abstracts published in 2015 and 2016 reported the structure-modifying effects on cartilage thickness and bone marrow lesions (BMLs) on MRI on 12-month follow-up, using post-hoc analyses of the same study.42,43

In another clinical trial in which Sprifermin was administered up to 300 g for advanced knee OA, it was reported in 2016 that no significant benefits were detected for cartilage outcomes on histology, synovitis, effusion, BMLs on MRI and JSW on X-ray. However, the study was underpowered as MRI was only available in 30 out of 52 patients and the follow-up period was only 24 weeks, which may be too short for capturing the structure-modifying effects.44

In a 5-year, phase 2 dose-finding, multicenter randomized clinical trial [FGF18 Osteoarthritis Randomized Trial with administration of Repeated Doses (FORWARD) study], the effects of Sprifermin on changes in total femorotibial joint cartilage thickness (n=549) on MRI was evaluated at 2-year follow-up (NCT01919164). Hochberg et al reported in 2019 that three once-weekly IA injection of 100 g sprifermin provided a significant improvement in total femorotibial joint cartilage thickness [0.05 mm (95% CI, 0.03 to 0.07 mm)] for participants administered every 6 months and [0.04 mm (95% CI, 0.02 to 0.06 mm)] for participants administered every 12 months, compared with the placebo saline injection provided every 6 months (0.02 mm).45 No significant improvement in total WOMAC scores was detected, compared with placebo. The most frequently reported treatment-emergent adverse event was arthralgia and showed no difference from the placebo group (43%). An exploratory analysis of the same study at 3 year-follow-up (n=442) reveals significant differences (0.05 mm [95% CI, 0.030.07 mm]) in total femorotibial joint cartilage thickness over MRI between Sprifermin (100 g of Sprifermin every 6 months) and placebo (saline every 6 months).45 However, the clinical significance of a 0.05-mm increase of cartilage thickness in this study remains unclear in terms of reducing risk for knee replacement, delaying time towards knee replacement, or both.46 No significant change in total WOMAC scores in this study may be attributed to using intra-articular saline injections as a control since the IA saline injection may act as an active placebo,47 masking symptomatic benefits. In addition, a large number of patients with low baseline pain and/or high baseline cartilage thickness may result in a potential floor effect on symptoms as 32% of this study had <40/100 points on WOMAC pain score at baseline and 50% had medial minimum joint space width (mJSW) >4.0 mm on baseline X-rays. Therefore, analysis of a more selective subgroup, featuring baseline characteristics associated with rapid structural and symptomatic OA progression should be investigated. In a 2019 ACR conference abstract, it was reported that in a subgroup at risk (n=161) of structural and symptomatic progression with a baseline medial or lateral mJSW between 1.5 and 3.5 mm and WOMAC pain score of 4090 out of 100, WOMAC pain was significantly improved on 3 year follow-up [8.8 (22.4, 4.9)] in the group administered with the 100 g Sprifermin (n=34) compared with the placebo (n=33)48 suggesting that, in this subgroup, the drug effect reaches the absolute minimal clinically important improvement for the WOMAC pain subscore which ranges 69.49

In a recent 2020 paper using a post-hoc analysis of the same data from the FORWARD study, thinning/thickening scores and ordered values of femorotibial cartilage thickness change on MRI over 24 months were analyzed by applying location-independent (ie not region-specific) analysis methodology in the knee joint.50 With administration of 100g Sprifermin every 6 months cartilage thickening is more than double [856m (717 to 996) vs 356m (313 to 398)] and cartilage thinning almost reduced to [432m (521 to 343) vs 335m (381 to 288)] that in healthy reference subjects from the Osteoarthritis Initiative dataset (n=82). The authors concluded that the finding supported the evidence of substantial structure-protective action of Sprifermin. However, as this is a post-hoc analysis, further study will be required to confirm its structure-modifying effect.

At a molecular level, the regulation of Wnt signalling determines osteoblast and chondrocyte lineage specification and their homeostasis.51 Increased Wnt signaling predisposes MSCs to an osteogenic lineage fate and induces generation of metalloproteinases which can cause cartilage degradation in OA.52 Increased expression and activation of the Wnt pathway in articular cartilage chondrocytes in OA similarly promotes cartilage degradation, while elevated Wnt signalling in subchondral bone enhances bone formation and sclerosis.5355 Therefore, pharmacological modulation of Wnt signaling might have potential benefits in repairing osteochondral dysregulation detected in OA disease process. Moreover, increased Wnt signaling in the synovium may potently lead to the OA progression via increased production of MMPs as well as activation of osteoclast differentiation and enhanced subchondral bone turnover.56,57

Lorecivivint (SM04690) is a small-molecule CLK/DYRK1A inhibitor that blocks Wnt signalling at the transcriptional level.58 It showed induction of chondrogenesis and reduction in cartilage degradation in preclinical studies.5860 In a 52-week, multicenter, phase-2 trial (n=455) (NCT02536833), the primary end point, a significant improvement in the WOMAC pain score compared with placebo at week 13, was not met, compared with IA placebo saline injection, However, at 52-week follow-up, intra-articular administration of 0.07 mg demonstrated a significant benefit in pain and functional scores [between-group difference versus placebo, 8.73, 95% CI (17.44, 0.03) and 10.26, 95% CI (19.82, 0.69)], as well as improvement in mJSW on X-rays [between-group difference versus placebo, +0.39 mm, 95% CI (0.06, 0.72)] in patients with unilateral knee OA. Serious adverse events were reported in 17 (3.7%) patients.61 The most common SAEs included infections and cardiac disorders and were deemed unrelated to the study drug by the investigators.62

Another phase-2 trial evaluated in 700 patients for 24 weeks was completed (NCT03122860) where the 0.07 mg lorecivivint treatment group demonstrated more favorable reductions in both WOMAC indices as compared with placebo.63 Recently, the investigators reported the safety data after the combined analysis of the two trials, which included 848 Lorecivivint-treated and 360 control subjects in total. The incidence of adverse effects or serious adverse effects was similar in treatment (41.3% and 2.4%) and control groups (38.3% and 1.1%), respectively. The most commonly reported AE in both groups was arthralgia (7.6% vs 7.2%).64 Two small phase-2 (NCT03727022, NCT03706521) and three phase-3 (NCT03928184, NCT04385303, NCT04520607) trials are still active.

Transforming growth factor- (TGF-) induces extracellular matrix protein synthesis and modulates cartilage development. A variety of TGF- signalling pathways are crucial for early cartilage growth, maintaining cartilage homeostasis in later life and may also possess anti-inflammatory and immunosuppressive properties.65 Impaired TGF- function in cartilage might be related to an increased susceptibility to OA.66 However, the biological effect of TGF- is under complex control, and may switch from being protective in normal joints to detrimental in OA as a result of changes in the predominant cell-surface receptors and intra-cellular signalling pathways in various joint tissues (cartilage, bone, synovium).67 In addition, osteocyte TGF- signaling could regulate the osteogenic and osteoclastic activity of mesenchymal stem cells and may be associated with the remodeling of subchondral bone in advanced OA.68

TissueGene-C (TG-C) uses a cell-mediated cytokine gene therapy approach and includes non-irradiated allogeneic human chondrocytes and irradiated allogeneic human GP2-293 cells in a ratio of 3:1, retrovirally transduced to promote TGF-beta1 transcription (hChonJb#7 cells).6971 A recent study reported as a possible mechanism of action that TG-C induced an M2 macrophage-dominant pro-anabolic micro-environment in a rat model, thereby providing a beneficial effect on cartilage regeneration.72 At one-year follow-up after a single IA administration, there were significant improvements in pain, sports activities and quality of life but structure-modifying effects on the cartilage were insignificant (n=156).73 In a phase-2 trial (NCT01221441) including 57 patients in the treatment group and 29 patients in the placebo group, the TG-C administration caused less progression (47.9% vs 34.6%; adjusted RR 0.7, 95% CI 0.51.1) of cartilage damage than placebo over 12-months.69 In a phase-3 trial (NCT02072070) which included 163 patients, symptomatic benefit was detected.74

The two pivotal phase-3 trials (NCT03203330, NCT03291470) had been on hold in April 2019 while the regulators were investigating chemistry, manufacturing, and control issues related with the potential mislabeling of ingredients.75 This clinical hold was lifted in April 2020, and trial enrollments have been reinitiated later in 2020.76 Recently, analysis of the safety data from an observational long-term safety follow-up trial showed that there is no evidence to suggest that injection of TG-C was associated with increased risk of cancer nor generated any long-term safety concerns over an average 10 years.71

Senescence is characterized mainly by altered responses to cellular stress and proliferation arrest of cells.77 Senescent cells (SnCs) are a newly implicated factor in the OA pathogenic process78 by promoting pathological age-related deterioration via the production of proinflammatory cytokines, chemokines, extracellular proteases, and growth factors (termed the senescence-associated secretory phenotype (SASP))79 and altering the function of neighbouring cells (termed secondary or paracrine senescence).80 Therefore, senotherapeutics which are directed at SnCs are an emerging therapy for treating diseases related to ageing. Senotherapeutics can be classified into of 3 types: 1) senolytics which kill and destroy SnCs selectively; 2) senomorphics which modulate or even reverse the phenotype of SnCs to those of young cells by blocking SASP; 3) senoinflammation, the immune system-mediated clearance of SnCs.81 Several senolytic pharmaceutical drugs such as Fisetin and UBX0101 are emerging.

Fisetin is a polyphenol extracted from fruits and vegetables and shows potential senolytic and anti-inflammatory activities.82 Fisetin inhibited IL-1-induced MMP13 and ADAMTS5 expression in human OA chondrocytes in vitro, and reduced cartilage damage along with subchondral bone thickening and synovitis in a mouse OA model induced by destabilization of the medial meniscus (DMM).83 Two phase-2 clinical trials (NCT 04210986, NCT04815902) are under investigation in patients with knee OA and estimated to be completed in 2022 and 2025, respectively.

UBX0101 is a small molecule inhibitor of the MDM2/p53 protein interaction, which possesses a potent senolytic candidate. In a preclinical study, UBX0101 improved chondrogenesis in human OA tissue in vitro, and in an anterior cruciate ligament transection (ACLT) OA model in mice UBX0101 attenuated SnCs by stimulating apoptosis, and reduced cartilage damage and joint pain.84 The amount SnCs in human OA synovial tissues positively correlated with knee pain, disease severity and synovitis severity.85 A phase-1 study (n=48) revealed that a single intra-articular injection of UBX0101 at different doses up to 4 mg had a favorable safety profile and dose-dependent, clinically meaningful improvements in pain on Numeric Rating Scale (010) [3.95 (95% CI, 4.74, 3.16)] and WOMAC function [1.05 (95% CI, 1.36,-0.74)] compared with placebo injection. Recently, UNITY Biotechnology announced 12-week data from UBX0101 Phase-2 Clinical Study (NCT04129944) which did not detect a significant change in pain and function in 183 patients with painful knee OA.86 A follow-up observational study of the previous trial (NCT04349956) was terminated in November 2020 due to failure to meet the trial outcomes.

Subchondral change in OA involves an uncoupled remodelling process, which is characterized by both increased osteoblast activation and bone formation but simultaneously macrophage infiltration and osteoclast formation.87 Activation of osteoclasts can result in pain genesis through developing acidic conditions at the osteochondral junction, thereby activating acid-sensing receptors of sensory neurons.88,89 Subchondral bone also undergoes remarkable alterations in both composition and structural organization, leading to adverse effects on the overlying articular cartilage.90 Therefore, targeting the pathways that modify subchondral bone turnover is an attractive option for DMOAD research.89 The pharmaceutical drugs in phase 2 and 3 stages of development for bone-driven endotype are summarized in Table 2.

Table 2 The Registered Phase 2/3 Clinical Trials on Compounds with Potential Disease-Modifying Effects on Subchondral Bone

Cathepsin K is a cysteine protease which induces bone resorption and cartilage damage through the breakdown of key bone matrix proteins.91,92 Cathepsin K knock out mice had attenuated cartilage damage in OA induced by DMM, and inhibition of Cathepsin K in rabbits by daily oral dosing with L-006235 reduced cartilage damage and subchondral bone remodelling in an ACLT model of OA.93,94

MIV-711 is a selective cathepsin K inhibitor, and in a 6-month phase 2 clinical trial (NCT02705625) (n=244), significantly reduced femoral bone disease progression and reduced cartilage loss, although there was no improvement in pain outcome.95 Infrequent musculoskeletal symptoms, infections and rashes were reported. A further 6-month open-level extension study showed the maintenance of structural benefit with symptomatic improvement (n=50).96 However, as most of the participants in the extension sub-study were selected because their symptoms did not worsen, a treatment benefit may be due to positive selection bias.95

Recombinant human PTH, teriparatide, is a 134 amino-acid fragment acquired from human PTH). Its anabolic action on bone production is used for osteoporosis management. In OA, it exhibits the ability to maintain articular cartilage health,97 stimulate the synthesis of extracellular matrix and induce chondrocyte proliferation in pre-clinical injury-induced OA models.98 PTH can increase subchondral bone mineral density, which could exert a negative effect on OA progression. In this sense, PTH could be an excellent drug in OA patients with osteoporosis and low subchondral sclerosis.99 Additionally, intermittent parathyroid hormone treatment attenuates OA pain in a DMM model, in association with inhibiting subchondral sensory innervation, subchondral bone deterioration, and articular cartilage degeneration.100 A phase-2 study is currently ongoing to evaluate the efficacy of PTH in knee OA participants (NCT03072147).

TPX-100 is a novel 23-amino-acid peptide derived from MEPE, a member of the Small Integrin-Binding Ligand, N-linked Glycoprotein (SIBLING) protein family, involved in subchondral bone remodeling.101 TPX-100 provided symptomatic improvements in patellofemoral OA knees administered with 4 weekly 200 mg injections compared with placebo injection in the contralateral knees (n=93), but only 14% of knees showed changes in cartilage thickness/volume measured on MRI over 12 months with no evidence of structural modification. No drug-related SAEs occurred in this study.102 Another 2020 OARSI conference abstract reported a statistically significant decrease in pathologic bone shape change in the femur at both 6 and 12 months using 3D femoral bone shape change.103

Antiresorptive drugs have shown reduction in bone remodeling and improvement in trabecular microarchitecture and bone mineralization. In clinical trials investigating the structure-modifying effects of bisphosphonates (alendronate, risedronate, zoledronic acid), the results are inconsistent across the studies and their outcomes presented a great heterogeneity.17,104 In a recent systematic review including preclinical studies (n=26) over the past two decades (20002020), these drugs showed better chondroprotective effects at high doses with a dose-dependent manner as well as depending on the timing of treatment initiation in relation to OA stage (time-dependency).105 Therefore, these agents may still be of potential benefits in certain OA endotypes with high rates of subchondral bone turnover. This phenotype-dependency has been demonstrated in pre-clinical research, where bisphosphonates are differentially effective in reducing pain and not only bone but also cartilage pathology in OA models with high versus low bone turnover.106109 Recently, clodronate (n=74)110 and neridronate (n=64)111 have been successfully used for the treatment of knee and hand OA, with an interesting efficacy on BMLs, although the sample sizes are small. An individual patient data meta-analysis for examining their efficacy in specific knee OA subtypes is still ongoing.112

In a multicentre, randomised controlled trial involving knee OA patients with significant knee pain and MRI-detected BMLs (n = 223), 2 annual infusions with 5 mg of zoledronic acid (the most potent of all bisphosphonates) did not significantly reduce cartilage volume loss, knee pain or BML size although the study was designed for detecting effects on the bone-driven subgroup with BMLs which may likely have potential benefits from this therapy.113 It was noted that more knee replacement procedures were performed in the zoledronic acid group compared with the placebo group (9% vs 2%) in contrast with other population-based studies.114,115

Another study involving Osteoarthritis Initiative (OAI) female participants (n=346) showed that bisphosphonate therapy may be protective of radiographic knee OA progression in nonoverweight patients with earlystage OA.116 Currently, a Phase 3 study (NCT04303026) to examine its effects in hip OA is ongoing. A phase 2 study examining the effects of another anti-resorptive, denosumab, in hand OA is expected to finish in 2021 (NCT02771860).

Vitamin D has a direct impact on cartilage by inducing proteoglycan synthesis in mature chondrocytes,117 and enhances chondrocyte viability and reduces their inflammatory cytokine synthesis through activating AMPK/mTOR and autophagy.118 Active vitamin D administration reduced cartilage degradation and inflammation in models of OA in mice and rats induced by meniscal injury/meniscectomy and ACLT.118120 Out of two recently published systematic reviews, one review showed the association of vitamin D deficiency with knee OA in patients but inconsistent evidence for its role in the prevention of incidence and progression of radiographic OA,121 while the other argued that inconsistent results may be attributed to factors such as severity of knee OA, baseline level of serum vitamin D, duration of treatment, and vitamin D dosages.122 There is a need for multicentric and well-conducted randomized studies using larger samples to determine its efficacy. A small Phase 4 clinical trial is currently active (NCT04739592).

Synovial inflammation (synovitis) is an important contributing factor to the OA pathogenesis through increased local production of pro-inflammatory cytokines, chemokines, and mediators of joint tissue damage123,124 which may be amenable to a range of anti-inflammatory drugs commonly used in inflammatory rheumatic diseases. The pharmaceutical drugs in phase 2 and 3 stages of development for inflammation-driven endotype are summarized in Table 3.

Diacerein is a purified anthraquinone derivative. It involves an inhibitory action on IL-1 and its signalling pathway, possesses an anticatabolic effect on OA tissues and reduces generation of metalloproteases.125 In animal models of OA (sheep meniscectomy, canine ACLT, rabbit ACLT and partial meniscectomy) diacerein has generally shown limited long-term effect on cartilage composition or pathology, but some evidence of reducing synovitis.126129 In a 2014 Cochrane review, the authors concluded that diacerein demonstrated only a minimal symptomatic improvement in patients with unclear benefits in JSW on X-rays, compared with placebo. Diarrhoea was the main adverse event with an absolute difference of 26%.130

The EMAs Pharmacovigilance Risk Assessment Committee suspended diacerein across Europe in 2013 due to its harms overweighing benefits,131 and then re-evaluated the drug in 2014, suggesting that it remain available with restrictions to limit risks of severe diarrhoea and hepatotoxicity.132 In 2016, the European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis (ESCEO) reported that diacerein had efficacy similar to that of NSAIDs with slower onset of action, suggesting that it might have some benefits for patients with contraindication to NSAID.133

Recently, results of a phase-3 clinical trial (NCT02688400) were reported where the authors explored the comparative efficacy and safety of diacerein vs celecoxib in patients with moderate and severe knee OA using a non-inferiority trial design [(6-months of diacerein 50 mg once daily for 1 month and twice daily thereafter (n = 187), or celecoxib 200 mg once daily (n = 193)]. Diacerein was non-inferior to celecoxib in reducing pain, stiffness, or functional limitations. The diacerein group had a higher number of emergent AEs (26.3%) compared with the celecoxib group (17.4%), mainly due to higher diarrhoea events (10.2% vs 3.7%). One patient in the diacerein group had three SAEs (abdominal pain, elevated transaminase and gamma-glutamyl transferase, collectively suggestive of hepatitis) which resolved spontaneously following drug withdrawal.134

In in vitro and in vivo preclinical studies, interleukin-1 (IL-1), tumor necrosis factor- (TNF-), IL-6, IL-15, IL-17, and IL-18 exhibit pro-inflammatory actions, leading to the initiation and progression of cartilage damage and joint inflammation. So far, IL-1 and TNF- have been the most extensively studied cytokines in pre-clinical research.135,136 Despite this favorable evidence in animal OA models, most clinical trials investigating the disease-modifying effects demonstrated by inhibitors of IL-1 and TNF- in OA patients failed to meet the primary and secondary endpoints such as in cases of Gevokizumab (XOMA-052),137 AMG108,138 Lutikizumab (ABT-981),139,140 anakinra,141 adalimumab142144 and etanercept.145 In a meta-analysis evaluating the efficacy of disease-modifying anti-rheumatic drugs in OA, neither IL1-inhibitors nor TNF-inhibitors possess symptomatic benefits irrespective of the joint site affected or the inflammatory phenotype (erosive or non-erosive OA).146

These failed trial results may suggest the implication of a more complicated interaction among various cytokines in the OA pathogenic process. One of the reasons for failure may be that the clinical trials were designed to detect an effect on symptoms rather than on joint structure, which is conversely the main outcome evaluated in preclinical studies, or that they are underpowered or have not followed participants for long enough to find meaningful structural effects such as proposed in the recent CANTOS trial.147 In a recent exploratory analysis of the CANTOS trial involving patients with elevated high-sensitivity C-reactive protein (hs-CRP) levels 2 mg/L and a history of myocardial infarction (n=10061), IL-1 inhibition using canakinumab may render a substantial reduction of THR/TKR rates as well as OA-related symptoms on an averaged 3.7 years follow-up.147 Although the study had some positives such as a large sample size and long-term follow-up, it was not primarily designed to investigate the DMOAD efficacy of canakinumab and many relevant OA outcomes were missing, necessitating further confirmatory studies.

IL-6 can increase the risk of radiographic OA and associated with knee cartilage damage,148 suggesting the potential role of low-level inflammation in the pathogenesis of OA. IL-6R blockage with tocilizumab contributes to cartilage preservation and increases bone volume in a mouse model of ischemic osteonecrosis,149 and reduced cartilage lesions, osteophyte formation and synovitis in DMM-induced OA in mice.150 However, male IL-6 knock out mice have increased cartilage damage and age-related OA.151 In local joint tissues, IL-6 classic signaling produces structure-protective effects, while trans-signaling leads to catabolic effects.152 This finding might suggest that selective inhibition of IL-6 trans-signaling could be a superior treatment strategy as this may inhibit deleterious IL-6 effects in OA, while maintaining protective IL-6 signaling via the classic pathway.153 Recently, in a phase-3 trial evaluating the efficacy of tocilizumab in hand OA for 12 weeks (n=104), it revealed no more effectiveness than placebo for pain relief (7.9 vs 9.9 on VAS score in the tocilizumab and placebo groups).154

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that potently and broadly suppresses proinflammatory cytokine activity. It also possesses chondroprotective effects, via reduced production of matrix metalloproteases155 as well as inhibition of chondrocyte apoptosis.156 Therefore, IL-10 could have potential benefits in OA management, both for pain improvement and suppression of the cartilage-damaging processes. Currently, there is a phase-2 clinical trial evaluating the safety and efficacy of a single injection of XT-150 (a plasmid DNA with a variant of human IL-10 transgene) in patients with knee OA (NCT04124042), and it is estimated to be complete in 2022.

In this section, we briefly put forward the reasons for failures in OA clinical trials and possible steps to overcome these barriers (Figure 2).

Figure 2 Reasons for DMOAD trial failures.

The drug will be required to demonstrate symptomatic benefits (pain and/or function) coupled with structural modifications to meet regulatory requirements as a disease-modifying agent.19,20 To date, no agent has been approved by the regulatory agencies.17 Some argue that the improvements in structural change (in the absence of any meaningful symptomatic benefits) should be a meaningful target for approval, in and of itself. However, this is unlikely to meet consumers needs as their primary reason for clinical presentation relates to symptomatic complaints.30

On the other hand, OA is a slowly progressive disease and only 14% of patients with incident OA have measurable disease progression over a 1-year period (Figure 2).157 Therefore, structure-modifying effects using targeted therapy would be optimal to delay or even avoid disease worsening and joint replacement. In OA, symptom-structure discordance is often described.158 Analysis of data from the Osteoarthritis Initiative revealed that changes in bone structure over 2 years do not translate into pain worsening until 4 years,159 suggesting that a structure-modifying drug may need longer follow-up to detect symptomatic benefit. In addition, a variety of disease outcomes using different OA subtypes (genotypes, phenotypes and endotypes) are needed to demonstrate the ability of a structure-modifying drug to directly predict for symptomatic benefits to overcome the regulatory hurdles.18

In addition, FDAs formal recognition of OA as a serious disease paves the way for using surrogate outcome measures for regulatory approval of DMOADs under accelerated approval regulations. However, two challenges need to be addressed: 1) selection/qualification of appropriate surrogate outcome measures, and 2) appropriate designs for post-marketing confirmatory studies. To overcome the first challenge, the Foundation for NIH (FNIH) OA Biomarkers Consortium initiative was established.160 For addressing the second challenge, Kraus et al proposed two major study design scenarios: 1) prospective trial continuation which continue all patients on initial drug allocation into the post-marketing approval trial until a failure threshold is achieved; and 2) separate post-marketing approval study which use different study population administered with active treatment only.161

The imaging standard in OA clinical trials has been radiographically measured mJSW which is notoriously unresponsive to change as well as possessing several other drawbacks such as issues with alignment, positioning and assuming JSW as the composite contribution of changes in other structures in this heterogeneous OA with multiple-tissue involvement.162,163 Therefore, utilization of this insensitive-to-change measure may limit our opportunity to detect any modification in what oftentimes is a slow-moving disease.

In 2015 OARSI published recommendations related to the applications of knee imaging in knee OA trials to set standards and improve quality assurance.164 Although a range of different MRI approaches have been developed to evaluate changes in overall joint structure,165167 further validation studies and evaluation of their clinimetrics are required to gain acceptance by regulatory authorities as a suitable surrogate endpoint which is the focus of the FNIH OA Biomarkers Consortium.160

In addition, the emergence of approved surrogate outcomes would allow pharmaceutical companies to examine the efficacy of the DMOADs in a shorter duration of clinical trials and reduce drug development costs. In this way, there is a possibility of instituting accelerated approval based on surrogate imaging endpoints and post-marketing approval studies to prove the longitudinal benefit-to-harm profile and the durability of the potential new therapies.161

In the study design for post-marketing approval which uses observational outcomes such as time-to-event of joint replacement surgery, considerable barriers exist in terms of need for large sample sizes due to low annual incidence rates (1.611.9%),14 long study follow-ups (>5 years at least),46 and the impact of non-disease and other subjective factors on the outcome (ie, comorbidities and/or age of the patient, costs, insurance cover, etc.).168,169 There is a lack of universal consensus criteria for guiding patient recommendations regarding joint replacement surgery, leading to differences even among treatment centres within the same region. These issues need to be adequately addressed by study design.161 There is a need for developing a criteria set to define appropriateness for total knee replacement or a virtual total knee replacement.170

Instead of utilizing the systemic route of administration which may produce undesirable systemic toxicity and off-target effects, many of the agents in the development pipeline are focused on an intra-articular route for drug delivery. This can also potentially enhance the local bioavailability, thereby maximizing therapeutic effects locally in the joint with a higher safety profile compared to systemic exposure.171 On the other hand, the marked placebo effect generated by local intraarticular administration is well-documented in the literature,172 making the assessment of symptom efficacy more challenging.30

Another issue related with the intra-articular therapy is that drugs have a short residence time within the joint.171,173 To overcome this barrier, a variety of drug delivery systems were proposed to prolong drug residence time while providing a stable concentration within the therapeutic window, leading to a reduction of side effects and better patient compliance.174 It remains unclear how long particular drugs have to remain in the joint for a meaningful symptomatic relief and/or structure-modification after an intra-articular administration. An ideal drug delivery system should comply with adequate disease modification, biocompatibility, and biodegradability while responding to its physiological environment.175

In the randomized clinical trials for IA drugs, saline is commonly used as the placebo in the control group. A recent meta-analysis examining the effects of IA saline in 50 clinical trials (n=4076) revealed significant improvement of pain severity on 0100 VAS up to 6 months [13.4 (21.7/5.1)] and WOMAC function sub-score [10.1 (12.2,-8.0)]. The pooled responder rate after saline injections using the OMERACT-OARSI criteria is 48% at 3 months and 56% at 6 months,47 challenging the concept of saline being a mere placebo.176 However, there is no evidence supporting hypotheses advocating the disease-modifying role of saline injection. Future scientifically robust studies which examined the effects of sham injections compared with saline injections are required to shed new light on this issue.

The IA therapies show a considerably larger therapeutic effect after the adjustment for the effects of IA saline, suggesting an inappropriate underestimating of the true effect of the active medication.177 Further research is required to determine the underlying mechanisms and the factors influencing the placebo response and ways to overcome it. In addition, the mechanisms of pain genesis in OA are poorly understood and thought to involve a complex interaction among local pathological processes in the OA joint and neuronal mechanisms and alterations of pain processing (ie central sensitization, especially in advanced OA).178 Further studies should focus on the effects of these interactions on the outcomes in the placebo-controlled clinical trials. It is also necessary to strictly report in each clinical trial what placebo has been used as well as the presence or absence of any additional blinded clinical evaluator, even more, if considering clinical trials with intra-articular therapies.

As OA is a heterogeneous disease with a combination of different endotypes in varying degree at different stages of the disease process, a one size fits all approach using a single therapeutic agent targeting a single target within a single endotype may be unlikely to succeed in the management of OA.179 Therefore, as in the oncology therapeutic area, combinations of drugs targeting different hallmarks of OA pathogenic process should be considered. Further research examining the potential synergistic action of combining anabolic therapies with those that downregulate catabolic factors will be required.

OA is well known for marked variations of disease expression,180 involves a variety of tissue pathologies as a whole joint disease16 and presents with different pathobiological manifestations,181 suggesting the potential value of personalised and precision medicine from the treatment perspective. Personalized medicine is used for treatment focusing on the patient based on their individual clinical characterization, considering the diversity of symptoms, severity, and genetic traits.182 In precision medicine, the molecular information maximizes the accuracy with which the patients are categorized and treated, typically applying large amounts of data for identification of patient subtypes which possess sharing specific relevant characteristics to predict diagnosis, progression, or treatment response, and to utilize appropriate therapeutic targets.183 The use of precision medicine in OA remains limited.

The implementation of private/ public initiatives, such as the Osteoarthritis Initiative, the FNIH biomarkers consortium, the European APPROACH ((Applied Public-Private Research enabling OsteoArthritis Clinical Headway)) project have contributed greatly to moving the field forward. Clinical phenotypes, endotypes, and molecular and imaging biomarkers are being identified, but the exact interplay among them and underlying mechanisms of each remain to be elucidated.24 While these biomarkers may have potential benefits in detecting those patients with the greatest risk for structural progression, their use still needs to be translated into more efficient clinical trial design and widespread clinical application.184

There remains an immense unmet need for effective and safe targeted interventions to inhibit both pain and disease progression. The complex overlapping interplay among the pathobiological OA processes and heterogeneity of clinical presentations of patients with OA, call for a universally accepted classification of phenotypes and endotypes for developing targeted disease-modifying therapy and providing the appropriate treatment in clinical setting. Although challenges exist towards the eventual management of OA by applying the concepts of personalized and precision medicine, the lessons learned through failed clinical trials, the ongoing developments of more advanced imaging and sophisticated biomarkers tools and effective drug delivery systems are leading to substantial progress in our field.

WMO is supported by the Presidential Scholarship of Myanmar for his PhD course. DJH is supported by the NHMRC Investigator Grant. VD is supported by a University of Sydney Postgraduate Award scholarship.

DJH provides consulting advice on scientific advisory boards for Pfizer, Lilly, TLCBio, Novartis, Tissuegene, Biobone. CL has provided consulting advice for Merck Serono and Galapagos Pharmaceuticals, and receives research funding from numerous pharmaceutical companies (Fidia Farmaceutici, Inter-K Peptide Therapeutics Ltd, Taisho Pharmaceutical Co. Ltd, Concentric Analgesics Inc, Cynata Therapeutics, CEVA Animal Health, Regeneus) through specific services/testing contract research agreements between and managed by The University of Sydney or the NSLHD. The authors report no other conflicts of interest in this work.

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2. OARSI TP-cCfOPo. OARSI White Paper- OA as a Serious Disease; 2016.

3. Safiri S, Kolahi AA, Smith E, et al. Global, regional and national burden of osteoarthritis 19902017: a systematic analysis of the Global Burden of Disease Study 2017. Ann Rheum Dis. 2020;79(6):819828. doi:10.1136/annrheumdis-2019-216515

4. Neogi T. The epidemiology and impact of pain in osteoarthritis. Osteoarthritis Cartilage. 2013;21(9):11451153. doi:10.1016/j.joca.2013.03.018

5. Murphy L, Schwartz TA, Helmick CG, et al. Lifetime risk of symptomatic knee osteoarthritis. Arthritis Rheum. 2008;59(9):12071213. doi:10.1002/art.24021

6. Hootman JM, Helmick CG, Barbour KE, Theis KA, Boring MA. Updated projected prevalence of self-reported doctor-diagnosed arthritis and arthritis-attributable activity limitation among US Adults, 20152040. Arthritis Rheumatol. 2016;68(7):15821587. doi:10.1002/art.39692

7. WHO. Chronic rheumatic conditions; Published 2021. https://www.who.int/chp/topics/rheumatic/en/. Accessed June 7, 2021.

8. Osteoarthritis and Allied Disorders. In: The Burden of Musculoskeletal Diseases in the United States (BMUS). Third ed. 2014.

9. Lo J, Chan L, Flynn S. A systematic review of the incidence, prevalence, costs, and activity and work limitations of amputation, osteoarthritis, rheumatoid arthritis, back pain, multiple sclerosis, spinal cord injury, stroke, and traumatic brain injury in the United States: a 2019 Update. Arch Phys Med Rehabil. 2021;102(1):115131. doi:10.1016/j.apmr.2020.04.001

10. Puig-Junoy J, Ruiz Zamora A. Socio-economic costs of osteoarthritis: a systematic review of cost-of-illness studies. Semin Arthritis Rheum. 2015;44(5):531541. doi:10.1016/j.semarthrit.2014.10.012

11. Leyland KM, Gates LS, Sanchez-Santos MT, et al. Knee osteoarthritis and time-to all-cause mortality in six community-based cohorts: an international meta-analysis of individual participant-level data. Aging Clin Exp Res. 2021;33(3):529545. doi:10.1007/s40520-020-01762-2

12. Bannuru RR, Osani MC, Vaysbrot EE, et al. OARSI guidelines for the non-surgical management of knee, hip, and polyarticular osteoarthritis. Osteoarthritis Cartilage. 2019;27(11):15781589. doi:10.1016/j.joca.2019.06.011

13. Kolasinski SL, Neogi T, Hochberg MC, et al. 2019 American college of rheumatology/arthritis foundation guideline for the management of osteoarthritis of the hand, hip, and knee. Arthritis Care Res. 2020;72(2):149162. doi:10.1002/acr.24131

14. Weinstein AM, Rome BN, Reichmann WM, et al. Estimating the burden of total knee replacement in the United States. J Bone Joint Surg Am. 2013;95(5):385392.

15. Shane Anderson A, Loeser RF. Why is osteoarthritis an age-related disease? Best Pract Res Clin Rheumatol. 2010;24(1):1526. doi:10.1016/j.berh.2009.08.006

16. Loeser RF, Goldring SR, Scanzello CR, Goldring MB. Osteoarthritis: a disease of the joint as an organ. Arthritis Rheum. 2012;64(6):16971707. doi:10.1002/art.34453

17. Oo WM, Yu SP, Daniel MS, Hunter DJ. Disease-modifying drugs in osteoarthritis: current understanding and future therapeutics. Expert Opin Emerg Drugs. 2018;23(4):331347. doi:10.1080/14728214.2018.1547706

18. Food and Drug Administration of the United States. Osteoarthritis: structural Endpoints for the Development of Drugs; 2018.

19. Food and Drug Administration of the United States. Draft guidance for industry: clinical development programs for drugs, devices, and biological products intended for the treatment of osteoarthritis (OA); 1999.

20. European Medicines Agency. Clinical investigation of medicinal products used in the treatment of osteoarthritis; 2010.

21. Felson DT. Identifying different osteoarthritis phenotypes through epidemiology. Osteoarthritis Cartilage. 2010;18(5):601604. doi:10.1016/j.joca.2010.01.007

22. Bierma-Zeinstra SM, Verhagen AP. Osteoarthritis subpopulations and implications for clinical trial design. Arthritis Res Ther. 2011;13(2):213. doi:10.1186/ar3299

23. Karsdal MA, Michaelis M, Ladel C, et al. Disease-modifying treatments for osteoarthritis (DMOADs) of the knee and hip: lessons learned from failures and opportunities for the future. Osteoarthritis Cartilage. 2016;24(12):20132021. doi:10.1016/j.joca.2016.07.017

24. Mobasheri A, Saarakkala S, Finnil M, Karsdal MA, Bay-Jensen A-C, van Spil WE. Recent advances in understanding the phenotypes of osteoarthritis. F1000Res. 2019;8:F1000Faculty Rev2091. doi:10.12688/f1000research.20575.1

25. DellIsola A, Allan R, Smith SL, Marreiros SS, Steultjens M. Identification of clinical phenotypes in knee osteoarthritis: a systematic review of the literature. BMC Musculoskelet Disord. 2016;17(1):425. doi:10.1186/s12891-016-1286-2

26. Van Spil WE, Kubassova O, Boesen M, Bay-Jensen AC, Mobasheri A. Osteoarthritis phenotypes and novel therapeutic targets. Biochem Pharmacol. 2019;165:4148. doi:10.1016/j.bcp.2019.02.037

27. Jameson JL, Longo DL. Precision medicine--personalized, problematic, and promising. N Engl J Med. 2015;372(23):22292234. doi:10.1056/NEJMsb1503104

28. Deveza LA, Nelson AE, Loeser RF. Phenotypes of osteoarthritis: current state and future implications. Clin Exp Rheumatol. 2019;37 Suppl 120(5):6472.

29. Mobasheri A, van Spil WE, Budd E, et al. Molecular taxonomy of osteoarthritis for patient stratification, disease management and drug development: biochemical markers associated with emerging clinical phenotypes and molecular endotypes. Curr Opin Rheumatol. 2019;31(1):8089. doi:10.1097/BOR.0000000000000567

30. Oo WM, Hunter DJ. Disease modification in osteoarthritis: are we there yet? Clin Exp Rheumatol. 2019;37 Suppl 120(5):135140.

31. Troeberg L, Nagase H. Proteases involved in cartilage matrix degradation in osteoarthritis. Biochim Biophys Acta. 2012;1824(1):133145.

32. Wang M, Sampson ER, Jin H, et al. MMP13 is a critical target gene during the progression of osteoarthritis. Arthritis Res Ther. 2013;15(1):R5R5. doi:10.1186/ar4133

33. Glasson SS, Askew R, Sheppard B, et al. Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis. Nature. 2005;434(7033):644648. doi:10.1038/nature03369

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Disease-modifying therapies for osteoarthritis | DDDT - Dove Medical Press

ABU DHABI, 22nd June, 2021 (WAM) -- The Ministry of Health and Prevention (MoHAP) and the Emirates Health Services (EHS) recently revealed innovative immunotherapy for cancer and viral infections in cooperation with Japans Kyoto University.

This came during the participation of the ministry and the EHS at the Arab Health 2021 which began in Dubai on 21st June and concludes on 24th June.

The treatment is based on the clinical application of the therapy using T cell preparation after it was discovered that such cells can fight cancer and viral infections. The T cell medicine will be produced using the iPS cell technology.

T Cell makes up a group of lymphocytes present in the blood and plays a major role in cellular immunity. It is possible to produce T cells in large numbers and store them in appropriate conditions to be administered to patients when needed.

Thus, by the success of this project, patients with cancer or viral infection may have great merit in which they can make very easy access to T cell therapy.

Strategic partnerships Dr. Youssef Mohamed Al Serkal, Director-General of the Emirates Health Services, spoke about the commitment of the ministry and the EHS to having strategic partnerships with the most prestigious medical research centres while keeping an eye on the sustainable investment in future healthcare services.

"Although the prevalence of cancer in the UAE is considered lower than in other parts of the world, we work hard to make a qualitative shift in cancer and viral infection healthcare," Al Serkal stated, adding, "This is part of our strategy to provide healthcare services in innovative and sustainable ways and implement the national strategy to reduce cancer mortality rates."

Al Serkal pointed out that the ministry and EHS support the National Cancer Control Programme and prepare a road map to achieve the target indicator. They also analyse the current status of cancer diseases and their diagnostic and therapeutic pathways, support research and studies on the control of cancer diseases and viral infections, and back workshops and educational and training activities. awareness campaigns, and innovative initiatives.

Dr. Kalthum Al Balushi, Director of Hospitals Department, said, "The ground-breaking treatment technology for cancer and viral infections, in cooperation with the Kyoto University, represents a paradigm shift in health services provided by the Ministry and the EHS."

The treatment is based on stimulating immune cells to fight cancer cells using pluripotent stem cells, which is a recent global trend that has begun to open great prospects for improving the quality of life of patients, Al Balushi added.

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MoHAP, EHS reveal immunotherapy for cancer, viral infections at Arab Health 2021 - WAM EN

BOSTON--(BUSINESS WIRE)--Gamida Cell Ltd. (Nasdaq: GMDA), an advanced cell therapy company committed to cures for blood cancers and serious hematologic diseases, today announced that the results of a Phase 3 clinical study of omidubicel have been published in Blood, the official journal of the American Society of Hematology. Omidubicel is an advanced cell therapy under development as a potential life-saving allogeneic hematopoietic stem cell transplant solution for patients with hematologic malignancies.

The results demonstrate that transplantation with omidubicel leads to faster neutrophil and platelet recovery compared to a standard umbilical cord blood graft, and results in fewer early bacterial and viral infections and less time in the hospital.

We are pleased that the data from this well-conducted international Phase 3 trial have been published in Blood, the highly respected, peer-reviewed journal of the American Society of Hematology, said Ronit Simantov, M.D., chief medical officer of Gamida Cell. The robust results of this clinical trial have demonstrated that omidubicel could provide an important new option for patients with hematologic malignancies in need of a bone marrow transplant.

Data from this study were previously presented at the Transplantation & Cellular Therapy Meetings of the American Society of Transplantation and Cellular Therapy and Center for International Blood & Marrow Transplant Research, and most recently during the Presidential Symposium at the 47th Annual Meeting of the European Society for Blood and Marrow Transplantation. The pivotal study was an international, multi-center, randomized Phase 3 trial designed to compare the safety and efficacy of omidubicel to standard umbilical cord blood transplant in patients with high-risk hematologic malignancies undergoing a bone marrow transplant.

Previous studies have shown that engraftment with omidubicel is durable, with some patients in the Phase 1/2 study now a decade past their transplant. The Phase 3 data reinforce omidubicels potential to be a new standard of care for patients who are in need of stem cell transplantation but do not have access to an appropriate matched donor, said Mitchell Horwitz, M.D., lead author of the paper and a professor of medicine at the Duke Cancer Institute.

The full Blood manuscript is available here: https://ashpublications.org/blood/article/doi/10.1182/blood.2021011719/476235/Omidubicel-Versus-Standard-Myeloablative-Umbilical.

Details of Phase 3 Efficacy and Safety Results Shared in Blood

The intent-to-treat analysis included 125 patients aged 1365 years with a median age of 41. Forty-four percent of the patients treated on study were non-Caucasian, a population known to be underrepresented in adult bone marrow donor registries. Patient demographics and baseline characteristics were well-balanced across the two study groups. Patients with acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome or lymphoma were enrolled at more than 30 clinical centers in the United States, Europe, Asia, and Latin America.

Gamida Cell previously reported in May 2020 that the study achieved its primary endpoint, showing that omidubicel demonstrated a statistically significant reduction in time to neutrophil engraftment, a measure of how quickly the stem cells a patient receives in a transplant are established and begin to make healthy new cells and a key milestone in a patients recovery from a bone marrow transplant. The median time to neutrophil engraftment was 12 days for patients randomized to omidubicel compared to 22 days for the comparator group (p<0.001).

All three secondary endpoints, details of which were first reported in December 2020, demonstrated a statistically significant improvement among patients who were randomized to omidubicel compared to patients randomized to standard cord blood graft. Platelet engraftment was significantly accelerated with omidubicel, with 55 percent of patients randomized to omidubicel achieving platelet engraftment at day 42, compared to 35 percent for the comparator (p = 0.028). Hospitalization in the first 100 days after transplant was also reduced in patients randomized to omidubicel, with a median number of days alive and out of hospital for patients randomized to omidubicel of 61 days, compared to 48 days for the comparator (p=0.005). The rate of infection was significantly reduced for patients randomized to omidubicel, with the cumulative incidence of first grade 2 or grade 3 bacterial or invasive fungal infection for patients randomized to omidubicel of 37 percent, compared to 57 percent for the comparator (p=0.027). Additional data reported in the manuscript included a comparison of infection density, or the number of infections during the first year following transplantation, which showed that the risk for grade 2 and grade 3 infections was significantly lower among recipients of omidubicel compared to control (risk ratio 0.5, p<0.001).

Data from the study relating to exploratory endpoints also support the clinical benefit demonstrated by the studys primary and secondary endpoints. There was no statistically significant difference between the two patient groups in incidence of grade 3/4 acute GvHD (14 percent for omidubicel, 21 percent for the comparator) or all grades chronic GvHD at one year (35 percent for omidubicel, 29 percent for the comparator). Non-relapse mortality was shown to be 11 percent for patients randomized to omidubicel and 24 percent for patients randomized to the comparator (p=0.09).

These clinical data results form the basis of a Biologics License Application (BLA) that Gamida Cell plans to submit to the U.S. Food and Drug Administration (FDA) in the fourth quarter of 2021.

About Omidubicel

Omidubicel is an advanced cell therapy under development as a potential life-saving allogeneic hematopoietic stem cell (bone marrow) transplants for patients with hematologic malignancies (blood cancers), for which it has been granted Breakthrough Status by the FDA. Omidubicel is also being evaluated in a Phase 1/2 clinical study in patients with severe aplastic anemia (NCT03173937). The aplastic anemia investigational new drug application is currently filed with the FDA under the brand name CordIn, which is the same investigational development candidate as omidubicel. For more information on clinical trials of omidubicel, please visit http://www.clinicaltrials.gov.

Omidubicel is an investigational therapy, and its safety and efficacy have not been established by the FDA or any other health authority.

About Gamida Cell

Gamida Cell is an advanced cell therapy company committed to cures for patients with blood cancers and serious blood diseases. We harness our cell expansion platform to create therapies with the potential to redefine standards of care in areas of serious medical need. For additional information, please visit http://www.gamida-cell.com or follow Gamida Cell on LinkedIn or Twitter at @GamidaCellTx.

Cautionary Note Regarding Forward Looking Statements

This press release contains forward-looking statements as that term is defined in the Private Securities Litigation Reform Act of 1995, including with respect to the potential for omidubicel to become a new standard of care and the anticipated submission of a BLA for omidubicel, which statements are subject to a number of risks, uncertainties and assumptions, including, but not limited to Gamida Cells ability to prepare regulatory filings and the review process therefor; complications in Gamida Cells plans to manufacture its products for commercial distribution; and clinical, scientific, regulatory and technical developments. In light of these risks and uncertainties, and other risks and uncertainties that are described in the Risk Factors section and other sections of Gamida Cells Annual Report on Form 20-F, filed with the Securities and Exchange Commission (SEC) on March 9, 2021, as amended on March 22, 2021, and other filings that Gamida Cell makes with the SEC from time to time (which are available at http://www.sec.gov), the events and circumstances discussed in such forward-looking statements may not occur, and Gamida Cells actual results could differ materially and adversely from those anticipated or implied thereby. Any forward-looking statements speak only as of the date of this press release and are based on information available to Gamida Cell as of the date of this release.

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Gamida Cell Announces Publication in Blood, the Journal of the American Society of Hematology, of the First Pivotal Trial to Evaluate a Cell Therapy...

Imagine being able to reverse blindness, cure multiple sclerosis (MS), or rebuild your heart muscles after a heart attack. For the past few decades, research into stem cells, the building blocks of tissues and organs, has raised the prospect of medical advances of this kind yet it has produced relatively few approved treatments. But that could be about to change, says Robin Ali, professor of human molecular genetics of Kings College London. Just as gene therapy went from being a fantasy with little practical value to becoming a major area of treatment, stem cells are within a few years of reaching the medical mainstream. Whats more, developments in synthetic biology, the process of engineering and re-engineering cells, could make stem cells even more effective.

Stem cells are essentially the bodys raw material: basic cells from which all other cells with particular functions are generated. They are found in various organs and tissues, including the brain, blood, bone marrow and skin. The primary promise of adult stem cells lies in regenerative medicine, says Professor Ali.

Stem cells go through several rounds of division in order to produce specialist cells; a blood stem cell can be used to produce blood cells and skin stem cells can be used to produce skin cells. So in theory you can take adult stem cells from one person and transplant them into another person in order to promote the growth of new cells and tissue.

In practice, however, things have proved more complicated, since the number of stem cells in a persons body is relatively limited and they are hard to access. Scientists were also previously restricted by the fact that adult stem cells could only produce one specific type of cell (so blood stem cells couldnt produce skin cells, for instance).

In their quest for a universal stem cell, some scientists initially focused on stem cells from human embryos, but that remains a controversial method, not only because harvesting stem cells involves destroying the embryo, but also because there is a much higher risk of rejection of embryonic stem cells by the recipients immune system.

The good news is that in 2006 Japanese scientist Shinya Yamanaka of Kyoto University and his team discovered a technique for creating what they call induced pluripotent stem cells (iPSC). The research, for which they won a Nobel Prize in 2012, showed that you can rewind adult stem cells development process so that they became embryo-like stem cells. These cells can then be repurposed into any type of stem cells. So you could turn skin stem cells into iPSCs, which could in turn be turned into blood stem cells.

This major breakthrough has two main benefits. Firstly, because iPSCs are derived from adults, they dont come with the ethical problems associated with embryonic stem cells. Whats more, the risk of the body rejecting the cells is much lower as they come from another adult or are produced by the patient. In recent years scientists have refined this technique to the extent that we now have a recipe for making all types of cells, as well as a growing ability to multiply the number of stem cells, says Professor Ali.

Having the blueprint for manufacturing stem cells isnt quite enough on its own and several barriers remain, admits Professor Ali. For example, we still need to be able to manufacture large numbers of stem cells at a reasonable cost. Ensuring that the stem cells, once they are in the recipient, carry out their function of making new cells and tissue remains a work in progress. Finally, regulators are currently taking a hard line towards the technology, insisting on exhaustive testing and slowing research down.

The good news, Professor Ali believes, is that all these problems are not insurmountable as scientists get better at re-engineering adult cells (a process known as synthetic biology). The costs of manufacturing large numbers of stem cells are falling and this can only speed up as more companies invest in the area. There are also a finite number of different human antigens (the parts of the immune system that lead a body to reject a cell), so it should be possible to produce a bank of iPSC cells for the most popular antigen types.

While the attitude of regulators is harder to predict, Professor Ali is confident that it needs only one major breakthrough for the entire sector to secure a large amount of research from the top drug and biotech firms. Indeed, he believes that effective applications are likely in the next few years in areas where there are already established transplant procedures, such as blood transfusion, cartilage and corneas. The breakthrough may come in ophthalmology (the treatment of eye disorders) as you only need to stimulate the development of a relatively small number of cells to restore someones eyesight.

In addition to helping the body repair its own tissues and organs by creating new cells, adult stem cells can also indirectly aid regeneration by delivering other molecules and proteins to parts of the body where they are needed, says Ralph Kern, president and chief medical officer of biotechnology company BrainStorm Cell Therapeutics.

For example, BrainStorm has developed NurOwn, a cellular technology using peoples own cells to deliver neurotrophic factors (NTFs), proteins that can promote the repair of tissue in the nervous system. NurOwn works by modifying so-called Mesenchymal stem cells (MSCs) from a persons bone marrow. The re-transplanted mesenchymal stem cells can then deliver higher quantities of NTFs and other repair molecules.

At present BrainStorm is using its stem-cell therapy to focus on diseases of the brain and nervous system, such as amyotrophic lateral sclerosis (ALS, also known as Lou Gehrigs disease), MS and Huntingtons disease. The data from a recent final-stage trial suggests that the treatment may be able to halt the progression of ALS in those who have the early stage of the disease. Phase-two trial (the second of three stages of clinical trials) of the technique in MS patients also showed that those who underwent the treatment experienced an improvement in the functioning of their body.

Kern notes that MSCs are a particularly promising area of research. They are considered relatively safe, with few side effects, and can be frozen, which improves efficiency and drastically cuts down the amount of bone marrow that needs to be extracted from each patient.

Because the manufacture of MSC cells has become so efficient, NurOwn can be used to get years of therapy in one blood draw. Whats more, the cells can be reintroduced into patients bodies via a simple lumbar puncture into the spine, which can be done as an outpatient procedure, with no need for an overnight stay in hospital.

Kern emphasises that the rapid progress in our ability to modify cells is opening up new opportunities for using stem cells as a molecular delivery platform. Through taking advantage of the latest advances in the science of cellular therapies, BrainStorm is developing a technique to vary the molecules that its stem cells deliver so they can be more closely targeted to the particular condition being treated. BrainStorm is also trying to use smaller fragments of the modified cells, known as exosomes, in the hope that these can be more easily delivered and absorbed by the body and further improve its ability to avoid immune-system reactions to unrelated donors. One of BrainStorms most interesting projects is to use exosomes to repair the long-term lung damage from Covid-19, a particular problem for those with long Covid-19. Early preclinical trials show that modified exosomes delivered into the lungs of animals led to remarkable improvements in their condition. This included increasing the lungs oxygen capacity, reducing inflammation, and decreasing clotting.

Overall, while Kern admits that you cant say that stem cells are a cure for every condition, there is a lot of evidence that in many specific cases they have the potential to be the best option, with fewer side effects. With Americas Food and Drug Administration recently deciding to approve Biogens Alzheimers drug, Kern thinks that they have become much more open to approving products in diseases that are currently considered untreatable. As a result, he thinks that a significant number of adult stem-cell treatments will be approved within the next five to ten years.

Adult stem cells and synthetic biology arent just useful in treatments, says Dr Mark Kotter, CEO and founder of Bit Bio, a company spun out of Cambridge University. They are also set to revolutionise drug discovery. At present, companies start out by testing large numbers of different drug combinations in animals, before finding one that seems to be most effective. They then start a process of clinical trials with humans to test whether the drug is safe, followed by an analysis to see whether it has any effects.

Not only is this process extremely lengthy, but it is also inefficient, because human and animal biology, while similar in many respects, can differ greatly for many conditions. Many drugs that seem promising in animals end up being rejected when they are used on humans. This leads to a high failure rate. Indeed, when you take the failures into account, it has been estimated that it may cost as much to around $2bn to develop the typical drug.

As a result, pharma companies are now realising that you have to insert the human element at a pre-clinical stage by at least using human tissues, says Kotter. The problem is that until recently such tissues were scarce, since they were only available from biopsies or surgery. However, by using synthetic biology to transform adult stem cells from the skin or other parts of the body into other types of stem cells, researchers can potentially grow their own cells, or even whole tissues, in the laboratory, allowing them to integrate the human element at a much earlier stage.

Kotter has direct experience of this himself. He originally spent several decades studying the brain. However, because he had to rely on animal tissue for much of his research he became frustrated that he was turning into a rat doctor.

And when it came to the brain, the differences between human and rat biology were particularly stark. In fact, some human conditions, such as Alzheimers, dont even naturally appear in rodents, so researchers typically use mice and rats engineered to develop something that looks like Alzheimers. But even this isnt a completely accurate representation of what happens in humans.

As a result of his frustration, Kotter sought a way to create human tissues. It initially took six months. However, his company, Bit Bio, managed to cut costs and greatly accelerate the process. The companys technology now allows it to grow tissues in the laboratory in a matter of days, on an industrial scale. Whats more, the tissues can also be designed not just for particular conditions, such as dementia and Huntingdons disease, but also for particular sub-types of diseases.

Kotter and Bit Bio are currently working with Charles River Laboratories, a global company that has been involved in around 80% of drugs approved by the US Food and Drug Administration over the last three years, to commercialise this product. They have already attracted interest from some of the ten largest drug companies in the world, who believe that it will not only reduce the chances of failure, but also speed up development. Early estimates suggest that the process could double the chance of a successful trial, effectively cutting the cost of each approved drug by around 50% from $2bn to just $1bn. This in turn could increase the number of successful drugs on the market.

Two years ago my colleague Dr Mike Tubbs tipped Fate Therapeutics (Nasdaq: FATE). Since then, the share price has soared by 280%, thanks to growing interest from other drug companies (such as Janssen Biotech and ONO Pharmaceutical) in its cancer treatments involving genetically modified iPSCs.

Fate has no fewer than seven iPSC-derived treatments undergoing trials, with several more in the pre-clinical stage. While it is still losing money, it has over $790m cash on hand, which should be more than enough to support it while it develops its drugs.

As mentioned in the main story, the American-Israeli biotechnology company BrainStorm Cell Therapeutics (Nasdaq: BCLI) is developing treatments that aim to use stem cells as a delivery mechanism for proteins. While the phase-three trial (the final stage of clinical trials) of its proprietary NurOwn system for treatment of Amyotrophic lateral sclerosis (ALS, or Lou Gehrigs disease) did not fully succeed, promising results for those in the early stages of the disease mean that the company is thinking about running a new trial aimed at those patients. It also has an ongoing phase-two trial for those with MS, a phase-one trial in Alzheimers patients, as well as various preclinical programmes aimed at Parkinsons, Huntingtons, autistic spectrum disorder and peripheral nerve injury. Like Fate Therapeutics, BrainStorm is currently unprofitable.

Australian biotechnology company Mesoblast (Nasdaq: MESO) takes mesenchymal stem cells from the patient and modifies them so that they can absorb proteins that promote tissue repair and regeneration. At present Mesoblast is working with larger drug and biotech companies, including Novartis, to develop this technique for conditions ranging from heart disease to Covid-19. Several of these projects are close to being completed.

While the US Food and Drug Administration (FDA) controversially rejected Mesoblasts treatment remestemcel-L for use in children who have suffered from reactions to bone-marrow transplants against the advice of the Food and Drug Administrations own advisory committee the firm is confident that the FDA will eventually change its mind.

One stem-cell company that has already reached profitability is Vericel (Nasdaq: VCEL). Vericels flagship MACI products use adult stem cells taken from the patient to grow replacement cartilage, which can then be re-transplanted into the patient, speeding up their recovery from knee injuries. It has also developed a skin replacement based on skin stem cells.

While earnings remain relatively small, Vericel expects profitability to soar fivefold over the next year alone as the company starts to benefit from economies of scale and runs further trials to expand the range of patients who can benefit.

British micro-cap biotech ReNeuron (Aim: RENE) is developing adult stem-cell treatments for several conditions. It is currently carrying out clinical trials for patients with retinal degeneration and those recovering from the effects of having a stroke. ReNeuron has also developed its own induced pluripotent stem cell (iPSC) platform for research purposes and is seeking collaborations with other drug and biotech companies.

Like other small biotech firms in this area, it is not making any money, so it is an extremely risky investment although the rewards could be huge if any of its treatments show positive results from their clinical trials.

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Investing in stem cells, the building blocks of the body - MoneyWeek

INTRODUCTION

Mitochondrial diseases are a heterogeneous group of devastating disorders characterized by respiratory chain dysfunction (1). Although mitochondrial disorders have distinct tissue and organ presentation, they seem to activate common stress responses evolved to mitigate the negative impact of respiratory deficiency on cellular and organismal metabolism (1). It appears that mitochondrial stress responses precede respiratory chain deficiency, thereby suggesting that they constitute an early event in pathogenesis of mitochondria-related diseases (2). This suggests that monitoring the activation and/or alteration of mitochondrial stress responses may provide early diagnostic markers in these conditions. Moreover, manipulation of mitochondrial stress responses may be beneficial for patients with mitochondrial disease and thus therapeutically exploited (3, 4).

Initially, the mitochondrial unfolded protein response (UPRmt) was postulated to be a common stress response to respiratory chain dysfunction (5). UPRmt constitutes a transcriptional program that up-regulates mitochondrial chaperones and proteases aimed to restore the loss of organelle proteostasis. Notwithstanding that UPRmt was first described to be triggered by the accumulation of misfolded proteins within the mitochondrial matrix in mammalian cells (5), most of the subsequent mechanistic studies were performed in Caenorhabditis elegans (6). In contrast, many aspects of the mammalian UPRmt signaling are less well understood. In mammalian cells, it is thought that mitochondrial proteotoxic stress leads to CHOP [CCAAT/enhancer binding protein (C/EBP) homology protein] up-regulation resulting in up-regulated transcription of UPRmt-responsive genes (5, 7). The CHOP-binding sites in the UPRmt gene promoters are presumably flanked by two conserved regions named the mitochondrial UPR elements 1 and 2 (MURE1 and MURE2) (7, 8). The role of CHOP in governing transcription of UPRmt genes is however controversial as the transcription factors that bind to MURE1 and MURE2 elements have not been identified (7, 9). Nevertheless, multiple studies confirmed up-regulation of the CHOP mRNA in cells derived from patients with various mitochondrial disorders, as well as mitochondrial disease models (2, 1012). This illustrates that although CHOP plays a pivotal role in mammalian mitochondrial stress responses, the underpinning mechanisms of its actions in the context of mitochondrial dysfunction are still obscure.

Recently, it became clear that unlike in C. elegans, mammalian UPRmt may not be the primary response to mitochondrial dysfunction but rather function as a part of more complex mitochondrial stress response (1114). Mammalian cells treated with mitochondrial toxins exhibit transcriptional reprogramming mimicking the integrated stress response (ISR) arm of the UPR, which is centered on the activating transcription factor 4 (ATF4) (13, 14). Consistent with this, studies carried out in models with defects in different steps of mitochondrial DNA (mtDNA) expression and protein synthesis revealed activation of ISR transcriptional signatures (11, 12). ISR hallmarks are increased eIF2 phosphorylation, reduction in ternary eIF2:tRNAiMet:guanosine 5-triphosphate (GTP) complex levels, and subsequent inhibition of global protein synthesis that is paralleled by selectively induced translation of a subset of inhibitory upstream open reading frame (uORF) containing stress-responsive mRNAs, including ATF4, CHOP, and GADD34 (15). CHOP induction during ISR is thought to lead to cell death via induction of Growth Arrest and DNA Damage-Inducible Protein 34 (GADD34)mediated eIF2a dephosphorylation and activation of Endoplasmic Reticulum Oxidoreductase 1 Alpha (ERO1A) endoplasmic reticulum (ER) oxidase (16).

CHOP is a multifunctional transcription factor that dimerizes with members of the C/EBP and ATF/cyclic adenosine 3,5-monophosphate response element binding protein families (17). Although up-regulated in response to a wide variety of stresses such as growth arrest and DNA damage, amino acid and glucose deprivation, hypoxia, and ER stress, the role of CHOP in cellular physiology is incompletely understood. CHOP is considered to induce apoptosis, but its transcriptional targets largely overlap with those of ATF4, including genes promoting cell survival and growth (16, 18). These findings highlight the intricate interaction partnerdependent roles of CHOP under different stresses and in various tissues. They also point out the importance of understanding the context-dependent role of CHOP under different physiological conditions. In the context of mitochondrial respiratory chain dysfunction, the role of CHOP is particularly important as CHOP was proposed to be the main transcription factor that conveys specificity of the mitochondrial stress response (5).

Here, we aimed to decipher the role of CHOP in the regulation of the mitochondrial stress response. As a model for the most common cause of mitochondrial diseases, namely, loss of mitochondrial translation, we used mice deficient in the mitochondrial aspartyl transfer RNA (tRNA) synthase DARS2 specifically in heart and skeletal muscle (DARS2 KO) (2). We demonstrate a beneficial role of CHOP in mitochondrial mutants as its loss leads to a marked shortening of life span in DARS2/CHOP double knockout (DKO) as compared to DARS2 KO animals. The beneficial effects of CHOP appear to be independent of UPRmt activation but rather mediated by attenuation of harmful overactivation of the ISR and a consequent metabolic imbalance. We also provide mechanistic evidence that these effects stem from the interplay between CHOP, ATF4, and C/EBP in regulation of mitochondrial ISR targets.

To determine the in vivo function of CHOP in the context of mammalian mitochondrial dysfunction, we intercrossed whole-body Chop/ mice (CHOP KO) with heart and skeletal muscle-specific DARS2-deficient mice (Dars2fl/fl; Ckmm-Cre+/tg; DARS2 KO) (fig. S1, A and B) (2). The resulting animals deficient in both CHOP (whole body) and DARS2 (heart and skeletal muscle) (Dars2fl/fl; Ckmm-Cre+/tg; Chop/ and DKO) were born in Mendelian ratios (fig. S1C). We previously showed that DARS2 depletion mediated by Ckmm-Cre expression induces dilated cardiomyopathy preceding any pathological phenotypes in skeletal muscle (2). Hence, we monitored the effects of CHOP loss on pathologies caused by DARS2 abrogation in the heart.

Approximately from 2 weeks of age, a large number of DKO mice became increasingly susceptible to sudden death during a routine ear-clipping handling for genotyping. This procedure was tolerated well up to postnatal day 13 (P13) by mice of all four genotypes; hence P13 (1) was defined as the early stage of heart dysfunction in DKO animals (DKOE). It appeared that the deterioration of the health status of DKO mice characterized by lower spontaneous cage activity, piloerection, unsteady gait, and overall droopiness is a very rapid process as the interval from the first apparent symptoms to death of the mice at around P17 (2) was between 24 and 48 hours. This interval was defined as the late/terminal stage in DKO mice (DKOL). Consequently, the life expectancy of DKOs was severely reduced (>60%) compared to DARS2 KOs, signifying the essential role for CHOP in adaptation to impaired mitochondrial protein synthesis in heart (Fig. 1A). CHOP deficiency in the absence of DARS2 resulted in dilated cardiomyopathy (Fig. 1B and fig. S1, D to F) characterized by increased expression of mRNAs encoding cardiac hypertrophy markers Nppa and Nppb (Fig. 1C). Although no gross morphological changes were observed upon hematoxylin and eosin (H&E) staining, ultrastructural analyses suggested a disrupted myocardial organization, characterized by severely disorganized sarcomeric structures, expected to cause disturbances in contractile function of DKOL hearts (Fig. 1, D and E). Therefore, DKOL animals display very similar pathological changes, as compared to the terminal stage DARS2 KO mice (2), whereby the onset of these pathologies is markedly accelerated upon CHOP loss.

(A) Kaplan-Meyer survival curves for wild-type (WT; n = 36), CHOP KO (n = 35), DARS2 KO (n = 47), and DKO animals (n = 60). The life span of DKO in comparison to DARS2 KO mice is significantly decreased (P < 0.0001; log-rank test and Gehan-Breslow-Wilcoxon test). The viability of CHOP KO mice was WT-like in a 12-month follow-up. (B) Heart gross morphology. (C) Fold changes of the cardiac hypertrophy markers Nppa and Nppb obtained from the RNA sequencing dataset at P17 (2) (n = 4). Bars represent means SD [multivariate analysis of variance (MANOVA) followed by one-way ANOVA and Tukeys multiple comparisons test, **P < 0.05, ***P < 0.001, and ****P < 0.0001]. (D) H&E staining; (n = 3) at P17 (2). Scale bars, 50 m. (E) Transmission electron microscopybased analyses of cardiac tissue biopsies; (n = 1) at P17 (2). Scale bars, 2 m.

We next sought to identify pathways that are affected by the CHOP deficiency in the context of DARS2 KO. To this end, we compared global changes in mRNA levels to corresponding changes in the proteome in CHOP KO, DARS2 KO, and DKOL versus control hearts using the anota2seq algorithm (19). Scatter plots comparing mRNA and protein changes in DARS2 KO hearts revealed alterations in protein levels that were mainly independent of the mRNA levels, thus arguing for a prevalent impact of translational and/or protein stability changes on the proteome (Fig. 2A, fig. S2A, and table S1). In contrast, DKOL animals primarily showed congruent changes in mRNA and protein levels, which accounted for ~75% of detected alterations in protein levels (Fig. 2A, fig. S2A, and table S1).

(A) Scatter plots of total mRNA and protein fold changes (FC) comparing CHOP KO, DARS2 KO, or DKOL to WT. The numbers of significantly regulated genes are indicated for translation/protein stability (red), and mRNA abundance (green). RNA sequencing and quantitative proteomics were performed on hearts of animals at P17 (2) (n = 4). (B) A GO network of overrepresented terms among genes regulated via changes in translation/protein stability (up-regulated, light red; down-regulated, dark red) and mRNA abundance (up-regulated, light green; down-regulated, dark green) in DKO versus WT. Nodes represent identified GO terms, while the pie chart within each node indicates the proportion of genes regulated. (C and D) Heatmap of protein expression (P) and total mRNA (T) log2 fold changes of (C) the OXPHOS subunits grouped in respective complexes and (D) OXPHOS assembly factors (n = 4). (E) In organello translation assay (left) of cardiac mitochondria at P17 (2). De novo protein synthesis was determined after 1 hour of 35S-methionine pulse labeling; protein turnover was assessed after 3 hours of the cold chase. Coomassie brilliant bluestained gel was used as a loading control. Relative protein synthesis and turnover rates (right) (n = 3). (F) Oxygen consumption of intact cardiac mitochondria at P17 (2). State 3: adenosine 5-diphosphate (ADP)stimulated respiration using CI or CI + CII substrates. State 4: Respiration upon addition of oligomycin. ETS, maximum respiration upon mitochondrial uncoupling (CI) and after addition of rotenone (CII) (n = 3 to 4). Bars represent means SD (MANOVA followed by one-way ANOVA and Tukeys multiple comparisons test, *P < 0.05, **P < 0.01, and ***P < 0.001).

Gene Ontology (GO) analysis performed using ClueGO (20) and annotation from the GO consortium (21) on genes whose expression was reduced indicated that oxidative phosphorylation, electron transport, complex I assembly, adenosine 5-triphosphate (ATP) biosynthesis, fatty acid oxidation, and heart contraction are predominantly disrupted in DKO hearts (Fig. 2B). This is consistent with the impairment of mitochondrial energy production and heart failure in DKO animals and similar to other models of mitochondrial cardiomyopathy (11). In contrast, translation, tRNA metabolism, mitochondrial RNA, and glutathione metabolism were primarily up-regulated pathways (Fig. 2B). We observed further perturbations in apoptotic pathways, amino acid catabolism, and purine nucleotide metabolism that contained a combination of up- and down-regulated gene expression changes (Fig. 2B).

A general down-regulation of steady-state levels of individual oxidative phosphorylation (OXPHOS) subunits detected in DARS2 KO hearts was further decreased in DKOL animals (Fig. 2C and fig. S2B). Intriguingly, while in DARS2 KO animals, most of the changes in the levels of OXPHOS subunits were not accompanied by alterations in mRNA abundance, numerous OXPHOS subunit-encoding genes exhibited congruent changes in mRNA and protein levels in DKOL animals (Fig. 2C). These include three of four subunits of succinate dehydrogenase (SDH; complex II), a complex fully encoded by nuclear DNA, usually up-regulated upon mitochondrial translation defects. This was further confirmed using an enzyme-histochemical assay, showing that substantial cyclooxygenase (COX) deficiency observed in DKOL animals is not accompanied by a compensatory SDH up-regulation (fig. S2C), as observed in DARS2 and other mitochondrial mutants (2, 22). Furthermore, while we detected a general compensatory up-regulation of OXPHOS assembly factors in DARS2 KO hearts, many were either unaltered or down-regulated in DKOL samples (Fig. 2D).

Although Dars2 deletion primarily interferes with mitochondrial protein synthesis, at P17, only a moderately dysbalanced mitochondrial translation was observed in DARS2 KO (Fig. 2E). In contrast, mitochondrial de novo protein synthesis in DKOL mice was significantly decreased and severely dysregulated, whereas the protein turnover remained unaffected (Fig. 2E). The exaggerated translation defect observed in DKOL animals was not caused by a decrease in mtDNA or mtRNA levels (fig. S2, D and E). Some mtRNAs were up-regulated (e.g., mt-COX3 and mt-ND1) in both DARS2 KO and DKO hearts, possibly as a compensatory response to defective protein synthesis (Fig. 2E and fig. S2E).

Severe dysregulation of mitochondrial translation in DKOL was accompanied with a strong decrease in the respiration capacity of all inducible states in mitochondria isolated from DKOL hearts (Fig. 2F). In contrast, no major defects in DARS2 KO heart mitochondria respiration were observed, thus suggesting compensation for the mitochondrial protein synthesis defect (Fig. 2F).

Unexpectedly, a comparable defect at the level of assembled respiratory chain complexes and supercomplexes was detected in DARS2 KO and DKOL mice despite higher levels of individual OXPHOS subunits in DARS2 KO (Fig. 2C and fig. S2F). These data suggest that, at early stages of DARS2 deficiency, nascent nuclear-encoded OXPHOS subunits are not efficiently incorporated in respiratory chain complexes in DARS2 KO hearts and are likely turned over at higher rates. Although DKOL and DARS2 KO mitochondria have comparable levels of respiratory chain supercomplexes (fig. S2F), DKOL mitochondria fail to sustain normal respiration (Fig. 2F). This suggests that the OXPHOS activity is further indirectly affected by CHOP deficiency that might lead to disruption of mitochondrial integrity or supply of critical metabolites.

CHOP deficiency in the context of mitochondrial dysfunction is expected to blunt the mitochondrial stress response (5). Therefore, by analyzing changes in the transcriptome, we compared pathways that are affected in DARS2-deficient hearts before and after CHOP depletion (table S2).

In DARS2 KO heart, relatively few mRNAs changed their expression, and most were up-regulated. Notably, using Cytoscape plug-in iRegulon, we demonstrated that two-thirds of these transcripts overlapped with an ISR signature activated by ATF4, which was also identified as the most prominent regulator of gene expression in DARS2 KO hearts (Fig. 3A and tables S2 and S3) (18, 23, 24). The most up-regulated transcripts in DARS2 KO hearts encoded enzymes involved in one-carbon metabolism, serine biosynthesis, and trans-sulfuration, as well as Gdf15 and Fgf21 (Fig. 3, A and B, and table S2), the two cytokines shown to be excreted from tissues upon OXPHOS deficiency (25, 26). Similar changes (Fig. 3A) were previously described in other cellular and in vivo models for mitochondrial OXPHOS defects, confirming that DARS2 deficiency causes a stress response relevant for many mitochondrial disease states (1114).

(A) Heatmap of total mRNA fold changes (log2) of significantly changed ATF4 target genes [as predicted by Cytoscape plug-in iRegulon (23, 24)], in DARS2 KO animals compared to WT controls. Black boxes above DKO and below CHOP KO rows indicate their respective significantly changed transcripts as compared to WT controls (n = 4). (B) Fgf21 log2 raw expression counts (sequenced reads, +0.5) as this gene was not detected in multiple WT and CHOP KO samples and hence was excluded during data filtering. Of note, these samples will obtain negative log2 values (n = 4). (C) Western blot analysis (left) and quantification of ISR markers (right). HSC70 was used as a loading control. Antibodies used were raised against proteins indicated in panels. Experiments were performed on cardiac lysates of mice at P17 (2) (n = 3). (D) p-eIF2/eIF2 ratio quantified from (C). (B to D) Bars represent means SD (MANOVA followed by one-way ANOVA and Tukeys multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). (E) Western blot analysis and quantification of UPRmt markers in WT, CHOP KO, DARS2 KO, and DKO animals at P17 (2). Antibodies used were raised against proteins indicated in panels. HSC70 was used as a loading control. Bars represent means SD; no significant differences were detected (MANOVA: Wilks test, P = 0.176; Hotelling-Lawleys test, P = 0.183; Pollais test, P = 0.232) (n = 3). (F) Heatmap of total mRNA fold changes (log2) for the selected alleged CHOP target genes involved in apoptosis (n = 4).

The ISR activation in DARS2 KO hearts was confirmed by increased eIF2 phosphorylation, accompanied by up-regulation of ATF4 (Fig. 3, C and D). These effects were further potentiated by CHOP loss, whereby induction of both eIF2 phosphorylation and ATF4 was more pronounced in DKOL relative to DARS2 KO hearts (Fig. 3, C and D). Transcript and protein levels of almost all ATF4 targets were highly up-regulated in DKOL as compared to DARS2 KO animals (Fig. 3, A to C). Consistently, further analysis of binding motifs in genes up-regulated in DKOL hearts established ATF4 as the most prominent signature (table S3) (23, 24). The most up-regulated transcripts in DARS2 KO and DKOL showed a notable overlap. To this end, of the top 11 most up-regulated transcripts, 8 overlapped, despite the 40-fold difference in the number of overall changes between the two models (table S2). The only difference was that these transcripts were, on average, more than 10-fold more up-regulated in DKOL than in DARS2 KO hearts (table S2). In contrast, UPRmt markers were not significantly changed in DARS2 KO or DKOL animals, adding evidence that UPRmt is neither an early nor prominent stress response in mammalian cells and tissues upon mitochondrial OXPHOS dysfunction (Fig. 3E). Instead, our data suggest a central role for ISR and ATF4-dependent regulation in the context of mitochondrial dysfunction in vivo and point to an unexpected role of CHOP in the suppression of the transcriptional overactivation of ATF4 targets.

CHOP is proposed to be involved in the regulation of apoptosis upon ER stress, although the exact mechanism remains controversial, as exogenously expressed CHOP has also been reported to positively regulate genes involved in protein synthesis and not apoptosis (16, 18). Henceforth, we analyzed changes in the expression levels of various apoptotic genes reported to be CHOP targets (27). Notably, proapoptotic members of the B-cell lymphoma 2 (BCL-2) family (Puma/Bbc3, Bid, Bax, and Bim/Bcl2l11) and genes encoding proteins involved in the activation or execution of apoptosis (Dr5/Tnfrsf10b, Casp3, and Ero1l) were not suppressed but often further up-regulated upon loss of CHOP in DARS2-deficient animal (Fig. 3F). Similarly, the steady-state level of proapoptotic protein BCL2-associated X protein (BAX) was up-regulated, and we observed a higher cleavage of caspase 3 in DKOL hearts as compared to control animals (fig. S3A). These results suggest that, unexpectedly, apoptosis may be up-regulated in DARS2-deficient hearts upon CHOP depletion and thus contribute to the detrimental phenotype observed in DKOL mice.

As we observed major changes in the abundance of proteins involved in amino acid metabolism, we next measured amino acid levels by liquid chromatographytandem mass spectrometry. While only minor perturbations in amino acid levels were observed in DARS2 KO hearts, most amino acids were significantly up-regulated in DKO mice (fig. S3B). Of note, serine, glutamine, glutamate, and aspartate levels were not significantly changed in either DARS2 KO or DKOL relative to control hearts (fig. S3B). The unaltered serine levels, despite the increased levels of enzymes involved in serine synthesis [Phosphoglycerate dehydrogenase (PHGDH), Phosphoserine Aminotransferase 1 (PSAT1) , and Phosphoserine Phosphatase (PSPH)], suggest an increased flux of serine-derived one-carbon units for further methylation reactions into the one-carbon cycle. Similarly, glutamine and glutamate are likely used to replenish tricarboxylic acid cycle intermediates and aspartate production that is essential for nucleotide synthesis and cell proliferation (28, 29). Increased levels of citrate and isocitrate in DKOL, but not DARS2 KO, hearts suggest that glutamine primarily undergoes reductive metabolism (fig. S3C), as seen in the patient-derived cell lines harboring mtDNA mutations (30). Increased citrate levels can propagate intracellular acidosis, leading to hypocalcemia caused by reduced availability of Ca2+, further contributing to reduced contractility of the heart through a vicious circle of the excitation-contraction-metabolism impairment (31). Additional effects of elevated citrate levels on the regulation of metabolic enzyme and/or chromatin dynamics by acetylation may further contribute to accelerated pathological phenotypes observed in DKOL hearts.

Next, we tested whether mitochondrial stressinduced ISR has a beneficial or detrimental role in conditions of mitochondrial dysfunction. For these analyses, we took advantage of two cell models for mitochondrial respiratory chain dysfunction: (i) mouse skin fibroblasts with severe mitochondrial dysfunction caused by the loss of COX10 (COX10 KO), an early assembly factor of the respiratory cytochrome c oxidase (32); and (ii) mouse embryonic fibroblasts (MEFs) treated with actinonin, an inhibitor of mitochondrial peptide deformylase causing impairment in mitochondrial translation (33).

In the COX10 KO cells, a robust activation of the ISR was detected as evidenced by increased levels of phosphorylated eIF2, ATF4, and ATF4 targets (Fig. 4A and fig. S4A). To test whether increased ATF4 levels are a direct result of ISR activation, we incubated COX10 KO cells with the ISR inhibitor (ISRIB) (34). This treatment abrogated ATF4 induction and attenuated up-regulation of its downstream targets at both transcript and protein levels (Fig. 4A and fig. S4A). The phosphorylation of eIF2 remained unchanged (Fig. 4A), which was expected as ISRIB bolsters guanine-nucleoside exchange activity of eIF2B without affecting on phospho-eIF2 levels (34). Similarly, increased ATF4 levels induced by actinonin treatment were suppressed by ISRIB (Fig. 4B). Mirroring the results from DKOL mice, loss of CHOP combined with mitochondrial dysfunction induced by actinonin treatment greatly increased ATF4 protein and transcript levels, and expression of ATF4 targets Shmt2, Pycr1, and Mthfd2 (Fig. 4, B and C).

(A) Western blot analysis (left) and relative protein levels (right) of ISR markers and ATF4 downstream targets in immortalized COX10 KO and WT fibroblasts upon 48-hour treatment with DMSO () or ISRIB (+). (B) Western blot analysis of WT and CHOP KO MEFs treated for 48 hours with DMSO () or actinonin (+) in the presence (+) or absence () of ISRIB during the last 4 hours before protein isolation. (C) Relative transcript levels in WT and CHOP KO MEFs treated for 48 hours with DMSO (control) or actinonin. Tbp expression was used for normalization (n = 3). (D) Growth curves of respective exponential growth phases of WT and CHOP KO MEFs treated with DMSO (control), actinonin, and +/ISRIB, respectively. Curves were determined using linear regression (n = 3). Bars represent means SD. (E) Western blot analysis of heart lysates from 4-week-old WT and DARS2 KO animals treated with control (DMSO) and ISRIB, according to the experimental setup presented in the schematic illustration (top). Animals are treated with daily injections of saline (control) or ISRIB solution for 7 days (blue boxes), starting at P19, and euthanized at P27 (red line) (n = 3). (F) Quantification of ISR markers (top), OXPHOS subunits (bottom), and p-eIF2/eIF2 ratio (right) from the Western blot analysis at (E). (A, B, and F) Antibodies used were raised against proteins indicated in panels. HSC70 was used as a loading control. (A, C, and F) Bars represent means SD (MANOVA followed by one-way ANOVA and Tukeys multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Prevention of ISR overactivation in CHOP KO MEFs by ISRIB treatment resulted in a partial rescue of the proliferation defect induced by actinonin (Fig. 4D). In turn, wild-type (WT) cells treated with actinonin and CHOP KO cells grown under control conditions showed minor growth defects, which were not further affected by ISRIB (Fig. 4D). Therefore, CHOP deficiency, only in conditions of mitochondrial dysfunction, results in a detrimental ISR activation, which can be partially rescued by ISRIB treatment.

To assess the effect of ISRIB treatment in vivo, DKOL mice and respective controls were injected with ISRIB (5 g/g) for up to 7 or 12 days, starting from 1 week of age (fig. S4B). Unfortunately, neither protocol resulted in the suppression of ATF4 levels or downstream targets in either DKO or DARS2 KO animals nor did it affect steady-state levels of OXPHOS subunits (fig. S4, C and D). However, this is not unexpected given the fact that ISRIB inhibits low-level ISR activity but does not affect strong ISR signaling (35), as observed in DKO mice. In contrast, a 7-day treatment of DARS2 KO animals with ISRIB, starting from P20, resulted in an apparent reduction of ISR markers (Fig. 4, E and F). Nevertheless, ISRIB-mediated suppression of ISR in DARS2 KO animals up to 4 weeks of age was not beneficial as it also prevented compensatory complex II (CII) up-regulation.

One of the hallmarks of the acute ISR is suppression of global protein synthesis, accompanied by translational activation of some uORF-containing mRNAs (15). To further understand the consequences of ISR activation in our model, we measured the global protein synthesis rate at P6, P13, and P17 in vivo in DKO and control hearts (36). At P6, cytoplasmic translation of all four genotypes was similar, in agreement with no phenotypes observed at this time point (fig. S5A). Coinciding with increased eIF2 phosphorylation, a 70% decrease in general protein synthesis was detected in mice at P13 (DKOE; Fig. 5A and fig. S5B). Within a few days, this effect seems to be reversed as we detected fully recovered protein synthesis rates in DKOL hearts at P17 (Fig. 5B and fig. S5C). This was despite unaltered eIF2 phosphorylation levels and activation of ATF4 and its targets that were comparable between DKOE and DKOL hearts (fig. S5D). These findings suggested a transition from acute to prolonged ISR, characterized by recovery of global protein synthesis and ongoing translation of ISR-sensitive mRNAs (37). These distinctions in global protein synthesis levels reflected different phenotypes of DKOE and DKOL mice. In the acute ISR, when global translation is strongly down-regulated, DKOE (P13 1) animals cope better with the mitochondrial translation defect when compared to DARS2 KO animals (Fig. 5C). This is illustrated by the unaffected levels of OXPHOS complexes and supercomplexes in DKOE animals (Fig. 5D and fig. S5E). However, these effects are reversed when DKO animals reach the prolonged ISR stage, which is characterized by partial recovery of global mRNA translation and sustained ATF4-mediated transcriptional reprograming (fig. S5D). This reactivation of normal translation is likely to result in ER stress, and further energy crisis as protein synthesis is highly energy demanding (38). Consistently, we detected increased levels of the ER-chaperone binding immunoglobulin protein (BIP) in P17 DKOL hearts, which mirrored findings in DARS2-deficient hearts at the terminal state of 6 weeks of age (Fig. 5E). Levels of several ER Ca2+ transporter proteins were also profoundly disturbed [Ryanodine receptors (RyR), Sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2), and The inositol 1,4,5-trisphosphate receptor type 2 (IP3RII)], which may explain defects in the conductive system of the heart (Fig. 5F). Perturbed Ca2+ homeostasis due to the dysregulation of the ER Ca2+ transporters and increased Ca2+ release by ERO1-stimulated IP3R activation may also contribute to ER stress leading to the development of fatal cardiomyopathy (Fig. 5, E and F). Therefore, although strong activation of ISR, as seen in DKOE animals, brings brief protection from the mitochondrial dysfunction, it cannot be sustained over prolonged period of time and results in a detrimental switch to a prolonged ISR program leading to additional ER stress, loss of Ca2+ homeostasis, and premature death.

(A and B) The relative protein synthesis rate of animals injected with puromycin at (A) P13 and (B) P17. Bars represent means SD (one-way ANOVA and Tukeys multiple comparisons test, **P < 0.01 and ***P < 0.001) (n = 4). (C) De novo synthesis in mitochondria isolated from WT, CHOP KO, DARS2 KO, and DKOE and DKOL animals after 1 hour of 35S-methionine pulse labeling followed by SDS-PAGE. Coomassie bluestained gel was used as a loading control. (D) Blue native polyacrylamide gel electrophoresis (BN-PAGE) and subsequent Western blot analysis of OXPHOS complexes and supercomplexes in mitochondria isolated from WT, CHOP DO, DARS2 KO, and early (DKOE) and late-stage (DKOL) DKO animals. Subunit-specific antibodies (left) were used to detect respective complexes and supercomplexes (right) (n = 3). (E) Western blot analysis of BIP levels in WT, CHOP KO, DARS2 KO, and DKO at P17 (2) (top) and WT and DARS2 KO at 6 weeks (bottom) (n = 3). (F) Western blot analysis proteins involved in the Ca2+ metabolism in WT, CHOP KO, DARS2 KO, and DKOL at P17 (2) (n = 3). (E and F) HSC70 was used as a loading control (n = 3).

The prolonged activation of ISR in DKOL hearts may have adverse effects on cellular and organismal fate. GADD34, a regulatory subunit of the enzyme dephosphorylating eIF2, is thought to function as ISR rheostat acting to restore protein synthesis and block excessive ATF4 activation (15). Unexpectedly, although CHOP was proposed to be a primary Gadd34 transcriptional activator (16), DKOL animals at P17 showed a significant up-regulation of Gadd34 transcripts to similar levels as those observed in terminal, 6-week-old DARS2 KO animals (Fig. 6A). This result suggests that CHOP may play a GADD34-independent role in the suppression of the overactivation of ATF4 induction and ATF4-mediated transcriptional reprogramming.

(A) Relative Gadd34 transcript levels at P17 (2) WT, CHOP KO, DARS2 KO, and DKO animals, as well as in 6-week-old WT and DARS2 KO mice. Bars represent means SD, samples were normalized to WT mice of the respective age (P17: one-way ANOVA, *P < 0.05, **P < 0.01, and ***P < 0.001; 6 weeks: unpaired Students t test) (n = 4). (B) Coimmunoprecipitation (co-IP) of CHOP from WT, CHOP KO, DARS2 KO, and DKOL hearts. The CHOP and C/EBP interaction was monitored with Western blotting using an antibody against C/EBP. One percent of the input fractions was used as loading controls. Asterisks indicate the immunoglobulin G heavy and light chains. (C) Western blot analysis (left) and quantification (right) of the three CEBP isoforms LAP1, LAP2, and LIP in CHOP KO MEFs treated for 48 hours with actinonin along with the respective control (n = 3). (D) Western blot analysis (left) and quantification (right) of steady-state levels of ISR markers in actinonin-treated (48 hours) CHOP KO MEFs expressing the CEBP LIPL120T mutant variant along with the respective controls (n = 3). (E) Western blot analysis of the ATF4 and three CEBP isoforms in actinonin-treated (48 hours) CHOP KO MEFs expressing the CEBP LIP WT and CEBP LIPL120T mutant variant along with the WT cells and respective controls (n = 4). (C to E) Antibodies used were raised against proteins indicated in the panels. HSC70 was used as a loading control. (A, C, and D) Bars represent means SD (MANOVA followed by one-way ANOVA and Tukeys multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001) (n = 3).

As a prerequisite for DNA binding, CHOP needs to heterodimerize with other transcription factors (17). To this end, to identify CHOP-interacting partners that may play a role in mitochondrial stress responses, we immunoprecipitated CHOP from DARS2 KO heart extracts, followed by mass spectrometry (table S4). Notably, besides CHOP, only six proteins were identified. Among those, the most enriched protein and the only transcription factor was C/EBP (table S4). These results were confirmed by Western blot analysis following coimmunoprecipitation (co-IP) against CHOP (Fig. 6B). Notably, CHOP and C/EBP appear to interact only upon mitochondrial dysfunction (i.e., DARS2 KO), despite similar levels of C/EBP in WT and DARS2 KO hearts (Fig. 6B and fig. S6A). The mass spectrometry analysis of C/EBP immunoprecipitates corroborated these results (table S5). In DKO hearts, C/EBP instead interacted with ATF4 and ATF3 (table S5). Previously, the induction of Atf3 was detected in the terminal stages mitochondrial stress responses along with UPRmt (12).

Further interplay of the three proteins is illustrated by the fact that mitochondrial dysfunction in C/EBP-deficient cells exacerbated the ISR stress and led to ATF4 activation similar to CHOP KO (fig. S6B). Interaction of CHOP with C/EBP was previously proposed in the context of mitochondrial dysfunction, wherein CHOP/C/EBP dimers are thought to bind and activate the promoters of UPRmt-responsive genes (5). Consistent with these results, we propose that C/EBP-CHOP heterodimers might act as suppressors of ATF4 overactivation upon mitochondrial dysfunction.

C/EBP is primarily regulated at the translational level and exists in three different isoforms, two activating [Liver-enriched activator protein (LAP1) and LAP2], and one inhibitory [Liver-enriched inhibitor protein (LIP)] (39). The C/EBP target genes are presumably positively regulated by LAP1/2 proteins, whereas LIP binding is thought to repress the transcription of respective promoter (39), although recently more complex functions have been proposed for C/EBP LIP in vivo (40). To further dissect the interplay between CHOP and C/EBP, we assessed the levels of all three C/EBP isoforms in different models of mitochondrial dysfunction. COX10 KO cells with strong chronic mitochondrial dysfunction presented an increase of all C/EBP isoforms (fig. S6C). Acute mitochondrial dysfunction caused by actinonin treatment in MEFs or DARS2 deficiency in heart had a milder effect on the levels of LAP isoforms (Fig. 6C and fig. S6D). Still, C/EBP LIP levels were strongly increased by actinonin treatment in WT cells (Fig. 6C). Notably, this effect was strongly blunted in CHOP-deficient cells and DKOL mice, indicating that an increase in C/EBP LIP levels is dependent on the CHOP presence (Fig. 6C and fig. S6D). In general, the CHOP presence seems to have a positive effect on the C/EBP levels in MEFs, indicating a regulation opposite to that of ATF4.

Under ER stress, CHOP and C/EBP LIP are shown to act in concert to exert their respective functions in the nucleus (41). According to the proposed model, CHOP depends on the interaction with C/EBP LIP to enter the nucleus, while the interaction with CHOP is thought to mask the nuclear export signal (NES) of C/EBP LIP, thereby reducing its exclusion from the nucleus and subsequent proteasomal degradation (41). To test whether C/EBP LIP plays a role in the direct regulation of the mitochondrial dysfunctioninduced ISR, we expressed mutant LIPL120T, carrying a leucine-to-threonine substitution predicted to disrupt NES (42), in CHOP KO cells treated with actinonin (Fig. 6D). The expression of LIPL120T in CHOP KO cells resulted in intense ablation of basal and actinonin-induced ATF4 mRNA and protein levels and a marked decrease in the mRNA and protein levels of its downstream targets, even in the absence of mitochondrial insult (Fig. 6D and fig. S6E). Moreover, expression of LIPL120T mutant resulted in decreased expression of the endogenous C/ebp gene (fig. S6E). Intriguingly, moderate overexpression of WT C/EBP LIP in CHOP KO cells resulted in a mild further increase of ATF4 levels upon mitochondrial dysfunction (Fig. 6E). In contrast, C/EBP LIPL120T mutant suppresses ATF4 while also decreasing endogenous C/EBP levels (Fig. 6E). These results also suggest that mutant C/EBP LIPL120T does not require CHOP for its action.

It has been shown that ER stress leads to interdependent translocation and retention of C/EBP and CHOP inside the nucleus (41). Therefore, we next investigated the effects of mitochondrial stress on subcellular localization of C/EBP, CHOP, and ATF4. In both WT and CHOP KO cells, C/EBP and ATF4 were detected mainly in the nucleus (fig. S6F). The expression of either WT or mutant C/EBP LIP did not affect the subcellular localization of ATF4 in CHOP KO cells (Fig. 7A). Therefore, the ATF4 levels in CHOP-deficient cells appear not to be regulated through alterations in subcellular localization of LIP. Alternatively, in the absence of CHOP, C/EBP LIPL120T may bind ATF4 and prevent its translocation to the nucleus, thus promoting its degradation. To test this hypothesis, we incubated WT and CHOP KO cells in the presence or absence of the proteasome inhibitor MG132. In control conditions, both ATF4 and C/EBP were rapidly degraded, and only a modest fraction was retained and transported to the nucleus (Fig. 7B and fig. S6G). The rate of turnover, however, appeared not to be affected by mitochondrial function or CHOP deficiency (Fig. 7B and fig. S6G). In turn, mitochondrial dysfunction, induced by actinonin treatment, induced translocation of ATF4 to the nucleus and promoted activation of ISR. Of note, the expression of LIPL120T mutant resulted in lower levels of ATF4 in all fractions (Fig. 7B and fig. S6G). Overall, these results suggest that fine-tuning of mitochondrial stress responses is dependent on CHOP:C/EBP LIP interaction but not their subcellular localization nor their potential effects on the nuclear translocation of ATF4.

(A) Cell fractionation followed by the Western blot analysis of the ATF4 and three C/EBP isoforms in actinonin-treated (48 hours) WT or CHOP KO MEFs expressing WT C/EBP LIP or C/EBP LIPL120T mutant. (B) Cell fractionation followed by the Western blot analysis of the ATF4 and three C/EBP isoforms in actinonin-treated (48 hours) CHOP KO MEFs expressing WT C/EBP LIP and C/EBP LIPL120T mutant along with the WT cells and respective controls. The MG132 (15 M) was applied in the last 6 hours of the actinonin treatment. Elevated protein ubiquitination reflects proteasome inhibition. (A and B) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and H3K4me3 were used as loading controls and to determine quality of fractionation (n = 3). (C) CHOP levels increase early upon mitochondrial dysfunction leading to its association with C/EBP. The interaction with C/EBP likely promotes translocation of CHOP to the nucleus where it negatively regulates Atf4 levels and transcription of downstream ISR targets. Abrogation of CHOP results in increased ATF4:C/EBP association and transcription of ISR-regulated genes, created with BioRender.com.

Understanding of the mitochondrial stress response in mammals remains incomplete. In the present study, we uncovered an intricate interplay between three transcription factors regulating the mitochondrial stress response: CHOP, C/EBP, and ATF4. Contrary to its previously proposed role as a transcriptional activator of UPRmt, we present strong evidence that CHOP, through its interaction with C/EBP, attenuates prolonged ISR and mitochondrial cardiomyopathy through regulation of ATF4 levels (Fig. 7). Our results argue that upon mitochondrial dysfunction, the interaction of CHOP with C/EBP is needed for the adjustment of an ATF4-regulated transcriptional program. Very early upon DARS2 depletion, Chop is increasingly expressed (2) and forms a complex with C/EBP, which might facilitate the translocation of CHOP:C/EBP heterodimers to the nucleus. Regulation of ATF4 levels by C/EBP isoform LIP inhibition was proposed during ultraviolet (UV) stress, but CHOP was shown not to play a role in this context (43).

Similar to CHOP, C/EBP is a pleiotropic transcription factor that contributes to the regulation of homeostasis in several tissues, including bone, skin, and fat (40). We showed that in the context of mitochondrial dysfunction, the C/EBP accumulates in the cell (in particular, LIP isoform) and dimerizes with CHOP to presumably prevent overactivation of an ATF4-mediated response. In the absence of CHOP, C/EBP dimerizes with ATF4, which correlates with further induction of ISR. Our data suggest that C/EBP also dimerizes with ATF3 when CHOP is absent in DKO animals. ATF3 is shown to be activated during the second stage of ISR (12, 44). Once expressed, ATF3 binds promoters of ISR-responsive genes, leading to a subsequent suppression of transcription back toward the basal level (44). It is possible that also in the DKO animals, ATF3:C/EBP interaction is part of the feedback loop intended to suppress the ATF4 overactivation. In contrast, the interaction of ATF4 with C/EBP positively activates targeted genes under different conditions (45), which might have a deleterious outcome leading to, e.g., skeletal muscle atrophy (46). In contrast, we show that a dominant-negative C/EBP LIPL120T fully suppresses Atf4 and C/ebp overexpression upon mitochondrial dysfunction and down-regulates even basal levels of these transcription factors. Our findings thus suggest that C/EBP acts as a promiscuous transcription factor in the context of mitochondrial dysfunction, whereby differential transcriptional activity and associated functional outcomes are determined via interactions with CHOP and ATF4 (Fig. 7C). Further work is however required to dissect precise mechanisms of the observed interplay between CHOP, ATF4, and C/EBP.

CHOP is a transcription factor that is ubiquitously expressed at very low levels but quickly activated by a variety of insults such as ER stress, amino acid deprivation, glucose starvation, and UV irradiation (47). To date, CHOP was mostly studied in the context of ER stress, where it was proposed to regulate many pro- and anti-apoptotic genes in the late phase of ISR (47, 48). While numerous functions related to cell proliferation, differentiation, and development have been described for this transcription factor, in unstressed conditions, CHOP-deficient mice do not present any conspicuous phenotype (48, 49). Nevertheless, these mice seem to be protected from transient renal insufficiency caused by acute tubular necrosis (49). CHOP depletion seems to be beneficial in various other conditions, e.g., by delaying the onset of metabolic disease in several diabetic models (50), protecting livers from diet-induced hepatosteatosis (51), or delaying the onset of brain ischemia-induced neuronal cell death (52). Collectively, these studies suggest that loss of CHOP often leads to beneficial effects by delaying apoptosis in vivo. Unexpectedly, in mitochondrial mutants, CHOP depletion does not seem to decrease levels of proteins involved in the activation of apoptosis, as even the proposed bona fide CHOP targets BH3 interacting-domain death agonist (BID), Bcl-2-like protein 11 (BIM), ERO1A, and Tribbles homolog 3 (TRIB3) further increase their levels in DKO mutants.

We also provide evidence that CHOP loss is detrimental in mitochondrial mutants as it leads to early-onset fatal mitochondrial cardiomyopathy. This is, at least in part, mediated by the overactivation of ISR that is paralleled by inhibition of global protein synthesis and appears to be beneficial for a short time as DKOE animals maintain higher levels of OXPHOS complexes and balanced mitochondrial translation. However, loss of CHOP mitigates sustained suppression of protein synthesis in vivo that results in rapid loss of OXPHOS complexes and mitochondrial respiration. This is likely to affect mitochondrial import capacity leading to vicious cycle of damaging events. Simultaneously, mRNA translation rates are restored in DKOL around P17, coinciding with a detrimental phenotype. This is partly reminiscent of a transition from the acute to prolonged ISR in the cellular model of ER stress (37). During the acute ISR phase, global translation is reduced, and only a subset of stress-responsive mRNAs are translated, whereas the prolonged ISR is characterized by recovery of global translation while still allowing execution of acute ISR translational programs (37). While the prolonged ISR appears to have a beneficial effect in vitro by preventing cell death under conditions of ER stress (37), we show that in vivo, mitochondrial dysfunction in the heart impedes a sustained chronic ISR program. To this end, recovery of protein synthesis escalates ER stress possibly by increasing ER load. Recovery of global translation is also expected to significantly increase the energy demand and thereby result in energy depletion caused by massively reduced respiratory capacity due to DARS2 loss. According to the energy starvation hypothesis, suboptimal ATP supply predisposes for the contractile dysfunction observed during heart failure (53). It was shown that even very few cardiomyocytes with severe mitochondrial dysfunction are sufficient to promote ventricular arrhythmias, which lead to heart failure (54). Considering the severe impairment of electron transport chain (ETC) function in DKO mice, the occurrence of cardiac arrhythmias in those animals, contributing to the pathology, seems likely.

The pathology observed in DKOL animals is not a DARS2-specific phenomenon but a prevalent cardiac phenotype in mutants affecting mitochondrial gene expression and translation, as shown by a comparative study of five different models (11). At the molecular level, we demonstrated markedly increased serine synthesis and remodeling of the one-carbon cycle in hearts of DARS2 KO, DKOL mice, and cell culture models, attributable to OXPHOS deficiency and not to the loss of DARS2 in particular. Moreover, similar changes are described in other models and different tissues (11, 13, 14, 55). The vast majority of these alterations have been attributed to ATF4, which has been identified as a major regulator of amino acid metabolism feeding into the folate cycle during ISR induced by different stress signals including mitochondrial dysfunction (13, 14, 56). Although ATF4 may be activated by several different pathways, such as nuclear respiratory factor 2 (NRF2) stabilization or mechanistic (previously mammalian) target of rapamycin (mTOR) signaling (57, 58), we showed that ATF4 up-regulation caused by mitochondrial OXPHOS deficiency could be successfully prevented by suppression of the ISR.

In conclusion, we found a regulatory mechanism that fine-tunes the activation of the ISR upon mitochondrial dysfunction. We showed that CHOP is needed to prevent excessive activation of the ATF4-mediated stress response that results in cardiotoxic effects. This is mediated by CHOP interaction with C/EBP, which likely promotes CHOP:C/EBP heterodimer translocation to the nucleus. Our results also highlight an unforeseen opportunity of exploring a therapeutic intervention targeting ATF4 activity in various mitochondrial diseases.

DARS2 KO (Dars2fl/fl; Ckmm-Cre+/tg) mice were generated as previously described (2). WT control animals (Dars2fl/fl; Ckmm-Cre+/+ and Dars2+/fl; Ckmm-Cre+/+) were also obtained from this breeding. CHOP KO [B6.129S(Cg)-Ddit3tm2.1Dron/J] mice were obtained from the Jackson laboratory. Those mice are characterized by a Chop::LacZ KO allele, resulting in the whole-body KO of Chop (Chop/) (49).

Conditional Dars2-floxed mice (Dars2fl/fl) were crossed to CHOP KO mice (Chop/) to obtain CHOP-deficient animals with floxed Dars2 alleles (Dars2fl/fl; Chop/). Triple transgenic mice were generated by intercrossing of CHOP-deficient animals with floxed Dars2 alleles (Dars2fl/fl; Chop/), with transgenic mice harboring one copy of the Cre recombinase under control of the striated muscle creatine kinase (Ckmm) promoter (Ckmm-Cre+/tg). Resulting heterozygous triple transgenic mice (Dars2+/fl; Ckmm-Cre+/tg; Chop+/) and CHOP-deficient animals with floxed Dars2 alleles (Dars2fl/fl; Chop/) were used to lastly generate CHOP KO (Dars2+/fl; Ckmm-Cre+/+; Chop/ and Dars2fl/fl; Ckmm-Cre+/+; Chop/) and DKO (Dars2fl/fl; Ckmm-Cre+/tg; Chop/) mice. Genotyping for the Dars2 allele was performed as previously described (2). Genotyping for the Ckmm-Cre and Chop alleles was performed following the instructions of the Jackson laboratory using the protocol 22415 along with the primers oIMR3884, oIMR3885, and oIMR3886 for the Chop allele and the protocol Tg(Ckmm-Cre)5Khn along with the primers oIMR1085, oIMR6754, oIMR8744, and oIMR8745 for the Ckmm-Cre allele, respectively (www.jax.org). One- to 6-week-old animals were used in experiments approved and authorized by the Animal Ethics Committee of North-Rhein Westphalia (Landesamt fr Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen) following the German and European Union regulations. Animal work was performed in conformity with the recommendations and guidelines of the Federation of European Laboratory Animal Science Associations.

Immortalized MEFs and fibroblasts were cultured in standard conditions, at 37C and 5% CO2. The cell culture medium was composed of Dulbeccos modified Eagles medium [glucose (4.5 g/liter), GlutaMAX, and sodium pyruvate; Gibco Life Technologies] supplemented with 10% Fetal Bovine Serum Premium, South American Origin (Biowest) and penicillin-streptomycin (Pen-Strep) (Gibco Life Technologies). In conditions of mitochondrial dysfunction (induced either genetically or by treatment), the medium was additionally supplemented with uridine (50 g/ml). At 90% confluency, cells were split cell typedependently in ratios ranging from 1:4 to 1:20.

Generation of immortalized MEF lines. Embryos from embryonic day 13.5 of intercrossed CHOP KO (Chop/) mice were used to isolate primary MEFs (59). Immortalization was achieved by transformation with the SV40 T antigen.

Drug treatments. For induction of mitochondrial dysfunction by actinonin treatment, 80% confluent cells were treated for 48 hours with 100 M actinonin (Sigma-Aldrich). Proteasome was inhibited with 15 M MG132 for the last 6 to 8 hours of treatment as indicated. Inhibition of the ISR was achieved by 4- or 48-hour 1 M ISRIB (Sigma-Aldrich) treatments of 90% confluent cells. All compounds were solubilized in dimethyl sulfoxide (DMSO). Untreated cells were supplemented with corresponding amounts of the solvent. Treatments were renewed on a daily basis.

Transfection. Transfection of plasmids conferring hygromycin resistance (pTK-Hyg LIP, pTK-Hyg LIPwestern, pTK-Hyg LAP, and pTK-Hyg C/EBP) was performed with Lipofectamine 2000 or Lipofectamine LTX (Invitrogen) according to the manufacturers instructions using the forward transfection procedure. Seventy-two hours after transfection, the culture medium was replaced by hygromycin-supplemented (100 g/ml) medium for negative selection of untransfected cells. Transfected cells were maintained in hygromycin-supplemented (100 g/ml) medium.

Cell growth estimation. To estimate differences in cell growth caused by CHOP deficiency and/or mitochondrial dysfunction, an equal number of cells were seeded and treated as indicated. The numbers of cells were determined at the indicated time points using the Countess Automatic Cell Counter (Invitrogen) combined with trypan blue staining.

Freshly collected hearts were immediately transferred into 10 ml of prechilled mito-isolation buffer (MIB) [100 mM sucrose, 50 mM KCl, 1 mM EDTA, 20 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, and 0.2% bovine serum albumin (BSA) free from fatty acids (pH adjusted to 7.2)] supplemented with 1 g of subtilisin (Sigma-Aldrich) per mg of tissue. Approximately 20 long strokes of a Potter S (Sartorius) homogenizer at 1000 rpm were required for homogenization. After centrifugation (800g, 5 min, 4C), the mitochondria-containing supernatant was transferred into a fresh tube. Pelleted mitochondria (8500g, 5 min, 4C) were resuspended in 30 ml of MIB and subjected to a third centrifugation step (700g, 5 min, 4C). Last, mitochondria were pelleted (8500g, 5 min, 4C) and resuspended in 100 l of macrophage inflammatory protein without BSA. Protein concentration of mitochondria was determined using Bradford reagent (Sigma-Aldrich) according to the manufacturers instructions. Mitochondria were either immediately used (respirometry or in organello translation) or snap-frozen and stored at 80C.

High-resolution respirometry using an Oxygraph-2k (OROBOROS Instruments) and a carbohydrate substrate-uncoupler-inhibitor titration protocol was conducted to determine mitochondrial oxygen consumption rates. First, the respiration medium (120 mM sucrose, 50 mM KCl, 20 mM tris-HCl, 1 mM EGTA, 4 mM KH2PO4, 2 mM MgCl2, and 0.1% BSA) was added to the Oxygraph chamber, and air equilibration was performed. Next, 25 g of freshly isolated cardiac mitochondria was added. The respiration medium was supplemented with 2 mM pyruvate, 0.8 mM malate, 2 mM glutamate, and 2 mM adenosine 5-diphosphate (ADP) to assess CI-dependent respiration. By providing additional 4 mM succinate, convergent CI- and CII-dependent respiration was determined. Inhibition of ATP-synthase-complex V (CV) by addition of oligomycin (1.5 g/ml) allowed evaluating the coupling efficiency. The maximal capacity of the electron transfer system (ETS) was assessed by titration of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (0.5 M increments). Maximal capacity of the ETS of CII solely could be determined by inhibition of CI through addition of 0.5 M rotenone. Last, inhibition of CIII by supplementation of 2.5 M antimycin A allowed the determination of the residual oxygen consumption.

De novo mitochondrial translation was assessed by incubation (1 hour, 37C, on rotating wheel) of 1.5 mg of freshly isolated mitochondria in 1 ml of 35S-translation buffer [100 mM mannitol, 10 mM Na-succinate, 80 mM KCl, 5 mM MgCl2, 1 mM KH2PO4, 25 mM Hepes (pH 7.4), 5 mM ATP, 200 M GTP, 6 mM creatine phosphate, creatine kinase (60 g/ml), cysteine (60 g/ml), tyrosine (60 g/ml), amino acids (60 g/ml) (Ala, Arg, Asp, Asn, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, and Val), 35S-methionine (7 l/ml)]. Subsequently, mitochondria were pelleted (12,000g, 2 min) and resuspended in 1 ml of nonradioactive translation buffer containing methionine instead of 35S-methionine. Half of the sample (pulse fraction) was pelleted again, resuspended in 100 l of SDSpolyacrylamide gel electrophoresis (PAGE) loading buffer [50 mM tris-HCl (pH 6.8), 2% SDS (w/v), 10% glycerol (v/v), 1% -mercaptoethanol, 12.5 mM EDTA, and 0.02% bromophenol blue], and lysed (30 min, room temperature) before transfer at 20C. For the cold chase allowing to estimate the protein turnover, the remaining 500 l of resuspended mitochondria was incubated for 3 hours at 37C on a rotating wheel. Subsequently, the chase fraction was pelleted, resuspended in 100 l of SDS-PAGE loading buffer, and lysed as the pulse sample before.

Separation of mitochondrial proteins was achieved by SDS-PAGE. Ten microliters per sample was loaded on a 15-cm-long, 15% polyacrylamide gel and run in a SE600X Chroma Deluxe Dual Cooled Vertical Protein Electrophoresis Unit (Hoefer) overnight at 80 V continuously. After fixing (50% methanol and 10% acetic acid) for 30 min, staining in Coomassie solution, and destaining (20% methanol and 10% acetic acid) of the polyacrylamide gel, the latter one was placed on Whatman paper (GE Healthcare) and dried (2 hours, 80C) in a gel dryer. For detection of radioactive signals of de novo synthetized proteins, Amersham Hyperfilm MP (GE Healthcare) was exposed to the dried polyacrylamide gel.

Cellular protein lysates. Washed cell pellets were resuspended in cold radioimmunoprecipitation assay buffer [150 mM NaCl, 1% Triton X-100 (v/v), 0.5% Na-deoxycholate (w/v), 0.1% SDS (w/v), 50 mM tris-HCl (pH 7.4), 50 mM NaF, and 2 mM EDTA] supplemented with 1 protease inhibitor cocktail (Sigma-Aldrich) and 1 PhosSTOP phosphatase inhibitor cocktail (Roche). Next, cells were incubated 30 min on ice with brief vortexing every 10 min. Following 2 45-s sonication, the lysates were cleared (10 min, 20,000g, 4C) and transferred into fresh tubes.

Cardiac tissue protein lysates. Homogenization of 25 mg of cardiac tissue samples in 400 l of cold organ lysis buffer [50 mM Hepes (pH 7.4), 50 mM NaCl, 1% Triton X-100 (v/v), 0.1 M NaF, 10 mM EDTA, 0.1% SDS (w/v), 10 mM Na-orthovanadate, 2 mM phenylmethylsulfonyl fluoride, 1 protease inhibitor cocktail (Sigma-Aldrich), and 1 PhosSTOP phosphatase inhibitor cocktail (Roche)] was performed with the Precellys CK 14 (Bertin Technologies) (5000 rpm, 30 s). Cleared protein lysates (45 min, 20,000g, 4C) were transferred into fresh tubes. Determination of protein concentration was performed with Bradford reagent (Sigma-Aldrich) according to the manufacturers instructions. Protein lysates were stored at 80C.

SDSpolyacrylamide gel electrophoresis. Protein samples were dissolved in SDS-PAGE loading buffer [50 mM tris-HCl (pH 6.8), 2% SDS (w/v), 10% glycerol (v/v), 1% -mercaptoethanol, 12.5 mM EDTA, and 0.02% bromophenol blue] before denaturation. Depending on the required range of protein sizes, the proteins were separated on 8 to 15% acrylamide gels [stacking gel: 5% acrylamide-bisacrylamide (37.5:1), 12.5 mM tris-HCl, 0.1% SDS (w/v), 0.25% Ammonium persulfate (APS), and 0.25% Tetramethylethylenediamine (TEMED) (pH 6.8); separating gel: 8 to 15% acrylamide-bisacrylamide (37.5:1), 37.5 mM tris-HCl, 0.1% SDS (w/v), 0.1% APS, and 0.1% TEMED (pH 8.8)] in running buffer [25 mM tris-HCl, 250 mM glycine, and 0.1% SDS (w/v) (pH 8.3)].

Western blot. Transfer of proteins on a nitrocellulose membrane by Western blot was conducted in transfer buffer (30 mM tris-HCl, 240 mM glycine, 0.037% SDS, and 20% methanol) at 400 mA for 2 hours at 4C. For a first evaluation of the transfer, shortly washed membranes (dH2O) were stained with Ponceau S solution (Sigma-Aldrich). Depending on the antibody requirements, destaining and blocking of membranes were performed for 1 hour either in 5% milk-PBST (Phosphate-Buffered Saline/Tween) or 3% BSA-TBST (Tris-Buffered Saline/Tween) on a gently shaking platform before subsequent immunodecoration with the indicated antibodies according to the manufacturers instructions. Secondary horseradish peroxidasecoupled antibodies (1:5000) were incubated for 1 hour before detection by Pierce ECL Western blotting substrate (Thermo Fisher Scientific). Densitometry-based quantification of Western blots was performed with ImageJ and Image Studio Lite Software.

Blue native polyacrylamide gel electrophoresis (BN-PAGE) was performed on the basis of the NativePAGE Novex Bis-Tris Gel System (Invitrogen) according to the manufacturers instructions. For analysis of mitochondrial supercomplexes, 10 g of mitochondria was lysed with 4% of digitonin. Analysis of individual mitochondrial complexes was conducted after lysis of 10 g of mitochondria in 1% n-dodecyl--D-maltoside (DDM). After completion of lysis (15 min on ice), lysates were cleared (30 min, 20,000g, 4C), and the resulting supernatant was loaded on a 4 to 16% bis-tris gradient gel. Subsequently, proteins were transferred to an Amersham Hybond polyvinylidene difluoride membrane (GE Healthcare) by Western blot and subsequently immunodecorated with indicated antibodies.

Independently normalized label-free proteomics and RNA sequencing data were scaled before analysis using the anota2seq algorithm (version 1.4.2) (19). Furthermore, datasets were reduced to genes identified on both platforms resulting in a total of 2556 mRNAs for analysis. Analysis of changes in protein levels and total mRNA was performed using the anota2seqAnalyze function to identify differences between CHOP KO, DARS2 KO, and DKO compared to WT. Changes were considered significant when passing the following parameters within the anota2seqSelSigGenes function: maxPAdj = 0.15, minSlopeTranslation = 1, maxSlopeTranslation = 2, selDeltaPT = log2(1.2), selDeltaP = 0, and selDeltaT = 0. Changes in translation or protein stability, as well as changes in mRNA abundance, were characterized using the anota2seqRegModes() function. GO analysis (60) was performed in Cytoscape (v 3.8.0) (23) using the ClueGO (v 2.5.7) app (20). Within ClueGO, four gene lists were provided corresponding to the identified modes for regulation of gene expression using anota2seq (i.e., translation/protein stability and mRNA abundance) divided into up- and down-regulated mRNAs. GO term inclusion parameter was set to a 5 gene overlap and <4% of total genes present in the GO term. For the resulting network, GO term grouping and fusion parameters were enabled, and only GO terms with a false discovery rate of <5% were displayed. Furthermore, anota2seq was applied on the full RNA sequencing dataset (14,174 protein coding transcripts) following the same approach as above. Master regulators among significantly up-regulated total mRNAs in the DARS2 KO versus WT comparison were detected using iRegulon (v1.3) with default settings (24).

The Q5 Site-Directed Mutagenesis Kit (New England Biolabs) was used to introduce a point mutation (L120T) in the pTK-Hyg LIP plasmid (41). For primer design, the New England Biolabs (NEB) online design software NEBaseChanger was used. All three steps described in the protocol [exponential amplification, Kinase, Ligase & DpnI treatment (KLD) reaction, and transformation] were performed as indicated in the manual.

Protein synthesis was determined using the nonradioactive technique called surface sensing of translation described in (61). This assay is based on the incorporation of the structural analogue of tyrosyl-tRNA puromycin in nascent polypeptide chains and subsequent detection of puromycylated proteins using an anti-puromycinspecific antibody.

Briefly, mice were injected at the indicated time points intraperitoneally with 0.04 mol of puromycin dissolved in phosphate-buffered saline (PBS) per gram of body weight. Thirty minutes after injection, the animals were euthanized, and collected tissues were snap-frozen in liquid nitrogen. Subsequently, protein lysates of the collected tissues were prepared and processed by SDS-PAGE and Western blot. The relative signal intensity of the anti-puromycinspecific antibody is proportional to the relative protein synthesis rates at the time point of puromycin injection.

Briefly, mice were injected intraperitoneally with 5 g of ISRIB (stock solution: 5 mg/ml in DMSO, dissolved in PBS up to the weight-dependent injection volume of 30 to 50 l) per gram of body weight or the corresponding amount of PBS-dissolved solvent (DMSO) on a daily basis for the indicated time periods. One day after the last injection, the animals were euthanized, and collected tissues were snap-frozen in liquid nitrogen. Subsequently, protein lysates of the collected tissues were prepared and processed by SDS-PAGE and Western blot.

Numerical data are expressed as means SD. Statistical analysis was performed using the indicated statistical tests. If not indicated differently, statistical significance was considered for P < 0.05. With exception of multivariate analysis of variance (MANOVA) and omics analyses, all statistical tests were performed, and graphs were plotted using GraphPad Prism 8.0 software. MANOVA was performed with XLSTAT version 2020.3 software.

Acknowledgments: We wish to thank the CECAD Imaging and Proteomics Core Facilities for excellent support. Funding: The work was supported by Aleksandra Trifunovics grants of the Deutsche Forschungsgemeinschaft [DFG; German Research Foundation (SFB 1218)Projektnummer 269925409 and TR 1018/8-1] and the Center for Molecular Medicine Cologne, University of Cologne. S.K. received scholarship from the Cologne Graduate School of Ageing Research (CGA). I.T. acknowledges Senior Scholar Award from Le Fonds de recherche du QubecSant (FRQS) and support from Canadian Institutes for Health Research (MOP-363027) and Joint Canada-Israel Health Research Program (JCIHRP) (108589-001) to I.T. and O.L. O.L.s lab was supported by grants from the Swedish Research Council (2016-02891), the Swedish Cancer Society (19 0314), and the Wallenberg Academy Fellows program (2013.0181). M.H.s laboratory is supported by NIH R01 DK060596 grant. Author contributions: Conceptualization: A.T., S.K., C.O., K.Sz., O.L., I.T., and M.H. Data curation: S.K., C.O., A.T., S.B., O.L., and K.Sz. Formal analysis: S.K., C.O., A.T., S.B., O.L., and K.Sz. Funding acquisition: A.T., S.K., O.L., I.T., and M.H. Investigation: S.K., C.O., A.K., K.Se., K.Sz., C.L., S.B., and O.L. Visualization: S.K., C.O., A.T., O.L., I.T., and M.H. Writing: A.T., S.K., C.O., O.L., and I.T. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Further information and requests for resources and reagents should be addressed to and will be fulfilled by A.T. Mouse and cell lines requests include signing of material transfer agreement.

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Adaptation to mitochondrial stress requires CHOP-directed tuning of ISR - Science Advances

Aging is the ultimate risk factor for most diseases, such as cancer, neurodegenerative, cardiovascular, diabetes, degenerative fibrosis and many others. When we are young, we are typically healthy, despite a predisposition that will lead inevitably to a specific degenerative condition. However, the degenerative processes do not kick in until a certain age, when we are older. It looks like when we are younger, the body can compensate cumulative stress and damage caused to our cells in the tissues, allowing to maintain that equilibrium, called homeostasis, that keeps our organs functional and healthy. However, over time this buffering capacity becomes thinner and thinner, until things wear off: our tissues stop working as they used to. These changes are typically caused by an initial small number of rare but bad cells, that progressively increase over time, causing additional damage to the good cells that eventually stop working efficiently, causing a vicious cycle. Eventually the bad cells take over leading to the onset of a disease.

Our body is equipped with a number or regenerative and healing functions. Some are intrinsic in every cell, such as DNA repair mechanisms that are triggered when something compromises the integrity of our genomic structures. These are important functions that enable a cell, for example when it replicates, to repair errors and other damages that might have happened to our DNA. For example, two large proteins called ATM and ATR, involved in the cellular response to DNA damage, are responsible to maintain genomic instability caused by intrinsic and external DNA-damaging agents, such as UV light or various chemicals and toxins. A lack of functions of these proteins results in progressive neurodegeneration, immunodeficiency, predisposition to malignancy or radiation sensitivity. Mutations on the genes encoding these proteins can cause premature aging and premature development of these diseases, but this occurs also naturally, over time.

Cells also have an intrinsic immune system, producing factors called interferons employed by the cells as antiviral agents and to modulate other immune functions. It can be triggers by a viral infection so when a cell is infected will release interferons, protecting the neighbor cells against potential infection. Interferons can also suppress growth of blood vessels preventing tumors to get nutrients and growing. They can also activate immune cells so they can better fight viruses, tumors and others agents. Unfortunately, an age-related decline or impaired innate interferon functions in the cells results in a number of negative consequences in the body, such as increased susceptibility of the elderly to infections, tumors and damage.

In the body there are several cell types responsible to keep the tissues in check. The immune system is specialized to recognize remove and remember damaging agents. Those could be external, such as virus, bacteria or parasites, or internal, such as tumorigenic cells or senescent cells (see below). The immune system is a very sophisticated network of cell types, intercommunicating with each other to maintain the body clean from damaging factors. As we age the immune system also ages and loses capacity to recognize or responding to these damaging agents. It also become exhausted by an increasing chronic inflammation that progressively accumulate as we age, phenomenon also called inflammaging.

Another important repairing mechanism is the regenerative tissue functions, driven by the stem cells. Those cells are progenitor cells, often dormant in a quiescent state in the tissue and waiting to be activated by some damage. Stem cells are critical because once activated they can generate a progeny of daughter cells capable of re-growing the damaged tissue back to its original structure and function. Stem cells have another important function: they can regenerate themselves, in a process called self-renewal. This is important so that the new repaired tissue can repeat the process if a new damage occurs. The regenerative capacity of our body is remarkable, allowing our tissues to keep their integrity, health and functions. However, over time also stem cells age or respond to the aged microenvironment where they live (called the niche), and they become less efficient to repair tissues or to self-renewing. As a result, our tissues change, become atrophic, fibrotic or dysfunctional leading eventually to diseases.

In regenerative medicine, the application of stem cells resulted of the generation of multiple new therapeutic opportunities. A promising area uses stem cells to generate bioengineering strategies to grow new tissues in a petri dish to be then transplanted in the body to repair damaged tissues. Some applications are already in clinical use, such as for skin grafts. Many others are on their way, either in preclinical development or in clinical trials for many different tissue types and for different clinical indications.

Another promising stem cells application is the direct transplantation into damaged tissues, where they can grow and engraft repairing. However, as we age stem cells become less efficient. What if we If we could rejuvenate them? We could restore their capacity to repair our tissues and maintain homeostasis. Promising and exciting strategies are advancing in that direction. For example, we and others showed that it is possible to reprogram epigenetically a cell so it can become the younger and healthier version of itself (Sarkar et al., 2020). This is a mechanism that every cell has encoded in its DNA, but normally works only in the germline (the sperm and the egg) during the embryogenesis to make sure that the cellular clock is turned back to zero, before initiating the cellular programs to generate the embryo. This important for example to prevent making old newborn babies. This intrinsic rejuvenative mechanism is locked in the other somatic cells of the body. We found it is possible to re-activate it transiently and safely, without changing the identity of the cell, enabling to push back the cellular clock of aged human cells to make them healthier and restore their functions. These technologies are under development to be translated into therapeutics with the promise that one day could rejuvenate the aged cells in the body so they can become the younger version of themselves, repeating the process over time when needed.

Among many of the drivers of the aging process, there is one that seems to stands out as the lower hanging fruit among the emerging space of the longevity therapeutics. This is cellular senescence. Every damage that occurs to the cells in our body can push the cells to stop what they are doing and activate a safety mechanism that locks them into an arrested state called cellular senescence. Senescent cells cannot replicate anymore preventing them to cause additional damage, such as becoming cancer cells. All sort of damage can trigger this response leading to cellular senescence such as, oxidative stress, mitochondrial dysfunctions, DNA damage, viral infection, cigarette smoking, pollutions, chemicals, etc. They all can induce that safety lock and push damage cells to become senescent.

Senescent cells dont die easily but they stick around in the tissue, accumulating slowly over time. Importantly, cellular senescence is a pleiotropic mechanism, meaning it can be both good or bad. When we are young, we can efficiently get rid of senescent cells. The body uses them positively such as for tissue repair, wound healing or tissue remodeling. However, as we age, and our immune system ages (partially trough cellular senescence, a phenomenon called immune-senescence), our body become less efficient in removing senescent cells, which then start to accumulate.

Being able to make a new generation of drugs that are very selective for senescent cells, will enable the promise to achieve rejuvenative clinical results in humans similarly to what we found in preclinical results. On that end, we recently published a targeted strategy with the goal to advance the field in that direction (Doan et al., 2020). Using a prodrug, we engineered a small molecule to generate a selective senolytic compound to develop a targeted therapy. This prodrug is inactive in non-senescent cells but activated by senescent cells, taking advantage of an enzymatic function of those cells. In geriatric mice this prodrug showed to be well tolerated but also efficacious to clear senescent cells, resulting in restored cognitive functions, muscle functions, stem cells functions, vitality and overall health. As we advance senolytic drugs to the clinic to treat age-related diseases, it is very important to be mindful that elderly individuals, who are frail, with co-morbidities and exposed to multiple medications, will not well tolerate drugs that are not safe and effective. Importantly, not all senescent cells are the same. They are rare, interspersed in the tissues but are also very heterogeneous. Being able to hit the right senescent cells, in the right diseased tissue will be key to enable effective therapies. Developing drugs that are very potent, selective and potent and safe will be pivotal.

The longevity therapeutics space is emerging, but is already disrupting the medical industry. The goal of longevity therapeutics is not just to add years to life, extending lifespan. The true goal is to add life to years and extend health span. A target that gets closer every day.

Marco Quarta is CEO, Rubedo Life Sciences.

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The Rise of Longevity Therapeutics - Pharmaceutical Executive

During a virtual Targeted Oncology Case-Based event, Pashna N. Munshi, MD, associate clinical director, Stem Cell Transplant and Cellular Immunotherapy Program, assistant professor of Medicine, Georgetown University School of Medicine at MedStar Georgetown University Hospital, discussed the case of a 48-year-old male patient with acute graft-versus-host-disease (GVHD).

Targeted OncologyTM: What factors contribute to the risk of acute GVHD in a patient like this one?

MUNSHI: A lot of donor-recipient factors and other conditions increase the risk of acute GVHD. [These include] gender matching, human leukocyte antigen disparity, degree of mismatch, and having an older donor. Theres also [blood group] incompatibility and definitely CMV mismatched status. Though now that the FDA has approved letermovir [Prevymis] for patients who are undergoing allogeneic transplant if they have a CMV-positive donor,1 were seeing very little CMV reactivation. That has been a bit of a game changer for the good.

Patients have an increased risk of GVHD if they receive a transplant from a peripheral blood stem cell source versus a bone marrow graft, because the peripheral blood has more T cells in its composition. The myeloablative regimens [are associated with greater risk of GVHD than] reduced-intensity regimens.2

Do you agree with these poll results? Would you start with systemic therapy for this patient?

It can get a little tricky whether you want to give patients systemic steroids or wait and see if something gentler might work. I tend to agree that, at this point, the patient needs to immediately start with systemic steroids, because there are 2 organ systems involved. Once the lower gastrointestinal [GI] tract gets involved, it surely portends a poor prognosis if the grade becomes worse. And they become refractory to steroids very quickly: 50% of these patients will eventually not respond to steroids.

How would you stage this patients GVHD?

There are many criteria for staging GVHD. The criteria that most clinical trials use are the Mount Sinai Acute GVHD International Consortium [MAGIC] criteria.3 They are adapted from the Glucksberg criteria, which are very similar.4

Three organ systems [are involved in] acute GVHD: the skin, the liver, and the GI tract. Skin involvement is graded on the basis of the body surface area involved. Liver involvement is graded on the basis of the total bilirubin level. Upper GI involvement is graded on the basis of anorexia, nausea, and vomiting, and it just comes in stage 0 or stage I, depending on if its persistent or not. To determine lower GI tract involvement, we measure stool volume, especially when patients are admitted to the hospital. But once they go home, we cant do that, so we ask them how many times a day they have diarrhea. Is it watery? Is it muddy? Whats the volume? Is it large or small?

The patient can characterize the stool and tell their doctor how many times per day: 4 times, 5 times, 6 times. This patient is having 4 episodes per day; that puts them in stage I lower GI GVHD. But with a 60% body rash, that puts them in stage III skin GVHD. So really getting up there with skin, but not so much yet for GI. Once each organ [involvement is] staged, theres an aggregate score based on the combination of these organs. Then we come up with the grade.

In this patient, with a stage III rash, stage I upper GI, and stage I lower GI GVHD, they have a total score of a grade 2 acute GVHD. This is still in the mild to moderate zone. Anything above grade 2 is considered very severe GVHD.

Would you recommend that this patient receive systemic steroids?

In the scheme of things, somebody who didnt have symptoms and now is having active symptoms, especially with lower GI tract involvement, definitely needs high-dose steroids to get in there and [stop] the inflammation.

On what would you base a prognosis for this patient?

We can risk stratify these patients on the basis of the stage of organ involvement.5 Broadly, they can be at a standard risk or at a high risk [of poor response to treatment, mortality, and transplant-related mortality]. The patient is at high risk once they have very active GI involvement [or] if they have 2 organs involved. This is one more reason to think about starting these patients early on steroids. Why is this important? Because once a patient has high-risk GVHD, the chance of response to steroids is even lower, and once they dont respond to steroids, there is a higher [risk of] transplant-related mortality. The probability of transplant-related mortality is 44% for patients with high-risk acute GVHD flares, [versus] 22% for patients with low-risk GVHD [P < .001]. These are a few things to think about. Act very swiftly if a patient has 2-organ involvement, especially the lower GI tract.

Can biomarkers guide treatment decisions in this case?

In the field of GVHD, biomarkers are a very exciting advancement. We want a prognostic model of which patients will get GVHD. Can biomarkers in the blood [help] prevent GVHD and improve transplant outcomes?

A large prospective trial was done through the Bone and Marrow Transplant Clinical Trials Network where a set of 6 biomarkers were tested at several time points after the transplant.6 They saw that they could predict when GVHD happened by using these biomarkers. They could see that as the levels of these biomarkers increased, the patients had higher scores of GVHD. Once treatment was started, if specific biomarkers went down it was predictive of response at day 28 [56% vs 17%; odds ratio, 6.32; P = .001] and also predictive of [decreased] transplant-related mortality by day [180 (49% vs 87%; P < .0001)]. If all these biomarkers went up aggressively, overall survival was lower [P < .0001].

The MAGIC Consortium also tried to test biomarkers.7 They looked at 2 biomarkers, REG3Athe regenerating islet-derived 3-alpha, which is specific for the GI tract and ST2. Looking at these 2 biomarkers, they came up with an algorithm of prediction. On the basis of how these biomarkers responded at the time of GVHD and to treatment, they could predict mortality by 6 months. In clinical practice, it is difficult to use this day in and day out. We still use our clinical skills to assess the degree of GVHD. But all patients eventually get treated the same waywith high-dose steroidsdespite biomarkers being elevated or not.

At this point, [biomarker data] may tell us an association rather than a causality. Were not openly using biomarkers to guide our practice, but I think were learning to use them a bit more and knowing that theres something out there that could be used as a predictive tool. It is an exciting development.

Are there alternatives to systemic steroids?

Steroids remain the mainstay. We need to see if we can move to other therapies that are coming down the pipeline.

Data from the REACH1 [NCT02953678] and REACH2 [NCT02913261] trials led to ruxolitinib [Jakafi] approval.8,9 If we can use ruxolitinib in an up-front setting, [maybe we] can use the newly approved rho-kinase or ROCK2 inhibitors as well.10 We want to think about steroid-sparing agents. Maybe biomarkers can guide us in the future for that. But right now, in terms of, Do I start my patient on treatment? or Will they respond to this treatment, I find that [biomarkers are] still not a very useful tool because at the end of the day, the patients all still need to be started on steroids.

The minute you see that your patient is not responding to steroids, very quickly start them on a JAK2 inhibitor.

How do you dose steroids?

This patient received 2 mg/kg of prednisone per day for 14 days. Two mg/kg is a very high dose. The standard is 1 to 2 mg/kg.11 There are data to show that 2 mg isnt any different from 1 mg.12 But a lot of times, if its a very active, severe flare, we will use 2 mg/kg. Im not sure if I would have done 2 mg/kg in this case, but its certainly not out of the realm of treating these patients.

The goals of primary therapy for acute GVHD are to stabilize the organ manifestations, or improve them, and limit long-term treatment toxicity. We want to improve functional capacity and prevent any reduction in quality of life. First-line therapy is always with corticosteroids. Now ruxolitinib is approved for second-line therapy.8 There have been data to show that it can improve overall survival.

How do you taper glucocorticosteroids after achieving initial response?

If the patient is taking 2 mg/kg of steroids, an average 70-kg person, thats over 100 mg of steroids. After 2 weeks, they probably are not getting up from a seated position anymore with all the muscle wasting that can happen.

[As soon as they start to show improvement, it would be safe to start to taper the dose.] Traditionally, [the patient receives the full dose for] at least a week or 10 days. Then it is traditional to decrease the dose 10% every 5 to 7 days, gently coming down, making sure that the patient is not having any flares.

Describe the multidisciplinary teambased approach that you use for acute GVHD.

The incidence of acute GVHD in the patient population is anywhere from 30% to 50%, despite the best [efforts at] prophylaxis. Most patients will get some form of acute GVHDit can go up to even 80%. This [necessitates] a multidisciplinary team approach. If the patient is having diarrhea, theyre having malnourishment. Theres nausea or anorexia, so theyre not eating on top of that. Then theres skin rash, so the risk of infections and cellulitis. Theyre in pain. A dermatologist probably should be involved at some point. A nutrition team is also needed. If theyre on high-dose steroids, physical therapy should be involved up front. So early involvement of a whole team is very important. Thats usually how I treat my patients and usually how centers of excellence continue to treat active patients with GVHD after transplantation.

How do you determine if a patients GVHD is steroid refractory?

The strict definition of steroid refractoriness or resistance is if theres progression of acute GVHD within 3 to 5 days of starting high-dose steroids, or theres failure to improve within 1 week of starting these steroids, or theres incomplete response after more than 28 days of any immunosuppressive treatment.13 So, by and large, in 3 days or a maximum of 7 days, [it will be clear] if the patients GVHD is going to be steroid refractory or not.

Steroid dependence is [defined as when] the patients GVHD initially responded to steroids, but the disease flares when the dose is tapered, so they cannot be taken off the steroids.

Steroid intolerance is when the patient develops [unacceptable toxicity from steroids such as] uncontrolled diabetes or myopathies. Then it becomes hard to keep them on steroids.

What are the treatment options for patients with steroid-refractory GVHD?

Ruxolitinib now has been FDA approved for steroid-refractory acute GVHD, and its a category 1 definition.8,11 Ibrutinib [Imbruvica] has also been approvedits only FDA-approved indication is for chronic GVHD.14 There are many other treatment options [in the National Comprehensive Cancer Network guidelines].11 Oncologists always end up using some combination or other depending on which of these different immune suppression medications they are comfortable using.

What new treatments are in the pipeline?

In terms of BTK inhibitors, I dont think theres anything other than ibrutinib at this time point. There are many JAK inhibitors being studied.15 Baricitinib is another JAK inhibitor thats actively being studied for chronic GVHD, as well as for pulmonary GVHD.16 Then there are other rho-kinase inhibitors, called ROCK2 inhibitors. This is really making waves. Were very excited about this drug because the response rates are very high, about 70%.10 Its a smaller study, but clearly it has antifibrotic pathways. So I think thats going to be used much more in the up-front setting.

Then theres also alpha-1 antitrypsin, which targets the liver and macrophages and has very promising results from trials done at Dana-Farber Cancer Institute and Michigan.17 So I think were going to see very different characteristics of how to approach GVHD.

What data support the use of ruxolitinib in this setting?

The REACH1 study led to the approval of ruxolitinib for steroid-refractory acute GVHD.9,18 In this phase 2 trial, patients with steroid-refractory acute GVHD got ruxolitinib (5 mg twice a day) with or without a calcineurin inhibitor. They were allowed to remain on steroids. The primary end point of this trial was overall response rate [ORR] at day 28. They also looked at response rates at day 56 and day 100, biomarkers, failure-free survival, and durability of these responses. The ORR at day 28 was very high: 54.9%.18 The best ORR, which was at any given time during the treatment, which was as high as 73.2%. The median time to response was 7 days. So this was very quick. The median duration of response was 345 days, with more than 6 months follow-up. Nonrelapse mortality at 6 months was 44.4%. There were deaths from infections, etc, but not related directly to ruxolitinib.

Subsequently there was a phase 3 trial, REACH2.19 They looked at higher doses of ruxolitinib in steroid-refractory acute GVHD. They started off with 10 mg [of ruxolitinib] twice a day. This study had a similar primary end point of ORR at day 28. This was compared with best available therapy. This was done in Europe, so [the comparison was to the] best available therapy used in Europe, like anti-thymocyte globulin, sirolimus [Rapamune], etanercept [Enbrel], photopheresis, or other therapies; all things that we would use in the United States as well. They looked at similar key secondary end points, [including] duration of response at day 56.

The ORR for ruxolitinib was 62% at day 28, compared with the best available therapy arm, which was 39% [odds ratio, 2.64; 95% CI, 1.65 to 4.22; P < .001].19 Durable overall response at day 56 [was higher in the ruxolitinib group than it was in the control group (40% vs 22%, odds ratio, 2.38; 95% CI, 1.43-3.94; P < .001)].19

The lower grade acute GVHD, which was grade 2, had the highest complete response rate with ruxolitinib: 50.9% compared with just 26.4% with best available therapy.19 This is quite remarkable to have a complete response in GVHD so quickly. When you get to higher grades of GVHD, the complete response rate for ruxolitinib is not as impressive; its less than 30%. But its still much higher than the [response rates of] other therapies we would have otherwise treated these patients with in steroid-refractory disease. The key point is to diagnose steroid refractoriness early. Then get ruxolitinib in there to break the cycle and break the progression of organ grade to something higher.

The loss of response wasnt statistically significant. The estimated cumulative incidents for the loss of response at 6 months was 10% in ruxolitinib compared with 39% in the control arm.19 So patients continued to maintain responses, which, again, is what we want to see. We dont want to see flares if they come off steroids.

[Of the 4 organ systems involved in GVHD], the skin responses were the best with ruxolitinib. Lower GI and liver GVHD did have good responses, but the responses were not as remarkable. Ruxolitinib is an ideal drug in this setting, on the basis of the organ responses.

A secondary end point was failure-free survival, basically indicating a time point from randomization to either nonrelapse-related death or any new GVHD. This was not statistically significant because it was not designed to compare ruxolitinib survival outcomes with control therapy. But there were 5.0 months median failure-free survival with ruxolitinib compared with 1.0 month with control [hazard ratio for relapse or progression of hematologic disease, nonrelapse-related death, or addition of new systemic therapy for acute GVHD, 0.46; 95% CI, 0.35-0.60]. That tells you that the responses were maintained, and the treatment was still working.

[Most of the adverse events associated with ruxolitinib] were expected; the bone marrow is recovering so its a bit fragile. [The most common was] thrombocytopenia. You can reduce the dose of ruxolitinib down to 5 mg adjusted accordingly or support patients with transfusions. CMV reactivation was also common. But again, with letermovir, that happens less and less.

References:1. Merck receives FDA approval of Prevymis (letermovir) for prevention of cytomegalovirus (CMV) infection and disease in adult allogeneic stem cell transplant patients. News release. Merck. November 9, 2017. Accessed April 7, 2021. https://bit.ly/3fS6S0Q

2. Scott BL. Long-term follow up of BMT CTN 0901, a randomized phase 3 trial comparing myeloablative (MAC) to reduced intensity conditioning (RIC) prior to hematopoietic cell transplantation (HCT) for acute myeloid leukemia (AML) or myelodysplasia (MDS) (MAvRIC Trial). Biol Blood Marrow Transplant. 2020;26(3):S11. doi:10.1016/j.bbmt.2019.12.07

3. Harris AC, Young R, Devine S, et al. International, multicenter standardization of acute graft-versus-host disease clinical data collection: a report from the Mount Sinai Acute GVHD International Consortium. Biol Blood Marrow Transplant. 2016;22(1):4-10. doi:10.1016/j.bbmt.2015.09.001

4. Martino R, Romero P, Subira M, et al. Comparison of the classic Glucksberg criteria and the IBMTR Severity Index for grading acute graft-versus-host disease following HLA-identical sibling stem cell transplantation. International Bone Marrow

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Munshi Explains Staging, Prognosis, and Treatment for a Patient With Acute Graft-vs-Host-Disease - Targeted Oncology

Researchers used a pig model of heart attacks, which more closely resembles the human heart in size and physiology, and thus has high clinical relevance to human disease.

Researchers used a pig model of heart attacks, which more closely resembles the human heart in size and physiology, and thus has high clinical relevance to human disease.In a large-animal study, researchers have shown that heart attack recovery is aided by injection of heart muscle cells derived from human induced pluripotent stem cell line, or hiPSCs, that overexpress cyclin D2. This research, published in the journal Circulation, used a pig model of heart attacks, which more closely resembles the human heart in size and physiology, and thus has higher clinical relevance to human disease, compared to studies in mice.

An enduring challenge for bioengineering researchers is the failure of the heart to regenerate muscle tissue after a heart attack has killed part of its muscle wall. That dead tissue can strain the surrounding muscle, leading to a lethal heart enlargement.

Heart experts thus have sought to create new tissue applying a patch of heart muscle cells or injecting heart cells to replace damaged muscle. Similarly, they have tried to stimulate division of existing heart muscle cells near the damaged area. This current study, led by researchers at the University of Alabama at Birmingham, shows progress in both goals.

After the experimental heart attack, heart tissue around the infarction site was injected with about 30 million bioengineered human cardiomyocytes that were differentiated from hiPSCs. These cells also overexpress cyclin D2, part of a family of proteins involved in cell division.

Compared to control human cardiomyocytes, the cyclin D2-cardiomyocytes showed enhanced potency to repair the heart. They proliferated after injection, and by four weeks, the hearts had less pathogenic enlargement, reduced size of dead muscle tissue and improved heart function.

Intriguingly, the cyclin D2-cardiomyocytes stimulated not only their own proliferation, but also proliferation of existing heart muscle cells around the infarction site of the pig heart, as well as showing angiogenesis, the development of new blood vessels.

These results suggest that the cyclin D2-cardiomyocyte transplantation may be a potential therapeutic strategy for the repair of infarcted hearts, said study leader Jianyi Jay Zhang, M.D., Ph.D., the chair of Biomedical Engineering, a joint department of the UAB School of Medicine and the UAB School of Engineering.

This ability of the graft cyclin D2-cardiomyocytes to stimulate the proliferation of nearby existing heart cells suggested paracrine signaling, a type of cellular communication where a cell produces a signal that induces changes in nearby cells.

Exosomes small blebs or tiny vesicles that are released by human or animal cells and contain proteins and RNA from the cells that release them are one common form of paracrine signaling.

Zhang and colleagues found that exosomes that they purified from the cyclin D2-cardiomyocyte growth media indeed promoted proliferation of cultured cardiomyocytes. In addition, the treated cardiomyocytes were more resistant to programmed cell death, called apoptosis, induced by low oxygen levels. The exosomes also induced proliferation of various other cell types, including human umbilical vein endothelial cells, human vascular smooth muscle cells and 7-day-old rat cardiomyocytes that have almost undetectable proliferation.

Part of the cargo that exosomes carry are microRNAs, or miRNAs. These short pieces of RNA have the ability to interact with messenger RNA in target cells, and they are robust players of gene regulation in cells. Humans have more than 2,000 miRNAs with different RNA sequences, and these are thought to regulate a third of the genes in the genome.

So, the researchers documented which microRNAs were present in exosomes from the cyclin D2-overexpressing cardiomyocytes and in exosomes from non-overexpressing cardiomyocytes. As expected, they found differences.

Jianyi Jay Zhang, M.D., Ph.D.Together, the exosomes from both types of cells contained 1,072 different miRNAs, and 651 were common to the two exosome groups. However, 332 miRNAs were found only in the cyclin D2-overexpressing cardiomyocytes, and 89 miRNAs were specific for the non-overexpressing cardiomyocytes. In preliminary work of characterizing the effects of specific miRNAs, one particular miRNA from the cyclin D2-overexpressing exosomes was shown to stimulate proliferation when delivered into rat cardiomyocytes.

Thus, as the therapeutic potential of exosomes for improving cardiac function becomes more evident, combining an exosome-mediated delivery of proliferative miRNAs with transplantation of cyclin D2-overexpressing cardiomyocytes, or cell products, could become a new promising strategy for upregulating proliferation of the recipient cardiomyocytes and reducing cardiac fibrosis, Zhang said. Altogether, our data suggest that cardiac cell therapy, involving cardiomyocytes with enhanced proliferation capacity, may become an efficacious future strategy for myocardial repair and prevention of congestive heart failure in patients with acute myocardial infarctions.

UAB Department of Biomedical Engineering co-authors with Zhang, in the study Cyclin D2 overexpression enhances the efficacy of human induced pluripotent stem cell-derived cardiomyocytes for myocardial repair in a swine model of myocardial infarction, are Meng Zhao, Yuji Nakada, Yuhua Wei, Weihua Bian, Anton V. Borovjagin, Yang Zhou and Gregory P. Walcott.

Additional co-authors are Yuxin Chu and Min Xie, Division of Cardiovascular Disease, UAB Department of Medicine; Wuqiang Zhu, Department of Cardiovascular Diseases, Physiology and Biomedical Engineering, Mayo Clinic Arizona, Scottsdale; Thanh Nguyen, UAB Informatics Institute; and Vahid Serpooshan, Emory University and Georgia Institute of Technology, Atlanta.

Support came from National Institutes of Health grants HL114120, HL131017, HL149137 and HL134764.

At UAB, Zhang holds the T. Michael and Gillian Goodrich Endowed Chair of Engineering Leadership.

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Heart attack recovery aided by injecting heart muscle cells that overexpress cyclin D2 - The Mix

KENILWORTH, N.J.--(BUSINESS WIRE)--Merck & Co. (NYSE: MRK), known as MSD outside the United States and Canada, today announced positive results from the pivotal neoadjuvant/adjuvant Phase 3 KEYNOTE-522 trial investigating KEYTRUDA, Mercks anti-PD-1 therapy, in combination with chemotherapy as pre-operative (neoadjuvant) treatment and then continuing as a single agent (adjuvant) treatment after surgery. KEYNOTE-522 met its dual primary endpoint of event-free survival (EFS) for the treatment of patients with high-risk early-stage triple-negative breast cancer (TNBC). Based on an interim analysis conducted by the independent Data Monitoring Committee (DMC), neoadjuvant KEYTRUDA plus chemotherapy followed by adjuvant KEYTRUDA as monotherapy showed a statistically significant and clinically meaningful improvement in EFS compared with neoadjuvant chemotherapy alone. As previously communicated, KEYNOTE-522 met its other dual primary endpoint of pathological complete response (pCR). The safety profile of KEYTRUDA in this trial was consistent with that observed in previously reported studies; no new safety signals were identified.

KEYTRUDA is the first immunotherapy to show positive results for event-free survival in patients with high-risk early-stage TNBC, a particularly aggressive form of breast cancer, said Dr. Roy Baynes, senior vice president and head of global clinical development, chief medical officer, Merck Research Laboratories. The improvement in pathological complete response rates initially observed following pre-operative treatment was encouraging, and now that we are seeing the data mature after four years to include a statistically significant improvement in event-free survival, we look forward to working with the FDA and other global authorities to bring this new option to patients as quickly as possible. We are grateful to the study participants who are critical to our efforts to advance potential treatment options for patients with TNBC.

An analysis of pCR from KEYNOTE-522 was presented at the European Society for Medical Oncology (ESMO) 2019 Congress and published in the New England Journal of Medicine. Findings showed a statistically significant increase in pCR for KEYTRUDA plus chemotherapy versus chemotherapy alone as neoadjuvant therapy in patients with early-stage TNBC, regardless of PD-L1 status. As previously announced, the company received a Complete Response Letter (CRL) from the FDA in March 2021 regarding Mercks supplemental Biologics License Application (sBLA) seeking approval for KEYTRUDA for the treatment of patients with high-risk early-stage TNBC based on these pCR data and early interim EFS findings. The CRL followed the FDAs Oncologic Drugs Advisory Committee meeting that voted 10-0 that a regulatory decision should be deferred until further data were available from KEYNOTE-522.

The KEYTRUDA clinical development program for TNBC encompasses several internal studies and external collaborative trials, including the ongoing studies KEYNOTE-242 and KEYNOTE-355.

Merck has an expansive clinical development program investigating KEYTRUDA in earlier lines of therapy including in neoadjuvant, adjuvant and locally advanced settings, with approximately 20 registrational studies ongoing.

About KEYNOTE-522

KEYNOTE-522 is a Phase 3, randomized, double-blind trial (ClinicalTrials.gov, NCT03036488), evaluating a regimen of neoadjuvant KEYTRUDA in combination with chemotherapy followed by adjuvant KEYTRUDA as monotherapy versus a regimen of neoadjuvant chemotherapy followed by adjuvant placebo. The dual primary endpoints are pCR and EFS. The secondary endpoints include pCR rate using alternative definitions (i.e., no invasive or noninvasive residual cancer in breast or nodes) at the time of definitive surgery, overall survival, EFS in patients whose tumors express PD-L1 (Combined Positive Score [CPS] 1), safety and patient-reported outcomes. The study enrolled 1,174 patients who were randomized 2:1 to receive either:

About Triple-Negative Breast Cancer (TNBC)

Triple-negative breast cancer is an aggressive type of breast cancer that characteristically has a high recurrence rate within the first five years after diagnosis. While some breast cancers may test positive for estrogen receptors, progesterone receptors or overexpression of human epidermal growth factor receptor 2 (HER2), TNBC tests negative for all three. Approximately 15-20% of patients with breast cancer are diagnosed with TNBC. TNBC tends to be more common in women who are younger than 40 years of age, who are African American or who have a BRCA1 mutation.

About KEYTRUDA (pembrolizumab) Injection, 100 mg

KEYTRUDA is an anti-PD-1 therapy that works by increasing the ability of the bodys immune system to help detect and fight tumor cells. KEYTRUDA is a humanized monoclonal antibody that blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2, thereby activating T lymphocytes which may affect both tumor cells and healthy cells.

Merck has the industrys largest immuno-oncology clinical research program. There are currently more than 1,400 trials studying KEYTRUDA across a wide variety of cancers and treatment settings. The KEYTRUDA clinical program seeks to understand the role of KEYTRUDA across cancers and the factors that may predict a patient's likelihood of benefitting from treatment with KEYTRUDA, including exploring several different biomarkers.

Selected KEYTRUDA (pembrolizumab) Indications in the U.S.

Melanoma

KEYTRUDA is indicated for the treatment of patients with unresectable or metastatic melanoma.

KEYTRUDA is indicated for the adjuvant treatment of patients with melanoma with involvement of lymph node(s) following complete resection.

Non-Small Cell Lung Cancer

KEYTRUDA, in combination with pemetrexed and platinum chemotherapy, is indicated for the first-line treatment of patients with metastatic nonsquamous non-small cell lung cancer (NSCLC), with no EGFR or ALK genomic tumor aberrations.

KEYTRUDA, in combination with carboplatin and either paclitaxel or paclitaxel protein-bound, is indicated for the first-line treatment of patients with metastatic squamous NSCLC.

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with NSCLC expressing PD-L1 [tumor proportion score (TPS) 1%] as determined by an FDA-approved test, with no EGFR or ALK genomic tumor aberrations, and is stage III where patients are not candidates for surgical resection or definitive chemoradiation, or metastatic.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with metastatic NSCLC whose tumors express PD-L1 (TPS 1%) as determined by an FDA-approved test, with disease progression on or after platinum-containing chemotherapy. Patients with EGFR or ALK genomic tumor aberrations should have disease progression on FDA-approved therapy for these aberrations prior to receiving KEYTRUDA.

Head and Neck Squamous Cell Cancer

KEYTRUDA, in combination with platinum and fluorouracil (FU), is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent head and neck squamous cell carcinoma (HNSCC).

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent HNSCC whose tumors express PD-L1 [combined positive score (CPS) 1] as determined by an FDA-approved test.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with recurrent or metastatic HNSCC with disease progression on or after platinum-containing chemotherapy.

Classical Hodgkin Lymphoma

KEYTRUDA is indicated for the treatment of adult patients with relapsed or refractory classical Hodgkin lymphoma (cHL).

KEYTRUDA is indicated for the treatment of pediatric patients with refractory cHL, or cHL that has relapsed after 2 or more lines of therapy.

Primary Mediastinal Large B-Cell Lymphoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with refractory primary mediastinal large B-cell lymphoma (PMBCL), or who have relapsed after 2 or more prior lines of therapy. KEYTRUDA is not recommended for treatment of patients with PMBCL who require urgent cytoreductive therapy.

Urothelial Carcinoma

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC) who are not eligible for cisplatin-containing chemotherapy and whose tumors express PD-L1 (CPS 10), as determined by an FDA-approved test, or in patients who are not eligible for any platinum-containing chemotherapy regardless of PD-L1 status. This indication is approved under accelerated approval based on tumor response rate and duration of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC) who have disease progression during or following platinum-containing chemotherapy or within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.

KEYTRUDA is indicated for the treatment of patients with Bacillus Calmette-Guerin (BCG)-unresponsive, high-risk, non-muscle invasive bladder cancer (NMIBC) with carcinoma in situ (CIS) with or without papillary tumors who are ineligible for or have elected not to undergo cystectomy.

Microsatellite Instability-High or Mismatch Repair Deficient Cancer

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR)

This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with MSI-H central nervous system cancers have not been established.

Microsatellite Instability-High or Mismatch Repair Deficient Colorectal Cancer

KEYTRUDA is indicated for the first-line treatment of patients with unresectable or metastatic MSI-H or dMMR colorectal cancer (CRC).

Gastric Carcinoma

KEYTRUDA, in combination with trastuzumab, and fluoropyrimidine- and platinum-containing chemotherapy, is indicated for the first-line treatment of patients with locally advanced unresectable or metastatic HER2-positive gastric or gastroesophageal junction (GEJ) adenocarcinoma. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with recurrent locally advanced or metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test, with disease progression on or after two or more prior lines of therapy including fluoropyrimidine- and platinum-containing chemotherapy and if appropriate, HER2/neu-targeted therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Esophageal Carcinoma

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic esophageal or gastroesophageal junction (GEJ) (tumors with epicenter 1 to 5 centimeters above the GEJ) carcinoma that is not amenable to surgical resection or definitive chemoradiation either:

Cervical Carcinoma

KEYTRUDA is indicated for the treatment of patients with recurrent or metastatic cervical cancer with disease progression on or after chemotherapy whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Hepatocellular Carcinoma

KEYTRUDA is indicated for the treatment of patients with hepatocellular carcinoma (HCC) who have been previously treated with sorafenib. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Merkel Cell Carcinoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with recurrent locally advanced or metastatic Merkel cell carcinoma (MCC). This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Renal Cell Carcinoma

KEYTRUDA, in combination with axitinib, is indicated for the first-line treatment of patients with advanced renal cell carcinoma (RCC).

Tumor Mutational Burden-High

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic tumor mutational burden-high (TMB-H) [10 mutations/megabase] solid tumors, as determined by an FDA-approved test, that have progressed following prior treatment and who have no satisfactory alternative treatment options. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with TMB-H central nervous system cancers have not been established.

Cutaneous Squamous Cell Carcinoma

KEYTRUDA is indicated for the treatment of patients with recurrent or metastatic cutaneous squamous cell carcinoma (cSCC) that is not curable by surgery or radiation.

Triple-Negative Breast Cancer

KEYTRUDA, in combination with chemotherapy, is indicated for the treatment of patients with locally recurrent unresectable or metastatic triple-negative breast cancer (TNBC) whose tumors express PD-L1 (CPS 10) as determined by an FDA-approved test. This indication is approved under accelerated approval based on progression-free survival. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Selected Important Safety Information for KEYTRUDA

Severe and Fatal Immune-Mediated Adverse Reactions

KEYTRUDA is a monoclonal antibody that belongs to a class of drugs that bind to either the programmed death receptor-1 (PD-1) or the programmed death ligand 1 (PD-L1), blocking the PD-1/PD-L1 pathway, thereby removing inhibition of the immune response, potentially breaking peripheral tolerance and inducing immune-mediated adverse reactions. Immune-mediated adverse reactions, which may be severe or fatal, can occur in any organ system or tissue, can affect more than one body system simultaneously, and can occur at any time after starting treatment or after discontinuation of treatment. Important immune-mediated adverse reactions listed here may not include all possible severe and fatal immune-mediated adverse reactions.

Monitor patients closely for symptoms and signs that may be clinical manifestations of underlying immune-mediated adverse reactions. Early identification and management are essential to ensure safe use of antiPD-1/PD-L1 treatments. Evaluate liver enzymes, creatinine, and thyroid function at baseline and periodically during treatment. In cases of suspected immune-mediated adverse reactions, initiate appropriate workup to exclude alternative etiologies, including infection. Institute medical management promptly, including specialty consultation as appropriate.

Withhold or permanently discontinue KEYTRUDA depending on severity of the immune-mediated adverse reaction. In general, if KEYTRUDA requires interruption or discontinuation, administer systemic corticosteroid therapy (1 to 2 mg/kg/day prednisone or equivalent) until improvement to Grade 1 or less. Upon improvement to Grade 1 or less, initiate corticosteroid taper and continue to taper over at least 1 month. Consider administration of other systemic immunosuppressants in patients whose adverse reactions are not controlled with corticosteroid therapy.

Immune-Mediated Pneumonitis

KEYTRUDA can cause immune-mediated pneumonitis. The incidence is higher in patients who have received prior thoracic radiation. Immune-mediated pneumonitis occurred in 3.4% (94/2799) of patients receiving KEYTRUDA, including fatal (0.1%), Grade 4 (0.3%), Grade 3 (0.9%), and Grade 2 (1.3%) reactions. Systemic corticosteroids were required in 67% (63/94) of patients. Pneumonitis led to permanent discontinuation of KEYTRUDA in 1.3% (36) and withholding in 0.9% (26) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 23% had recurrence. Pneumonitis resolved in 59% of the 94 patients.

Pneumonitis occurred in 8% (31/389) of adult patients with cHL receiving KEYTRUDA as a single agent, including Grades 3-4 in 2.3% of patients. Patients received high-dose corticosteroids for a median duration of 10 days (range: 2 days to 53 months). Pneumonitis rates were similar in patients with and without prior thoracic radiation. Pneumonitis led to discontinuation of KEYTRUDA in 5.4% (21) of patients. Of the patients who developed pneumonitis, 42% of these patients interrupted KEYTRUDA, 68% discontinued KEYTRUDA, and 77% had resolution.

Immune-Mediated Colitis

KEYTRUDA can cause immune-mediated colitis, which may present with diarrhea. Cytomegalovirus infection/reactivation has been reported in patients with corticosteroid-refractory immune-mediated colitis. In cases of corticosteroid-refractory colitis, consider repeating infectious workup to exclude alternative etiologies. Immune-mediated colitis occurred in 1.7% (48/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (1.1%), and Grade 2 (0.4%) reactions. Systemic corticosteroids were required in 69% (33/48); additional immunosuppressant therapy was required in 4.2% of patients. Colitis led to permanent discontinuation of KEYTRUDA in 0.5% (15) and withholding in 0.5% (13) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 23% had recurrence. Colitis resolved in 85% of the 48 patients.

Hepatotoxicity and Immune-Mediated Hepatitis

KEYTRUDA as a Single Agent

KEYTRUDA can cause immune-mediated hepatitis. Immune-mediated hepatitis occurred in 0.7% (19/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.4%), and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 68% (13/19) of patients; additional immunosuppressant therapy was required in 11% of patients. Hepatitis led to permanent discontinuation of KEYTRUDA in 0.2% (6) and withholding in 0.3% (9) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, none had recurrence. Hepatitis resolved in 79% of the 19 patients.

KEYTRUDA with Axitinib

KEYTRUDA in combination with axitinib can cause hepatic toxicity. Monitor liver enzymes before initiation of and periodically throughout treatment. Consider monitoring more frequently as compared to when the drugs are administered as single agents. For elevated liver enzymes, interrupt KEYTRUDA and axitinib, and consider administering corticosteroids as needed. With the combination of KEYTRUDA and axitinib, Grades 3 and 4 increased alanine aminotransferase (ALT) (20%) and increased aspartate aminotransferase (AST) (13%) were seen, which was at a higher frequency compared to KEYTRUDA alone. Fifty-nine percent of the patients with increased ALT received systemic corticosteroids. In patients with ALT 3 times upper limit of normal (ULN) (Grades 2-4, n=116), ALT resolved to Grades 0-1 in 94%. Among the 92 patients who were rechallenged with either KEYTRUDA (n=3) or axitinib (n=34) administered as a single agent or with both (n=55), recurrence of ALT 3 times ULN was observed in 1 patient receiving KEYTRUDA, 16 patients receiving axitinib, and 24 patients receiving both. All patients with a recurrence of ALT 3 ULN subsequently recovered from the event.

Immune-Mediated Endocrinopathies

Adrenal Insufficiency

KEYTRUDA can cause primary or secondary adrenal insufficiency. For Grade 2 or higher, initiate symptomatic treatment, including hormone replacement as clinically indicated. Withhold KEYTRUDA depending on severity. Adrenal insufficiency occurred in 0.8% (22/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.3%), and Grade 2 (0.3%) reactions. Systemic corticosteroids were required in 77% (17/22) of patients; of these, the majority remained on systemic corticosteroids. Adrenal insufficiency led to permanent discontinuation of KEYTRUDA in <0.1% (1) and withholding in 0.3% (8) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement.

Hypophysitis

KEYTRUDA can cause immune-mediated hypophysitis. Hypophysitis can present with acute symptoms associated with mass effect such as headache, photophobia, or visual field defects. Hypophysitis can cause hypopituitarism. Initiate hormone replacement as indicated. Withhold or permanently discontinue KEYTRUDA depending on severity. Hypophysitis occurred in 0.6% (17/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.3%), and Grade 2 (0.2%) reactions. Systemic corticosteroids were required in 94% (16/17) of patients; of these, the majority remained on systemic corticosteroids. Hypophysitis led to permanent discontinuation of KEYTRUDA in 0.1% (4) and withholding in 0.3% (7) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement.

Thyroid Disorders

KEYTRUDA can cause immune-mediated thyroid disorders. Thyroiditis can present with or without endocrinopathy. Hypothyroidism can follow hyperthyroidism. Initiate hormone replacement for hypothyroidism or institute medical management of hyperthyroidism as clinically indicated. Withhold or permanently discontinue KEYTRUDA depending on severity. Thyroiditis occurred in 0.6% (16/2799) of patients receiving KEYTRUDA, including Grade 2 (0.3%). None discontinued, but KEYTRUDA was withheld in <0.1% (1) of patients.

Hyperthyroidism occurred in 3.4% (96/2799) of patients receiving KEYTRUDA, including Grade 3 (0.1%) and Grade 2 (0.8%). It led to permanent discontinuation of KEYTRUDA in <0.1% (2) and withholding in 0.3% (7) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement. Hypothyroidism occurred in 8% (237/2799) of patients receiving KEYTRUDA, including Grade 3 (0.1%) and Grade 2 (6.2%). It led to permanent discontinuation of KEYTRUDA in <0.1% (1) and withholding in 0.5% (14) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement. The majority of patients with hypothyroidism required long-term thyroid hormone replacement. The incidence of new or worsening hypothyroidism was higher in 1185 patients with HNSCC, occurring in 16% of patients receiving KEYTRUDA as a single agent or in combination with platinum and FU, including Grade 3 (0.3%) hypothyroidism. The incidence of new or worsening hypothyroidism was higher in 389 adult patients with cHL (17%) receiving KEYTRUDA as a single agent, including Grade 1 (6.2%) and Grade 2 (10.8%) hypothyroidism.

Type 1 Diabetes Mellitus (DM), Which Can Present With Diabetic Ketoacidosis

Monitor patients for hyperglycemia or other signs and symptoms of diabetes. Initiate treatment with insulin as clinically indicated. Withhold KEYTRUDA depending on severity. Type 1 DM occurred in 0.2% (6/2799) of patients receiving KEYTRUDA. It led to permanent discontinuation in <0.1% (1) and withholding of KEYTRUDA in <0.1% (1). All patients who were withheld reinitiated KEYTRUDA after symptom improvement.

Immune-Mediated Nephritis With Renal Dysfunction

KEYTRUDA can cause immune-mediated nephritis. Immune-mediated nephritis occurred in 0.3% (9/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.1%), and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 89% (8/9) of patients. Nephritis led to permanent discontinuation of KEYTRUDA in 0.1% (3) and withholding in 0.1% (3) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, none had recurrence. Nephritis resolved in 56% of the 9 patients.

Immune-Mediated Dermatologic Adverse Reactions

KEYTRUDA can cause immune-mediated rash or dermatitis. Exfoliative dermatitis, including Stevens-Johnson syndrome, drug rash with eosinophilia and systemic symptoms, and toxic epidermal necrolysis, has occurred with antiPD-1/PD-L1 treatments. Topical emollients and/or topical corticosteroids may be adequate to treat mild to moderate nonexfoliative rashes. Withhold or permanently discontinue KEYTRUDA depending on severity. Immune-mediated dermatologic adverse reactions occurred in 1.4% (38/2799) of patients receiving KEYTRUDA, including Grade 3 (1%) and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 40% (15/38) of patients. These reactions led to permanent discontinuation in 0.1% (2) and withholding of KEYTRUDA in 0.6% (16) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 6% had recurrence. The reactions resolved in 79% of the 38 patients.

Other Immune-Mediated Adverse Reactions

The following clinically significant immune-mediated adverse reactions occurred at an incidence of <1% (unless otherwise noted) in patients who received KEYTRUDA or were reported with the use of other antiPD-1/PD-L1 treatments. Severe or fatal cases have been reported for some of these adverse reactions. Cardiac/Vascular: Myocarditis, pericarditis, vasculitis; Nervous System: Meningitis, encephalitis, myelitis and demyelination, myasthenic syndrome/myasthenia gravis (including exacerbation), Guillain-Barr syndrome, nerve paresis, autoimmune neuropathy; Ocular: Uveitis, iritis and other ocular inflammatory toxicities can occur. Some cases can be associated with retinal detachment. Various grades of visual impairment, including blindness, can occur. If uveitis occurs in combination with other immune-mediated adverse reactions, consider a Vogt-Koyanagi-Harada-like syndrome, as this may require treatment with systemic steroids to reduce the risk of permanent vision loss; Gastrointestinal: Pancreatitis, to include increases in serum amylase and lipase levels, gastritis, duodenitis; Musculoskeletal and Connective Tissue: Myositis/polymyositis rhabdomyolysis (and associated sequelae, including renal failure), arthritis (1.5%), polymyalgia rheumatica; Endocrine: Hypoparathyroidism; Hematologic/Immune: Hemolytic anemia, aplastic anemia, hemophagocytic lymphohistiocytosis, systemic inflammatory response syndrome, histiocytic necrotizing lymphadenitis (Kikuchi lymphadenitis), sarcoidosis, immune thrombocytopenic purpura, solid organ transplant rejection.

Infusion-Related Reactions

KEYTRUDA can cause severe or life-threatening infusion-related reactions, including hypersensitivity and anaphylaxis, which have been reported in 0.2% of 2799 patients receiving KEYTRUDA. Monitor for signs and symptoms of infusion-related reactions. Interrupt or slow the rate of infusion for Grade 1 or Grade 2 reactions. For Grade 3 or Grade 4 reactions, stop infusion and permanently discontinue KEYTRUDA.

Complications of Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

Fatal and other serious complications can occur in patients who receive allogeneic HSCT before or after antiPD-1/PD-L1 treatment. Transplant-related complications include hyperacute graft-versus-host disease (GVHD), acute and chronic GVHD, hepatic veno-occlusive disease after reduced intensity conditioning, and steroid-requiring febrile syndrome (without an identified infectious cause). These complications may occur despite intervening therapy between antiPD-1/PD-L1 treatment and allogeneic HSCT. Follow patients closely for evidence of these complications and intervene promptly. Consider the benefit vs risks of using antiPD-1/PD-L1 treatments prior to or after an allogeneic HSCT.

Increased Mortality in Patients With Multiple Myeloma

In trials in patients with multiple myeloma, the addition of KEYTRUDA to a thalidomide analogue plus dexamethasone resulted in increased mortality. Treatment of these patients with an antiPD-1/PD-L1 treatment in this combination is not recommended outside of controlled trials.

Embryofetal Toxicity

Based on its mechanism of action, KEYTRUDA can cause fetal harm when administered to a pregnant woman. Advise women of this potential risk. In females of reproductive potential, verify pregnancy status prior to initiating KEYTRUDA and advise them to use effective contraception during treatment and for 4 months after the last dose.

Adverse Reactions

In KEYNOTE-006, KEYTRUDA was discontinued due to adverse reactions in 9% of 555 patients with advanced melanoma; adverse reactions leading to permanent discontinuation in more than one patient were colitis (1.4%), autoimmune hepatitis (0.7%), allergic reaction (0.4%), polyneuropathy (0.4%), and cardiac failure (0.4%). The most common adverse reactions (20%) with KEYTRUDA were fatigue (28%), diarrhea (26%), rash (24%), and nausea (21%).

In KEYNOTE-054, KEYTRUDA was permanently discontinued due to adverse reactions in 14% of 509 patients; the most common (1%) were pneumonitis (1.4%), colitis (1.2%), and diarrhea (1%). Serious adverse reactions occurred in 25% of patients receiving KEYTRUDA. The most common adverse reaction (20%) with KEYTRUDA was diarrhea (28%).

Excerpt from:
Merck Announces Phase 3 KEYNOTE-522 Trial Met Dual Primary Endpoint of Event-Free Survival (EFS) in Patients With High-Risk Early-Stage...

CRISPR is revolutionary. Its also a total brute.

The classic version of the gene editing wunderkind literally slices a gene to bits just to turn it off. Its effective, yes. But its like putting an electrical wire through a paper shredder to turn off a misbehaving light bulb. Once the wires are cut, theres no going back.

Why not add a light switch instead?

This month, a team from the University of California, San Francisco (UCSF) reimagined CRISPR to do just that. Rather than directly acting on genesirrevocably dicing away or swapping genetic lettersthe new CRISPR variant targets the biological machinery that naturally turns genes on or off.

Translation? CRISPR can now flip a light switch to control geneswithout ever touching them directly. It gets better. The new tool, CRISPRoff, can cause a gene to stay silent for hundreds of generations, even when its host cells morph from stem cells into more mature cells, such as neurons. Once the sleeping beauty genes are ready to wake up, a complementary tool, CRISPRon, flips the light switch back on.

This new technology changes the game so now youre basically writing a change [into genes] that is passed down, said author Dr. Luke Gilbert. In some ways we can learn to create a version 2.0 of CRISPR-Cas9 that is safer and just as effective.

The crux is something called epigenetics. Its a whole system of chemicals and proteins that controls whether a gene is turned on or off.

If that sounds confusing, lets start with what genes actually look like inside a cell and how they turn on. By turning on, I mean that genes are made into proteinsthe stuff that builds our physical form, controls our metabolism, and makes us tick along as living, breathing humans.

Genes are embedded inside DNA chains that wrap very tightly around a core proteinkind of like bacon-wrapped asparagus. For genes to turn on, the first step is that they need a bunch of proteins to gently yank the DNA chain off the asparagus, so that the genes are now free-floating inside their cellular space capsule, called the nucleus.

Once that chunk of bacon-y DNA is free, more proteins rush over to grab onto the gene. Theyll then roll down the genes nucleotides (A, T, C, and G) like a lawn mower. Instead of mulch, however, this biological machine spews out a messenger that tells the cell to start making proteinsmRNAs. (Yup, the same stuff that makes some of our Covid-19 vaccines.) mRNA directs our cells protein factory to start production, and voil, that gene is now turned on!

Anything that disrupts this process nukes the genes ability to turn into proteins, essentially shutting it off. Its enormously powerfulbecause one single epigenetic machine can control hundreds or thousands of genes. Its a master light switch for the genome.

The team started with a CRISPR system that has a neutered Cas9. This means that the protein normally involved in cutting a gene, Cas9, can no longer snip DNA, even when tethered to the correct spot by the other component, the guide RNA bloodhound. They then tacked on a protein thats involved in switching off genes to this version of CRISPR.

Heres the clever part: the protein is designed to hijack a natural epigenetic process for switching genes off. Genes are often shut down through a natural process called methylation. Normally, the process is transient and reversible on a gene. CRISPRoff commandeers this process, in turn shutting down any targeted gene but for a far longer period of timewithout physically ripping the gene apart.

Thanks to epigenetics enhancing power, CRISPRoff lets researchers go big. In one experiment targeting over 20,000 genes inside immortalized human kidney cells with CRISPRoff, the team was able to reliably shut those genes off.

Not satisfied with a one-way street, the team next engineered a similar CRISPR variant, with a different epigenetics-related protein, dubbed CRISPRon. In cells inside petri dishes, CRISPRon was able to override CRISPRoff, and in turn, flip the genes back on.

We now have a simple tool that can silence the vast majority of genes, said study author Dr. Jonathan Weissman. We can do this for multiple genes at the same time without any DNA damage and in a way that can be reversed.

Even crazier, the off switch lasted through generations. When the team turned off a gene related to the immune system, it persisted for 15 monthsafter about 450 cellular generations.

The edits also lasted through a fundamental transformation, that is, a cells journey from an induced pluripotent stem cell (iPSC) to a neuron. iPSCs often start as skin cells, and are rejuvenated into stem cells through a chemical bath, when they then take a second voyage to become neurons. This process often wipes away epigenetic changes. But to the authors surprise, CRISPRoffs influence remained through the transformations. In one experiment, the team found that shutting off a gene related to Alzheimers in iPSCs also reduced the amount of subsequently encoded toxic proteins in the resulting neurons.

What we showed is that this is a viable strategy for silencing Tau and preventing that protein from being expressed, said Weissman, highlighting just one way CRISPRoffand controlling the epigenome in generalcan alter medicine.

This isnt the first time someones tried to target the epigenome with CRISPR. The same team previously experimented with another set of CRISPR variants that tried the same thing. The difference between the two is time and stability. With the previous setup, scientists struggled to keep the light switch off for a single generation. The new one has no trouble maintaining any changes through multiple divisionsand transformationsin the genome.

A reliable CRISPR tool for epigenetics is insanely powerful. Although we have drugs that work in similar ways, theyre far less accurate and come with a dose of side effects. For now, however, CRISPRoff and CRISPRon only work in cells in petri dishes, and the next step towards genomic supremacy would be to ensure they work in living beings.

If thats the case, it could change genetic editing forever. From reprogramming biological circuits in synthetic biology to hijacking or reversing ones to prevent disease, epigenetic reprogramming offers a way to do it all without ever touching a gene, nixing the threat of mutationswhile leading to lasting effects through generations.

I think our tool really allows us to begin to study the mechanism of heritability, especially epigenetic heritability, which is a huge question in the biomedical sciences, said study author Dr. James Nuez.

Image Credit: nobeastsofierce/Shutterstock.com

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A New CRISPR Tool Flips Genes On and Off Like a Light Switch - Singularity Hub

By Guest Blogger Rich Whittle, Bioastronautics & Human Performance Lab at Texas A&M UniversityThe recent landing of the probe Perseverance on Mars, and the excitement generated by the high-resolution images currently being broadcast back to Earth, has inevitably started people thinking about human exploration of the Red Planet. However, the challenges faced by a manned journey to Mars are much more than just technical, but reflect some fundamental aspects of human physiology. In this guest article, Rich Whittle of the Bioastronautics and Human Performance Lab at Texas A&M University, reflects on some of the key issues.

NASA has ambitious plans to begin manned exploration of Mars, although its current focus is on sending the next man and first woman to the Moon as part of the Artemis programme, which will establish a permanent human presence there in the coming decade. A key part of this latter objective is to place a spaceship called Gateway in orbit around the Moon, from which landers will take astronauts to the surface and support their activities. Gateway will also conduct a wide variety of human and scientific missions, and in particular study the physiological effects of long journeys into space, in preparation for that first manned voyage to Mars.

The human body has evolved over hundreds of thousands of years to flourish on the surface of the Earth, and it is perhaps not surprising that the stresses of spaceflight pose unique physiological and medical problems. In fact, many of the basic issues associated with spaceflight, such as hypoxia, dysbarism, acceleration, and thermal support, have been well studied through aviation and diving medicine in the years prior to spaceflight. But while the last 50 years of manned space exploration have shown that humans can adapt to space, remaining productive for up to 1 year and possibly longer, there are still many problems associated with the prolonged exposure to a unique combination of stressful stimuli including acceleration, radiation, and weightlessness. The latter condition is a critical feature of spaceflight and has significant effects on human physiology, many of which were quite unexpected at the beginning of space exploration.

Scientists have known for a long time that the human body responds in specific ways to the microgravity environment of spaceflight. For example, a person who is inactive for an extended period loses overall strength, as well as muscle and bone mass. Unsurprisingly spaceflight has a similar effect, resulting in loss of bone mineral density (BMD), and increasing the risk of bone fractures in astronauts. It is predicted that a third of astronauts will be at risk for osteoporosis during a predicted 7-month long human mission to Mars. It is however possible to compensate for this loss of muscle and bone mass using resistive exercise devices that NASA has developed to allow for more intense workouts in zero gravity.

Overall, the pathophysiological adaptive changes that occur during spaceflight, even in well-trained, highly selected, and healthy individuals, have been likened to an accelerated aging process, and are being studied in research groups around the world. My own research at Texas A&M focuses on changes to the cardiovascular system caused by the microgravity environment of spaceflight. In an upright position under the Earths standard 1G gravity, arterial blood pressure is lower above the heart and higher below the heart. But in a weightless environment the body experiences a uniform arterial pressure, which decreases the cardiac workload, and reduces the need for blood pressure regulatory mechanisms. As a result, the muscles of the heart and blood vessels begin to atrophy, and consequently some astronauts experience orthostatic intolerance, the difficulty or inability to stand because of light headedness after return to Earth. During spaceflight, cardiovascular changes are noticeable immediately after the onset of weightlessness, with astronauts exhibiting characteristically puffy faces, stuffed noses, and chicken legs, as approximately 2L of fluid is shifted from the legs towards the head.

These fluid shifts affect not only the cardiovascular system but also the brain, eyes, and other neurological functions. The apparent increase in fluid within the skull is potentially linked to a collection of pathologies of the eye known as Spaceflight Associated Neuro-ocular Syndrome (SANS). This is principally manifested through a hyperopic shift in visual acuity, which in some cases does not resolve on return to Earth.

We believe that many of these problems can be overcome through effective countermeasures during spaceflight, and are often reversible after landing. Physical exercise programs are the main countermeasure used during spaceflight to protect the cardiovascular system. The technology involved has advanced from a rowing ergometer used in the early Skylab missions, through a motorized treadmill used in the ISS. This has been recently joined by a device for performing resistive exercise, and now rowing ergometers are once again being looked at for longer duration missions to Mars due to their small footprint.

However, some astronauts have returned from the ISS with unexpectedly stiff arteries, of a magnitude expected from 10 20 years of normal aging. Arterial stiffening is often linked to an increased blood pressure and elevated risk for cardiovascular disease. Additionally, other studies have suggested that insulin resistance occurs during spaceflight, possibly due to reduced physical activity, which could lead to increased blood sugar and increased risk of developing type 2 diabetes. These results suggest that the astronauts exercise routine did not always counteract the effect of the microgravity environment and indicated that further countermeasures might be needed to help maintain astronaut health. Here at Texas A&M we are looking at both lower body negative pressure (LBNP) and artificial gravity generated through short radius centrifugation as exciting new countermeasures that could be used in long duration spaceflight.

As we begin to further understand the effects of spaceflight on human physiology, scientists are now starting to study some of the underlying cellular mechanisms using model organisms, cell cultures, organs on a chip and stem cells. And because many of the observed changes seen in space, such as cardiovascular dysfunction due to inflammation, lack of exercise, intracranial hypertension, and hormonal and metabolic changes, resemble those caused by aging or illnesses, the research we conduct may have important applications on Earth. Hopefully, our push for manned exploration of the planets of the solar system will lead to tangible benefits to the health and well-being of humans on our home planet.

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The Physiological Challenges of Spaceflight - Cambridge Wireless

Synthego, the genome engineering company, today announced the launch of Eclipse, a new high-throughput cell engineering platform designed to accelerate drug discovery and validation by providing highly predictable CRISPR-engineered cells at scale through the integration of engineering, bioinformatics, and proprietary science. The launch of this unique CRISPR-based platform is driving the companys growing impact in biopharma R&D, reinforcing Synthegos position as the genome engineering leader.

CRISPR-engineered cells have a wide range of applications in research and development across disease areas, including in neuroscience and oncology. Synthego created the Eclipse Platform to enhance disease modeling, drug target identification and validation, and accelerate cell therapy manufacturing.

"By industrializing cell engineering, Synthegos Eclipse Platform will enable economies of scale, turning a historically complex process into one that is flexible, reliable, and affordable, said Bill Skarnes, Ph.D., professor and director of Cellular Engineering at The Jackson Laboratory and Synthego advisory board member. Offering CRISPR edits at scale, similar to what Synthego did with sgRNA reagents, puts researchers on the cusp of being able to study thousands of genes, and examine hundreds of variants of those genes. This will allow scientists to more faithfully model the complexity of a human disease, which could lead to the development of therapeutic drugs or next-generation gene therapies for many serious diseases.

To ensure the success of any type of edit, Eclipse uses machine learning to apply experience from several hundred thousand genome edits across hundreds of cell types. With this machine learning, combined with automation, the new platform can reduce costs and increase the scalability of engineered cell production. The Eclipse Platform is modular in design, allowing for fast deployment of upgrades or add-ons. It is engineered to use a cell-type agnostic process and immediately benefit researchers working with induced pluripotent stem (iPS) cells and immortalized cell lines.

We are living in a new era of life sciences innovation one that has added to DNA sequencing and being able to read out of biology, now being able to write into and engineer biology. We created our Eclipse Platform at the convergence of science and technology to make genome editing more precise, scalable, and accessible, said Paul Dabrowski, CEO and co-founder of Synthego. We are excited to expand our impact on advancing the life sciences innovation with the launch of this unique CRISPR-based platform.

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Synthego Launches Eclipse Platform to Accelerate Research and Development of Next-generation Medicines - The Scientist

Advances in the treatment of patients with leukemias and lymphomas have led to a significant improvement in survival, which has increased the need for bone marrow transplant as a later-line therapy, said Mehrdad Abedi, MD, who added that Orca-T, a high precision cell therapy, confers significant antitumor activity, minimizes the incidence of acute and chronic graft-vs-host disease (GVHD), and causes less adverse effects (AEs) compared with standard bone marrow transplant among these patients.

There is a lot of research [ongoing]; the holy grail of research is trying to figure out whether we can separate the graft-vs-leukemia or graft-vs-lymphoma effect from GVHD, said Abedi. [The Orca-T trial] is basically looking at the past 10 years of research in this area to try to identify the cells that are good from those that are bad.

During the 2021 Transplantation and Cellular Therapy (TCT) Meetings, findings from an analysis of 2 studies demonstrated a significant reduction in cases of GVHD, a higher GVHD relapse-free survival rate, and a lack of treatment-related mortalities with Orca-T when historically compared with hematopoietic stem cell transplant (HSCT).

Additionally, the median time to neutrophil engraftment, median time to platelet engraftment, and median time from day 0 to hospital discharge was shortened with Orca-T compared with HSCT.

In an interview with OncLive during the 2021 TCT Meetings, Abedi, a professor of cancer, hematology/oncology, and internal medicine in the Department of Internal Medicine, Division of Hematology and Oncology at the UC Davis Comprehensive Cancer Center, discussed the challenges of standard transplant, how Orca-T could overcome some of those limitations, and the potential future of transplant in hematologic malignancies.

Abedi: HSCT has been around for more than 50 years in one form or another. It has been used mostly for patients with blood cancers, [such as] leukemia and lymphoma. Allogeneic bone marrow transplants, where we use cells from a donor, are very effective. [They are associated with] a very high response rate for patients who have no other options and whose disease is going to [recur] without transplant.

The problem [with allogeneic bone marrow transplant] is that it [is associated with] AEs. When we give donor cells to patients after high-dose chemotherapy, [which is given] so that the patients body doesnt reject [the cells], even though the cells are a match for the patient, they can still [develop] severe GVHD.

The acute form of GVHD can be life threatening, whereas the chronic form can become a nuisance for the rest of a patients life. A lot of patients suffer [from GVHD] to the point where they regret going through transplant. Fortunately, that is not everybody, but it is still a problem that needs to be solved and an unmet need for the field.

This research has been focused on [the question of]: Are there specific cells in the graft we give to the patients immune cells that can cause GVHD? Can we separate those cells from those that are responsible for causing graft-vs-leukemia effects?

Basically, Orca Bio approached UC Davis a few years ago to [start] collaborative research with our Good Manufacturing Practice [GMP] facility and to produce these products.

Each graft of [the Orca-T] product has stem cells and immune cells in it. The stem cells are what we need to maintain the graft and [allow the product to] stay in the patient for a long time. The immune cells are the ones that can cause graft-vs-leukemia [effects], which is what we want.

From the work that Robert Negrin, [MD] at Stanford University and many other investigators [did], it is very clear that there is a population of T cells called T-regulatory cells that can prevent GVHD. There are other populations, such as the nave T cells or conventional T cells that can cause GVHD. It looks like a smaller number of [those cells] may actually be helpful; they can cause graft-vs-leukemia, but not GVHD.

The graft is basically designed so that we can give stem cells, but they get rid of a lot of conventional T cells that can cause rapid GVHD. [The graft provides] a small amount of conventional T cells that can cause graft-vs-leukemia effects, as well as the regulatory T cells that can prevent GVHD. UC Davis got involved [with this work] because we have a very robust GMP facility. We helped Orca Bio design these protocols and manufacture the cells. Now, [Orca Bio] has moved on to their own facility. I have been involved [with this research] for the past few years.

[We] havent looked at the QOL data, but there have been several short-term and long-term benefits so far that we have seen.

We give high-dose chemotherapy that can get rid of all [the patients] stem cells and leukemia or lymphoma cells. Then, we give the graft; however, after the graft is given, it takes a couple of weeks usually to engraft [before] new cells [develop]. In between, the patients are sick because of the effects of the chemotherapy; patients also have low blood counts.

[With Orca-T] we have seen that the engrafting [occurs] a little bit earlier. For everyday [sooner] the engraftment [occurs], there is less risk of complication and suffering for the patient. That has been a major difference [with Orca-T].

Also, we have noticed that patients in general are doing better [with Orca-T compared with traditional transplant]. When we give high-dose chemotherapy, it is the same whether the patient is on a clinical trial or not. They may still get sick [with Orca-T] because of the effects of chemotherapy.

However, there is also inflammation [that can occur] as the donor cells are trying to establish themselves in the body because some of them attack the body. That inflammation also adds to the problems with chemotherapy. We havent seen that inflammation [with Orca-T]. We have seen some effects from the chemotherapy, but because we dont have the inflammation, other AEs that we see with the standard of care arent seen with Orca-T.

In general, patients do better [with Orca-T vs standard of care], and that has been a universal experience with all of our patients who have gone through the trial so far.

[Patients] just look and feel better. When they are discharged, they are not as sick compared with patients [receiving] the standard of care.

With the standard of care, after we give the graft, we have to give patients a new medication to prevent GVHD because it is such a lethal problem. If we dont put patients on immunosuppressive medications to prevent GVHD, there is a very high chance that they get and die from GVHD. Those immunosuppressive medications are critical.

The way the Orca-T graft is designed is that we dont need as many of those [immunosuppressive] medications. For example, there is a medication called methotrexate that we give on days 1, 3, 6, and 11 after [standard] transplant. That medication can cause a lot of other AEs, such as mouth sores, delayed engraftment, and kidney [problems], that can make the patient miserable. With Orca-T, we dont have to [give methotrexate], which by itself is a huge improvement.

We do give immunosuppressive medications, such as tacrolimus [Envarsus XR] or sirolimus [Rapamune] to these patients, but we dont give 2 or 3 medications as we usually do with the standard of care. Less immunosuppressive medications mean probably less infection, but more importantly, less AEs. Thats [a factor that has made] a big difference in patient experience.

That is a very loaded question. There are a lot of new drugs coming, some of which are very targeted to treat leukemia or lymphoma. Thus far, we havent seen any curative [benefit] with those drugs, so in most situations, we will still need an allogeneic stem cell transplant for patients.

The exceptions [to that] were rare diseases, such as chronic myeloid leukemia [CML], where [an oral medication was approved] and we dont need to transplant patients. That is an example of a targeted treatment that may exclude [the need to] transplant patients. That would be great because transplant is not a trivial procedure and it has a lot of AEs.

However, [CML] is a very specific disease with 1 gene that causes the disease. In most leukemias and lymphomas, multiple genes are involved, so we dont think single-target treatments will get rid of the disease. We still think that allogeneic transplant will be around for a long time.

In fact, if you look at any center in the country, the volume of transplant has substantially increased over time because patients are living longer. Patients end up being able to go to a transplant vs before when many were dying in the middle of their treatment and not getting to transplant.

That being said, there are 2 directions [we can go in]. One is to try to decrease the AEs associated with transplant. This [Orca-T cell therapy] is very effective [in doing this] by targeting the graft-vs-leukemia effect and preventing GVHD. The second direction is to use these allogeneic cells to specifically target the tumor cells. That is why gene modifications, CAR T-cell therapies, and other approaches are coming. They still use allogeneic cells from a donor, but now they are directing them to specifically go after tumor cells.

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Orca-T Offers an Alternative to HSCT With Improved Patient Experience - OncLive

Scientists have created small, synthetic living machines that self-organize from single cells, move quickly through different environments without the need for muscle cells, can remember their experiences, heal themselves when damaged, and exhibit herd behaviors.

Earlier, scientists have developed swarms of robots from synthetic materials and moving biological systems from muscle cells grown on precisely shaped scaffolds. But until now the creation of a self-directed living machine has remained beyond reach.

Biologists and computer scientists from Tufts University and the University of Vermont have created novel, tiny self-healing living machines from frog cells (Xenopus laevis) that they call Xenobots. These can move around, push a payload, and even exhibit collective behavior in a swarm.

In an article titled A cellular platform for the development of synthetic living machinespublished in the journal Science Robotics, the researchers report a method for creating these of Xenobots from frog cells. This cellular platform can be used to study self-organization, collective behavior, and bioengineering and provide versatile, soft-body, living machines for applications in biomedicine and environmental biology.

The next version of Xenobots have been createdtheyre faster, live longer, and can now record information [Doug Blackiston, Tufts University]

We are witnessing the remarkable plasticity of cellular collectives, which build a rudimentary new body that is quite distinct from their defaultin this case, a frog despite having a completely normal genome, says Michael Levin, Distinguished Professor of Biology and director of the Allen Discovery Center at Tufts University, and corresponding author of the study. In a frog embryo, cells cooperate to create a tadpole. Here, removed from that context, we see that cells can re-purpose their genetically encoded hardware, like cilia, for new functions such as locomotion. It is amazing that cells can spontaneously take on new roles and create new body plans and behaviors without long periods of evolutionary selection for those features.

Xenobots can move around in a coordinated manner with the help of cilia present on their surface. These cilia grow through normal tissue patterning and do not require complicated architectural procedures such as scaffolding or microprinting, making the high-throughput production of Xenobots possible. And while the frog cells are organizing themselves into Xenobots, they are amenable to surgical, genetic, chemical, and optical stimulation. The researchers show that the Xenobots can maneuver through water, heal after damage and exhibit predictable collective behaviors.

The scientists also provide a proof of principle for a programmable molecular memory using a light-controlled protein that can record exposure to a specific wavelength of light.

Compared to their first edition, Xenobots 1.0, that were millimeter-sized automatons constructed in a top down approach by manual placement of tissue and surgical shaping of frog skin and cardiac cells to produce motion, this updated version of Xenobots 2.0 takes a bottom up approach. The biologists took stem cells from frog embryos and allowed them to self-assemble and grow into spheroids, where some of the cells after a few days differentiated to produce ciliatiny hair-like projections that move back and forth or rotate in a specific way. Cilia act like legs to help the new spheroidal Xenobots move rapidly across a surface.

In a way, the Xenobots are constructed much like a traditional robot. Only we use cells and tissues rather than artificial components to build the shape and create predictable behavior. Says Doug Blackiston, PhD, senior scientist and co-first author on the study with research technician, Emma Lederer. On the biology end, this approach is helping us understand how cells communicate as they interact with one another during development, and how we might better control those interactions.

Scientists at UVM ran computer simulations that modeled different shapes of the Xenobots and analyzed its effects on individual and collective behavior. Robotics expert, Joshua Bongard, PhD, and a team of computer scientists used an evolutionary algorithm on the Deep Green supercomputer cluster at UVMs Vermont Advanced Computing Core to simulate the behavior of the xenobots under numerous random environmental conditions. These simulations identified Xenobots that excelled at working together in swarms to gather large piles of debris in a field of particles.

We know the task, but its not at all obviousfor peoplewhat a successful design should look like. That is where the supercomputer comes in and searches over the space of all possible Xenobot swarms to find the swarm that does the job best, says Bongard. We want Xenobots to do useful work. Right now, were giving them simple tasks, but ultimately were aiming for a new kind of living tool that could, for example, clean up microplastics in the ocean or contaminants in soil.

Xenobots can quickly collect garbage working together in a swarm to sweep through a petri dish and gather larger piles of iron oxide particles. They can also cover large flat surfaces and travel through narrow capillaries.

The Tufts scientists engineered the Xenobots with a memory capability to record one bit of information, using a fluorescent reporter protein called EosFP that glows green but when exposed to blue light at 390nm wavelength, the protein emits red light instead.

The researchers injected the cells of the frog embryos with messenger RNA coding for the EosFP protein before the stem cells were excised to create the Xenobots so that the mature Xenobots have a built-in fluorescent switch which can record exposure to blue light.

To test the memory capacity, the investigators, allowed 10 Xenobots to swim around a surface on which one spot is illuminated with a beam of blue light. After two hours, they found that three bots emitted red light. The rest remained their original green, effectively recording the travel experience of the bots. This molecular memory in Xenobots could be harnessed to detect the presence of radioactive contamination, chemical pollutants, drugs, or a disease condition.

When we bring in more capabilities to the bots, we can use the computer simulations to design them with more complex behaviors and the ability to carry out more elaborate tasks, said Bongard. We could potentially design them not only to report conditions in their environment but also to modify and repair conditions in their environment.

The biological materials we are using have many features we would like to someday implement in the botscells can act like sensors, motors for movement, communication and computation networks, and recording devices to store information, says Levin. One thing the Xenobots and future versions of biological bots can do that their metal and plastic counterparts have difficulty doing is constructing their own body plan as the cells grow and mature, and then repairing and restoring themselves if they become damaged. Healing is a natural feature of living organisms, and it is preserved in Xenobot biology.

Cells in a biological robot can also absorb and break down chemicals and work like tiny factories, synthesizing and excreting chemicals and proteins.

Xenobots were designed to exhibit swarm activity, moving about on cilia legs. [Doug Blackiston, Tufts University]

Originally posted here:
Scientists Create Living Machines That Move, Heal, Remember and Work in Groups - Genetic Engineering & Biotechnology News

MIAMI, Feb. 19, 2021 (GLOBE NEWSWIRE) -- Longeveron Inc. (NASDAQ: LGVN) ("Longeveron" or "Company"), a clinical stage biotechnology company developing cellular therapies for chronic aging-related and life-threatening conditions, today announced that Geoff Green, Chief Executive Officer of Longeveron, has been invited to give a presentation titled “A Regenerative Medicine Approach to Aging Frailty” at the Intercontinental Summit on Aging & Gerontology being held virtually on March 8, 2021. He will discuss Longeveron’s ongoing clinical research program in Aging Frailty and Alzheimer’s disease, in addition to the regenerative medicine investigational clinical research field in general for diseases and conditions associated with aging.

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CEO of Longeveron Inc. to Present at the Intercontinental Summit on Aging & Gerontology

Stem cells are undifferentiated cells which are capable of differentiating into any type of cell that make-up the human body and thus, are capable of producing non-regenerative cells such as neural and myocardial cells.

Statistics:

The global stem cells market is estimated to account forUS$ 9,941.2 Mnin terms of value in2020and is expected to reachUS$ 18,289.9 Mnby the end of2027.

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GlobalStem CellsMarket: Drivers

Approval and launch of new products is expected to propel growth of the global stem cells market over the forecast period. For instance, in December 2019, BioRestorative Therapies, Inc. received a Notice of Allowance on its patent application for a method of generating brown fat stem cells from Israeli Patent Office.

Moreover, increasing number of stem cell banking resource centers is also expected to aid in growth of the market. For instance, in March 2020, Stemlife Berhad, a cord blood bank in Malaysia, started a Stem Cell Banking Resource Center in Jerudong Park Medical Center, Brunei.

Quick Buy Stem Cells Market Research Report: https://www.coherentmarketinsights.com/insight/buy-now/4222

Statistics:

Adult stem cells held dominant position in the global stem cells market in 2019, accounting for81.2%share in terms of value, followed by Human Embryonic Stem Cells and Induced Pluripotent Stem Cells, respectively

Figure 1. GlobalStem CellsMarket Share (%), by Value, by Cell Type, 2019.

GlobalStem CellsMarket: Restraints

High cost of stem cell therapy is expected to hinder growth of the global stem cells market. For instance, Bioinformant a research firm engaged in stem cell research, reported that the cost of stem cell therapy ranges between US$ 5,000-8,000 per patient and in some cases it may rise as much as US$ 25,000 or more depending on the complexity of the procedure.

Moreover, restrictions on research activities related to stem cells had hampered the growth of embryonic stem cells historically and resulted in its meager share in the total market in spite of its advantages over adult stem cells.

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GlobalStem CellsMarket: Opportunities

R&D in stem cell donation is expected to offer lucrative growth opportunities for players in the global stem cells market. For instance, in March 2020, researchers from Dankook University and Catholic University, South Korea, reported investigation of the types and degrees of physical and psychological discomfort experienced by hematopoietic stem cell donors before, during, and after the donation process.

Moreover, adoption of online distribution channel is also expected to aid in growth of the global stem cells market. For instance, The US Direct-to-Consumer Marketplace for Autologous Stem Cell Interventions, published in the journal Perspectives in Biology and Medicine, in 2018, the number of new stem cell businesses with websites doubled on average every year between 2009 and 2014, in the U.S.

The global stem cells market was valued atUS$ 9,112.0 Mnin2019and is forecast to reach a value ofUS$ 18,289.9 Mnby2027at aCAGR of 9.1%between2020 and 2027.

Figure 2. GlobalStem CellsMarket Value (US$ Mn), and Y-o-Y Growth (%), 2019-2027

Market Trends/Key Takeaways

Adoption of stem cells for the treatment of various diseases is expected to propel growth of the global stem cells market. For instance, in January 2020, researchers at University of Houston developed biologic cardiac pacemaker-like cells by taking fat stem cells and reprogramming them as an alternative treatment for heart conditions such as conduction system disorders and heart attacks.

Moreover, increasing investment in stem cell therapies is also expected to aid in growth of the market. For instance, in July 2018, the Emory Orthopaedics & Spine Center, in collaboration with Sanford Health, Duke University, Andrews Institute, and Georgia Institute of Technology, received US$ 13 million grant from the Marcus Foundation for a multicenter clinical trial studying stem cell options for treating osteoarthritis. The Phase 3 trial was initiated in March 2019, and is expected to complete by December 2021.

GlobalStem CellsMarket: Competitive Landscape

Major players operating in the global stem cells market include Advanced Cell Technology, Inc., Angel Biotechnology Holdings PLC, Bioheart Inc., Lineage Cell Therapeutics., BrainStorm Cell Therapeutics, Inc., California Stem Cell Inc., Celgene Corporation, Takara Bio Europe AB, Cellular Engineering Technologies, Cytori Therapeutics Inc., Osiris Therapeutics, and STEMCELL Technologies Inc.

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GlobalStem CellsMarket: Key Developments

Major players in the market are focused on adopting collaboration and partnership strategies to expand their product portfolio. For instance, in September 2018, STEMCELL Technologies signed an exclusive license agreement with Brigham and Womens Hospital for rights to commercialize technologies for the generation of human pluripotent stem cell-derived kidney organoids.

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We can absolutely cut the number of cancer deaths down so that one day in our lifetimes it can be a rare thing for people to die of cancer, said Patrick Hwu, MD, president and CEO of Moffitt Cancer Center in Florida and among gene therapys pioneers. It still may happen here and there, but itll be kind of like people dying of pneumonia. Its like, He died of pneumonia? Thats kind of weird. I think cancer can be the same way.

The excitement returned in spades in 2017 when the FDA signed off on a gene-therapy drug for the first time, approving the chimeric antigen receptor (CAR) T-cell treatment tisagenlecleucel (Kymriah; Novartis) for the treatment of B-cell precursor acute lymphoblastic leukemia. At last, scientists had devised a way to reprogram a persons own T cells to attack tumor cells.

Were entering a new frontier, said Scott Gottlieb, MD, then the FDA Commissioner, in announcing the groundbreaking approval.

Gottlieb wasnt exaggerating. The growth in CAR T-cell treatments is exploding. Although only a handful of cell and gene therapies are on the market, FDA officials predicted in 2019 that the agency will receive more than 200 investigational new drug applications per year for cell and gene therapies, and that by 2025, it expects to have accelerated to 10 to 20 cell and gene therapy approvals per year.1

Essentially, you can kill any cancer cell that has an antigen that is recognized by the immune cell, Hwu said. The key to curing every single cancer, which is our goal, is to have receptors that can recognize the tumor but dont recognize the normal cells. Receptors recognizing and then attacking normal cells is what can cause toxicity.

Cell therapy involves cultivating or modifying immune cells outside the body before injecting them into the patient. Cells may be autologous (self-provided) or allogeneic (donor-provided); they include hematopoietic stem cells and adult and embryonic stem cells. Gene therapy modifies or manipulates cell expression. There is considerable overlap between the 2 disciplines.

Juliette Hordeaux, PhD, senior director of translational research for the University of Pennsylvanias gene therapy program, is cautious about the FDAs predictions, saying shed be thrilled with 5 cell and/or gene therapy approvals annually.

For monogenic diseases, there are only a certain number of mutations, and then well plateau until we reach a stage where we can go after more common diseases, Hordeaux said.

Safety has been the main brake around adeno-associated virus vector (AAV) gene therapy, added Hordeaux, whose hospitals program has the institutional memory of both Jesse Gelsingers tragic death during a 1999 gene therapy trial as well as breakthroughs by Carl June, MD, and others in CAR T-cell therapy.

Sometimes there are unexpected toxicity [events] in trials.I think figuring out ways to make gene therapy safer is going to be the next goal for the field before we can even envision many more drugs approved.

In total, 3 CAR T-cell therapies are now on the market, all targeting the CD19 antigen. Tisagenlecleucel was the first. Gilead Sciences received approval in October 2017 for axicabtagene ciloleucel (axi-cel; Yescarta), a CAR T-cell therapy for adults with large B-cell non-Hodgkin lymphoma. Kite Pharma, a subsidiary of Gilead, received an accelerated approval in July 2020 for brexucabtagene autoleucel (Tecartus) for adults with relapsed or refractory mantle cell lymphoma.

On February 5, 2021, the FDA approved another CD19-directed therapy for relapsed/refractory large B-cell lymphoma, lisocabtagene maraleucel (liso-cel; JCAR017; Bristol Myers Squibb). The original approval date was missed due to a delay in inspecting a manufacturing facility (see related article).

Idecabtagene vicleucel (ide-cel; bb2121; Bristol Myers Squibb) is under priority FDA review, with a decision expected by March 31, 2021. The biologics license application seeks approval for ide-cel, a B-cell maturation antigendirected CAR therapy, to treat adult patients with multiple myeloma who have received at least 3 prior therapies.2

The number of clinical trials evaluating CAR T-cell therapies has risen sharply since 2015, when investigators counted a total of 78 studies registered on the ClinicalTrials.gov website. In June 2020, the site listed 671 trials, including 357 registered in China, 256 in the United States, and 58 in other countries.3

Natural killer (NK) cells are the research focus of Dean Lee, MD, PhD, a physician in the Division of Hematology and Oncology at Nationwide Childrens Hospital. He developed a method for consistent, robust expansion of highly active clinical-grade NK cells that enables repeated delivery of large cell doses for improved efficacy. This finding led to several first-in-human clinical trials evaluating adoptive immunotherapy with expanded NK cells under an FDA Investigational New Drug application. He is developing both genetic and nongenetic methods to improve tumor targeting and tissue homing of NK cells. His eff orts are geared toward pediatric sarcomas.

The biggest emphasis over the past 20 to 25 years has been cell therapy for cancer, talking about trying to transfer a specific part of the immune system for cells, said Lee, who is also director of the Cellular Therapy and Cancer Immunology Program at Nationwide Childrens Hospital, at The Ohio State University Comprehensive Cancer Center Arthur G. James Cancer Hospital, and at the Richard J. Solove Research Institute.

The Pivot Toward Treating COVID-19 and Other Diseases

However, Lee said, NKs have wider potential. This is kind of a natural swing back. Now that we know we can grow them, we can reengineer them against infectious disease targets and use them in that [space], he said.

Lee is part of a coronavirus disease 2019 (COVID-19) clinical trial, partnering with Kiadis, for off-the-shelf K-NK cells using Kiadis proprietary platforms. Such treatment would be a postexposure preemptive therapy for treating COVID-19. Lee said the pivot toward treating COVID-19 with cell therapy was because some of the very early reports on immune responses to coronavirus, both original [SARS-CoV-2] and the new [mutation], seem to implicate that those who did poorly [overall] had poorly functioning NK cells.

The revolutionary gene editing tool CRISPR is making its initial impact in clinical trials outside the cancer area. Its developers, Jennifer Doudna, PhD, and Emmanuelle Charpentier, PhD, won the Nobel Prize in Chemistry 2020.

For patients with sickle cell disease (SCD), CRISPR was used to reengineer bone marrow cells to produce fetal hemoglobin, with the hope that the protein would turn deformed red blood cells into healthy ones. National Public Radio did a story on one patient who, so far, thanks to CRISPR, has been liberated from the attacks of SCD that typically have sent her to the hospital, as well from the need for blood transfusions.4

Its a miracle, you know? the patient, Victoria Gray of Forest, Mississippi, told NPR.

She was among 10 patients with SCD or transfusion-dependent beta-thalassemia treated with promising results, as reported by the New England Journal of Medicine.5 Two different groups, one based in Nashville, which treated Gray,5 and another based at Dana-Farber Cancer Institute in Boston,6 have reported on this technology.

Stephen Gottschalk, MD, chair of the department of bone marrow transplantation and cellular therapy at St Jude Childrens Research Hospital, said, Theres a lot of activity to really explore these therapies with diseases that are much more common than cancer.

Animal models use T cells to reverse cardiac fibrosis, for instance, Gottschalk said. Using T cells to reverse pathologies associated with senescence, such as conditions associated with inflammatory clots, are also being studied.

Hordeaux said she foresees AAV being used more widely to transmit neurons to attack neurodegenerative diseases.

The neurons are easily transduced by AAV naturally, she said. AAV naturally goes into neurons very efficiently, and neurons are long lived. Once we inject genetic matter, its good for life, because you dont renew neurons.

Logistical Issues

Speed is of the essence, as delays in producing therapies can be the difference between life and death, but the approval process takes time. The process of working out all kinks in manufacturing also remains a challenge. Rapid production is difficult, too, because of the necessary customization of doses and the need to ensure a safe and effective transfer of cells from the patient to the manufacturing center and back into the patient.7

Other factors that can slow down launches include insurance coverage, site certification, staff training, reimbursement, and patient identification. The question of how to reimburse has not been definitively answered; at this point, insurers are being asked to issue 6- or even 7-figure payments for treatments and therapies that may not work.8

CAR T, I think, will become part of the standard of care, Gottschalk said. The question is how to best get that accomplished. To address the tribulations of some autologous products, a lot of groups are working with off -the-shelf products to get around some of the manufacturing bottlenecks. I believe those issues will be solved in the long run.

References

1. Statement from FDA Commissioner Scott Gottlieb, MD, and Peter Marks, MD, PhD, director of the Center for Biologics Evaluation and Research on new policies to advance development of safe and effective cell and gene therapies. News release. FDA website. January 15, 2019. https://www.fda.gov/news-events/press-announcements/statement-fda-commissioner-scott-gottlieb-md-and-peter-marks-md-phd-director-center-biologics. Accessed January 13, 2021.

2. Bristol Myers Squibb provides regulatory update on lisocabtagene maraleucel (liso-cel). News release. Bristol Myers Squibb; November 16, 2020. Accessed January 11, 2021. https://news.bms.com/news/details/2020/Bristol-Myers-Squibb-Provides-Regulatory-Update-on-Lisocabtagene-Maraleucel-liso-cel/default.aspx

3. Wei J, Guo Y, Wang Y. et al. Clinical development of CAR T cell therapy in China: 2020 update. Cell Mol Immunol. Published online September 30, 2020. doi:10.1038/s41423-020-00555-x

4. Stein R. CRISPR for sickle cell diseases shows promise in early test. Public Radio East. November 19, 2019. Accessed January 11, 2021. https://www.publicradioeast.org/post/crisprsickle-cell-disease-shows-promise-early-test

5. Frangoul H, Altshuler D, Cappellini MD, et al. CRISPR-Cas9 gene editing for sickle cell disease and -Thalassemia. N Engl J Med. Published online December 5, 2020. DOI: 10.1056/NEJMoa2031054

6. Esrick EB, Lehmann LE, Biffi A, et al. Post-transcriptional genetic silencing of BCL11A to treat sickle cell disease. N Engl J Med. Published online December 5, 2020. doi:10.1056/NEJMoa2029392

7. Yednak C. The gene therapy race. PwC. February 5, 2020. Accessed January 11, 2021. https://www.pwc.com/us/en/industries/healthindustries/library/gene-therapy-race.html

8. Gene therapies require advanced capabilities to succeed after approval. PwC website. Accessed January 11, 2021. https://www.pwc.com/us/en/industries/health-industries/library/commercializing-gene-therapies.html

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The Untapped Potential of Cell and Gene Therapy - AJMC.com Managed Markets Network

Introduction

Extracellular vesicles (EVs) including exosomes, microvesicles, and apoptotic bodies are produced and released by almost all types of cell. EVs vary in size, properties, and secretion pathway depending on the originating cell.1,2 Exosomes are small EVs (sEVs) which are formed by a process of inward budding in early endosomes to form multivesicular bodies (MVBs) with an average size of 100 nm, and released into the extracellular microenvironment to transfer their components.3,4 Microvesicles are composed of lipid components of the plasma membrane and their sizes range from 1001000 nm, whereas apoptotic bodies result from programmed cell death.5 Initially, EVs were considered to maintain cellular waste through release of unwanted proteins and biomolecules; later, these organelles were considered important for intercellular communications through various cargo molecules such as lipids, proteins, DNA, RNA, and microRNAs (miRNAs).6 Previously, it was suggested that EVs play a critical role in normal cells to maintain homeostasis and prevent cancer initiation. Inhibition of EVs secretion causes accumulation of nuclear DNA in the cytoplasm, leading to apoptosis.1 The induction of apoptosis is the principal event of the reactive oxygen species (ROS) dependent DNA damage response.7,8

Several studies reported that exosomes are synthesized by means of two major pathways, the endosomal sorting complexes required for transport (ESCRT)-dependent and ESCRT-independent, and the processes are highly regulated by multiple signal transduction cascades.18 Exosomes released from the cell through normal exocytosis mechanisms are characterized by vesicular docking and fusion with the aid of SNARE complexes. Exosomes are considered to be organelle responsible for garbage disposal agents. However, at a later stage, these secretory bodies play a critical role in maintaining the physiological and pathological conditions of the surrounding cells by transferring information from donor cells to recipient cells. Exosome development begins with endocytosis to form early endosomes, later forming multivesicular endosomes (MVEs), and finally generating late endosomes by inward budding. MVEs merge with the cell membrane and release intraluminal endosomal vesicles that become exosomes into the extracellular space.9,10 Exosome biogenesis is dependent on various critical factors including the site of biogenesis, protein sorting, physicochemical aspects, and transacting mediators.11

Exosomes contain various types of cargo molecules including lipids, proteins, DNAs, mRNAs, and miRNAs. Most of the cargo is involved in the biogenesis and transportation ability of exosomes.12,13 Exosomes are released by fusion of MVBs with the cell membrane via activation of Rab-GTPases and SNAREs. Exosomes are abundant and can be isolated from a wide variety of body fluids and also cell culture medium.14 Exosomes contain tetraspanins that are responsible for cell penetration, invasion, and fusion events. Exosomes are released onto the external surface by the MVB formation proteins Alix and TSG101. Exosome-bound proteins, annexins and Rab protein, govern membrane transport and fusion whereas Alix, flotillin, and TSG101 are involved in exosome biogenesis.15,16 Exosomes contain various types of proteins such as integral exosomal membrane proteins, lipid-anchored outer and inner membrane proteins, peripheral surface and inner membrane proteins, exosomal enzymes, and soluble proteins that play critical roles in exosome functions.11

The functions of exosomes depend on the origin of the cell/tissue, and are involved in the immune response, antigen presentation programmed cell death, angiogenesis, inflammation, coagulation, and morphogen transporters in the creation of polarity during development and differentiation.1720 Ferguson and Nguyen reported that the unique functions of exosomes depend on the availability of unique and specific proteins and also the type of cell.21 All of these categories influence cellular aspects of proteins such as the cell junction, chaperones, the cytoskeleton, membrane trafficking, structure, and transmembrane receptor/regulatory adaptor proteins. The role of exosomes has been explored in different pathophysiological conditions including metabolic diseases. Exosomes are extremely useful in cancer biology for the early detection of cancer, which could increase prognosis and survival. For example, the presence of CD24, EDIL3, and fibronectin proteins on circulating exosomes has been proposed as a marker of early-stage breast cancer.22 Cancer-derived exosomes promoted tumor growth by directly activating the signaling pathways such as P13K/AKT or MAPK/ERK.23 Tumor-derived exosomes are significantly involved in the immune system, particularly stimulating the immune response against cancer and delivering tumor antigens to dendric cells to produce exosomes, which in turn stimulates the T-cell-mediated antitumor immune response.24 Exosomal surface proteins are involved in immunotherapies through the regulation of the tumor immune microenvironment by aberrant cancer signaling.25 A study demonstrated that exosomes have the potential to affect health and pathology of cells through contents of the vesicle.26 Exosomes derived from mesenchymal stem cells exhibit protective effects in stroke models following neural injury resulting from middle cerebral artery occlusion.27 The structural region of the exosome facilitate the release of misfolded and prion proteins, and are also involved in the propagation of neurodegenerative diseases such as Huntington disease, Alzheimers disease (AD), and Parkinsons disease (PD).28,29

Exosomes serve as novel intercellular communicators due to their cell-specific cargo of proteins, lipids, and nucleic acids. In addition, exosomes released from parental cells may interact with target cells, and it can influence cell behavior and phenotype features30 and also it mediate the horizontal transfer of genetic material via interaction of surface adhesion proteins.31 Exosomes are potentially serving as biomarkers due to the wide-spread and cell-specific availability of exosomes in almost all body fluids.13 Therefore, exosomes are exhibited as delivery vehicles for the efficient delivery of biological therapeutics across different biological barriers to target cells.3234

In this review, first, we comprehensively describe the factors involved in exosome biogenesis and the role of exosomes in intercellular signaling and cell-cell communications, immune responses, cellular homeostasis, autophagy, and infectious diseases. In addition, we discuss the role of exosomes as diagnostic markers, and the therapeutic and clinical implications. Finally, we discuss the challenges and outstanding developments in exosome research.

The extracellular vesicles play critical role in inter cellular communication by serving as vehicles for transfer of biomolecules. These vesicles are generally classified into microvesicles, ectosomes, shedding vesicles, or microparticles. MVs bud directly from the plasma membrane, whereas exosomes are represented by small vesicles of different sizes that are formed as the ILV by budding into early endosomes and MVBs and are released by fusion of MVBs with the plasma membrane (Figure 1). Invagination of late endosomal membranes results in the formation of intraluminal vesicles (ILVs) within large MVBs.35 Biogenesis of exosomes occurs in three ways including vesicle budding into discrete endosomes that mature into multivesicular bodies, which release exosomes upon plasma membrane fusion; direct vesicle budding from the plasma membrane; and delayed release by budding at intracellular plasma membrane-connected compartments (IPMCs) followed by deconstruction of IPMC neck(s).11 The mechanisms of biogenesis of exosomes are governed by various types of proteins including the ESCRT proteins Hrs, CHMP4, TSG101, STAM1, VPS4, and other proteins such as the Syndecan-syntenin-ALIX complex, nSMase2, PLD2, and CD9.14,3639 After formation, the MVB can either fuse with the lysosome to degrade its content or fuse with the plasma membrane to release the ILVs as exosomes. The release of exosomes to the extracellular milieu is driven by proteins of the Rab-GTPase family including RAB2B, 5A, 7, 9A, 11, 27, and 35. SNARE family proteins VAMP7 and YKT6 have also been implicated in the release.14,38,4042 Biogenesis of exosomes is influenced by several external factors including cell type, cell confluency, serum conditions, and the presence and absence of cytokines and growth factors. In addition, biogenesis is also regulated by the sites of exosomes, protein sorting, physico-chemical aspects, and trans-acting mediators (Figure 2). For example, THP-1 cells were cultured in RPMI-1640 cell culture medium supplemented with 10% FCS secreted low level of exosomes compared to cells grown on cell culture medium supplemented with 1% FCS (Figure 3). The exogenous factor like serum starvation influences biogenesis and secretion of exosomes.

Figure 1 Biogenesis and cargoes of exosomes.

Figure 2 Effect of various factors on biogenesis of exosomes.

Figure 3 Serum deprivation causes an increase of the number of cellular exosomes in THP-1 cells. Panel (A); 10% FCS. Panel (B); 1% FCS. Panel (C) Quantification of exosomes using DLS and NTA.

Exosome release depends on expression of Rab27 or Ral. For example, exosomes released from the MVB significantly decrease in cells depleted of Rab2741 or Ral.43 The most efficient EV-producing cell types have yet to be determined44 and few reports suggest that immature dendritic cells produce limited amounts of EVs45,46 whereas mesenchymal stem cells secrete vast amounts, relevant for the production of EV therapeutics on a clinical scale.47,48 A few proteins play a critical role in the biogenesis of EVs, such as Rab27a and Rab27b.49 Over expression of Rab27a and Rab27b produce significant amounts of EVs in cancer cells. For example, overexpression of Rab27a and Rab27b in breast cancer cells,50 hepatocellular carcinoma cells,51 glioma cells,52 and pancreas cancer cells53 produces significant levels of EVs. Although all types of cells secrete and release EVs, cancer cells seem to produce higher levels than normal cells.54 Furthermore, the presence of invadopodia that are docking sites for Rab27a-positive MVBs induces secretion of EVs, and also enhances secretion of EVs in cancer cells.55 Thus, inhibition of invadopodia formation greatly reduces exosome secretion into conditioned media. This evidence demonstrates that cancer cells potentially release more EVs than non-cancer cells.

The rate of origin of exosomes from the plasma membrane of stem cells is vigorous, at rates equal to the production of exosomes,56 which is consistent with a report suggesting that stem cells bud ~50100 nm-diameter vesicles directly from the plasma membrane.57 Plasma membrane-derived exosomes contain selectively enriched protein and lipid markers in leukocytes.58 Plasma membrane exosomal budding is also observed for glioblastoma exosomes.59 Conventional transmission electron microscopy revealed that certain cell types contain deep invaginations of the plasma membrane that are indistinguishable from MVBs.6062 Certain cell types secrete exosomes containing cargo proteins, which primarily bud from the plasma membrane, and exosome composition is determined predominantly by intracellular protein trafficking pathways, rather than by the distinct mechanisms of exosome biogenesis.63 Biogenesis of exosomes is regulated by syndecan heparan sulphate proteoglycans and their cytoplasmic adaptor syntenin. Syntenin interacts directly with ALIX through LYPX (n) L motifs.64 Glycosylation is an essential factor in the biogenesis of exosomes and N-linked glycosylation directs glycoprotein sorting into EMVs.65 Collectively, these reports suggest that exosomes are made at both plasma and endosome membranes rather than endosome alone. Oligomerization is a critical factor for exosomal protein sorting and it was found to be sufficient to target plasma membrane proteins to exosomes. High-order oligomeric proteins target them to exosomes.66 Further, plasma membrane anchors support exosomal protein budding. For example, budding of CD63 and CD9 from the plasma membrane is much more efficient than endosome-targeted budding of CD63 and CD9.63 Protein clustering is another factor that induces membrane scission.67

Physico-chemical properties determine budding efficiency and are crucial factors of exosome biogenesis, a fundamental process involving the budding of vesicles that are 30200 nm in size. In particular, lipids are critical players in exosome biogenesis, especially those able to form cone and inverse cone shapes. Generally, exosome membranes contain phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositols (PIs), phosphatidic acid (PA), cholesterol, ceramides, sphingomyelin, glycosphingolipids, and a number of lower abundance lipids.68,69 Exosomes have a rich content of PE and PS, which increase budding efficiency and promote exosome genesis and release. PA promotes exosome biogenesis and PLD2 is involved in the budding of certain exosomal cargoes.70 Besides these factors, ceramide is an important lipid molecule regulating exosome biogenesis and facilitating membrane curvature, which is essential for vesicular budding. Inhibition of an enzyme that generates ceramide impairs exosome biogenesis.71

The next critical factor is trans-acting mediators that are involved in the biogenesis of exosomes through regulating plasma membrane homeostasis, intracellular protein trafficking pathways, MVB maturation and trafficking, IPMC biogenesis, vesicle budding, and scission.11 For example, Rab proteins regulate exosome biogenesis via endosomes and the plasma membrane by determining organelle membrane identity, recruiting mechanistic effectors, and mediating organelle dynamics.72 The functions of Rab proteins in the control and biogenesis of exosomes depends on cell type. MVB biogenesis is regulated by Rab27a, Rab27b, their effectors Slp4, Slac2b, and Munc13-4, and also Rab 35 and Rab 11.73 Loss of Rab27 function leads to a ~5075% drop in exosome production, and is also involved in assembling the plasma membrane microdomains involved in plasma membrane vesicle budding, by regulating plasma membrane PIP2 dynamics.74 Overall, Rab27 proteins control exosome biogenesis at both endosomes and plasma membranes. In addition, Rab35 also contributes to exosome biogenesis by regulating PIP2 levels of plasma membrane, and its loss leads to a reduction of exosome release by ~50%.75 Gurunathan et al76 reported that yeast produces two classes of secretory vesicles, low density and high density, and dynamin and clathrin are required for the biogenesis of these two different types of vesicle.

The Ral family is involved in the biogenesis of exosomes, and inhibition of Ral causes an accumulation of MVBs near the plasma membrane and a ~50% decrease in the vesicular secretion of exosomes and exosomal marker proteins.43 Ral GTPases function through various effectors proteins, including Arf6 and the phospholipase PLD2, which are involved in exosomal release of SDCs.37 The ESCRT complex machinery (0 through III) are involved in MVB biogenesis on a major level including membrane deformation, sealing, and repair during a wide array of processes. The major contributions of the ESCRT complex to the biogenesis of vesicles are the recognition and sequestration of ubiquitinated proteins to specific domains of the endosomal membrane via ubiquitin binding subunits of ESCRT-0. After interaction with the ESCRT-I and -II complexes, the total complex will then combine with ESCRT-III, a protein complex that is involved in promoting the budding process. Finally, following cleaving of the buds to form ILVs, the ESCRT-III complex separates from the MVB membrane using energy supplied by the sorting protein Vps4.77 In addition, other proteins such as Alix, which is associated with several ESCRT (TSG101 and CHMP4) proteins, are involved in endosomal membrane budding and abscission, as well as exosomal cargo selection via interaction with syndecan.39 Another important factor, autophagy, is critically involved in exosome secretion. Autophagy related (Atg) proteins coordinate initiation, nucleation, and elongation during autophagosome biogenesis in the presence of ESCRT-III components including CHMP2A and VPS4. For instance, the absence of Atg5 in cancer cells causes a reduction in exosome production.78 Conversely, CRISPR/Cas9-mediated knockout of Atg5 in neuronal cells increases the release of exosomes and exosome-associated prions from neuronal cells.79

Exosomes play a critical role in the physiologic regulation of mammary gland development and are important mediators of breast tumorigenesis.80 Biogenesis of exosomes occurs in all cell types; however, production depends on cell type. For example, breast cancer cells (BCC) produce increased numbers of exosomes compared to normal mammary epithelial cells. Studies revealed that patients with BC have increased numbers of MVs in their blood.81 Kavanagh et al reported that several fold changes were observed from exosomes isolated from triple negative breast cancer (TNBC) chemoresistant therapeutic induced senescent (TIS) cells compared with control EVs.82 TIS cells release significantly more EVs compared with control cells, containing chemotherapy and key proteins involved in cell proliferation, ATP depletion, and apoptosis, and exhibit the senescence-associated secretory phenotype (SASP). Cannabidiol (CBD), inhibits exosome and microvesicle (EMV) release in three different types of cancer cells including prostate cancer (PC3), hepatocellular carcinoma (HEPG2), and breast adenocarcinoma (MDA-MB-231). All three different cell lines show variability in the release of exosomes in a dose-dependent manner. These variabilities are all due to mitochondrial function, including modulation of STAT3 and prohibitin expression. This study suggests that the anticancer agent CBD plays critical role in EMV biogenesis.83 Sulfisoxazole (SFX) inhibits sEV secretion from breast cancer cells through interference with endothelin receptor A (ETA) through the reduced expression of proteins involved in the biogenesis and secretion of sEV, and triggers co-localization of multivesicular endosomes with lysosomes for degradation.84 Secreted EVs from human colorectal cancer cells contain 957 vesicular proteins. The direct protein interactions between cellular proteins play a critical role in protein sorting during EV formation. SRC signaling plays a major role in EV biogenesis, and inhibition of SRC kinase decreases the intracellular biogenesis and cell surface release of EVs.85 Proteomic analysis revealed that the exosomes released from imatinib-sensitive GIST882 cell line exhibit 764 proteins. The authors found that significant amount of proteins belong to protein release function and involved in the classical pathway and overlap to a high degree with proteins of exosomal origin.86 Exosomes secreted by antigen-presenting cells contain high levels of MHC class II proteins and costimulatory proteins, whereas exosomes released from other cell types lack these proteins.1,87

The biogenesis of exosomes depends on a percentage of confluency of approximately 6090%, which influences the yield and functions of EVs.44 Gal et al88 observed a 10-fold decreased level of cholesterol metabolism in confluent cell cultures compared to cells in the preconfluent state. The high level of cholesterol content in confluent cells leads to a decreased level of EVs in prostate cancer.68 The major reason behind for the reduced level of vesicle production is contact inhibition, which triggers confluent cells to enter quiescence and/or alters their characteristics compared to actively dividing cells.89,90 Exogenous stimulation could influence the condition of the cells including the phenotype and efficacy of secretion. Previously, several studies demonstrated that various external factors increase biogenesis of EVs such as Ca2+ ionophores,91 hypoxia,9294 and detachment of cells,95 whereas lipopolysaccharide reduces biogenesis and release of EVs.96 Furthermore, serum, which supports adherence of the cells, plays a critical role in the biogenesis of EVs.97 For example, FCS has noticeable effects on cultured cells; however, the effects depend on cell type and differentiation status.97,98 To avoid the immense amounts of vesicles present in FCS, the use of conditioned media has been suggested. Culture viability and health status of cells are important aspects for producing an adequate amount of vesicles with proper cargo molecules such as protein and RNA.99,100 Exogenous stress, such as starvation, can induce phenotypic alterations and changes in proliferation. These changes cause alterations in the cells metabolism and eventually lead to low yields.101,102

Cellular stresses, such as hypoxia, inflammation, and hyperglycemia, influence the RNA and protein content in exosomes. To examine these factors, the effects of cellular stresses on endothelial cells were studied.99 Endothelial cells were exposed to different types of cellular stress such as hypoxia, tumor necrosis factor- (TNF-)-induced activation, and high glucose and mannose concentrations. The mRNA and protein content of exosomes produced by these cells were compared using microarray analysis and a quantitative proteomics approach. The results indicated that endothelial cell-derived exosomes contain 1354 proteins and 1992 mRNAs. Several proteins and mRNAs showed altered levels after exposure of their producing cells to cellular stress. Interestingly, cells exposed to high sugar concentrations had altered exosome protein composition only to a minor extent, and exosome RNA composition was not affected. Low-intensity ultrasound-induced (LIUS) anti-inflammatory effects have been achieved by upregulation of extracellular vesicle/exosome biogenesis. These exosomes carry anti-inflammatory cytokines and anti-inflammatory microRNAs, which inhibit inflammation of target cells via multiple shared and specific pathways. A study suggested that exosome-mediated anti-inflammatory effects of LIUS are feasible and that these techniques are potential novel therapeutics for cancers, inflammatory disorders, tissue regeneration, and tissue repair.103 Another factor, called manumycin-A (MA), a natural microbial metabolite, was analyzed in exosome biogenesis and secretion in castration-resistant prostate cancer (CRPC) C4-2B, cells. The effect of MA on cell growth was observed, and the results revealed that there was no effect on cell growth. However, MA attenuated the ESCRT-0 proteins Hrs, ALIX, and Rab27a, and exosome biogenesis and secretion by CRPC cells. The inhibitory effect of MA on exosome biogenesis and secretion was primarily mediated via targeted inhibition of Ras/Raf/ERK1/2 signaling. These findings suggest that MA is a potential drug candidate for the suppression of exosome biogenesis and secretion by CRPC cells.104

Methods of isolation of exosomes play critical roles in functions and delivery. Although several methods such as ultracentrifugation, density gradient centrifugation, chromatography, filtration, polymer-based precipitation, and immunoaffinity have been adopted to isolate pure exosomes without contamination, there is still a lack of consistency and agreement.105 Isolation of exosomes along with non-exosomal materials and damaged exosomal membranes creates artifacts and alters the protein and RNA profiles. Since exosomes are obtained from a variety of sources, the composition of proteins/lipids influences the sedimentation properties and isolation. Thus, precise and consistent techniques are warranted for the isolation, purification, and application of exosomes.

Although several functions of exosomes have been explored, the precise function of exosomes remains a mystery. Historically, exosomes have been known to function as cellular garbage bags, recyclers of cell surface proteins, cellular signalers, intercellular signaling and cell-cell communications, immune responses, cellular homeostasis, autophagy, and infectious diseases.106 (Figure 4) ECVs are secreted cell-derived membrane particles involved in intercellular signaling and cell-cell communications, and contain immense bioactive information. Most cell types produce exosomes and release these into the extracellular environment, circulating through different bodily fluids such as urine, blood, and saliva and transferring their cargo to recipient cells. These vesicles play a significant role in various pathological conditions, such as different types of cancer, neurodegenerative diseases, infectious diseases, pregnancy complications, obesity, and autoimmune diseases, as reviewed elsewhere.107 Exosomes play a significant role in intercellular communication between cells by interacting with target cells via endocytosis.108 More specifically, exosomes are involved in cancer development, survival and metastasis of tumors, drug resistance, remodeling of the extracellular matrix, angiogenesis, thrombosis, and proliferation of tumor cells.94,109111 Exosomes contribute significantly to tumor vascularization and hypoxia-mediated inter-tumor communication during cancer progression, and premetastatic niches, which are significant players in cancer.16,94,109,112 Exosomes derived from hepatic epithelial cells increase the expression of enhancer zeste homolog 2 (EZH2) and cyclin-D1, and subsequently promotes G1/S transition.113

Figure 4 Multifunctional aspects biological functions of exosomes.

Conventionally, cells communicate with adjacent cells through direct cell-cell contact through gap junctions and cell surface protein/protein interactions, whereas cells communicating with distant cells do so through secreted soluble factors, such as hormones and cytokines, to facilitate signal propagation.114 Cells also communicate through electrical and chemical signals.115 Several studies have suggested that exosomes play vital roles in intercellular communication by serving as vehicles for transferring various cellular constituents, such as proteins, lipids, and nucleic acids, between cells.6,116118 Exosomes function as exosomal shuttle RNAs in which some exosomal RNAs from donor cells functions in recipient cells,6 a form of genetic exchange. Recently, researchers found that cells communicating with other cells through exosomes carrying cell-specific cargoes of proteins, lipids, and nucleic acids may employ novel intercellular communication mechanisms.30 Exosomes exert influences through various mechanistic approaches, such as direct stimulation of target cells via surface-bound ligands; transfer of activated receptors to recipient cells; and epigenetic reprogramming of recipient cells.119,120 Exosomes play critical roles in immunoregulation, including antigen presentation, immune activation, immune suppression, and immune tolerance via exosome-mediated intercellular communication. Mesenchymal stem cell (MSC)-derived exosomes play significant roles in wound healing processes.121 Exosomes from platelet-rich plasma (PRP) inhibit the release of TNF-. PRP-Exos significantly decreases the apoptotic rate of osteoarthritis (OA) chondrocytes compared with activated PRP (PRP-As).122 Extracellular vesicle (ECV)-modified polyethylenimine (PEI) complexes enhance short interfering RNA (siRNA) delivery by forming non-covalent complexes with small RNA molecules, including siRNAs and anti-miRs, in both conditions, in vitro and in vivo.123 Non-GSC glioma cells were treated with GSC-released exosomes. The results showed that GSC-released exosomes increase proliferation, neurosphere formation, invasive capacities, and tumorigenicity of non-GSC glioma cells through the Notch1 signaling pathway and stemness-related protein expressions.124

Exosomal miR-1910-3p promotes proliferation and migration of breast cancer cells in vitro and in vivo through downregulation of myotubularin-related protein 3 and activation of the nuclear factor-B (NF-B) and wnt/-catenin signaling pathway, and promotes breast cancer progression.125 Human hepatic progenitor cell (CdH)-derived exosomes (EXOhCdHs) play a crucial role in maintaining cell viability and inhibit oxidative stress-induced cell death. Experimental evidence suggests that inhibition of exosome secretion treatment with GW4869 results in the acceleration of reactive oxygen species (ROS) production, thereby causing a decrease in cell viability.126 Tumor-derived EXs (TDEs) are vehicles that enable communication between cells by transferring bioactive molecules, also delivering oncogenic molecules and containing different molecular cargoes compared to EXs delivered from normal cells. They can therefore be used as non-invasive biomarkers for the early diagnosis and prognosis of most cancers, including breast and ovarian cancers.127 Exosomes released by ER-stressed HepG2 cells significantly enhance the expression levels of several cytokines, including IL-6, monocyte chemotactic protein-1, IL-10, and tumor necrosis factor- in macrophages. ER stress-associated exosomes mediate macrophage cytokine secretion in the liver cancer microenvironment, and also indicate the potential of treating liver cancer via an ER stress-exosomal-STAT3 pathway.128 Mesenchymal stem cell-derived exosomal miR-223 protects neuronal cells from apoptosis, enhances cell migration and increases miR-223 by targeting PTEN, thus activating the PI3K/Akt pathway. In addition, exosomes isolated from the serum of AD patients promote cell apoptosis through the PTEN-PI3K/Akt pathway and these studies indicate a potential therapeutic approach for AD.129 A mouse model of diabetes demonstrated that mesenchymal stromal cell-derived exosomes ameliorate peripheral neuropathy through increased nerve conduction velocity. In addition, MSC-derived exosomes substantially suppress proinflammatory cytokines.130

Exosomes derived from activated astrocytes promote microglial M2 phenotype transformation following traumatic brain injury (TBI). miR-873a-5p significantly inhibits LPS-induced microglial M1 phenotype transformation.131 Several studies reported that exosomes are involved in cancer progression and metastasis; however, this depends on the type of cells the exosomes were derived from. For example, human umbilical vein endothelial cells (HUVEC) were treated with exosomes derived from HeLa cells (ExoHeLa), and the expression of tight junctions (TJ) proteins, such as zonula occludens-1 (ZO-1) and Claudin-5, was significantly reduced compared with exosomes from human cervical epithelial cells. Thus, permeability of the endothelial monolayer was increased after the treatment with ExoHeLa. Mice studies have shown that injection of ExoHeLa into mice increased vascular permeability and tumor metastasis. The results from this study demonstrated that HeLa cell-derived exosomes promote metastasis by triggering ER stress in endothelial cells and break down endothelial integrity. Such effects of exosomes are microRNA-independent.132 Exosomes mediate the gene expression of target cells and regulate pathological and physiological processes including promoting angiogenesis, inhibiting ventricular remodeling and improving cardiac function, as well as inhibiting local inflammation and regulating the immune response. Accumulating evidence shows that exosomes possess therapeutic potential through their anti-apoptotic and anti-fibrotic roles.

The functions of exosomes in immune responses are well established and do not cause any severe immune responses. A mouse study demonstrated that administration of a low dose of mouse or human cell-derived exosomes for extended periods of time caused no severe immune reactions.133 The function of exosomes in immune regulation is regulated by the transfer and presentation of antigenic peptides. Exosomes contain antigen-presenting cells (APCs) carrying peptide MHC-II and costimulatory signals and directly present the peptide antigen to specific T cells to induce their activation.134 For example, intradermal injection of APC-derived exosomes with MHC-II loaded with tumor peptide delayed tumor progression and growth.135 Exosome-derived immunogenic peptides activate immature mouse dendritic cells and indirectly activate APCs, and induce specific CD4+ T cell proliferation.136 Exosomes containing IFNa and IFNg, tumor necrosis factor a (TNFa), and IL from macrophages promoted dendritic cell maturation, CD4+ and CD8+ T cell activation, and the regulation of macrophage IL expression.137 The cargo of exosomes, such as DNA and miRNA, regulate the innate and adaptive immune responses. Exosomes are able to regulate the immune response by controlling gene expression and signaling pathways in recipient cells through transfer of miRNAs, and eventually control dendritic cell maturation.138 Exosomes containing miR-212-3p derived from tumors down-regulate the MHC-II transcription factor RFXAP (regulatory factor X associated protein) in dendritic cells, possibly promoting immune evasion by cancer cells.139 Exosomes containing miR-222-3p down regulate expression of SOCS3 (suppressor of cytokine signaling 3) in monocytes, which is involved in STAT3-mediated M2 polarization of macrophages.140 In mice, exosomes stimulate adaptive immune responses, including the activation of dendritic cells, with the uptake of breast cancer cell-derived exosomal genomic DNA and activation of cGAS-STING signaling and antitumor responses.141 The priming of dendritic cells is associated with the uptake of exosomal genomic and mitochondrial DNA (mtDNA) from T cells, inducing type I IFN production by cGAS-STING signaling.142 Inhibition of EGFR leads to increased levels of DNA in the exosomes and induces cGAS-STING signaling in dendritic cells, contributing to the overall suppression of tumor growth.143 Conversely, uptake of tumor-derived exosomal DNA by circulating neutrophils was shown to enhance the production of tissue factor and IL-8, which play a role in promoting tumor inflammation and paraneoplastic events.144 Melanoma-derived exosomes containing PD-L1 (programmed cell death ligand 1) suppress CD8+ T cell antitumor function and cancer cell-derived exosomes block dendritic cell maturation and migration in a PD-L1-dependent manner. Engineered cancer cell-derived exosomes promote dendritic cell maturation, resulting in increased proliferation of T cells and antitumor activity.145147

Inflammation is an important process for maintaining homeostasis in cellular systems. Systemic inflammation is an essential component in the pathogenesis of several diseases.148,149 Exosomes seem to play a crucial role in inflammation processes through cargo molecules, such as miRNA and proteins, which act on nearby as well as distant target tissues. Exosomes play a vital role in intercellular communication between cells via endocytosis and are associated with modulation of inflammation, coagulation, angiogenesis, and apoptosis.20,150153 Exosomes derived from dendritic cells, B lymphocytes, and tumor cells release exosomes that can regulate immunological memory through the surface expression of antigen-presenting MHC I and MHC II molecules, and subsequently elicit T cell activation and maturation.134,137,154156 Exosomes play a crucial role in carrying and presenting functional MHC-peptide complexes to modulate antigen-specific CD8+ and CD4+ responses.157,158 Exosomes containing miR-Let-7d influence the growth of T helper 1 (Th1) cells and inhibit IFN- secretion.159 Exosomes derived from choroid plexus epithelial cells containing miR-146a and miR-155 upregulate the expression of inflammatory cytokines in astrocytes and microglia.160 Exosomes containing miR-181c suppress the expression of Toll-like receptor 4 (TLR-4) and subsequently lower TNF- and IL-1 levels in burn-induced inflammation.161 Exosomal miR-155 from bone marrow cells (BMCs) increases the level of TNF- and subsequently enhances innate immune responses in chronic inflammation.162 Exosomes containing miR-150-5p and miR-142-3p derived from dendritic cells (DCs) increase expression of interleukin 10 (IL-10) and a decrease in IL-6 expression.163 Exosomal miR-138 can protect against inflammation by decreasing the expression level of NF-B, a transcription factor that regulates inflammatory cytokines such as TNF- and IL-18.164 HIF-1-inducing exosomal microRNA-23a expression from tubular epithelial cells mediates the cross talk between tubular epithelial cells and macrophages, promoting macrophage activation and triggering tubulointerstitial inflammation.165 A rat model study demonstrated that bone marrow mesenchymal stem cell (BMSC)-derived exosomes reduced inflammatory responses by modulating microglial polarization and maintaining the balance between M2-related and M1-related cytokines.165 Melatonin-stimulated mesenchymal stem cell (MSC)-derived exosomes improve diabetic wound healing through regulating macrophage M1 and M2 polarization by targeting the PTEN/AKT pathway, and significantly suppressed the pro-inflammatory factors IL-1 and TNF- and reduced the relative gene expression of IL-1, TNF-, and iNOS. Increasing levels of anti-inflammatory factor IL-10 are associated with increasing relative expression of Arg-1.166

Immunomodulators are essential factors for the prevention and treatment of disorders occurring due to an over high-spirited immune response, such as the SARS-CoV-2-triggered cytokine storm leading to lung pathology and mortality seen during the ongoing viral pandemic.167 MSC-secreted extracellular vesicles exhibit immunosuppressive capacity, which facilitates the regulation of the migration, proliferation, activation, and polarization of various immune cells, promoting a tolerogenic immune response while inhibiting inflammatory responses.168 Collagen scaffold umbilical cord-derived mesenchymal stem cell (UC-MSC)-derived exosomes induce collagen remodeling, endometrium regeneration, increasing the expression of the estrogen receptor /progesterone receptor, and restoring fertility. Furthermore, exosomes modulate CD163+ M2 macrophage polarization, reduce inflammation, increase anti-inflammatory responses, facilitate endometrium regeneration, and restore fertility through the immunomodulatory functions of miRNAs.169 Exosomes released into the airways during influenza virus infection trigger pulmonary inflammation and carry viral antigens and it facilitate the induction of a cellular immune response.170 Shenoy et al171 reported that exosomes derived from chronic inflammatory microenvironments contribute to the immune suppression of T cells. These exosomes arrest the activation of T cells stimulated via the T cell checkpoint (TCR). Exosomes secreted by normal retinal pigment epithelial cells (RPE) by rotenone-stimulated ARPE-19 cells induce apoptosis, oxidative injury, and inflammation in ARPE-19 cells. Exosomes secreted under oxidative stress induce retinal function damage in rats and upregulate expression of Apaf1. Overexpression of Apaf1 in exosomes secreted under oxidative stress (OS) can cause the inhibition of cell proliferation, increase in apoptosis, and elicitation of inflammatory responses in ARPE-19 cells. Exosomes derived from ARPE-19 cells under OS regulate Apaf1 expression to increase apoptosis and to induce oxidative injury and inflammatory response through a caspase-9 apoptotic pathway.172 Collectively, these findings highlight the critical role of exosomes in inflammation and suggest the possibility of utilizing exosomes as an inducer to attenuate inflammation and restore impaired immune responses in various diseases including cancer.

The endomembrane system of eukaryotic cells is a complex series of interconnected membranous organelles that play vital roles in protecting cells from adverse conditions, such as stress, and maintaining cell homeostasis during health and disease.173 To preserve cellular homeostasis, higher eukaryotic cells are equipped with various potent self-defense mechanisms, such as cellular senescence, which blocks the abnormal proliferation of cells at risk of neoplastic transformation and is considered to be an important tumor-suppressive mechanism.174,175 Exosomes contribute to reduce intracellular stress and preservation of cellular homeostasis through clearance of damaged or toxic material, including proteins, lipids, and even nucleic acids. Therefore, exosomes serve as quality controller in cells.176 The vesicular transport system plays pivotal roles in the maintenance of cell homeostasis in eukaryote cells, which involves the cytoplasmic trafficking of biomolecules inside and outside of cells. Several types of membrane-bound organelles, such as the Golgi apparatus, endoplasmic reticulum (ER), endosomes and lysosomes, in association with cytoskeleton elements, are involved in the intracellular vesicular system. Molecules are transported through exocytosis and endocytosis to maintain homeostasis through the intracellular vesicular system and regulate cells responses to the internal and external environment. To maintain homeostasis and protect cells from various stress conditions, autophagy is an intracellular vesicular-related process that plays an important role through the endocytosis/lysosomal/exocytosis pathways through degradation and expulsion of damaged molecules out of the cytoplasm.177179 Autophagy, as an intracellular waste elimination system, is a synchronized process that actively participates in cellular homeostasis through clearance and recycling of damaged proteins and organelles from the cytoplasm to autophagosomes, and then to lysosomes.38,180182 Cells maintain homeostasis by autophagosomes, which are vesicles derived from autophagic and endosomal compartments. These processes are involved in adaption to nutrient deprivation, cell death, growth, and tumor progression or suppression. Autophagy flux contributes to maintaining homeostasis in the tumor microenvironment of endothelial cells. To support this concept, a study provided evidence suggesting that depletion of Atg5 in ECs could intensify the abnormal function of tumor vessels.183 Exosome secretion plays a crucial role in maintaining cellular homeostasis in exosome-secreting cells. As a consequence of blocking exosome secretion, nuclear DNA accumulates in the cytoplasm, thereby causing the activation of cytoplasmic DNA sensing machinery. Blocking exosome secretion aggravates the innate immune response, leading to ROS-dependent DNA damage responses and thus inducing senescence-like cell-cycle arrest or apoptosis in normal human cells. Thus, cells remove harmful cytoplasmic DNA, protecting them from adverse effects.182 Salomon and Rice reported that the involvement of exosomes in placental homeostasis and pregnancy disorders. EVs of placental origin are found in a variety of body fluids including urine and blood. Moreover, the number of exosomes throughout gestation is higher in complications of pregnancy, such as preeclampsia and gestational diabetes mellitus, compared to normal pregnancies.184

The endolysosomal system is critically involved in maintaining homeostasis through the highly regulated processes of internalization, sorting, recycling, degradation, and secretion. For example, endocytosis allows the internalization of various receptor proteins into cells, and vesicles formed from the plasma membrane fuse and deliver their membrane and protein content to early endosomes. Similarly, significant amounts of internalized content are recycled back to the plasma membrane via recycling endosomes,76 while the remaining material is sequestered in ILVs in late endosomes, also known as multivesicular bodies.185,186 Tetraspanin proteins, such as CD63 and CD81, are regulators of ILV formation. Once ILVs are formed, MVBs can degrade their cargo by fusing with lysosomes or, alternatively, MVBs can secrete their ILVs by fusing with the plasma membrane and release their content into extracellular milieu.187190 Exosomes play an important role in regulating intracellular RNA homeostasis by promoting the release of misfolded or degraded RNA products, and toxic RNA products. Y RNAs are involved in the degradation of structured and misfolded RNAs. Further studies have demonstrated that proteins involved in RNA processing are abundant in exosomes, and the half-lives of secreted RNAs are almost twice as short as those of intracellular mRNAs. These studies suggest that cells maintain intracellular RNA homeostasis through the release of distinct RNA species in extracellular vesicles.191193 Exosomes reduce cholesterol accumulation in Niemann-Pick type C disease, a lysosomal storage disease in which cells accumulate unesterified cholesterol and sphingolipids within the endosomal and lysosomal compartment.194

Autophagy is the intracellular vesicular-related process that regulates the cell environment against pathological and stress conditions. In order to maintain homeostasis and protect the cells against stress conditions, internal vesicles or secreted vesicles serve as a canal to degrade and expel damaged molecules out of the cytoplasm.38,181,182 Autophagy protects the cell from various stress conditions and maintains cellular homeostasis, regulating cell survival and differentiation through clearance and recycling of damaged proteins and organelles from the cytoplasm to autophagosomes, and then to lysosomes.180 Several studies have demonstrated that proteins are involved in controlling tumor cell function and fate, and mediate crosstalk between exosome biogenesis and autophagy. Coordination between exosome-autophagy networks serves as a tool to conserve cellular homeostasis via the lysosomal degradative pathway and/or secretion of cargo into the extracellular milieu.176,195 Autophagy is a multi-step process that occurs by initiation, membrane nucleation, maturation and finally the fusion of autophagosomes with lysosomes. The autophagy process is not only linked with endocytosis but is also linked with the biogenesis of exosomes. For example, subsets of the autophagy machinery involved in the biogenesis of exosomes and the autophagic process itself appear dispensable.78,196 Crosstalk between exosomal and autophagic pathways has been reported in a growing number of diseases. Proteomic studies were performed to analyze the involvement of key proteins in the interconnection between exosome and autophagy pathways. They found that almost all proteins were identified; however, their involvement differed between them. Among 100 proteins, four proteins were highly ranked including HSPA8 (3/100), HSP90AA1 (8/100), VCP (24/100), and Rab7A (81/100). These data suggest an interconnection between the exosome and autophagy.197,198 Endosomal autophagy plays a significant role in the interconnection between exosomes and autophagy. Stress is a major factor for autophagy. In particular, the starvation of cells is a key inducer of autophagy, and induces enlargement of MVB structures and a co-localization of Rab11 and LC3 in these structures, an indication that autophagy-related processes are associated with the MVB.199 The sorting of autophagy-related cargo into MVBs is dependent on Hsc70 (HSPA8), VPS4, and TSG101, and independent on LAMP-2A, thereby excluding a role for, the lysosome.200 Several proteins are involved in the regulation and biogenesis of secretory autophagy compartments such as GRASPs, LC3, Rab8a, ESCRTs, and SNAREs, along with several Atg proteins.181,201,202 Autophagosomes could fuse with MVBs to form amphisomes and release vesicles to the external environment.203

Autophagy and exosome biogenesis and function are interconnected by microRNA. Over-expression of miR-221/222 inhibits the level of PTEN and activates Akt signaling, and subsequently reduces the expression of hallmarks that positively relate to autophagy including LC3, ATG5 and Beclin1, and increases the expression of SQSTM1/p62.204 MiR-221/222 from human aortic smooth muscle cell (HAoSMC)-derived exosomes inhibit autophagy in HUVECs by modulating the PTEN/Akt signaling pathway. miRNA-223 attenuates hypoxia-induced apoptosis and excessive autophagy in neonatal rat cardiomyocytes and H9C2 cells via the Akt/mTOR pathway, by targeting poly(ADP-ribose) polymerase 1 (PARP-1) through increased autophagy via the AMPK/mTOR and Akt/mTOR pathways205 ATG5 mediates the dissociation of vacuolar proton pumps (V1Vo-ATPase) from MVBs, which prevents acidification of the MVB lumen and allows MVB-PM fusion and exosome release. Accordingly, knockout of ATG5 or ATG16L1 significantly reduces exosome release and attenuates the exosomal enrichment of lipidated LC3B. These findings demonstrate that autophagic mechanisms possibly regulate the fate of MVBs and subsequent exosome biogenesis.78 Bone marrow MSC (BMMSC)-derived exosomes contain a high level of miR-29c, which regulates autophagy under hypoxia/reoxygenation (H/R) conditions.206 Human umbilical cord MSC-derived exosomes (HucMDEs) promote hepatic glycolysis, glycogen storage, and lipolysis, and reduce gluconeogenesis. Additionally, autophagy potentially contributes to the effects of HucMDE treatment and increases formation of autophagosomes and the autophagy marker proteins BECN1, MAP, and 1LC3B. These findings suggest that HucMDEs improve hepatic glucose and lipid metabolism in T2DM rats by activating autophagy via the AMPK pathway.207 Liver fibrosis is a serious disorder caused by prolonged parenchymal cell death, leading to the activation of fibrogenic cells, extracellular matrix accumulation, and eventually liver fibrosis. Exosomes derived from adipose-derived mesenchymal stem cells (ADSCs) have been used to deliver circular RNAs mmu_circ_0000623 to treat liver fibrosis. The findings from this study suggest that Exos from ADSCs containing mmu_circ_0000623 significantly suppress CCl4-induced liver fibrosis by promoting autophagy activation. Autophagy inhibitor treatment significantly reverses the treatment effects of Exos.208 Inhibition of autophagy by PDGF and its downstream molecule SHP2 (Src homology 2-containing protein tyrosine phosphatase 2) increased hepatic stellate cell (HSC)-derived EV release. Disruption of mTOR signaling abolishes PDGF-dependent EV release. Activation of mTOR signaling induces the release of MVB-derived exosomes by inhibiting autophagy, as well as microvesicles, through activation of ROCK1 signaling. Furthermore, deletion of SHP2 attenuates CCl4 or BDL-induced liver fibrosis.209 The therapeutic effects of exosomes containing high concentrations of mmu_circ_0000250 were analyzed in diabetic mice. The findings indicated that a high concentration of mmu_circ_0000250 had a better therapeutic effect on wound healing when compared with wild-type exosomes from ADSCs. The results also showed that exosome treatment with mmu_circ_0000250 increased angiopoiesis in wounded skin and suppressed apoptosis by inducing miR-128-3p/SIRT1-mediated autophagy.210 A study showed that mice treated with differentiated cardiomyocyte (iCM) exosomes exhibited significant cardiac improvement post-myocardial infarction, with significantly reduced apoptosis and fibrosis. Apoptosis was associated with reduced levels of hypoxia and inhibition of exosome biogenesis. iCM-exosome-treated groups showed upregulation of autophagosome production and autophagy flux. Hence, these findings indicate that iCM-Ex can improve post-myocardial infarction cardiac function by regulating autophagy in hypoxic cardiomyocytes.211 Exosomes of hepatocytes play a crucial role in inhibiting hepatocyte apoptosis and promoting hepatocyte regeneration. Mesenchymal stem cell-derived hepatocyte-like cell exosomes (MSC-Heps-Exo) were injected into a mouse hepatic Ischemia/reperfusion (I/R) I/R model through the tail. The results demonstrated that MSC-Heps-Exo effectively relieve hepatic I/R damage, reduce hepatocyte apoptosis, and decrease liver enzyme levels. A possible mechanism of reduced hepatic ischemia/reperfusion injury is the enhancement of autophagy.212

Exosomes play a critical role in viral infections, particularly of retroviruses and retroviruses, and use preexisting pathways for intracellular protein trafficking and formation of infectious particles. Exosomes and viruses share several features including biogenesis, uptake by cells, and the intracellular transfer of RNAs, mRNAs, and cellular proteins. Some features are different, including self-replication after infection of new cells, regulation of viral expression, and complex viral entry mechanisms.213,214 Exosomes secreted from virus-infected cells carry mostly cargo molecules such as viral proteins, genomic RNA, mRNA, miRNA, and genetic regulatory elements.215218 These cargo molecules are involved in the alteration of recipient cell behavior, regulating cellular responses, and enabling infection by various types of viruses such as human T-cell lymphotropic virus (HTLV), hepatitis C virus (HCV), dengue virus, and human immunodeficiency virus (HIV).215 Exosomes communicate with host cells through contact between exosomes and their recipient cells, via different kinds of mechanisms. Initially, the transmembrane proteins of exosomes build a network directly with the signaling receptors of target cells and then join with the plasma membrane of recipient cells to transport their content to the cytosol. Finally, the exosomes are incorporated into the recipient cells.219221 A report suggested that disruption of exosomal lipid rafts leads to the inhibition of internalization of exosomes.95 Exosomes derived from HIV-infected patients contain the trans-activating response element, which is responsible for HIV-1 replication in recipient cells through downregulation of apoptosis.222 While exosomes serving as carrier molecules, exosomes contain miRNAs that induce viral replication and immune responses either by direct targeting of viral transcripts or through indirect modulation of virus-related host pathways. In addition, exosomes have been found to act as nanoscale carriers involved in HIV pathogenesis. For example, exosomes enhance HIV-1 entry into human monocytic and T cell lines through the exosomal tetraspanin proteins CD9 and CD81.223 Influenza virus infection causes accumulation of various types of microRNAs in bronchoalveolar lavage fluid, which are responsible for the potentiation of the innate immune response in mouse type II pneumocytes. Serum of influenza virus-infected mice show significant levels of miR-483-3p, which increases the expression of proinflammatory cytokine genes and inflammatory pathogenesis of H5N1 influenza virus infection in vascular endothelial cells.224 Exosomes are involved in the transmission of inflammatory, apoptotic, and regenerative signals through RNAs. Chen et al investigated the potential functions of exosomal RNAs by RNA sequencing analysis in exosomes derived from clinical specimens of healthy control (HC) individuals and patients with chronic hepatitis B (CHB) and acute-on-chronic liver failure caused by HBV (HBV-ACLF). The results revealed that the samples contained unique and distinct types of RNAs in exosomes.225 Zika virus (ZIKV) infection causes severe neurological malfunctions including microcephaly in neonates and other complications associated with Guillain-Barr syndrome in adults. Interestingly, ZIKV uses exosomes as mediators of viral transmission between neurons and increases production of exosomes from neuronal cells. Exosomes derived from ZIKV-infected cells contained both ZIKV viral RNA and protein(s) which are highly infectious to nave cells. ZIKV uses neutral Sphingomyelinase (nSMase)-2/SMPD3 to regulate production and release of exosomes.226

During infections, viruses replicate in host cells through vesicular trafficking through a sequence of complexes known as ESCRT, and assimilate viral constituents into exosomes. Exosomes encapsulate viral antigens to maximize infectivity by hiding viral genomes, entrapping the immune system, and maximizing viral infection in uncontaminated cells. Exosomes can be used as a source of viral antigens that can be targeted for therapeutic use. A Variety of infectious diseases caused by viruses such as HCV, ZIKV, West Nile virus (WNV), and DENV enter into the host cells using clathrin-mediated or receptor-mediated endocytosis. For example, HCV infects host cells by specific targeting of cells through cellular contact, and hepatocyte-derived exosomes that contain HCV RNA can stimulate innate immune cells.217,227230 Exosomes show structural and molecular similarity to HIV-1 and HIV-2, which are enclosed by a lipid bilayer, and in the vital features of size and density, RNA species, and macro biomolecules including carbohydrates, lipids, and proteins. HIV-infected cells release enriched viral RNAs containing exosomes derived from HIV-infected cells and are enhanced with viral RNAs and Nef protein.6,38,231236 Izquierdo-Useros et al reported that both exosomes and HIV-1 express sialyllactose-containing gangliosides and interact with each other via sialic-acid-binding immunoglobulin-like lectins (Siglecs)-1. Siglecs-1 stimulates mature dendritic cell (mDC) capture and storage of both exosomes and HIV-1 in mDCs.237 Exosomes released from HIV-infected T cells contain transactivation response (TAR) element RNA, which stimulate proliferation, migration, and invasion of oral/oropharyngeal and lung cancer cells.238 Nuclear VP40 from Ebola virus VP40 upregulates cyclin D1 levels, resulting in dysregulated cell cycle and EV biogenesis. Synthesized extracellular vesicles contain cytokines and EBOV proteins from infected cells, which are responsible for the destruction of immune cells during EBOV pathogenesis.239 HIV enters into the host cells through human T-cell immunoglobin mucin (TIM) proteins. TIMs are a group of proteins (TIM-1, TIM-3, and TIM-4) that promote phagocytosis of apoptotic cells.240 TIM-4 is involved in HIV-1 exosome-dependent cellular entry mechanisms. Substantiating this hypothesis, neural stem cell (NSC)-derived exosomes containing TIM-4 protein increase HIV-1 exosome-dependent cellular entry into host cells, and antibody against TIM4 inhibits exosome-mediated entry of HIV in various types of cell.241

Exosomes show immense promise in biomedical applications due to their potential in drug delivery, the carriage of biomolecular markers of many diseases, and cellular protection. In addition, they can be used in non-invasive diagnostics or minimum invasive diagnostics.150 Detection of biomarkers is vital for early diagnosis of cancer and also critical for treatment. Several studies have documented the importance of exosomes in a variety of diseases, although further examination of the biology and functions of exosomes is warranted due to the continuing emergence of new diseases in the present world. The complex cargo of exosomes facilitates the exploration of a variety of diagnostic windows into disease detection, monitoring, and treatment. Exosomes are found in all biological fluids and are secreted by all cells, rendering them attractive for use through minimally invasive liquid biopsies, and they have the potential for use in longitudinal sampling to follow disease progression.242 Exosomes are produced and secreted by almost all body fluids, including blood, urine, saliva, breast milk, cerebrospinal fluid, semen, amniotic fluid, and ascites. These exosomes contain micro RNAs, proteins, and lipids serving as diagnostic markers.120 Exosomes are used in diagnostic applications in various kinds of diseases, such as cardiovascular diseases (CVDs),243 diseases of the central nervous system (CNS),244 cancer,245 and other prominent diseases including in the liver,246 kidney,247 and lung.248 Exosomes are potentially used to detect cancer-associated mutations in serum and also for the transfer of genomic DNA from donor cells to recipient cells.249 Exosomes carrying specific miRNAs or groups of miRNAs can be used as diagnostic markers to detect cancer. For example, exosomes containing oncogenic Kras, which have tumor-suppressor miRNAs-100, seem to have high diagnostic value, which could facilitate the differentiation of the expression pattern between cancer cells and normal cells.250,251 Similarly, miR-21 is considered to be diagnostic marker for various types of cancer including glioblastomas and pancreatic, colorectal, colon, liver, breast, ovarian, and esophageal cancers.252 Tumor suppressor miRNAs, such as miR-146a and miR-34a, function as diagnostic tools to detect liver, breast, colon, pancreatic, and hematologic malignancies.251 Exosomes containing GPC1 (glypican 1) are used as diagnostic markers to detect pancreatic, breast, and colon cancer.253,254

Exosomes play critical roles in various types of disease, and particularly in cancer progression and resistance to therapy. The unique biogenesis of exosomes and their biological features have generated excitement for their potential use as biomarkers for cancer.255 Generally, exosomes are produced and secreted by most cells and contain all the biological components of a cell. Hence, exosomes are found in all biological fluids and provide excellent opportunities for use as biomarkers.242 Surface proteins of exosomes are involved in the regulation of the tumor immune microenvironment and the monitoring of immunotherapies. Hence, exosome proteins play a critical role in cancer signaling.256 Exosomes from patients with metastatic pancreatic cancer show a higher mutant Kras allele frequency than exosomes from patients with local disease. In addition, the exosomes also accumulate a significantly higher level of cancer cell-specific DNA such as cytoplasmic DNA.8,257 Exosomes protect DNA and RNA from enzymatic degradation by encapsulation and stability in exosomes. The enhanced stability and retention of exosomes in liquid biopsies increases the availability and performance of exosomes as cancer biomarkers.258 Cancer cells contain cargo molecules, such as nucleic acid, proteins, metabolites, and lipids that are relatively different from normal cells, which is a contributing factor for their candidacy as cancer biomarkers. Exosomes isolated and purified from patient plasma samples enriched for miR-10b-5p, miR-101-3p, and miR-143-5p have been identified as potential diagnostic markers for gastric cancer with lymph node metastasis, gastric cancer with ovarian metastasis, and gastric cancer with liver metastasis, respectively.259 Kato et al analyzed the expression of CD44 protein and mRNA from cell lysates and exosomes from prostate cancer cells.260 Exosomes from serum containing CD44v8-10 mRNA was used as a diagnostic marker for docetaxel resistance in prostate cancer patients. The study was performed to evaluate plasma exosomal mRNA-125a-5p and miR-141-5p miRNAs as biomarkers for the diagnosis of prostate cancer from 19 healthy individuals and 31 prostate cancer patients. In comparing the miR-125a-5p/miR-141-5p level ratio, prostate cancer patients had significantly higher levels of miR-125a-5p/miR-141-5p. The findings from this study demonstrated that plasma exosomal expression of miR-141-3p and miR-125a-5p are markers of specific tumor traits associated with prostate cancer.261 Serum samples from 81 patients with gastric cancer showed that exosomes contained significant levels of long non-coding RNA (lncRNA) H19, which could be a diagnostic marker for gastric cancer.262 Plasma exosomes are suitable candidates as biomarkers for various diseases. For instance, plasma exosome lncRNA expression profiles were examined in esophageal squamous cell carcinoma (ESCC) patients. The findings suggest that five different types of lncRNAs were at significantly higher levels in exosomes from ESCC patients than in non-cancer controls. These lncRNAs may serve as highly effective, noninvasive biomarkers for ESCC diagnosis.263 Differential expression of lncRNAs, such as LINC00462, HOTAIR, and MALAT1, are significantly upregulated in hepatocellular carcinoma (HCC) tissues. The exosomes of the control group had a larger number of lncRNAs with a high amount of alternative splicing compared to hepatic disease patients.264 To demonstrate exosomes as a non-invasive cancer diagnostic tool, RNA-sequencing analysis was performed between three pairs of non-small-cell lung cancer (NSCLC) patients and controls from Chinese populations. The results show that circ_0047921, circ_0056285, and circ_0007761 were significantly expressed and that these exosomal circRNAs are promising biomarkers for NSCLC diagnosis.265 Exosomes were isolated from the serum of 34 patients with acute myocardial infarction (AMI), 31 patients with unstable angina (UA), and 22 healthy controls. The isolated exosomes exhibited higher levels of miR-126 and miR-21 in the patients with UA and AMI than in the healthy controls.266 Xu et al designed a study to examine tumor-derived exosomes as diagnostic biomarkers. In this study exosome miRNA microarray analysis was performed in the peripheral blood from four lung adenocarcinoma patients, including two with metastasis and two without metastasis. The results found that miR-4436a and miR-4687-5p were upregulated in the metastasis and non-metastasis group, while miR-22-3p, miR-3666, miR-4448, miR-4449, miR-6751-5p, and miR-92a-3p were downregulated. Exosomes containing miR-4448 have served as a diagnostic marker of patients with adenocarcinoma metastasis. Increased understanding of exosome biogenesis, structure, and function would enhance the performance of biomarkers in various kinds of disease diagnosis, prognosis, and surveillance.267

Exosomes have unique features such as ease of handling, molecular composition, and critical immunogenicity, and it is particularly easy to use them to transfer genes and proteins into cells. These unique characteristic features can inhibit angiogenesis and cancer metastasis, which are the two main targets of cancer therapy.268,269 Exosomes have potential therapeutic applications in a variety of diseases due to their potential capacity as vehicles for the delivery of therapeutic agents (Figure 5). Exosomes from colon cancer cells contain the highly immunogenic antigens MelanA/Mart-1 and gp100, serving as an indicator of tumor origin in particular organelles. Animal studies have demonstrated that tumor-derived antigen-containing exosomes induce potent antitumor T-cell responses and tumor regression.270 Exosomes containing tumor antigens are able to stimulate CD4+and CD8+T cells, and antigen-presenting exosomes inhibit tumor growth.135,271,272 MSC-derived exosomes exhibit the immunomodulatory and cytoprotective activities of their parent cells.273,274 Similarly, exosomes derived from bone marrow show protective roles in myocardial ischemia/reperfusion injury,109 hypoxia-induced pulmonary hypertension,275 and brain injury,276,277 and inhibit breast cancer growth via vascular endothelial growth factor down-regulation and miR-16 transfer in mice.278 Mesenchymal cell- and epithelial cell-derived exosomes exhibit tolerance and without any undesired side effects in patients and also act as therapeutic agents themselves.48,279 Exosomes engineered with ligands containing RGD peptide are used to induce signaling in specific cell types, and doxorubicin-loaded exosomes derived from dendritic cells show therapeutic responses in mammary tumor-bearing mice.46 Exosomal microRNAs are able to control other cells, and the delivery of miRNA or siRNA payload promotes anticancer activity in mammary carcinoma and glioma.280,281 Rabies virus glycoprotein (RVG)-modified dendritic cell-derived exosomes suppress the expression of BACE1 in the brain, which indicates the therapeutic potential of exosomes to target AD.282 Furthermore, these exosomes stimulated neurite outgrowth in cultured astrocytes by transferring miR-133b between cells.27 Immunotherapy is able to induce tumor-targeting immunity or an antitumor host immune response. For example, tumor-associated antigen-loaded mature autologous dendritic cells increase survival of metastatic castration-resistant patients.283 Exosome therapy induces upregulation of CD122 molecules in CD4+ T cells, whereas the lymphocyte pool is stable. Multiple vaccinations with exosomes increase circulating CD3-/CD56+ natural killer (NK) cells.284 An in vitro study demonstrated that adipose stem cell-derived exosomes up-regulate the peroxisome proliferator-activated receptor gamma coactivator 1, phosphorylate the cyclic AMP response element binding protein, and ameliorate abnormal apoptotic protein levels.285 Exosomes are used as potential carriers to carry anti-inflammatory drugs. Curcumin-encapsulated exosomes show significant anti-inflammatory activity, and exosomes are also used to deliver anti-inflammatory drugs to the brain through a noninvasive intranasal route.286,287 Turturici et al reported that specific progenitor cell-derived EVs contain biological cargo that promotes angiogenesis and tissue repair, and modulates immune functions.288

Figure 5 Therapeutic potential and versatile clinical implications of exosomes.

Generally, exosomes serve as vehicles for the delivery of drugs and are also actively involved as therapeutic agents. Conversely, injected exosomes enter into other cells and deliver functional cargo molecules very efficiently and rapidly, with minimal immune clearance and are well tolerated.16,21,245,289,290 Intravenous administration of human MSC-derived exosomes supports neuroprotection in a swine model of traumatic brain injury.291 In vitro and in vivo models demonstrate that exosomes from human-induced pluripotent stem cell-derived mesenchymal stromal Cells (hiPSC-MSCs) protect the liver against hepatic ischemia/reperfusion injury through increasing the level of proliferation of primary hepatocytes, activity of sphingosine kinase, and synthesis of sphingosine-1-phosphate (S1P).292 Exosomes derived from macrophages show potential for use in neurological diseases because of their easy entry into the brain by crossing the blood-brain barrier (BBB). Catalase-loaded exosomes displayed a neuroprotective effect in a mouse model of PD and exosomes loaded with dopamine entered into the brain better in comparison to free dopamine.33,293 Treatment of tumor-bearing mice with autologous exosomes loaded with gemcitabine significantly suppressed tumor growth and increase longevity, and caused only minimal damage to normal tissues. The study demonstrated that autologous exosomes are safe and effective vehicles for targeted delivery of GEM against pancreatic cancer.294

Generally, lipid-based nanoparticles such as liposomes or micelles, or synthetic delivery systems have been adopted to transport active molecules. However, the merits of synthetic systems are limited due to various factors including inefficiency, cytotoxicity and/or immunogenicity. Therefore, the development of natural carrier systems is indispensable. One of the most prominent examples of such natural carriers are exosomes, which are used to transport drug and active biomolecules. Exosomes are more compatible with other cells because they carry various targeting molecules from their cells of origin. Exosomes are nano-sized membrane vesicles derived from almost all cell types, which carry a variety of cargo molecules from their parent cells to other cells. Due to their natural biogenesis and unique qualities, including high biocompatibility, enhanced stability, and limited immunogenicity, they have advantages as drug delivery systems (DDSs) compared to traditional synthetic delivery vehicles. For instance, extracellular vesicles, including exosomes, carry and protect a wide array of nucleic acids and can potentially deliver these into recipient cells.6 EVs possess inherent targeting properties due to their lipid composition and protein content enabling them to cross biological barriers, and these salient features exploit endogenous intracellular trafficking mechanisms and trigger a response upon uptake by recipient cells.45,295297 The lipid composition and protein content of exocytic vesicles have specific tropism to specific organs.296 The integrin of exosomes determines the ability to alter the pharmacokinetics of EVs and increase their accumulation in various type of organs including brain, lungs, or liver.117 For example, EVs containing Tspan8 in complex with integrin alpha4 were shown to be preferentially taken up by pancreatic cells.298 Similarly, the lipid composition of EVs influences the cellular uptake of EVs by macrophages.299 EVs derived from dendritic cell achieved targeted knockdown by fusion between expression of Lamp2b and neuron-specific RVG peptide by using siRNA in neuronal cell.45 EVs loaded with Cre recombinase protein were able to deliver functional CreFRB to recipient cells through active and passive mechanisms in the presence of endosomal escape, enhancing the compounds chloroquine and UNC10217832A.300 EVs from cardiosphere-derived cells achieved targeted delivery by fusion of the N-terminus of Lamp2b to a cardiomyocyte-specific peptide (CMP).301 RVG-exosomes were used to deliver anti-alpha-synuclein shRNA minicircle (shRNA-MC) therapy to the alpha-synuclein preformed-fibril-induced mouse model of parkinsonism. This therapy decreased alpha-synuclein aggregation, reduced the loss of dopaminergic neurons, and improved clinical symptoms. RVG exosome-mediated therapy prolonged the effectiveness and was specifically delivered into the brain.302 Zhang et al evaluated the effects of umbilical cord-derived macrophage exosomes loaded with cisplatin on the growth and drug resistance of ovarian cancer cells. High loading efficiency of cisplatin was achieved by membrane disruption of exosomes by sonication.303 Incorporation of cisplatin into umbilical cord blood-derived M1 macrophage exosomes increased cytotoxicity 3.3-fold in drug-resistant A2780/DDP cells and 1.4-fold in drug-sensitive A2780 cells, compared to chemotherapy alone. Loading of cisplatin into M2 exosomes increased cytotoxicity by nearly 1.7-fold in drug-resistant A2780/DDP cells and 1.4-fold in drug-sensitive A2780 cells. The findings suggest that cisplatin-loaded M1 exosomes are potentially powerful tools for the delivery of chemotherapeutics to treat cancers regardless of drug resistance. Shandilya et al developed a chemical-free and non-mechanical method for the encapsulation and intercellular delivery of siRNA using milk-derived exosomes through conjugation between bovine lactoferrin with poly-L-lysine, wherein lactoferrin as a ligand was captured by the GAPDH present in exosomes, loading siRNA in an effortless manner.304 Targeted drug delivery was achieved with low immunogenicity and toxicity using exosomes derived from immature dendritic cells (imDCs) from BALB/c mice by expressing the fusion protein RGD. Recombinant methioninase (rMETase) was loaded into tumor-targeting iRGD-Exos. The findings suggest that the iRGD-Exos-rMETase group exhibited significant antitumor activity compared to the rMETase group.305 Several diseases show high inflammatory responses; therefore, amelioration of inflammatory responses is a critical factor. The inflammatory responses in various disease models can be attenuated through introduction of super-repressor IB (srIB), which is the dominant active form of IB, and can inhibit translocation of nuclear factor B into the nucleus. Intraperitoneal injection of purified srIB-loaded exosomes (Exo-srIBs) showed diminished mortality and systemic inflammation in septic mouse models.306 Systemic administration of macrophage-derived exosomes modified with azide and conjugated with dibenzocyclooctyne-modified antibodies of CD47 and SIRP (aCD47 and aSIRP) through pH-sensitive linkers can actively and specifically target tumors through distinguishing between aCD47 and CD47 on the tumor cell surface.307 SPION-decorated exosomes prepared using fusion proteins of cell-penetrating peptides (CPP) and TNF- (CTNF-)-anchored exosomes coupled with superparamagnetic iron oxide nanoparticles (CTNF--exosome-SPIONs) significantly enhanced tumor cell growth inhibition via induction of the TNFR I-mediated apoptotic pathway. Furthermore, in vivo studies in murine melanoma subcutaneous cancer models showed that TNF--loaded exosome-based vehicle delivery enhanced cancer targeting under an external magnetic field and suppressed tumor growth with mitigating toxicity.308 Yu et al309 developed a formulation of erastin-loaded exosomes labeled with folate (FA) to form FA-vectorized exosomes loaded with erastin (erastin@FA-exo) to target triple-negative breast cancer (TNBC) cells with overexpression of FA receptors. Erastin@FA-exo increased the uptake efficiency of erastin and also significantly inhibited the proliferation and migration of MDA-MB-231 cells compared with erastin@exo and free erastin. Interestingly, erastin@FA-exo promoted ferroptosis with intracellular depletion of glutathione and ROS generation. Plasma exosomes (Exo) loaded with quercetin (Exo-Que) improved the drug bioavailability, enhanced the brain targeting of Que and potently ameliorated cognitive dysfunction in okadaic acid (OA)-induced AD mice compared to free quercetin by inhibiting phosphorylated tau-mediated neurofibrillary tangles.310 Spinal cord injury (SCI) causes paralysis of the limbs. To determine the role of resveratrol in SCI, exosomes derived from resveratrol-treated primary microglia were used as carriers which are able to enhance the solubility of resveratrol and enhance penetration of the drug through the BBB, thereby increasing its concentration in the CNS. The findings demonstrated that Exo + Res are highly effective at crossing the BBB with good stability, suggesting they have potential for enhancing targeted drug delivery and recovering neuronal function in SCI therapy, and is likely associated with the induction of autophagy and inhibition of apoptosis via the PI3K signaling pathway.311 Delivery of miR-204-5p by exosomes inhibits cancer cell proliferation and tumor growth, and induces apoptosis and chemoresistance by specifically suppressing the target genes of miR-204-5p in human cancer cells.312 Engineered exosomes with RVG peptide on the surface for neuron targeting and NGF-loaded exosomes (NGF@ExoRVG) were efficiently delivered into ischemic cortex, with a burst release of encapsulated NGF protein and de novo NGF protein translated from the delivered mRNA. The delivered NGF protein showed high stability and a long retention time, and also reduced inflammation by reshaping microglia polarization, promoted cell survival, and increased the population of double cortin-positive cells, a neuroblast marker.313 Intranasal delivery of mesenchymal stem cell-derived extracellular vesicles exerts immunomodulatory and neuroprotective effects in a 3xTg model of AD by activation of microglia cells and increased dendritic spine density.314 Exosome-encapsulated paclitaxel showed efficacy in the treatment of multi-drug resistant cancer cells and it overcomes MDR in cancer cells.315,316 Saari et al found that the loading of Paclitaxel to autologous prostate cancer cell-derived EVs increased its cytotoxic effect.316 Exosome loaded doxorubicin (exoDOX) avoids undesired and unnecessary heart toxicity by partially limiting the crossing of DOX through the myocardial endothelial cells.317 Studies from in vitro and in vivo demonstrate that exosome loaded doxorubicin showed that exosomes did not decrease the efficacy of DOX and there is no cardiotoxicity in DOX-treated mice.318

The intrinsic properties of exosomes have been exploited to control various types of diseases, including neurodegenerative conditions and cancer, through promoting or restraining the delivery of proteins, metabolites, and nucleic acids into recipient cells effectively, eventually altering their biological response. Furthermore, exosomes can be engineered to deliver diverse therapeutic payloads to the target site, including siRNAs, antisense oligonucleotides, chemotherapeutic agents, and immune modulators. The natural lipid and protein composition of exosomes increases bioavailability and minimizes undesirable side effects to the recipients. Due to the availability of exosomes in biological fluid, they can be easily used as potential biomarkers for diagnosis of diseases. Exosomes are naturally decorated with numerous ligands on the surface that can be beneficial for preferential tumor targeting.282 Due to their unique properties, including superior targeting capabilities and safety profile, exosomes are the subject of clinical trials as cancer therapeutic agents.284 Exosomes derived from DCs loaded with tumor antigens have been used to vaccinate cancer patients with the goal of enhancing anti-tumor immune responses.284,319,320

Due to the potential level of various types of cargoes and salient features, exosomes are involved in intercellular messaging and disease diagnosis. As a result of dedicated studies, exosomes have been identified as natural drug delivery vehicles. However, we still face challenges regarding the purity of exosomes due to the lack of standardized techniques for their isolation and purification, inefficient separation methods, difficulties in characterization, and lack of specific biomarkers.321 The first challenge is the use of conventional methods, which are laborious for isolation and purification, time consuming, and vulnerable to contamination by other impurities, which will affect drug delivery processes. The second challenge is the various cellular origins of exosomes, which could affect specific applications. For example, in the application of exosomes in cancer therapy, we should avoid the use of exosomes derived from cancer cells, due to their oncogenic properties. Finally, exosomes have variable properties due to extraction from different types of cell and different cell culture techniques. Therefore, there is a necessity to address and overcome the challenges. There is also a need for an exosome consortium to develop common protocols for the development of rapid and precise methods of exosome isolation, and to assist the selection of sources that are dependent upon the specific therapeutic application. The most important challenge of exosome biology is the clinical translation of exosome-based research using different cell sources. Further characterization studies based on therapeutic applications are needed. Finally, important steps need to be taken to purify exosomes in a feasible, rapid, cost-effective, and scalable manner, which are free from downstream processing and have minimal processing times, that are specifically targeted to therapeutic applications and clinical settings.

The achievement of exosome therapy is based on success rate of clinical trials. Exosomes with size ranges from 60 to 200nm have been used as an active pharmaceutical ingredient or drug carrier in disease treatment. Exosomes derived from human and plant-derived exosomes are registered in clinical trials, but more complete reports are available for humanderived exosomes.322 There are two major exosomes from DCs and MSCs are frequently used in clinical trials, which potentially induce inflammation response and inflammation treatment. The more crucial aspect of exosomes in clinical trials needs to comply with good manufacturing practice (GMP) including upstream, downstream and quality control. Recently, France and USA conducted clinical trials using EVs containing MHCpeptide complexes derived from dendritic could alter tumor growth in immune competent mice and a Phase I anti-non-small cell lung cancer319,320 and several other clinical trial studies are shown in Table 1. Recent clinical case shows promising results with MSC-EVs derived from unrelated bone marrow donors for the treatment of a steroid-refractory graft-vs-host disease patient.279 Similarly, exosomes were used for the treatment of various types of diseases such as melanoma, non-small-cell-lung cancer, colon cancer and chronic kidney disease.284,319,320,323,324

Table 1 Summary of the Exosome Used in Clinical Trials (Source: clinicaltrials.com)

Exosomes are nano-sized membrane vesicles released by the fusion of an organelle of the endocytic pathway, a multivesicular body, with the plasma membrane. Since the last decade, exosomes have played a critical role in nanomedicine and studies related to exosome biology have increased immensely. Exosomes are secreted by almost all cell types and they are found in almost all types of body fluids. They function as mediators of cell-cell communications and play a significant role in both physiological and pathological processes. Exosomes carry a wide range of cargoes including proteins, lipids, RNAs, and DNA, which mediate signaling to recipient cells or tissues, making them a promising diagnostic biomarker and therapeutic tool for the treatment of cancers and other pathologies. In this review, we summarized what is known to date about the factors involved in exosome biogenesis and the role of exosomes in intercellular signaling and cell-cell communications, immune responses, cellular homeostasis, autophagy, and infectious diseases. Further, we reviewed the role of exosomes as diagnostic markers, and their therapeutic and clinical implications. Furthermore, we highlighted the challenges and outstanding developments in exosome research. The clinical application of exosomes is inevitable and they represent multicomponent biomarkers for several diseases including cancer and neurological diseases, etc. Recently, the mortality rate due to various types of cancers has increased. Therefore, therapies are essential to reduce mortality rates. At this juncture, we need sensitive, rapid, cost-effective, and large-scale production of exosomes to use as cancer biomarkers in diagnosis, prognosis, and surveillance. Furthermore, novel technologies are required for further tailoring exosomes as drug delivery vesicles with high drug pay loads, high specificity and low immunogenicity, and free of toxicity undesired side-effects. In addition, standardized and uniform protocols are necessary to isolate and purify exosomes for clinical applications, and more precise isolation and characterization procedures are required to increase understanding of the heterogeneity of exosomes, their cargo, and functions. There is an urgent need for information regarding the composition and mechanisms of action of the various substances in exosomes and to determine how to obtain highly purified exosomes at the right dosage for their clinical use. Currently, exosomes represent a promising tool in the field of nanomedicine and may provide solutions to a variety of todays medical mysteries.

The future direction of exosome research must focus on addressing the differential responses of communication between normal cells and cancer cells, how normal cells rapidly become cancerous, and how exosomes plays critical role in cancer progression via cell-cell communications. In vivo studies need to urgently address the critical factors such as biogenesis, trafficking, and cellular entry of exosomes originating from unmanipulated exosomes that control regulatory pathological functions. Further studies are required to decipher the mechanism of the cell-specific secretion and transport of exosomes, and the biological controls exerted by target cells. Exosomes represent a clinically significant nanoplatform. To substantiate this idea, numerous systematic in vivo studies are necessary to demonstrate the potency and toxicology of exosomes, which could help bring this novel idea a step closer to clinical reality. The most vital part of the system is to optimize the conditions for the engineering of exosomes that are non-toxic, for use in clinical trials. Furthermore, the translation of exosomes into clinical therapies requires their categorization as active drug components or drug delivery vehicles. Finally, future research should focus on the nanoengineering of exosomes that are tailored specifically for drug delivery and clinical efficacy.

Although we are the authors of this review, we would never have been able to complete it without the great many people who have contributed to the field of exosomes biogenesis, functions, therapeutic and clinical implications of exosomes aspects. We owe our gratitude to all those researchers who have made this review possible. We have cited as many references as permitted and apologize to the authors of those publications that we have not cited due to the limitation of references. We apologize to other authors who have worked on these aspects but whom we have unintentionally overlooked.

This study was supported by the KU-Research Professor Program of Konkuk University.

This work was supported by a grant from the Science Research Center (2015R1A5A1009701) of the National Research Foundation of Korea.

The authors report no conflicts of interest related to this work..

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[Full text] A Comprehensive Review on Factors Influences Biogenesis, Functions, Th | IJN - Dove Medical Press

REDWOOD CITY, Calif.--(BUSINESS WIRE)--Jasper Therapeutics, Inc., a biotechnology company focused on hematopoietic cell transplant therapies, today announced positive preliminary findings from its ongoing multicenter Phase 1 clinical trial of JSP191, a first-in-class anti-CD117 (stem cell factor receptor) monoclonal antibody, as a conditioning agent in older patients with myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) undergoing hematopoietic (blood) cell transplantation.

Data from the first six patients who received a single dose of JSP191 prior to transplantation showed successful engraftment in all six patients. Complete donor myeloid chimerism (equal or greater than 95%) was observed in five of six evaluable patients at 28 days, and all three evaluable patients had total donor chimerism equal or greater than 95% observed at day 90. In addition, at 28 days, three of five evaluable patients showed complete eradication of measurable residual disease (MRD) as measured by next-generation sequencing. Two of the five evaluable patients showed substantial reductions in MRD. No treatment-related serious adverse events were reported.

The findings were presented by lead investigator Lori Muffly, M.D., M.S., Assistant Professor of Medicine (Blood and Bone Marrow Transplantation) at Stanford Medicine, as a late-breaking abstract at the 2021 Transplantation & Cellular Therapy (TCT) Meetings of the American Society for Transplantation and Cellular Therapy (ASTCT) and the Center for International Blood & Marrow Transplant Research (CIBMTR).

These early clinical results are the first to demonstrate that JSP191 administered in combination with a standard non-myeloablative regimen of low-dose radiation and fludarabine is well tolerated and can clear measurable residual disease in older adults with MDS or AML undergoing hematopoietic cell transplantation a patient population with historically few options, said Kevin N. Heller, M.D., Executive Vice President, Research and Development, of Jasper Therapeutics. These patients could be cured by hematopoietic cell transplantation, but the standard-of-care myeloablative conditioning regimens used today are highly toxic and associated with high rates of morbidity and mortality particularly in older adults. Traditional lower intensity transplant conditioning regimens are better tolerated in older adults, but are associated with higher rates of relapse in MDS/AML patients with measurable residual disease. JSP191, a well-tolerated biologic conditioning agent that targets and depletes both normal hematopoietic stem cells and those that initiate MDS and AML, has the potential to be a curative option for these patients.

The open-label, multicenter Phase 1 study (JSP-CP-003) is evaluating the safety, tolerability and efficacy of adding JSP191 to the standard conditioning regimen of low-dose radiation and fludarabine among patients age 65 to 74 years with MDS or AML undergoing hematopoietic cell transplantation. Patients were ineligible for full myeloablative conditioning. The primary outcome measure of the study is the safety and tolerability of JSP191 as a conditioning regimen up to one year following a donor cell transplant.

We designed JSP191 to be given as outpatient conditioning and to have both the efficacy and safety profile required for use in newborn patients and older patients for successful outcomes, said Wendy Pang, M.D., Ph.D. Executive Director, Research and Translational Medicine, of Jasper Therapeutics. We are enthusiastic about the reduction of measurable residual disease seen in these patients, especially given that it is associated with improved relapse-free survival. We are excited to continue our research in MDS/AML, with plans for an expanded study. We are evaluating JSP191, the only antibody of its kind, in two ongoing clinical studies and are encouraged by the positive clinical data seen to date.

About MDS and AML

Myelodysplastic syndromes (MDS) are a group of disorders in which immature blood-forming cells in the bone marrow become abnormal and do not make new blood cells or make defective blood cells, leading to low numbers of normal blood cells, especially red blood cells.1 In about one in three patients, MDS can progress to acute myeloid leukemia (AML), a rapidly progressing cancer of the bone marrow cells.1 Both are diseases of the elderly with high mortality. Each year, about 5,000 patients with MDS and 8,000 people with AML in the G7 countries receive hematopoietic cell transplants. These transplants are curative but are underused due to the toxicity of the current high-intensity conditioning regimen, which includes the chemotherapy agents busulfan and fludarabine.

About JSP191

JSP191 (formerly AMG 191) is a first-in-class humanized monoclonal antibody in clinical development as a conditioning agent that clears hematopoietic stem cells from bone marrow. JSP191 binds to human CD117, a receptor for stem cell factor (SCF) that is expressed on the surface of hematopoietic stem and progenitor cells. The interaction of SCF and CD117 is required for stem cells to survive. JSP191 blocks SCF from binding to CD117 and disrupts critical survival signals, causing the stem cells to undergo cell death and creating an empty space in the bone marrow for donor or gene-corrected transplanted stem cells to engraft.

Preclinical studies have shown that JSP191 as a single agent safely depletes normal and diseased hematopoietic stem cells, including in animal models of SCID, myelodysplastic syndromes (MDS) and sickle cell disease (SCD). Treatment with JSP191 creates the space needed for transplanted normal donor or gene-corrected hematopoietic stem cells to successfully engraft in the host bone marrow. To date, JSP191 has been evaluated in more than 90 healthy volunteers and patients.

JSP191 is currently being evaluated in two separate clinical studies in hematopoietic cell transplantation. A Phase 1/2 dose-escalation and expansion trial is evaluating JSP191 as a sole conditioning agent to achieve donor stem cell engraftment in patients undergoing hematopoietic cell transplantation for severe combined immunodeficiency (SCID), which is potentially curable only by this type of treatment. Data presented at the 62nd American Society of Hematology (ASH) Annual Meeting showed that a single dose of JSP191 administered prior to stem cell transplantation in a 6-month-old infant was effective in establishing sustained donor chimerism followed by development of B, T and NK immune cells. No treatment-related adverse events were reported. A Phase 1 clinical study is evaluating JSP191 in combination with another low-intensity conditioning regimen in patients with MDS or AML undergoing hematopoietic cell transplantation. For more information about the design of these two ongoing clinical trials, visit http://www.clinicaltrials.gov (NCT02963064 and NCT04429191).

Additional studies are planned to advance JSP191 as a conditioning agent for patients with other rare and ultra-rare monogenic disorders and autoimmune diseases.

About Jasper Therapeutics

Jasper Therapeutics is a biotechnology company focused on the development of novel curative therapies based on the biology of the hematopoietic stem cell. The companys lead compound, JSP191, is in clinical development as a conditioning antibody that clears hematopoietic stem cells from bone marrow in patients undergoing a hematopoietic cell transplant. This first-in-class conditioning antibody is designed to enable safer and more effective curative hematopoietic cell transplants and gene therapies. For more information, please visit us at jaspertherapeutics.com.

1 https://www.cancer.org/cancer/myelodysplastic-syndrome/about/what-is-mds.html

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Jasper Therapeutics Announces Positive Data from Phase 1 Clinical Trial of JSP191 as Targeted Stem Cell Conditioning Agent in Patients with...

BOSTON--(BUSINESS WIRE)--Gamida Cell Ltd. (Nasdaq: GMDA), an advanced cell therapy company committed to cures for blood cancers and serious hematologic diseases, today announced the results of a Phase 3 clinical study of omidubicel presented in an oral session at the Transplantation & Cellular Therapy Meetings of the American Society of Transplantation and Cellular Therapy (ASTCT) and Center for International Blood & Marrow Transplant Research (CIBMTR), or the TCT Meetings. Omidubicel is an advanced cell therapy under development as a potential life-saving allogeneic hematopoietic stem cell transplant solution for patients with hematologic malignancies.

This clinical data set was from the international, multi-center, randomized Phase 3 study of omidubicel that was designed to evaluate the safety and efficacy of omidubicel in patients with high-risk hematologic malignancies undergoing a bone marrow transplant compared to a comparator group of patients who received a standard umbilical cord blood transplant. This is the first presentation of these data in a peer-reviewed conference. The full presentation is available on the Gamida Cell website.

The results of this global Phase 3 study of omidubicel in patients with hematologic malignancies show that omidubicel resulted in faster hematopoietic recovery, fewer bacterial and viral infections and fewer days in hospital, all of which are meaningful results and represent potentially important advancements in care when considering the patient experience following transplant, said Mitchell Horwitz, M.D., principal investigator and professor of medicine at the Duke Cancer Institute. The comparator, a transplant with umbilical cord blood, has been historically shown to result in low incidence of graft versus host disease (GvHD) in relation to other graft sources, and in this study, omidubicel demonstrated a GvHD profile similar to the comparator. Moreover, previous studies have shown that engraftment with omidubicel is durable, with some patients in the Phase 1/2 study receiving their transplant more than 10 years ago. The data presented at this meeting indicate that omidubicel has the potential to be considered a new standard of care for patients who are in need of stem cell transplantation but do not have access to a matched donor.

Details of Phase 3 Efficacy and Safety Results Shared at the TCT Meetings

Patient demographics including racial and ethnic diversity and baseline characteristics were well-balanced across the two study groups. The studys intent-to-treat analysis included 125 patients aged 1365 years with a median age of 41. Diseases included acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome or lymphoma. Patients were enrolled at more than 30 clinical centers in the United States, Europe, Asia, and Latin America.

Gamida Cell previously reported in May 2020 that the study achieved its primary endpoint, showing that omidubicel demonstrated a statistically significant reduction in time to neutrophil engraftment, a measure of how quickly the stem cells a patient receives in a transplant are established and begin to make healthy new cells, and a key milestone in a patients recovery from a bone marrow transplant. The median time to neutrophil engraftment was 12 days for patients randomized to omidubicel compared to 22 days for the comparator group (p<0.001).

All three secondary endpoints demonstrated a statistically significant improvement among patients who were randomized to omidubicel in relation to patients randomized to the comparator group (intent-to-treat). Platelet engraftment was significantly accelerated with omidubicel, with 55 percent of patients randomized to omidubicel achieving platelet engraftment at day 42, compared to 35 percent for the comparator (p = 0.028). The rate of infection was significantly reduced for patients randomized to omidubicel, with the cumulative incidence of first grade 2 or grade 3 bacterial or invasive fungal infection for patients randomized to omidubicel of 37 percent, compared to 57 percent for the comparator (p = 0.027). Hospitalization in the first 100 days after transplant was also reduced in patients randomized to omidubicel, with a median number of days alive and out of hospital for patients randomized to omidubicel of 60.5 days, compared to 48.0 days for the comparator (p = 0.005). The details of these data were first reported in December 2020.

Previously unpublished data from the study relating to exploratory endpoints also support the clinical benefit demonstrated by the studys primary and secondary endpoints. There was no statistically significant difference between the two patient groups related to grade 3/4 acute GvHD (14 percent for omidubicel, 21 percent for the comparator) or all grades chronic GvHD at one year (35 percent for omidubicel, 29 percent for the comparator). Non-relapse mortality was shown to be 11 percent for patients randomized to omidubicel and 24 percent for patients randomized to the comparator (p=0.09).

These clinical data results will form the basis of a Biologics License Application (BLA) that Gamida Cell expects to submit to the U.S. Food and Drug Administration (FDA) in the second half of 2021.

We believe that omidubicel has the potential to transform the field of hematopoietic bone marrow transplant by expanding access to this potentially curative cell therapy treatment for thousands of patients who are in need of a transplant but lack access to a matched related donor, said Julian Adams, Ph.D., chief executive officer of Gamida Cell. Sharing the results of the Phase 3 study of omidubicel with the transplant community is a major moment for Gamida Cell, and we are forever grateful to the patients who participated in this study, their caregivers, and the work of the investigators and their teams.

About Omidubicel

Omidubicel is an advanced cell therapy under development as a potential life-saving allogeneic hematopoietic stem cell (bone marrow) transplant solution for patients with hematologic malignancies (blood cancers). In both Phase 1/2 and Phase 3 clinical studies (NCT01816230, NCT02730299), omidubicel demonstrated rapid and durable time to engraftment and was generally well tolerated.1,2 Omidubicel is also being evaluated in a Phase 1/2 clinical study in patients with severe aplastic anemia (NCT03173937). The aplastic anemia investigational new drug application is currently filed with the FDA under the brand name CordIn, which is the same investigational development candidate as omidubicel. For more information on clinical trials of omidubicel, please visit http://www.clinicaltrials.gov.

Omidubicel is an investigational therapy, and its safety and efficacy have not been established by the FDA or any other health authority.

About Gamida Cell

Gamida Cell is an advanced cell therapy company committed to cures for patients with blood cancers and serious blood diseases. We harness our cell expansion platform to create therapies with the potential to redefine standards of care in areas of serious medical need. For additional information, please visit http://www.gamida-cell.com or follow Gamida Cell on LinkedIn or Twitter at @GamidaCellTx.

Cautionary Note Regarding Forward Looking Statements

This press release contains forward-looking statements as that term is defined in the Private Securities Litigation Reform Act of 1995, including with respect to timing of anticipated regulatory submissions, which statements are subject to a number of risks, uncertainties and assumptions, including, but not limited to the progress and expansion of Gamida Cells manufacturing capabilities and other commercialization efforts and clinical, scientific, regulatory and technical developments. In light of these risks and uncertainties, and other risks and uncertainties that are described in the Risk Factors section and other sections of Gamida Cells Annual Report on Form 20-F, filed with the Securities and Exchange Commission (SEC) on February 26, 2020, its Report on Form 6-K filed with the SEC on August 12, 2020, and other filings that Gamida Cell makes with the SEC from time to time (which are available at http://www.sec.gov), the events and circumstances discussed in such forward-looking statements may not occur, and Gamida Cells actual results could differ materially and adversely from those anticipated or implied thereby. Any forward-looking statements speak only as of the date of this press release and are based on information available to Gamida Cell as of the date of this release.

1 Horwitz M.E., Wease S., Blackwell B., Valcarcel D. et al. Phase I/II study of stem-cell transplantation using a single cord blood unit expanded ex vivo with nicotinamide. J Clin Oncol. 2019 Feb 10;37(5):367-374.

2 Gamida Cell press release, Gamida Cell Announces Positive Topline Data from Phase 3 Clinical Study of Omidubicel in Patients with High-Risk Hematologic Malignancies, issued May 12, 2020. Last accessed August 31, 2020.

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Gamida Cell Presents Efficacy and Safety Results of Phase 3 Study of Omidubicel in Patients with Hematologic Malignancies at the 2021 TCT Meetings of...