The stock price of Creative Medical Technology Holdings Inc (OTCMKTS: CELZ) a company that engages in stem cell research and developing applications to treat male sexual dysfunction and related issues increased by 80.77% yesterday as it went from $0.0026 to $0.0047 per share. One of the biggest triggers for the stock price increase is an announcement about the company announcing the successful application of ImmCelz immunotherapy for treatment of stroke.

In an animal model of ischemia stroke, the middle cerebral artery ligation model, administration of ImmCelz resulted in 34% reduction in infarct volume, whereas control bone marrow mesenchymal stem cells reduced infarct volume by 21%. And there were improvements in functional recovery were observed using the Rotarod test.

At 28 days after induction of stroke the animals receiving ImmCelz had superior running time (92% of non-stroke controls) compared to animals that received bone marrow mesenchymal stem cells (73% of non-stroke control). And animals that received saline had a running time that was 50% of non-stroke controls.

KEY QUOTES:

The regenerative potential of immune cells that have been programmed by stem cells is a fascinating and novel area of research. Conceptual advantages of using reprogrammed T cells include higher migratory ability due to smaller size, as well as ability to replicate and potentially formregenerative memory cells.

Dr.Amit Patel, coinventor of ImmCelz

This data, which is covered by our previous filed patents, such as no. 15/987739,Generation of autologous immune modulatory cells for treatment of neurological conditions, demonstrate that immune modulation via this stem cell based method may be a novel and superior way of addressing the$30 billion dollarmarket for stroke therapeutics. The fact that this technology, which has priority back to 2017, is demonstrating such stunning results, motivates us to consider filing an Investigational New Drug Application for use in stroke.

Dr.Thomas Ichim, coinventor of the patent and Chief Scientific Officer of Creative Medical Technology

While stroke historically has been a major area of unmet medical need, the rise in stroke cases , as well as the fact that younger people are increasingly falling victim to stroke, strongly motivates us to accelerate our developmental programs and to continue to explore participation of Big Pharma in this space. We are eager to replicate the existing experiments start compiling the dossier needed to take ImmCelz into humans using the Investigational New Drug Application (IND) route through the FDA.

Timothy Warbington, President and CEO of Creative Medical Technology

Disclaimer: This content is intended for informational purposes. Before making any investment, you should do your own analysis.

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Creative Medical Technology Stock Price Increased 80.77%: Why It Happened - Pulse 2.0

Bone Marrow Stem Cells Comments Off on Creative Medical Technology Stock Price Increased 80.77%: Why It Happened – Pulse 2.0 | December 17th, 2020

PHILADELPHIA, Dec. 17, 2020 /PRNewswire/ --Immunotherapies, such as checkpoint inhibitor drugs, have made worlds of difference for the treatment of cancer. Most clinicians and scientists understand these drugs act on what's known as the adaptive immune system, the T cells and B cells that respond to specific threats to the body.

New research from a team co-led by Penn Dental Medicine's George Hajishengallis suggests that the innate immune system, which responds more generally to bodily invaders, may be an important yet overlooked component of immunotherapy's success.

Their work, published in the journal Cell, found that "training" the innate immune system with -glucan, a compound derived from fungus, inspired the production of innate immune cells, specifically neutrophils, that were programmed to prevent or attack tumors in an animal model.

"The focus in immunotherapy is placed on adaptive immunity, like checkpoint inhibitors inhibit the interaction between cancer cells and T cells," says Hajishengallis. "The innate immune cells, or myeloid cells, have not been considered so important. Yet our work suggests the myeloid cells can play a critical role in regulating tumor behavior."

The current study builds on earlier work by Hajishengallis and a multi-institutional team of collaborators, which showed that trained immunity, elicited through exposure to the fungus-derived compound -glucan, could improve immune recovery after chemotherapy in a mouse model.

In that previous study, the researchers also showed that the "memory" of the innate immune system was held within the bone marrow, in hematopoietic stem cells that serve as precursors of myeloid cells, such as neutrophils, monocytes, and macrophages.

The team next wanted to get at the details of the mechanism by which this memory was encoded. "The fact that -glucan helps you fight tumors doesn't necessarily mean it was through trained immunity," says Hajishengallis.

To confirm that link, the researchers isolated neutrophils from mice that had received the innate immune training via exposure to -glucan and transferred them, along with cells that grow into melanoma tumors, to mice that had not received -glucan. Tumor growth was significantly dampened in animals that received cells from mice that had been trained.

-glucan is already in clinical trials for cancer immunotherapy, but the researchers say this finding suggests a novel mechanism of action with new treatment approaches.

"This is a breakthrough concept that can be therapeutically exploited for cancer immunotherapy in humans," Hajishengallis says, "specifically by transferring neutrophils from -glucan-trained donors to cancer patients who would be recipients."

Contact: Beth Adams, [emailprotected]

SOURCE Penn Dental Medicine

http://www.dental.upenn.edu

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Priming the Immune System to Fight Cancer - PRNewswire

Bone Marrow Stem Cells Comments Off on Priming the Immune System to Fight Cancer – PRNewswire | December 17th, 2020

Today, we take an in-depth look at an intriguing development concern with one product approved and on the market and more importantly a more lucrative candidate that appears on its way to approval in Europe. A full analysis on this Busted IPO follows in the paragraphs below.

Orchard Therapeutics (ORTX) is a London, United Kingdom-based biopharmaceutical company that IPO'd in 2018. The company is focused on developing gene therapies for rare conditions. In 2018, Orchard acquired GSK's rare disease gene therapy portfolio, which originated from a collaboration between GSK and the San Raffaele Telethon Institute for Gene Therapy. The company's unique approach involves inserting a working copy of the missing or malfunctioning gene into a patient's own blood stem cells. This approach circumvents the need for a bone marrow transplant since it leverages blood stem cells intrinsic capacity to self-renew in a patient's bone marrow and produce new blood cells of all types. The company's overarching goal is to permanently correct genetic disorders via a single treatment.

The company does have one approved product called Strimvelis, which is indicated for patients with severe combined immunodeficiency due to adenosine deaminase deficiency for whom no suitable human leukocyte antigen matched related stem cell donor is available; however, the drug has only been approved by the EMA and not the FDA. The company's pipeline is candidate rich, spanning a variety of indications that are compartmentalized into three categories: neurometabolic/neurodegenerative disorders, immunological disorders, and blood disorders. The company has a couple of late-stage candidates. Orchard Therapeutics has a market capitalization of roughly $450 million and trades for approximately $4.50 a share.

Pipeline

Source: Company Presentation

OTL-200

OTL-200 is an ex vivo autologous gene therapy in development to treat metachromatic leukodystrophy. The drug uses a modified virus to insert an operational copy of the ARSA gene into a patient's cells. OTL-200 has received rare pediatric disease designation from the FDA. MLD is a rare and deadly inherited disease. The disease is characterized by the accumulation of fats called sulfatides, which causes a breakdown in the protective fatty layer surrounding nerves in the central and peripheral nerve systems. It is estimated that 1 in 40,000 to 1 in 160,000 people have the disease worldwide. OTL-200 was developed in partnership with the San Raffaele Telethon Institute for Gene Therapy.

Source: Company Presentation

On October 16th, the company announced that it received a positive CHMP opinion for the drug, which recommended full marketing authorization for the treatment of early-onset metachromatic leukodystrophy patients in the European Union. The positive opinion will now be reviewed by the European Commission. A final decision is expected by the end of 2020. If approved, the company would be targeting a launch in the first half of 2021.

Furthermore, the company is pursuing a regenerative medicine advanced therapy designation, and it filed an investigational new drug application or IND in the U.S., which was accepted by the FDA on November 19th. Orchard has also applied for Regenerative Medicine Advanced Therapy designation for OTL-200 to help facilitate additional dialogue with the FDA

In addition, within the neurometabolic/neurodegenerative disorders category, it was announced in September that the European Medicines Agency has granted Priority Medicines designation to OTL-203 for the treatment of mucopolysaccharidosis type I. This comes not long after 203 received Orphan Drug designation in the U.S.

Source: Company Presentation

OTL-103

OTL-103 is an ex vivo autologous gene therapy in development to treat Wiskott-Aldrich syndrome. The drug uses a modified virus to insert a working copy of the WAS gene into a patient's cells. WAS is a rare, X-linked, recessive, inherited immune disorder, which is characterized by reoccurring severe infections, autoimmunity, eczema and severe bleeding episodes. The company has received Rare Pediatric Disease designation and Regenerative Medicine Advanced Therapy designation from the FDA. OTL-103 is being developed in partnership with the San Raffaele Telethon Institute for Gene Therapy.

Looking ahead, the company is preparing to file a BLA in the U.S. and an MAA in the EU for OTL-103 in WAS in 2021.

Source: Company Presentation

As of September 30th, 2020, Orchard Therapeutics had cash and cash equivalents of $41.1 million compared to $19 million on December 31st, 2019. Research and development expenses for the third quarter were $14.6 million compared to $28.4 million in Q3 of 2019. Selling, general and administrative expenses were $12.9 million in the quarter compared to $14.2 million in the same quarter of 2019. The company did $1.9 million in product revenue for the quarter compared to $1.9 million in Q3 of 2019. Overall, the company reported a net loss of $20.2 million compared to a net loss of $36.7 million in Q3 of 2019. Management stated in the latest quarterly update that it expects its current financial position to cover its anticipated operating and capital expenditure requirements into 2022.

The company is sparsely covered in the United States despite a healthy market cap as our most names in this space domiciled overseas. The most recent recommendation comes from Oppenheimer on September 14th. The firm lowered its price target from $26 a share to $16 a share, but it maintained its overweight rating. The analyst stated that the updated price target reflects a more subdued opinion on the pace of pipeline development for MPS-1 and MPS-IIIA. It reiterated this rating on November 19th.

Both Barclays ($13 price target, down from $15 previously) and Goldman Sachs ($9 price target, down from $13) reiterated Buy ratings on ORTX albeit revising the price targets lower. Finally, on May 22nd, JPMorgan lowered its price target from $26 a share to $17 a share and maintained its overweight rating. The analyst's note did seem upbeat despite the lowered price target in that the analyst thinks that the company's pipeline possesses "broad potential".

Two things prevent Orchard from being considered for a large holding. First, the company looks like it will have to raise additional capital in the very near future. I think the company will raise capital immediately after the next positive news event. Second, overseas biotech concerns don't ever seem to get the attention from analysts or valuation from investors that companies domiciled in the States do. That said, the company has multiple shots on goal and definable potential catalysts on the near-term potential. Add in the possible wildcard of being a potential buyout target at some point, and ORTX would seem worthy of a small "watch item" stake at this time.

Bret Jensen is the Founder of and authors articles for the Biotech Forum, Busted IPO Forum, and Insiders Forum.

Author's note: I present and update my best small-cap Busted IPO stock ideas only to subscribers of my exclusive marketplace, The Busted IPO Forum. Our model portfolio has crushed the return of the Russell 2000 since its launch in the summer of 2017. To join the Busted IPO Forum community, just click on the logo below.

Disclosure: I am/we are long ORTX. I wrote this article myself, and it expresses my own opinions. I am not receiving compensation for it (other than from Seeking Alpha). I have no business relationship with any company whose stock is mentioned in this article.

Originally posted here:
Our View On Orchard Therapeutics - Seeking Alpha

Bone Marrow Stem Cells Comments Off on Our View On Orchard Therapeutics – Seeking Alpha | December 17th, 2020

Osteonecrosis of the femoral head (ONFH) is a refractory disease characterized by compromised subchondral microcirculation, bone necrosis, and microfracture accumulation without sustained compensatory remodeling.1 Its complex etiology, variability in location (lateral, medial, or central), intra-bone edema, and inflammation add to the unpredictable prognosis. Although few patients regress spontaneously, the progressive nature and lack of curative treatment for ONFH thus far are the challenges faced in the management of ONFH.

Osteonecrosis of the femoral head typically affects relatively young, active individuals between 20 and 40 years old and follows an unrelenting course resulting in substantial loss of function.2 The Indian Society of Hip and Knee Surgeons' Registry stated that 49% of total hip arthroplasty procedures in India are due to an irreversible stage of ONFH.3 Osteonecrosis of the femoral head is idiopathic in most cases. Steroid and alcohol consumption are the second most common causes.4 The term silent hip refers to an asymptomatic hip in patients with ONFH of the contralateral hip and is at risk of developing ONFH.5

Hip and groin pain and limp when patients walk are primary indicators. Radiography, magnetic resonance imaging (MRI), and computed tomography are tools for diagnosis, prognosis, and decision-making regarding treatment of ONHF. Crescent formation, collapse and anterolateral sequestration on radiographs, and a double line presentation on T2-weighted MRI provide confirmation of ONFH diagnosis.

The Ficat and Arlet staging of ONFH from I to IV indicates the progressive involvement and progression of the femoral head toward arthritis.6 However, it does not allow prediction of the possibility of progression. Ficat and Arlet stage I with extensive involvement of the femoral head will have a high chance of further progression to collapse. Steinberg grades of ONFH allow prediction of the possibility of progression to collapse in a precollapse hip.7 The Association Research Circulation Osseous (ARCO) takes into consideration the location of the crescent, amount of cartilage depression, and the area and volume of the femoral head affected as reliable predictors of prognosis in early stage ONFH and is helpful for identifying a femoral head at risk of progression and collapse.8

The most common surgical intervention in early stage ONFH is core decompression.9 However, core decompression is notable for its lack of effectivity in preventing collapse in cases where progression is most likely to happen (ie, in cases where there is extensive involvement [more than 30%] of the anterolateral region of the femoral head and crescent sign).10 Among other surgical interventions, fibular graft (vascularized or nonvascularized) proximal femoral osteotomy has been described.11

Hernigou and Beaujean12 reported abnormalities in the mesenchymal stem cell pool, which is known for its regenerative potential, following insult to the affected hip. Gangji et al13 later reported qualitative and quantitative abnormalities of osteoblast cells within the proximal femur in ONFH patients. Thus, it is accepted that the regenerative and reparative capacity of bone in ONFH is severely compromised. However, more than two decades of experience using various orthobiologics has not been convincingly satisfying, and many groups have expressed limitations of these therapies.1416

The pathology of ONFH involves ischemic imbalance of bone remodeling due to relatively enhanced osteoclastic action and poor regenerative potential of osteogenic cells in the proximal femur. The supply of differentiated osteogenic cells (osteoblasts) over time would result in arrest of ONFH progression. Core decompression would allow revascularization, and debridement of necrotic bone decreases the time needed for creeping substitution of new bone over dead bone. With this theoretical conviction, the author planned to use and assess the efficacy of autologous bone marrowderived cultured osteoblasts following core decompression and debridement in the treatment of patients diagnosed with ARCO stages II and III ONFH.

The surgeries were conducted at various hospitals. Fifteen patients (13 male and 2 female), with a mean age of 32 years (range, 2161 years), presented with typical ONFH symptoms. Patients were diagnosed with ARCO stage II or III ONFH (9 bilateral and 6 unilateral, for a total of 24 hips) on radiograph and MRI, and were considered for a predesigned treatment protocol that involved implantation of autologous cultured osteoblasts following core decompression and debridement.

Patient consent for inclusion in the study was obtained. The types of ONFH were idiopathic (8 patients), corticosteroid-induced (6 patients), and traumatic (1 patient) (Table 1). Efficacy of the treatment was assessed based on changes on radiograph and MRI and modified Harris Hip Score (mHHS), Oxford Hip Score (OHS), and visual analog scale (VAS) score after treatment. In a few patients, computed tomography also was performed.

Table 1:

Patient Characteristics

Treatment was performed in 2 steps.

Step 1. Percutaneous bone marrow aspiration from the iliac crest was performed and collected in transport media containing anticoagulant. This was transported under temperature-monitored conditions and processed at a good manufacturing practicecertified cell processing facility to obtain a predefined osteoblast culture (4 to 5 weeks).

The ex vivo culture of osteoblasts using bone marrow from the patient involved isolation of osteoprogenitor cells, osteogenic differentiation, and then expansion. Immunophenotypic characterization was performed to ensure the cultured cells tested positive for CD44+ and CD151+ markers. Alizarin red stain test ensured the presence of calcium deposits within the osteoblasts. Alkaline phosphatase test was used as an indicator of ability to form type I collagen.

Thus, ex vivo cultured live osteoblast cells, not less than 45 million, were filled and packed in sterile vials with appropriate transport/culture medium and were made available for individualized treatment. The cell viability was ensured during transport as well as after implant until the cells were integrated.

Step 2. The surgical implantation was planned as per the availability of cultured and expanded osteoblasts (4 to 5 weeks). In the first 3 patients, the osteoblast implant was performed soon after core decompression, whereas for the remaining 12 patients, core decompression was followed by debridement with implantation.

The location of the necrotic zone and its size was approximated on MRI. The patient was placed on a fracture table, and the C-arm was positioned as for routine core decompression. The entry point of the guidewire (2.5 mm) was chosen around the vastus ridge to allow faster healing in the cancellous bone (Figure 1).

Figure 1:

Surgical process details. The arrow indicates the high entry point of the guidewire at the vastus ridge targeting the area of osteonecrosis. A, 1-cm distance from the superior cortex to prevent fracture. B, varus appearance of the proximal femur due to mild flattening of the femoral head in the anterolateral femur in early osteonecrosis of the femoral head. This is the earliest sign observed radiographically and is indicative of stage IIB.

The larger sagittal dimensions of the trochanteric area allowed for a posterior entry point to avoid a possible subtrochanteric fracture due to posterior cortical breaching during or after intervention. Special effort was made to avoid a subtrochanteric entry point. On no occasion was the posterior cortex of the femur violated. The guidewire was passed in the center of the lesion but at least 1 cm from the superior cortex. An 8-mm cannulated core drill (from the dynamic hip screw set) was used over the wire to make a tunnel until the necrotic zone. The steps of the surgical intervention are shown in Figure 2.

Figure 2:

Surgical steps. Anteroposterior C-arm image of the hip with guidewire (A). Lateral C-arm image of the hip with guidewire (B). Drilling with 8-mm dynamic hip screw core drill bit (C). Anteroposterior C-arm image during curettage (D).

The tip of the drill, when removed, showed necrotic bone (Figure 3A), which was later sent for histopathology. Bone curettes of various sizes and angles then were used to curette the sequestrum under imaging guidance. The end point of curettage was the removal of hard sclerotic bone from the femoral head. If there was a bony ridge that was difficult to curette, a reamer was used. The author attempted to leave 1 cm of subchondral bone intact to allow faster revascularization of the femoral head by removing dead sclerotic bone. Curettes were kept at least 1 cm from the joint line.

Figure 3:

Drill bit with debrided live and dead bone (A). Instruments used during surgery (B).

After curettage was complete, the tunnel was plugged using an allograft of appropriate size. All of the instruments used during surgery are shown in Figure 3B. At this point, the patient was tilted to attain a gravity-dependent position of the operative hip to avoid any backflow of the implanted cells. A spinal needle was inserted through the small hole made in the allograft plug, and the osteoblast cell gel mixture was slowly injected in the space within the femoral head. Patients were held in the same position for approximately 10 minutes to allow the cells to settle without spilling.

Postoperatively, patients were partial weight bearing for 4 weeks using a walker. They progressed to using a walking stick by week 6, and then full weight bearing was permitted by week 8. For patients treated for bilateral ONFH, use of a walker was encouraged until week 6. Physical exercises to regain muscle strength and all hip joint movements were encouraged as soon as possible.

All patients underwent regular follow-up during the rehabilitation period and thereafter at 6, 12, and 18 months, with all anteroposterior and lateral radiographs of the hip and magnetic resonance imaging completed at 18 months. Two patients were lost to follow-up thereafter, and 13 patients continued with regular follow-up visits; 3 patients had follow-up of 7 years.

At 18 months after implant, no disease progression was observed on radiographs and MRIs for all patients. Postoperative mHHS, OHS, and VAS scores improved, and all of the patients had resumed normal routine activities and daily chores. Analysis of variance for HHS, OHS, and VAS scores showed a statistically significant difference (individual as well as mean values) from baseline to 18 months after implantation (P<.5; Table 2). Three patients who underwent follow-up for 7 years after implantation were assessed via telephone for HHS and VAS scores. For 1 of these patients, HHS improved from 90 to 95, and VAS score improved from 3 to 1 at 18 months. For another patient, HHS improved from 85 to 95, and VAS score improved from 2 to 1 at 18 months. One of the patients who underwent follow-up for 7 years walked daily for 3 to 4 km.

Table 2:

Pain and Function-Related Scores

One male patient who was treated for bilateral ONFH with follow-up of 5 years showed good improvement in HHS (from 65 to 92.5) at the end of 18 months, and his VAS score improved from 9 at baseline to 3 in both hips at 18 months after treatment. At 5 years postoperatively, he reported pain only after sitting for several hours and was more comfortable using a cane when walking.

Another male patient was diagnosed with ARCO stage III of the right hip. He had extensive involvement of the central and lateral lesion (>50%) with crescent depression less than 2 mm. Although reports for direct comparison were not available at 6 years after treatment, radiographs showed no further progression, with intramedullary changes evident. The joint space was preserved, which is consistent with good clinical function (Figure 4).

Figure 4:

Patient M4. Preoperative magnetic resonance image of Association Research Circulation Osseous stage III of the right hip. Extensive involvement of the central and lateral regions (>50%), with the crescent having less than 2 mm of depression (A, B). Magnetic resonance image at 5 months after treatment (C). Preoperative anteroposterior radiograph (D). Anteroposterior radiograph at 6 years after treatment showing no further progression, with evident intramedullary changes. The joint space is preserved, which is consistent with good clinical function (E).

One female patient had a history of tuberculosis treated with anti-Koch therapyanti-tubercular therapy and corticosteroids for 9 months as the standard care. This patient presented with extensive bilateral femoral head involvement evident on radiograph and computed tomography scan. The crescent depression was 2 to 4 mm. She was diagnosed with ARCO stage II of the right hip and grade III of the left hip. Radiographs at 6 years postoperatively showed arrest of osteonecrosis progression with an otherwise high risk of collapse because the ONFH was steroid induced. Clinically, this patient was able to resume all of her routine activities, including a daily commute to work and regular yoga, floor exercises, and stationary cycling (Figure 5).

Figure 5:

Patient F1. Preoperative anteroposterior radiographs (A, B). Preoperative computed tomography scans. There is extensive bilateral femoral head involvement (>30%), with 2 to 4 mm of depression of the crescent (C, D). Anteroposterior radiographs 6 months after treatment (E, F). Anteroposterior radiograph 6 years after treatment. Both femoral heads show arrest of osteonecrosis progression in a patient at high risk for collapse (G).

One male patient who was receiving long-term steroid treatment had relatively moderate improvement in HHS, from a baseline of 65 to 80 at 18 months after treatment. A female patient with bilateral ONFH had ARCO stage III in the right hip and a small, centrally located lesion (<30%) in the left hip. On radiograph and MRI, the right hip showed more than 90% involvement of the lateral, central, and medial regions but no crescent. The decision was made not to treat the left hip because it was deemed to have minimum possibility of progression. At 6 years after treatment, there was regression of necrosis. The patient has done well clinically and had a successful childbirth (Figure 6).

Figure 6:

Patient F2. Preoperative anteroposterior radiographs of the right hip showing more than 90% involvement of the medial, central, and lateral regions. There is no crescent (A, B). Preoperative magnetic resonance images (C, D). Anteroposterior radiograph at 3 months after treatment (E). Anteroposterior radiograph at 6 years after treatment (F).

Overall, the short-term and long-term results of autologous cultured osteoblast treatment along with routine procedures have been satisfactory. None of the 8 patients who underwent follow-up for 5 to 7 years showed any signs of disease progression, and none of the patients required repeat treatment or total arthroplasty.

Among invasive procedures, core decompression has been the standard of care for early stages of ONFH; however, varying degrees of improvement have been reported. Yoon et al17 and Rajagopal et al18 reported treatment of ONFH with core decompression was viable only in early stages, with the effect lasting for 2 to 3 years.

Among the biologics, platelet-rich plasma, growth factors, and bone marrow aspirate concentrate (BMAC) have been widely used along with conventional techniques such as core decompression or bone grafts.19,20 Several contributions in terms of understanding the clinical application and efficacy of biologics for the treatment of ONFH have been published during the past two decades.2123 Inherent limitations such as the absence of controlled studies, uncertainty, and heterogenicity of the composition of biologics have resulted in inconsistent results, and no treatment option has passed the regulatory approval process.24

In a recent study, Hauzeur et al25 reported obvious inefficacy of BMAC treatment in a randomized clinical trial comparing BMAC and core decompression vs core decompression alone. Their assessment criteria included clinical outcome, pain score, radiology, and the need for total hip arthroplasty.

Untreated bone marrow should be considered first-generation and processed bone marrow second-generation biological treatments for ONFH. The results using first- and second-generation biologics have been variable, and there are no long-term data and no formally approved products. Thus, curative treatment of ONFH, at least prior to the collapse stage, remains challenging.26

Kim et al27 were the first to report the clinical use of cultured osteoblasts in a single patient with bilateral ONFH (Ficat Arlet grade II); they reported a good outcome at 5 years without progression of disease. Later, Gangji et al28 compared the use of BMAC and autologous osteoblast cells in the treatment of avascular necrosis. They reported the group treated with osteoblast cells had twofold higher respondents at 36 months compared with the BMAC-treated group. These patients continued to have reduced pain until the end of the study period. Also, progression of disease from stage III to IV was more than 2 times higher in the BMAC-treated group compared with the osteoblast-treated group.28

The author proposes the evolution of biologics being used as first- and second-generation treatment, and the current modality of using autologous cultured osteoblasts as the latest and third-generation treatment. As such, the latter is the only modality that qualifies as cell-based therapy (Table 3).

Table 3:

Proposed Generations of Orthobiologics

Autologous cultured osteoblast implant is the most novel treatment modality for joint preservation. In the author's experience, 11 patients at 4 years, 6 patients at 5 years, and 3 patients at 7 years after transplant showed arrest of disease. Joint structure, biomechanics, strength, and function were regained in these patients, and they required no repeat treatment. Yet, unlike few other treatments, total arthroplasty still remains viable as a future option.

The assessment of ONFH progression on MRI after core decompression remains a sparsely studied subject. Therefore, radiographic and clinical examination during follow-up is crucial.

Autologous cultured osteoblast implantation is effective and safe for patients with ARCO stages II and III ONFH. This third-generation biologic can be considered a joint-preserving treatment in correctly chosen patients.

Patient Characteristics

Pain and Function-Related Scores

Proposed Generations of Orthobiologics

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Hip Preservation With Autologous Osteoblast Cell-Based Treatment in Osteonecrosis of the Femoral Head - Healio

Bone Marrow Stem Cells Comments Off on Hip Preservation With Autologous Osteoblast Cell-Based Treatment in Osteonecrosis of the Femoral Head – Healio | December 17th, 2020

Regeneron: Preventing Infection Among Households

Regeneron: A Phase 3, Randomized, Double-Blind, Placebo-Controlled Study Assessing the Efficacy and Safety of Anti-Spike SARS-CoV-2 Monoclonal Antibodies in Preventing SARS-Cov-2 Infection in Household Contacts of Individuals Infected with SARS-CoV-2

In this multisite trial, researchers are working to determine if monoclonal antibodies made by the drug company Regeneron Pharmaceuticals can prevent COVID-19 infection among people who have been exposed by someone in their household, but have not yet developed the disease. The trial is testing the same antibody cocktail given to President Donald Trump when he was hospitalized with COVID-19, though with a different use.

In this case, the antibodies are intended to prevent people from getting sick if they have a household member with COVID, Enfield said. So far, UVA has done a good job with recruitment, which is particularly tricky in this case as you have to find people who have been exposed to COVID in their household, but who do not yet have COVID.

UVA is recruiting 40 participants for the study, each of whom will receive four injections of either the antibodies or a placebo. Participants must have been exposed to COVID-19 by someone in their household within the previous 96 hours and continue to live with that person for a month.

Its been a rapid process, and a testament to the multidisciplinary team involved, from infectious disease clinicians and researchers to cell therapy, pulmonary critical care and several other departments, Sturek said. Its been all-hands-on-deck.

As results from these and other clinical trials continue to come in, Sturek also expressed hope that we will see widespread and effective vaccine distribution sooner, rather than later.

There is a lot on the horizon, from news around vaccines to getting the first wave of vaccines to high-risk people like health care workers, he said. Every day we learn something new, and its important to stay humble, to be able to adapt and change on the fly.

Fighting this pandemic has been a huge, multidisciplinary effort, and so many people joined in to help contribute and bring new treatments to our patients, and bring new research to the field. That doesnt get done without a huge team of nurses, clinical research coordinators, pharmacists, respiratory therapists and many, many others. It is impossible to overstate the importance of all of that teamwork.

See the article here:
Research That Saves Lives: Four COVID-19 Therapies Being Tested at UVA - University of Virginia

Bone Marrow Stem Cells Comments Off on Research That Saves Lives: Four COVID-19 Therapies Being Tested at UVA – University of Virginia | December 17th, 2020

Autologous hematopoietic stem cell transplant (AHSCT) induces a reduction in relapse rate and physical disability in patients with relapsing-remitting multiple sclerosis (RRMS) who respond inadequately to other treatments, a small study suggests.

The study, Selective cognitive dysfunction and physical disability improvement after autologous hematopoietic stem cell transplantation in highly active multiple sclerosis, was published in the journal Nature Scientific Reports.

AHSCT is an experimental approach to treat multiple sclerosis (MS) that is meant to rebuild a patients immune system in order to stop attacks on the brain and spinal cord.

The procedure begins with collecting a patients own (meaning autologous) healthy hematopoietic stem cells immature cells that can develop into all types of blood cells from the bone marrow. These cells are put back into the patient after a fairly non-aggressive combination of chemotherapy is given to kill the patients immune cells.

A team of researchers at the Vilnius University, in Lithuania, evaluated the effectiveness and safety of the AHSCT procedure in 24 patients (18 female, mean age 37.8 years) with highly active RRMS (mean disease duration of 8.6 years) who failed to respond to conventional therapies.

The aim of the study was to assess cognitive dysfunction and physical disability after AHSCT, to explore the potential factors influencing disability regression after the transplant, and to estimate the safety of low-dose immunosuppressive therapy in highly active relapsing MS patients.

Researchers assessed participants disability and cognition through changes in several functional measures, including the expanded disability status scale (EDSS) and the Brief International Cognitive Assessment for MS, which includes three cognitive domains measured by the symbol digit modalities test, brief visuospatial memory test revised, and California verbal learning test second edition.

Of the 24 patients, 13 (54.2%) completed a 24-month follow-up and were included in the efficacy analysis of AHSCT. From those, two (15.4%) had one relapse during the first year after AHSCT and three patients (23.1%) had one relapse during the second year after AHSCT.

The annualized relapse rate (ARR) was 2.7 one year before AHSCT and 1.9 at two years before AHSCT. After the AHSCT procedure, ARR dropped to 0.2 in the first year and to 0.3 in the second year. This represented an 89% reduction in ARR, when comparing the values at two years after AHSCT with those at two years before AHSCT.

The researchers also noted a reduction in disability progression (as measured by EDSS scores), with 84.6% of patients improving their disability score after AHSCT at month six and 76.9% at one year. Additionally, 76.9% of patients showed stable disability scores two years after the transplant.

The findings of EDSS improvement in almost 85% of the patients suggest that disability may be often at least temporarily reversible in patients with highly active [relapsing] MS if they receive suitable and well-timed treatment, the researchers wrote.

Using appropriate statistical models, researchers found that the clinical variable that explained the disability regression at months 6 and 12 after AHSCT was the disability progression over 6 months before AHSCT.

Improvements in cognition after AHSCT also were observed. Specifically, the scores of information processing speed and verbal learning, measured by the symbol digit modalities test, were significantly higher at month 12 after AHSCT (56.8) when compared to month three (48.3).

The score of brief visuospatial memory test revised that assesses visuospatial memory was slightly lower at month three (25.6) than before AHSCT (27.8), however, the difference was not significant.

The score of the California verbal learning test, which assesses verbal learning, was significantly higher at month 12 (63.6) than before AHSCT (55.2).

No new or active lesions were found on MRI after AHSCT, suggesting that all patients remained without radiological disease activity.

Furthermore, regarding safety, the incidence and severity of adverse events (side effects) after AHSCT were in the expected range and all were resolved. There were no transplant-related deaths reported.

Researchers noted several limitations to the studys findings, including the low sample size and the fact that the patientss assessment and follow-ups were provided at the same center without a comparative group.

Nonetheless, the outcomes are highly promising, as compared to conventional MS treatment, the researchers wrote. Further research is needed to replicate these findings and to assess long-term outcomes and safety of AHSCT.

Diana holds a PhD in Biomedical Sciences, with specialization in genetics, from Universidade Nova de Lisboa, Portugal. Her work has been focused on enzyme function, human genetics and drug metabolism.

Total Posts: 1,053

Patrcia holds her PhD in Medical Microbiology and Infectious Diseases from the Leiden University Medical Center in Leiden, The Netherlands. She has studied Applied Biology at Universidade do Minho and was a postdoctoral research fellow at Instituto de Medicina Molecular in Lisbon, Portugal. Her work has been focused on molecular genetic traits of infectious agents such as viruses and parasites.

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Stem Cell Transplant Reduces Relapses and Disability in RRMS... - Multiple Sclerosis News Today

Bone Marrow Stem Cells Comments Off on Stem Cell Transplant Reduces Relapses and Disability in RRMS… – Multiple Sclerosis News Today | December 16th, 2020

PHOENIX, Dec. 16, 2020 /PRNewswire/ --Creative Medical Technology Holdings Inc., (OTC CELZ) announced today positive preclinical data supporting the utilization of its ImmCelz cell based immunotherapy for treatment of stroke. In an animal model of ischemia stroke, the middle cerebral artery ligation model, administration of ImmCelz resulted in 34% reduction in infarct volume, whereas control bone marrow mesenchymal stem cells reduced infarct volume by 21%. Additionally, improvements in functional recovery where observed using the Rotarod test. At 28 days after induction of stroke the animals receiving ImmCelz had superior running time (92% of non-stroke controls) compared to animals which received bone marrow mesenchymal stem cells (73% of non-stroke control). Animals that received saline had a running time that was 50% of non-stroke controls.

"The regenerative potential of immune cells that have been programmed by stem cells is a fascinating and novel area of research." Said Dr. Amit Patel, coinventor of ImmCelz, and board member of the Company. "Conceptual advantages of using reprogrammed T cells include higher migratory ability due to smaller size, as well as ability to replicate and potentially form "regenerative memory cells."

"This data, which is covered by our previous filed patents, such as no. 15/987739, Generation of autologous immune modulatory cells for treatment of neurological conditions, demonstrate that immune modulation via this stem cell based method may be a novel and superior way of addressing the $30 billion dollar market for stroke therapeutics1." Said Dr. Thomas Ichim, coinventor of the patent and Chief Scientific Officer of the Company. "The fact that this technology, which has priority back to 2017, is demonstrating such stunning results, motivates us to consider filing an Investigational New Drug Application for use in stroke."

Creative Medical Technology Holdings possesses numerous issued patents in the area of cellular therapy including patent no. 10,842,815 covering use of T regulatory cells for spinal disc regeneration, patent no. 9,598,673 covering stem cell therapy for disc regeneration, patent no. 10,792,310 covering regeneration of ovaries using endothelial progenitor cells and mesenchymal stem cells, patent no. 8,372,797 covering use of stem cells for erectile dysfunction, and patent no. 7,569,385 licensed from the University of California covering a novel stem cell type.

"While stroke historically has been a major area of unmet medical need, the rise in stroke cases , as well as the fact that younger people are increasingly falling victim to stroke, strongly motivates us to accelerate our developmental programs and to continue to explore participation of Big Pharma in this space." Said Timothy Warbington, President and CEO of the Company. "We are eager to replicate the existing experiments start compiling the dossier needed to take ImmCelz into humans using the Investigational New Drug Application (IND) route through the FDA."

About Creative Medical Technology Holdings

Creative Medical Technology Holdings, Inc. is a commercial stage biotechnology company specializing in stem cell technology in the fields of urology, neurology and orthopedics and trades on the OTC under the ticker symbol CELZ. For further information about the company, please visitwww.creativemedicaltechnology.com.

Forward Looking Statements

OTC Markets has not reviewed and does not accept responsibility for the adequacy or accuracy of this release. This news release may contain forward-looking statements including but not limited to comments regarding the timing and content of upcoming clinical trials and laboratory results, marketing efforts, funding, etc. Forward-looking statements address future events and conditions and, therefore, involve inherent risks and uncertainties. Actual results may differ materially from those currently anticipated in such statements. See the periodic and other reports filed by Creative Medical Technology Holdings, Inc. with the Securities and Exchange Commission and available on the Commission's website atwww.sec.gov.

Timothy Warbington, CEO[emailprotected] CreativeMedicalHealth.com

Creativemedicaltechnology.comwww.StemSpine.comwww.Caverstem.comwww.Femcelz.com

1 Stroke Management Market Size Forecasts 2026 | Statistics Report (gminsights.com)

SOURCE Creative Medical Technology Holdings, Inc.

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Creative Medical Technology Holdings Announces Successful Application of ImmCelz Immunotherapy for Treatment of Stroke - PRNewswire

Bone Marrow Stem Cells Comments Off on Creative Medical Technology Holdings Announces Successful Application of ImmCelz Immunotherapy for Treatment of Stroke – PRNewswire | December 16th, 2020

Cancer researchers have created a new class of drugs to selectively target and destroy myeloid leukaemia cells with TET gene mutations.

Photomicrograph of bone marrow biopsy showing myeloblasts of acute myeloid leukemia (AML), a cancer of white blood cells.

Researchers have developed a novel class of targeted cancer drug that may be highly effective for the treatment of myeloid leukaemias. According to the team, their synthetic molecule, called TETi76, was able to selectively kill cells with TET2 gene mutations, one of the most common driver mutations in myeloid leukaemias.

Myeloid leukaemias are cancers derived from stem and progenitor cells in the bone marrow that give rise to all normal blood cells. These malignancies are normally treated with chemotherapy, either alone or in combination with targeted drugs; however, the significant side-effects associated with this treatment mean a more selective/targeted treatment is desirable.

In a new study published inBlood Cancer Discovery, researchers from the Cleveland Clinics Taussig Cancer Institute and Lerner Research Institute, both US, describe a new pharmacological strategy to preferentially target and eliminate leukaemia cells with TET2 mutations.

In preclinical models, we found that a synthetic molecule called TETi76 was able to target and kill the mutant cancer cells both in the early phases of disease what we call clonal haematopoiesis of indeterminate potential, or CHIP and in fully developed TET2 mutant myeloid leukaemia, said Dr Jaroslaw Maciejewski, a practicing haematologist and chair of the Cleveland Clinic Department of Translational Hematology & Oncology Research, who has been investigating the TET2 gene for the last decade.

TET genes encode DNA dioxygenase enzymes, which remove chemical groups from DNA molecules. Their activity ultimately changes what genes are expressed and can contribute to the development and spread of disease.

TET genes act as tumour suppressors, so loss-of-function mutations are common in haematological cancers, like leukaemias. While all members of the TET family are dioxygenases, TET2 is the most powerful. Genetic TET2 deficiency has been shown to skew differentiation of blood cells and clonal expansion of progenitor and stem cells. However, its related genes TET1 and TET3 provide residual enzymatic activity, sufficient to facilitate the survival of these progenitor cells harbouring cancerous mutations, thereby promoting the spread of the cancer, even when TET2 is inactive.

In their study, the research team designed TETi76 to replicate and amplify the effects of a natural molecule called 2-hydroxyglutarate (2HG), which inhibits the enzymatic activity of TET genes. They hoped to selectively eliminate TET2 mutant leukaemia cells centres by targeting their reliance on this residual DNA dioxygenase activity.

We took lessons from the natural biological capabilities of 2HG, explained Dr Babal Kant Jha, Maciejewskis collaborator from the Department of Translational Hematology & Oncology Research. We studied the molecule and rationally designed a novel small molecule, synthesised by our chemistry group headed by Dr James Phillips. Together, we generated TETi76 a similar, but more potent version capable of inhibiting not just TET2, but also the remaining disease-driving enzymatic activity of TET1 and TET3.

The researchers studied TETi76s effects in both preclinical disease and xenograft models (where human cancer cells are implanted into preclinical models). In both models, treatment with the novel TET inhibitor suppressed the clonal evolution of TET2 mutant cells.

While the team cautioned that additional studies would be critical to investigate the small molecules cancer-fighting capabilities in patients, Dr Jha said we are optimistic about our results, which show not just that TETi76 preferentially restricts the growth and spread of cells with TET2 mutations, but also gives survival advantage to normal stem and progenitor cells.

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Novel class of targeted cancer therapies could treat myeloid leukaemias - Drug Target Review

Bone Marrow Stem Cells Comments Off on Novel class of targeted cancer therapies could treat myeloid leukaemias – Drug Target Review | December 16th, 2020

Victoria Gray (second from left) with children Jamarius Wash, Jadasia Wash and Jaden Wash. Now that the gene-editing treatment has eased Gray's pain, she has been able be more active in her kids' lives and looks forward to the future. "This is really a life-changer for me," she says. Victoria Gray hide caption

Victoria Gray (second from left) with children Jamarius Wash, Jadasia Wash and Jaden Wash. Now that the gene-editing treatment has eased Gray's pain, she has been able be more active in her kids' lives and looks forward to the future. "This is really a life-changer for me," she says.

The last thing a lot of people want to do these days is get on a plane. But even a pandemic would not stop Victoria Gray. She jumped at the chance to head to the airport this summer.

"It was one of those things I was waiting to get a chance to do," says Gray.

She had never flown before because she was born with sickle cell disease. She feared the altitude change might trigger one of the worst complications of the devastating genetic disease a sudden attack of excruciating pain.

But Gray is the first person in the United States to be successfully treated for a genetic disorder with the help of CRISPR, a revolutionary gene-editing technique that makes it much easier to make very precise changes in DNA.

About a year after getting the treatment, it was working so well that Gray felt comfortable flying for the first time. She went to Washington, D.C., to visit her husband, who has been away for months on deployment with the National Guard.

"It was exciting. I had a window. And I got to look out the window and see the clouds and everything," says Gray, 35, of Forest, Miss.

Gray wore a mask the whole time to protect herself against the coronavirus, kept her distance from other people at the airport, and arrived happily in Washington, D.C., even though she's afraid of heights.

"I didn't hyperventilate like I thought I would," Gray says, laughing as she recounts the adventure in an interview with NPR.

NPR has had exclusive access to follow Gray through her experience since she underwent the landmark treatment on July 2, 2019. Since the last time NPR checked in with Gray in June, she has continued to improve. Researchers have become increasingly confident that the approach is safe, working for her and will continue to work. Moreover, they are becoming far more encouraged that her case is far from a fluke.

At a recent meeting of the American Society for Hematology, researchers reported the latest results from the first 10 patients treated via the technique in a research study, including Gray, two other sickle cell patients and seven patients with a related blood disorder, beta thalassemia. The patients now have been followed for between three and 18 months.

All the patients appear to have responded well. The only side effects have been from the intense chemotherapy they've had to undergo before getting the billions of edited cells infused into their bodies.

The New England Journal of Medicine published online this month the first peer-reviewed research paper from the study, focusing on Gray and the first beta thalassemia patient who was treated.

"I'm very excited to see these results," says Jennifer Doudna of the University of California, Berkeley, who shared the Nobel Prize this year for her role in the development of CRISPR. "Patients appear to be cured of their disease, which is simply remarkable."

Another nine patients have also been treated, according to CRISPR Therapeutics in Cambridge, Mass., and Vertex Pharmaceuticals in Boston, two companies sponsoring the research. Those individuals haven't been followed long enough to report any results, officials say.

But the results from the first 10 patients "represent an important scientific and medical milestone," says Dr. David Altshuler, Vertex's chief scientific officer.

The treatment boosted levels of a protein in the study subjects' blood known as fetal hemoglobin. The scientists believe that protein is compensating for defective adult hemoglobin that their bodies produce because of a genetic defect they were born with. Hemoglobin is necessary for red blood cells to carry oxygen.

Analyses of samples of bone marrow cells from Gray six months after getting the treatment, then again six months later, showed the gene-edited cells had persisted the full year a promising indication that the approach has permanently altered her DNA and could last a lifetime.

"This gives us great confidence that this can be a one-time therapy that can be a cure for life," says Samarth Kulkarni, the CEO of CRISPR Therapeutics.

Gray and the two other sickle cell patients haven't had any complications from their disease since getting the treatment, including any pain attacks or hospitalizations. Gray has also been able to wean off the powerful pain medications she'd needed most of her life.

Prior to the treatment, Gray experienced an average of seven such episodes every year. Similarly, the beta thalassemia patients haven't needed the regular blood transfusions that had been required to keep them alive.

"It is a big deal because we we able to prove that we can edit human cells and we can infuse them safely into patients and it totally changed their life," says Dr. Haydar Frangoul at the Sarah Cannon Research Institute in Nashville. Frangoul is Gray's doctor and is helping run the study.

For the treatment, doctors remove stem cells from the patients' bone marrow and use CRISPR to edit a gene in the cells, activating the production of fetal hemoglobin. That protein is produced by fetuses in the womb but usually shuts off shortly after birth.

The patients then undergo a grueling round of chemotherapy to destroy most of their bone marrow to make room for the gene-edited cells, billions of which are then infused into their bodies.

"It is opening the door for us to show that this therapy can not only be used in sickle cell and thalassemia but potentially can be used in other disorders," Frangoul says.

Doctors have already started trying to use CRISPR to treat cancer and to restore vision to people blinded by a genetic disease. They hope to try it for many other diseases as well, including heart disease and AIDS.

The researchers stress that they will have to follow Gray and many other patients for a lot longer to be sure the treatment is safe and that it keeps working. But they are optimistic it will.

Gray hopes so too.

"It's amazing," she says. "It's better than I could have imagined. I feel like I can do what I want now."

The last year hasn't always been easy for Gray, though. Like millions of other Americans, she has been sheltering at home with three of her children, worrying about keeping them safe and helping them learn from home much of the time.

"I'm trying to do the things I need to do while watch them at the same time to make sure they're doing the things they need to do," Gray says. "It's been a tough task."

But she has been able do other things she never got to do before, such as watch her oldest son's football games and see her daughter cheerleading.

"This is really a life-changer for me," she says. "It's magnificent."

She's now looking forward to going back to school herself, learning to swim, traveling more when the pandemic finally ends, and watching her children grow up without them worrying about their mother dying.

"I want to see them graduate high school and be able to take them to move into dorms in college. And I want to be there for their weddings just everything that the normal people get to do in life. I want to be able to do those things with my kids," she says. "I can look forward now to having grandkids one day being a grandmama."

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1st Patients To Get CRISPR Gene-Editing Treatment Continue To Thrive - NPR

Bone Marrow Stem Cells Comments Off on 1st Patients To Get CRISPR Gene-Editing Treatment Continue To Thrive – NPR | December 16th, 2020

Bone Regeneration Material Market: Introduction

Bone-regeneration techniques, either with autografts or allografts, represent a challenge for reconstructive surgery. Biomaterials are temporary matrices for bone growth and provide a specific environment and architecture for tissue development. Depending on the specific intended application of the matrix, whether for structural support, drug-delivery capability, or both, certain material categories may be more or less well suited to the final structure.

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Key Drivers and Restraints of Global Bone Regeneration Material Market

Increase in prevalence of degenerative joint diseases boost the market. Worldwide estimates of degenerative joint diseases indicate that 9.6% men and 18.0% women above 60 years have symptomatic osteoarthritis. According to expert opinions presented in the EULAR committee report, radiographic evidence of knee osteoarthritis in men and women over 65 years of age is found in 30% of the population.

In the absence of disease modifying therapy, a large number of patients with osteoarthritis progress to advance joint destruction. Surgery with bone grafts and substitutes play a major role in the management of osteoarthritis to avoid advanced joint destruction. According to the American College of Rheumatology, advances in biomaterial and tissue engineering are expected to create new opportunities to integrate surgical approaches in osteoarthritis.

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Increase in the number of orthopedic surgeries also fuels the market. According to the American Academy of Orthopaedic Surgeons (AAOS), approximately 129,000 total knee arthroplasty (TKA) surgeries were performed in the U.S. in 1990, and the number has increased to over 600,000 in 2010. The AAOS has projected that 3 million TKA procedures would be performed by 2030 in the U.S. alone. Moreover, spinal surgeries are becoming increasingly popular, and approximately 432,000 spinal fusions are performed in the U.S. each year. Bone grafts and substitutes are extensively used for the surgeries mentioned above. This is likely to fuel the bone regeneration material market.

Bone graft and substitutes are a long-term solution to bone problem treatment; however, these are expensive. No two patients or their customized bone grafts and substitutes treatments are exactly alike. Hence, the number of appointments, procedures, and costs vary accordingly. Surgeons charge US$ 35,000 to US$ 40,000 for a complex posterolateral lumbar spine fusion bone graft surgery. Most surgeons refer patients to specialty surgeons, neurologists, or orthopedic physicians, which increases the cost of procedure. Asia is price-sensitive and displays inhibitions with respect to investing in bone graft and substitutes, which are often only affordable to the elite population; therefore offering a comparatively smaller market.

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Cell-based Segment to Expand Significantly

Based on product type, the global bone regeneration material market can be divided into ceramic-based, polymer-based, growth factor-based, cell-based and others

The ceramic-based segment dominated the global market in 2019. It is projected to sustain its position during the forecast period. Ceramic-based bone grafts are widely used to reduce the need for iliac crest bone grafting. Rise in geriatric population with oral health issues across the world has augmented the number of bone graft surgeries performed in the last few years.

However, the cell based segment is projected to expand at a notable CAGR during the forecast period. Bone tissue engineering (BTE) using bone marrow stem cells has been suggested as a promising technique for reconstructing bone defect in order to overcome the drawbacks of bone graft materials.

Orthopedic surgery segment to dominate global bone regeneration material market

Based on application, the global bone regeneration material market can be segregated into orthopedic surgery, bone trauma, dental surgery and others.

In terms of revenue, the orthopedic surgery segment accounted for a prominent share of the market in 2019 owing to a rise in the geriatric population and increase in cases of orthopedic diseases. According to WHO, between 2015 and 2050, the proportion of the world's population over 60 years would nearly double from 12% to 22%. The number of people aged 60 years and older is estimated to outnumber children younger than 5 years by 2020. As per MVZ Gelenk-Klinik data, more than 2400 orthopedic surgical procedures are performed per year at the Gelenk Klinik Orthopaedic Hospital.

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North America to dominate global bone regeneration material market

In terms of region, the global bone regeneration material market can be divided into: North America, Europe, Asia Pacific, Latin America, and Middle East & Africa

North America accounted for a significant share of the bone regeneration material market in 2019, followed by Europe. Usage of new and innovative products in both premium and value segments among various bone grafts substitutes is projected to boost the bone regeneration material market in several countries in Europe and North America in the next few years. According to the Centers for Disease Control and Prevention (CDC), the total number of inpatient surgeries carried out in the U.S. were 51.4 million in 2014; of these 719,000 were total knee replacements and 332,000 were total hip replacement.

The market in developing countries in Asia Pacific is estimated to expand at a significant CAGR during the forecast period. The market in Asia Pacific is driven by an increase in population and time taken to accept new technologies. Increase in the number of patients and geriatric population are major factors that are expected to propel the market in Japan during the forecast period. According to the Gerontological Society of America, Japan has the highest proportion of geriatric population in the world. Hence, demand for orthopedic surgeries is estimated to be higher in Japan than that in other countries in Asia Pacific.

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Key Manufacturers Operating in Market

The global bone regeneration material market was highly fragmented in 2019. Key manufacturers operating in the global market are:

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Bone Regeneration Material Market: Cell-based Segment to Expand Significantly - BioSpace

Bone Marrow Stem Cells Comments Off on Bone Regeneration Material Market: Cell-based Segment to Expand Significantly – BioSpace | December 16th, 2020

Over two million babies, children, and adults in the United States are living with congenital heart disease--a range of birth defects affecting the heart's structure or function. Now, researchers at Gladstone Institutes and UC San Francisco (UCSF) have made inroads into understanding how a broad network of genes and proteins go awry in a subset of congenital heart diseases.

"We now have a better understanding of what genes are improperly deployed in some cases of congenital heart disease," says Benoit Bruneau, PhD, director of the Gladstone Institute of Cardiovascular Disease and a senior author of the new study. "Eventually, this might help us get a handle on how to modulate genetic networks to prevent or treat the disease."

Congenital heart disease encompasses a wide variety of heart defects, ranging from mild structural problems that cause no symptoms to severe malformations that disrupt or block the normal flow of blood through the heart. A handful of genetic mutations have been implicated in contributing to congenital heart disease; the first to be identified was in a gene known as TBX5. The TBX5 protein is a transcription factor--it controls the expression of dozens of others genes, giving it far-reaching effects.

Bruneau has spent the last 20 years studying the effect of TBX5 mutations on developing heart cells, mostly conducting research in mice. In the new study published inDevelopmental Cell, he and his colleagues turned instead to human cells, using novel approaches to follow what happens in individual cells when TBX5 is mutated.

"This is really the first time we've been able to study this genetic mutation in a human context," says Bruneau, who is also a professor in the Department of Pediatrics at UCSF. "The mouse heart is a good proxy for the human heart, but it's not exactly the same, so it's important to be able to carry out these experiments in human cells."

The scientists began with human induced pluripotent stem cells (iPS cells), which have been reprogrammed to an embryonic-like state, giving them--like embryonic stem cells--the ability to become nearly every cell type in the body.

Then, Bruneau's group used CRISPR-Cas9 gene-editing technology to mutate TBX5 in the cells and began coaxing the iPS cells to become heart cells. As the cells became more like heart cells, the researchers used a method called single-cell RNA sequencing to track how the TBX5 mutation changed which genes were switched on and off in tens of thousands of individual cells.

The experiment revealed many genes that were expressed at higher or lower levels in cells with mutated TBX5. Importantly, not all cells responded to the TBX5 mutation in the same way; some had drastic changes in gene expression while other were less affected. This diversity, the researchers say, reflects the fact that the heart is composed of many different cell types.

"It makes sense that some are more affected than others, but this is the first experimental data in human cells to show that diversity," says Bruneau.

Bruneau's team then collaborated with computational researchers to analyze how the impacted genes and proteins were related to each other. The new data let them sketch out a complex and interconnected network of molecules that work together during heart development.

"We've not only provided a list of genes that are implicated in congenital heart disease, but we've offered context in terms of how those genes are connected," says Irfan Kathiriya, MD, PhD, a pediatric cardiac anesthesiologist at UCSF Benioff Children's Hospital, an associate professor in the Department of Anesthesia and Perioperative Care at UCSF, a visiting scientist at Gladstone, and the first author of the study.

Several genes fell into known pathways already associated with heart development or congenital heart disease. Some genes were among those directly regulated by TBX5's function as a transcription factor, while others were affected in a less direct way, the study revealed. In addition, many of the altered genes were relevant to heart function in patients with congenital heart disease as they control the rhythm and relaxation of the heart, and defects in these genes are often found together with the structural defects.

The new paper doesn't point toward any individual drug target that can reverse a congenital heart disease after birth, but a better understanding of the network involved in healthy heart formation, as well as congenital heart disease may lead to ways to prevent the defects, the researchers say. In the same way that folate taken by pregnant women is known to help prevent neural tube defects, there may be a compound that can help ensure that the network of genes and proteins related to congenital heart disease stays balanced during embryonic development.

"Our new data reveal that the genes are really all part of one network--complex but singular--which needs to stay balanced during heart development," says Bruneau. "That means if we can figure out a balancing factor that keeps this network functioning, we might be able to help prevent congenital heart defects."

Reference: Kathiriya IS, Rao KS, Iacono G, et al. Modeling Human TBX5 Haploinsufficiency Predicts Regulatory Networks for Congenital Heart Disease. Developmental Cell. 2020. doi:10.1016/j.devcel.2020.11.020.

This article has been republished from the following materials. Note: material may have been edited for length and content. For further information, please contact the cited source.

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Network of Genes Involved in Congenital Heart Disease Identified - Technology Networks

Cardiac Stem Cells Comments Off on Network of Genes Involved in Congenital Heart Disease Identified – Technology Networks | December 16th, 2020

The agreement will accelerate the development and availability of new medical and pharmaceutical therapies to improve patients lives

Hamamatsu Photonics UK Ltd and Medical Technologies Innovation Facility (MTIF) are pleased to announce they have entered into a partnership agreement enabling customers the ability to view and utilize Hamamatsus Functional Drug Screening System (FDSS) CELL. This is the first FDSS/CELL to be made available in the UK in this way.

This new collaboration aims to leverage the photonics expertise, novel proprietary technology and applications of Hamamatsu, with the significant medical technology research and development capabilities of MTIF.

This is a high-end specialist piece of equipment utilised in the development of innovative medicines around the world. We are very excited to be able to provide customers with this capability, that complements our own research using this technically superb equipment. Says Professor John Hunt, Head of Strategic Research at MTIF and within Nottingham Trent University.

This partnership provides companies with a unique opportunity to use cutting edge high through-put technology to screen compounds for pharmacological activity. These capabilities are usually unavailable to all but the largest organisations. This collaboration allows organisations of every size the opportunity to accelerate their drug discovery programme. Says Professor Mike Hannay, Managing Director of the Medical Technologies Innovation Facility (MTIF) .

Hamamatsu has a long history in developing cutting edge scientific equipment for the life science market; our FDSS/CELL enables scientists, such as those working at MTIF, to make breakthroughs in the field of drug discovery and compound research. We are really excited about this new partnership between Hamamatsu and the team at MTIF helping to make such advanced instrumentation available to hundreds of potential users throughout the UK research community. Tim Stokes, Managing Director of Hamamatsu Photonics UK Ltd.

The FDSS/CELL is a compact, easy to use screening system that enables monitoring of GPCRs and ion channels for drug discovery and life science research. Screening various compounds at high throughput (96 / 384 well assays) is enabled by fluorescence or luminescence measurements using a highly sensitive Hamamatsu camera, which captures cell dynamics under the same conditions with no time lag between wells. It is also capable of recording changes in electrical potential in iPSC-derived neuronal and cardiac stem cells to gain a better understanding of toxic compound effects.

Through this new technical collaboration, HPUK and MTIF will organically integrate their respective advanced technologies and development capabilities to showcase this novel laboratory screening technology onsite at MTIF in Nottingham, UK.

Hamamatsu Photonics and MTIF aim to benefit the UK life science sector by accelerating the availability of new medical and pharmaceutical therapies. By aligning capabilities and ambitions, the parties will deliver benefit to clients by helping them to successfully navigate the complexities of discovering drug and cell therapy candidates.

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Industry News: Hamamatsu Photonics UK Ltd and the Medical Technologies Innovation Facility enter into a partnership agreement - SelectScience

Cardiac Stem Cells Comments Off on Industry News: Hamamatsu Photonics UK Ltd and the Medical Technologies Innovation Facility enter into a partnership agreement – SelectScience | December 16th, 2020

If youre like 95% of American adults, you had chickenpox as a kid. Before the United States started its widespread vaccination program in 1995, there were roughly four million cases of chickenpox every year. So, most people suffered through an infection with this highly contagious virus and its itchy, whole-body rash.

But unlike many childhood viruses, the varicella-zoster virus that causes chickenpox doesnt clear from the body when the illness ends. Instead it hangs around, taking up residence and lying dormant in the nerves, sometimes for decades, with the immune system holding it in check. In some people, it lives there harmlessly for the rest of their life. But in others, the virus can suddenly emerge and strike again, this time appearing as a different condition known as shingles.

Like chickenpox, shingles also causes a blistering rash, but this time it generally appears as a painful band around one side of your ribcage or on one side of your face. The first symptom for many people is pain or a burning sensation in the affected area. You may also have fever, a headache, and fatigue. Along with the rash and other temporary symptoms, shingles can also bring unpleasant, long-lasting, and sometimes permanent complications, such as skin infections, nerve pain in the area where the rash appeared, or even vision loss.

Experts dont fully understand this. One theory is that shingles occurs when your immune system loses its ability to keep the virus in check.

After you get chickenpox, your immune system is able to recognize the varicella-zoster virus thanks to specialized immune system cells, called B and T cells, that are able to remember the virus and quickly marshal an attack on it. Factors that weaken the immune system increase your risk of developing shingles. These include

While you may not be able to control certain factors that might trigger shingles, there are strategies you can use to prevent shingles. The most important is vaccination. Research shows that the shingles vaccine Shingrix is 90% effective in preventing an outbreak of shingles. Even if you do get shingles after being vaccinated, Shingrix greatly reduces your risk of developing persistent pain in the affected area, known as post-herpetic neuralgia.

In addition to getting vaccinated, its always a good idea to take steps to keep your body healthy, such as choosing healthy foods, staying active, and getting sufficient sleep. Its not clear if healthy lifestyle habits like these can prevent shingles, but even if they dont, theyre worthwhile because they will benefit your body in many other ways.

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Shingles: What triggers this painful, burning rash? - Harvard Health Blog - Harvard Health

Bone Marrow Stem Cells Comments Off on Shingles: What triggers this painful, burning rash? – Harvard Health Blog – Harvard Health | December 14th, 2020

As interest in chimeric antigen receptor (CAR) T-cell therapy continues to grow with more promising data coming out and approvals from the FDA in various hematologic malignancies, the role of this cellular therapy has yet to be defined in multiple myeloma, but recent data have inspired hope for this therapy in the relapsed/refractory population.

The B-cell maturation antigen (BCMA)directed CAR T-cell therapy idecabtagene vicleucel (ide-cel; bb2121) has generated excitement in this population following the submission of a Biologics License Application (BLA) in March 2020, seeking approval of ide-cel in patients with multiple myeloma who have received at least 3 prior therapies, including an immunomodulatory drug (IMiD), a proteasome inhibitor (PI), and an anti-CD38 antibody, and a Priority Review designation granted in September 2020. Following delays in the review due to coronavirus disease 2019, the Prescription Drug User Fee Act action date has been set as March 27, 2021.

Deep and durable responses were observed with ide-cel as treatment of heavily pretreated patients with relapsed/refractory multiple myeloma, according to updated results from the CRB-401 study presented by Yi Lin, MD, PhD, assistant professor of oncology and associate professor of medicine at Mayo Clinic, during the 2020 American Society of Hematology (ASH) Annual Meeting. The efficacy and safety findings were consistent with prior findings and supported a favorable clinical risk-benefit profile at target dose levels 150 x 106.1

The median overall survival with ide-cel was 34.2 months (95% CI, 19.2-not evaluable) among all patients in this triple-classexposed population, and half of the patients who had ongoing responses achieved a duration of response >2 years. The median progression-free survival (PFS) was 8.8 months (95% CI, 5.9-11.9). The objective response rate (ORR) overall was 75.8%, which included complete responses (CRs) in 38.7%.

These results from CRB-401 are comparable to the findings from the pivotal phase 2 KarMMa study (NCT03361748), which were presented earlier this year during the 2020 American Society of Clinical Oncology (ASCO) Virtual Scientific Program and support the Biologics License Application. The median OS for this study was 19.4 months, and the median PFS was 8.8 months. The ORR was 73%, which included a CR rate of 33%, and the median duration of response was 10.7 months.2

Ide-cel is being explored in several ongoing studies as well, including the phase 2 KarMMa-2 (NCT-3601078), phase 3 KarMMa-3 (NCT03651128), and phase 1 KarMMa-4 (NCT04196491) clinical trials. These phase 2 and 3 studies are evaluating ide-cel in patients with triple-classexposed disease, and the phase 1 study will explore the use of this CAR T-cell therapy in patients with high-risk newly diagnosed multiple myeloma.

These data have also set the stage for other BCMA-directed CAR T-cell therapies in development for the treatment of patients with multiple myeloma.

In an interview with Targeted Oncology, Lin discussed the updated findings from the CRB-401 study of ide-cel as treatment of patients with relapsed/refractory multiple myeloma.

TARGETED ONCOLOGY: What historical data have we seen with BCMA-directed CAR T-cell therapy in patients with relapsed/refractory multiple myeloma?

Lin: With the CAR T approach in multiple myeloma, the very first case report was actually with CD19-targeted CAR T because there was already experience with that particular antigen in leukemia and lymphomas. There's some ongoing effort in terms of dual targeting with CD19 and BCMA, but BCMA very quickly emerged as an ideal candidate for the myeloma space. This is an antigen that is more uniformly expressed on plasma cells, including myeloma cells, and maybe a small subset of mature B cells, but otherwise BCMA is not expressed on healthy tissues.

There have been some single-center clinical trials with the BCMA-targeted CAR T approach prior to the CRB-401 study, both with National Cancer Institute and the University of Pennsylvania with slightly different constructs. With those early phase 1 studies, there was a little bit more toxicity seen, although there was certainly some response, but the response wasn't particularly durable. CRB-401 is the first in a series of now industry-sponsored multicenter studies, in which we are now seeing a much more encouraging durable response rate and also a more favorable side effect profile as well. At ASH this year, I presented the longer follow-up on the phase 1 CRB-401 study. There is a pivotal phase 2 KarMMa study using the same CAR T construct that had been presented at ASCO earlier this year.

TARGETED ONCOLOGY: Please describe the design of the trial and what was different about the study.

Lin: The CRB-401 study has 2 parts. The first part is the dose-escalation part, and the second part is the dose expansion. The dose escalation is basically testing the range of a fixed dose of 50 million all the way up to 800 million of ide-cel CAR T cells in a relatively small number of patients, basically looking for signs of severe side effects to identify a safe dose. The dose expansion cohort is where we take the more promising doses in terms of response, and also safety profile, and test them in more patients to get a better safety signal, which is then moved forward for phase 2 testing in the KarMMa study.

In the dose-expansion portion of CRB-401, we required that each patient must have had exposure to an anti-CD38 antibody. That was allowed in a dose escalation but not required for everybody. [To be included in the study,] the patient must have had become refractory to the most recent line of treatment before they came on the study. The other thing that was different was that in the dose-escalation cohort, all patients had their myeloma cells in the bone marrow reviewed centrally by immunohistochemistry staining, and they were required to have at least 50% of these cells having BCMA expression in a dose-expansion cohort, to better understand the clinical efficacy and safety profiles of this treatment. We also included some patients that had BCMA expression below that to even levels that were not detectable by immunohistochemistry.

TARGETED ONCOLOGY: What were the results from this study?

Lin: The study [included] a total of 62 patients. The results from the first 33 patients were already published in the New England Journal of Medicine last year, and this year at ASH, data were presented for outcomes of the entire 62-patient cohort, with a median follow-up of now 18.1 months. What we have seen so far is in this entire treated patient cohort these are patients with very high-risk features of myeloma, and close to a third of these patients had high-risk cytogenetics, 37% of these patients had extra modularity plasma effect, and almost half of these patients needed some type of systemic therapy while their CAR T cells are being made. These patients, on average, had 6 lines of prior therapy, and in close to 70% or higher, these patients are either triple-refractory or were refractory to the most recent line of therapy.

For this group of patients that was treated overall, the safety signal was very tolerable, which is not surprising with CAR T therapy because these patients also do get lymphodepletion chemotherapy as part of the treatment with CAR T. We do see that low blood count is the most common side effect, including the more severe low blood counts, but on average, the recovery of these blood counts can be seen well under the first 3 months after CAR T infusion. The other most common side effects that we need to watch for with CAR T are cytokine release syndrome (CRS) and neurotoxicity. What we have seen in this study is that, on average, about 76% of these patients had some type of CRS. However, those that had grade 3 or higher, that is only [seen] in 6.5% of the patients, so much lower, and that's also reflected in the relative lower use of tocilizumab and steroids, as well, to manage the side effects. About 35% of these patients had some type of neurologic side effect, but again, only 1 patient had a more severe form of neurotoxicity. Compared to what we have seen with the CAR T experience in the lymphoma/leukemia space, this is a very, very encouraging safety profile.

We have also now seen that the ORR is quite high. It's 75.8% with a CR and stringent CR rate of about 38.7%. Many of these patients that had bone marrow that were evaluable for minimal residual disease (MRD) response were MRD-negative. We are seeing, since we tested many doses, that there is a dose-related increase in response with increasing [the] dose, and we have also seen that the duration of response is 10.3 months. When we look at the dose that was tested as well in those expansions [in] the 150 to 450 range, what we have seen is that the duration of response is comparable, so not significantly decreased, for patients with high-risk features like those with extramedullary disease for older patients, as well as patients who needed to get bridging therapy during treatment. The median PFS is 8.8 months, and the median OS is 34.2 months.

So far, the response rate, duration of response, and PFS seem to be comparable to what we also now see in the KarMMa study, which has less follow-up, but we are seeing a very nice median OS for a treatment in which we're just giving a 1 dose infusion and no follow-up maintenance therapy.

TARGETED ONCOLOGY: In terms of CAR T-cell therapy, how do you see this strategy impacting this patient population in the future?

Lin: I think there's definitely a role for this in the practice. The BLA for ide-cel has been submitted to the FDA, so we're anticipating review sometime in early 2021. This is very exciting because this could very well be the first CAR T for multiple myeloma. I think this would definitely be a treatment option for these patients. Based on how KarMMa is designed, we anticipate that the FDA approval will be in the space of patients who [have] had at least 3 lines of prior therapy and have been exposed to the currently approved 3 main backbones of treatmenta PI, IMiD, and the CD38 antibody. The full detail is pending final FDA review and the label. However, in that space, certainly looking at the demographic of the patient that's been treated so far as CRB-401 and KarMMa, that's a wider group of patients. Based on the fact that this is a treatment that is a basically living active cells, I perceive that the earlier that patient could get this therapy in the earliest possible approved indication, there would likely be potentially more benefit for the patients.

TARGETED ONCOLOGY: Do you think there is hope for this treatment in other hematologic malignancies outside of lymphomas and leukemias as well?

Lin: That is actually a very interesting question because what we're seeing in terms of the severity of CRS and neurotoxicity is a reflection of our evolving learning about how to manage the toxicity, as well. There is a component to the CAR design, the disease, the nature of the disease, the kinetics of the CAR T actions, in the manifestation of these symptoms. What we are seeing now, with even the prior CAR and next-generation CAR coming on, we will likely see an ongoing improvement in terms of a reduction of severity of these symptoms and also in the ways that we could manage these symptoms.

The fact that myeloma would be the next disease that has an FDA-approved CAR T also relates to the fact that the BCMA antigen is more restricted on the cell type where the malignancy is involved, similar to CD19 for lymphoid malignancy. We are seeing that there are some challenges, for example with acute myeloid leukemia or myeloid neoplasms where a number of antigens could overlap with stem cells, which we wouldn't want to try to hurt. There are some novel CAR approaches to try to overcome that, and those are in very early phase testing, so we'll need to see how those results evolve.

References

1. Lin Y, Raje NS, Berdeja JG, et al. Idecabtagene vicleucel (ide-cel, bb2121), a BCMA-directed CAR T cell therapy, in patients with relapsed and refractory multiple myeloma: updated results from phase 1 CRB-401 study. Presented at: 2020 ASH Annual Meeting & Exposition; December 5-8, 2020; Virtual. Abstract 131.

2. Munshi NC, Anderson Jr LD, Jagannath S, et al. Idecabtagene vicleucel (ide-cel; bb2121), a BCMA-targeted CAR T-cell therapy, in patients with relapsed and refractory multiple myeloma (RRMM): Initial KarMMa results.J Clin Oncol. 2020;38(suppl):8503. doi:10.1200/JCO.2020.38.15_suppl.8503

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Updated Findings Show Continued Efficacy for CAR T-Cell Therapy in Heavily Pretreated Myeloma - Targeted Oncology

Bone Marrow Stem Cells Comments Off on Updated Findings Show Continued Efficacy for CAR T-Cell Therapy in Heavily Pretreated Myeloma – Targeted Oncology | December 14th, 2020

The allogeneic off-the-shelf CD22-directed T-cell product, UCART22, showed early signs of activity and no evidence of unexpected toxicities at 2 dose levels for adult patients with relapsed/refractory CD22-positive B-cell acute lymphoblastic leukemia, according to the results of a study presented during the 2020 ASH Annual Meeting.1

In the phase 1 BALLI-01 (NCT04150497) dose-escalation and dose-expansion study, 2 patients at the 1 x 105 cells/kg dose achieved a complete remission (CR) with incomplete hematologic recovery on day 28. One of these patients attained a minimal residual disease (MRD)positive CR at day 42 followed by subsequent inotuzumab ozogamicin (Besponsa) and then transplant.

One patient at dose level 2, 1 x 106 cells/kg, experienced a significant bone marrow blast reduction at day 28, followed by disease progression.

No patients experienced dose-limiting toxicities (DLTs), immune effector cellassociated neurotoxicity syndrome (ICANS), graft-versus-host disease (GVHD), adverse effects (AE) of special interest (AESI), a UCART22-related AE that was grade 3 or higher, or a serious AE (SAE).

UCART22 showed no unexpected toxicities at the doses of 1 x 105 cells/kg and 1 x 106 cells/kg with fludarabine and cyclophosphamide lymphodepletion, lead study author Nitin Jain, MD, an assistant professor in the Department of Leukemia, The University of Texas MD Anderson Cancer Center, said in a virtual presentation during the meeting. Host immune recovery was observed early, and the addition of alemtuzumab [Lemtrada] to fludarabine and cyclophosphamide lymphodepletion is currently being explored with the goal to achieve deeper and more sustained T-cell depletion and to promote expansion and persistence of UCART22.

Standard treatment for adult patients with B-cell ALL includes multiagent chemotherapy with or without allogeneic stem cell transplant. However, 30% to 60% of patients with newly diagnosed B-cell ALL who achieve a CR will relapse, and the expected 5-year survival rate for those with relapsed/refractory disease is approximately 10%.

Previously, UCART19, when paired with lymphodepletion using fludarabine, cyclophosphamide, and alemtuzumab, was found to show efficacy in this patient population.2

CD22 is an FDA-approved therapeutic target in B-cell ALL. UCART22 is an immediately available, standardized, manufactured agent with the ability to re-dose, and its CAR expression redirects T cells to tumor antigens, Jain explained.

Moreover, through its mechanism of action, TRAC becomes disrupted using Transcription activator-like effector nucleases (Talen) technology to eliminate TCR from cell surface and reduce the risk of GVHD. CD52 is also disrupted with the use of Talen to eliminate sensitivity to lymphodepletion with alemtuzumab. Finally, there is a CD20 mimotope for rituximab (Rituxan) as a safety switch, Jain added.

UCART22 has also demonstrated in vivo antitumor activity in immune-compromised mice that were engrafted with CD22-positive Burkitt lymphoma cells in a dose-dependent manner.

In the dose-escalation/dose-expansion BALLI-01 study, investigators are enrolling up to 30 patients in a modified Toxicity Probability Interval design. There are 3 cohorts, which have 2 to 4 patients on each cohort: 1 x 105 cells/kg (dose level 1), 1 x 106 cells/kg (dose level 2), and 5 x 106 cells/kg. The focus of the dose-escalation phase of the trial was to determine the maximum-tolerated dose (MTD) and the recommended phase 2 dose (RP2D) before heading into the dose-expansion portion of the trial.

To be eligible for enrollment, patients must have been between 18 and 70 years old, have acceptable organ function, an ECOG performance status of 0 or 1, at least 90% of B-cell ALL blast CD22 expression, and had previously received at least 1 standard chemotherapy regimen and at least 1 salvage regimen.

End points of the trial included safety and tolerability, MTD/R2PD, investigator-assessed response, immune reconstitution, and UCART22 expansion and persistence.

The lymphodepletion regimens were comprised of fludarabine (at 30 mg/m2 x 4 days) plus cyclophosphamide (1 g/m2 x 3 days); the study has since been amended to include the regimen of fludarabine (at 30 mg/m2 x 3 days), cyclophosphamide (500 g/m2 x 3 days), and alemtuzumab (20 mg/day x 3 days) and is currently enrolling patients.

Following screening, lymphodepletion, and UCART22 infusion, patients underwent an observation period for DLTs with a primary disease evaluation at 28 days; additional efficacy evaluations occurred at 56 days and 84 days. Patients were followed for 2 years and continued to be assessed for long-term follow-up.

As of July 1, 2020, 7 patients were screened, of which 1 patient failed and 6 were therefore enrolled on the study. One patient discontinued therapy before receiving UCART22 due to hypoxia from pneumonitis that was linked with lymphodepletion. Five patients were treated with UCART22 at dose level 1 (n = 3) and dose level 2 (n = 2).

The median age of participants was 24 years (range, 22-52), 3 of the 5 patients were male, and 3 had an ECOG performance status of 0. The median number of prior therapies was 3 (range, 2-6), and there were a median 35% bone marrow blasts (range, 10%-78%) prior to lymphodepletion.

Three patients had complex karyotype and 2 had diploid cytogenetics. One patient each had the following molecular abnormalities: CRLF2, CRLF2 and JAK2, CDKN2A loss, KRAS and PTPN11, and IKZF1. Only 1 patient had undergone haploidentical transplant. Four patients previously received prior CD19- or CD22-directed therapy, including blinatumomab (Blincyto), inotuzumab ozogamicin (Besponsa), and CD19-directed CAR T-cell therapy. At study entry, 3 patients had refractory disease and 2 patients had relapsed disease.

Grade 3 or higher treatment-emergent AEs (TEAEs), which were unrelated to study treatment, included hypokalemia, anemia, increased bilirubin, and acute hypoxic respiratory failure. Also not related to UCART22, 3 patients experienced 4 treatment-emergent SAEs: porta-hepatis hematoma, sepsis, bleeding, and sepsis in the context of disease progression. No treatment discontinuations due to a treatment-related TEAE were reported.

The patient who achieved a CR followed by transplant was a 22-year-old male who had undergone 2 prior treatments for B-cell ALL and received UCART22 at a dose of 1 x 105 cells/kg. He did not experience CRS, ICANS, GVHD, nor a SAE, and all TEAEs were grade 1.

Jain also noted that host T-cell constitution was observed in all patients within the DLT observation period. UCART22 was also not detectable through flow cytometry or molecular analysis, the latter of which was at dose level 1 only.

References:

1. Jain N, Roboz GJ, Konopleva M, et al. Preliminary results of BALLI-O1: a phase I study of UCART22 (allogeneic engineered T cells expressing anti-CD22 chimeric antigen receptor) in adult patients with relapsed/refractory anti-CD22+ B-cell acute lymphoblastic leukemia (NCT04150497). Presented at: 2020 ASH Annual Meeting and Exposition; December 4-8, 2020; Virtual. Abstract 163.

2. Benjamin R, Graham C, Yallop D, et al. Preliminary data on safety, cellular kinetics and anti-leukemic activity of UCART19, an allogeneic anti-CD19 CAR T-cell product, in a pool of adult and pediatric patients with high-risk CD19+ relapsed/refractory b-cell acute lymphoblastic leukemia. Blood. 2018;132(suppl 1):896. doi:10.1182/blood-2018-99-111356.

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Early Signs of Activity and Tolerability Found in Allogeneic Product UCART22 for Patients with Relapsed/Refractory CD22+ B-Cell ALL - Cancer Network

Bone Marrow Stem Cells Comments Off on Early Signs of Activity and Tolerability Found in Allogeneic Product UCART22 for Patients with Relapsed/Refractory CD22+ B-Cell ALL – Cancer Network | December 14th, 2020

The Covid-19 can damage the heart both directly and indirectly, and lead to complications ranging from inflammation of the heart (myocarditis), injury to heart cells (necrosis), heart rhythm disorders (arrhythmias), heart attack, and muscle dysfunction that can lead to acute or protracted heart failure, experts said.

Covid-19 is a vascular disease that injures heart cells and muscle. It also leads to the formation of blood clots, both in the microvasculature and large vessels, which can block blood supply to the heart, brain and lungs and lead to stroke, heart attack and respiratory failure, said Dr Ravi R Kasliwal, chairman of clinical and preventive cardiology department at Medanta -The Medicity Hospital.

Also Read: Few Covid-19 deaths in Indias old-age homes, survey finds

A US study using MRI found cardiac abnormalities in 78 of 100 patients who had recently recovered from Covid-19, including 12 of 18 asymptomatic patients. Sixty patients had ongoing myocardial inflammation consistent with myocarditis, found the study, which was published in the Journal of American Medical Association Cardiology in July.

Even people with mild disease or no symptoms can develop life-threatening cardiovascular complications. Whats worrying is that this holds true for healthy adults with no pre-existing risk factors, which raise their risk of complications, said Dr Kasliwal, who recommends that everyone who has recovered from Covid-19 be screened for heart damage

Cardiac trouble

Extensive cardiac involvement is what differentiates Sars-CoV-2, the virus that causes Covid-19, from the six other coronaviruses that cause infection in humans, writes cardiologist Dr Eric J Topol, founder, director and professor of molecular medicine at the Scripps Research Translational Institute in La Jolla, California, in the journal Science.

The four human coronaviruses that cause cold-like symptoms have not been associated with heart abnormalities, though there have been isolated reports linking the Middle East Respiratory Syndrome (MERS) caused by MERS-CoV) with myocarditis, and cardiac disease with the Severe Acute Respiratory Syndrome (SARS) caused by Sars-CoV.

Also Read| Extraordinary uncertainties: Harvard prof on Covid-19, impact on mental health

Sars-CoV-2 is structurally different from Sars-CoV. The virus targets the angiotensin-converting enzyme 2 (Ace2) receptor throughout the body, facilitating cell entry by way of its spike protein, along with the cooperation of proteases. The heart is one of the many organs with high expression of Ace2. The affinity of Sars-CoV-2 to Ace2 is significantly greater than that of SARS, according to Dr Topol.

Topol notes the ease with which Sars-CoV-2 infects heart cells derived from induced pluripotent stem cells (iPSCs) in vitro, leading to a distinctive pattern of heart muscle cell fragmentation evident in autopsy reports. Besides directly infecting heart muscle cells, Sars-CoV-2 also enters and infects the endothelial cells that line the blood vessels to the heart and multiple vascular beds, leading to a secondary immune response. This causes blood pressure dysregulation, and activation of a proinflammatory response leading to a cytokine storm, which is a potentially fatal systemic inflammatory syndrome associated with Covid-19.

Persisting problems

Studies have found that injury to heart cells reflected in blood concentrations of a cardiac muscle-specific enzyme called troponin affects at least one in five hospitalised patients and more than half of those with pre-existing heart conditions, which raises the risk of death. Patients with higher troponin amounts also have high markers of inflammation (including C-reactive protein, interleukin-6, ferritin, lactate dehydrogenase), high neutrophil count, and heart dysfunction, all of which heighten immune response.

The heightened systemic inflammatory responses and diminished blood supply because of clotting, endotheliitis (blood vessel inflammation), sepsis, or hypoxemia (oxygen deprivation) because of acute lung infection leads to indirect cardiac damage, said Dr Kasliwal.

The cardiovascular damage associated with Sars-CoV-2 infection can persist beyond recovery. Since the virus affects the heart as much as the respiratory tract, further research is needed to understand why some people are more vulnerable to heart damage than others.

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Covid-19 can have impact on heart too, say experts - Hindustan Times

Cardiac Stem Cells Comments Off on Covid-19 can have impact on heart too, say experts – Hindustan Times | December 14th, 2020

Asian beauty woman putting on mask on face. Wearing corona virus prevention masks for long hours are ... [+] creating irritation, skin problems, acne pimples.

Its starts with a seemingly harmless blackhead. Then you notice redness and swelling that will inevitably birth a dreaded pimple. The next thing you know one zit has multiplied to cover almost an entire area of your face along the jawline or chin. Like most skin breakouts, once acne takes over your life, it will often feels like a losing battle.

My bout with acne took over three months of my quarantine life with wounds (okay, maybe scars is a better word for it) that remind me to never again take this skin pandemic lightly. The guesswork was tedious and for weeks seemed futile. There were trial and error attempts, in search of a balancing skincare routine that would clear skin without stripping it off its moisture. This is most important especially for those with combination skin. I even cheated on my trusted dermatologist, only to get a subpar facial that did nothing to improve skins condition. Said facialist, however, offer sound advice: Forego products that will leave your skin oily until the acne has subsided. This will take a while.

Before getting into the meaty details of this war against maskne, lets talk about causes. Maskne is basically acne brought about by wearing face masks for prolonged periods of time. Heat, dirt, sweat and bacteria, when covered by fabrics or other materials used to make face coverings, trigger skin flare ups. Even in cooler temperature, maskne continues to be a bane especially for sensitive skin thats dry and flaky. So yes, the war is far from over, but the good news is it can be won.

A GENTLE AND THOROUGH SKIN CLEANSE is your first step to beating this very frustrating skin condition. I looked far and wide for natural cleansers that would help to control the oil, sebum and dirt. Oil-based cleansers seemed to aggravate the flare up. Bar soaps left skin feeling tight, dry and flaky. Clean facial wash formulations with tea tree oil work wonders as an anti-microbial skin solution. Of the many choices on offer, an all-natural Philippine made Acne Defense Facial Wash from HUMAN NATURE fulfilled its promise of soothing a nasty flare up without drying skin.

Alpyn Beauty's Wild Huckleberry 8-Acid Polishing Peel

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M-61 Power Glow Toner exfoliates away impurities, refine pores, and smoothens skin texture to ... [+] prevent blemishes and quickly improve existing acne

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ZITSTICKA's Press Refresh Sheet Mask combines functional technology and skincare tailored to fight ... [+] acne. It is formulated with salicylic acid, glycolic acid, lactic acid, Niacinamide (to calm) and vitamin A.

Skin flare ups and breakouts in the past have taught me that the first thing to go when pimples take over are heavy creams and moisturizers. As much as we all love a good dose of hydration, war against maskne calls for heavy reinforcements like a hardworking, skin saving face mask. ZITSTICKAs Press Refresh Hydrogel Sheet Mask is a warrior that combines functional technology and skincare tailored to fight acne. It is formulated with salicylic acid, glycolic acid, lactic acid, Niacinamide (to calm) and vitamin A.

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Peace Out Healing Dots combine Hydrocolloid, Salicylic Acid, Vitamin A and Aloe Vera to clear up ... [+] acne-causing bacteria and reduce redness overnight.

Peace Out Acne Serum is formulated with Salicylic Acid, Niacinamide, Vitamin C & Zinc that work ... [+] together to clear blemishes, prevent them from returning

Maskne is stubborn and often requires a stroke of mad brilliance to beat. PEACE OUT, the leading anti acne skincare brand at Sephora, has changed the way we address breakouts with a full range of innovative skincare solutions. The Acne Healing Dot combines hydrocolloid polymer technology with clean, active ingredients to zap zits. Instead of applying gel or liquid formulas on the surface, PEACE OUT offers fun, stick on skin solutions for hardcore breakouts. I had to try it to believe. To use, simply apply the sticker directly on the affected areas and leave on for a couple of hours. Hydrocolloid dressings absorb moisture and toxins trapped underneath the skins surface, which effectively helps to reduce redness and swelling. Use of this technology also helps to speed up healing of acne. For best results, pair the Healing Dots with PEACE OUTs Acne Serum. Salicylic acid infused with Niacinamide and Vitamin C decongests pores, while reducing the appearance of dark spots and blemishes.

Heraux Molecular Anti-Inflammaging Serum shielding stem cells from the effects of stress and ... [+] promotES their overall youthful function.

Its also essential that you give skin the protective barrier it needs against stressors and irritants. This helps to form a layer that will keep it from inflammation and ultimately, breakouts. HERAUX Molecular Anti Inflammaging Serum uses the first and proprietary Biomimetic Lipid, HX-1, to target inflammaging or aging caused by inflammation. Through the discovery of two stem cell scientists, this breakthrough skincare formulation shields stem cells from the effects of stress and promotes their overall youthful function by modulating the protein that regulates regeneration versus inflammation. The layer has also helped me to protect skin and bring back its radiance and texture.

Ive been using Heraux for a month now, applying a pea-sized amount on thoroughly cleansed skin before getting on with my day. This potent, light serum leaves a matte finish on skin which is brilliant especially when you are paring down on makeup. Pores appear smaller and more importantly, skin barrier is less likely to react to friction or contact with face coverings. After a few weeks of continued use, skin was visibly more youthful and refreshed.

Femmue Lumiere Vital C Serum to help lighten post acne blemishes and dark spots.

Gradually bring back skin hydration into your routine with FEMMUE Ideal Creme Riche

Dull and dry skin with are some of the after effects of a maskne breakout. You can also expect unsightly blemishes and dark spots. To revive clear, smooth and radiant skin, FEMMUEs flower therapy serums and mask did not disappoint. Using modern botanicals, products like Lumiere Vital C Serum and Ideal Cream Riche are skin super healers that restore skins glow and texture. Lumiere is a multi-tasking lightweight formula that works as both an antioxidant and brightening solution. It stimulates collagen production while refining skin surface and lightening dark spots. It is also specially formulated to work beautifully with extra sensitive, acne-prone skin types. For a restorative nighttime skincare routine, pair this serum with FEMMUEs Ideal Crme Riche every other night. Apply a very thin layer all over your face, making sure to tap excess cream with facial tissue. I love to follow with a 10-minute LED Face Mask session just before dozing off.

Scelido Anti Bacterial and Anti Virus Masks are breathable, washable face coverings made with Aero ... [+] Silver fabric and Copper threads.

The best way to keep maskne from taking over your life again is to choose invest in a an antibacterial, breathable face mask. I love a stylish statement masks and have built quite a collection over the months. For long days that will require hours of wear, however, opt for cotton face masks that allow skin to breathe. Infusion of copper and metal ions into the fabric also help to fight off bacteria. SCELIDO Nano Antibacterial and Antivirus Cooling Mask uses ASKIN and Aero Silver fabric, which gives 99.9% antibacterial and UV protection. It is as effective in protecting wearers from viruses as a KF80 but is light, breathable, reusable and effective in preventing acne. Copper threads enhances protection level and seamless ergonomic design (with sizing from XS to Large) allows this facial covering to sit perfectly on skin without causing traction and irritation.

We all look forward to the day when masks and maskne become things of the past. War wounds, otherwise known as acne scars, are slowly beginning to fade. Till then, lessons from this battle remind us that nature truly is the best healer. And as we continue discover the skincare and self care routine that works best for us, any maskne survivor will tell you this: do your research, look at ingredients and dont go into guesswork.

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A Tried And Tested Guide On How To Win The War Against Maskne - Forbes

Skin Stem Cells Comments Off on A Tried And Tested Guide On How To Win The War Against Maskne – Forbes | December 13th, 2020

Author Thomas Thompson once characterized Houston circa 1945 as a city where medicine of the most mediocre sort was practiced, a city with a third-rate medical school, no heritage of scholarly thinking and where extinguishing life by violence was far more common than exploring methods to prolong it.

That all changed in less time than it takes to age a good bottle of wine, according to the author of the true crime classic Blood and Money.

In a swampy area six miles south of the heart of downtown, in fields where racoons and water moccasins lived, there sprang up a collection of medical facilities which, by 1970, had become one of the handful of distinguished medical centers in the world, Thompson wrote in Hearts: Of Surgeons and Transplants, Miracles and Disasters Along the Cardiac Front.

No one was more responsible for the transformation, of course, than Michael DeBakey and Denton Cooley, the pioneering surgeons whose innovations made Houston and the Texas Medical Center the epicenter for cardiovascular care, a place where the most cutting-edge therapies were practiced with the greatest skill, a place that drew patients from around the nation and world, both common man and heads of state.

The advances culminated in Cooley implanting the worlds first artificial heart in a person, a dream since the 1940s, a Kitty Hawk type of advance. The story made headlines around the world and, even though the device was never used again, its legacy can be seen in the mechanical cardiac parts people now take for granted valves, pacemakers and, most of all, support devices that help diseased hearts better pump blood.

But the achievements started long before that. In medical school. DeBakey invented the so-called roller pump, which made it possible to provide a surgical patient with a continuous flow of blood. DeBakeys invention would would become the essential component of the heart-lung machine that maintained the patients vital functions during procedures, ushering in the era of open heart surgery.

In 1952, DeBakey performed the first successful operation on an aneurysm a ballooning of the arterial wall by replacing the affecting area with a graft from a cadaver artery. The following year he performed the first successful surgery to remove blood clots and plaque from the inner lining of blood vessels that deliver blood to the brain and head, an advance that would go on to spare countless patients from devastating strokes.

Indeed, though DeBakey was known mostly as a heart surgeon, many experts consider such vascular procedures his greatest achievement. He made the aorta, the vessel that carries blood from the heart throughout the body, a treatable entity. Until then, aortic aneurysms and tears, were almost universally fatal.

Around that time, DeBakey created the first Dacron grafts one of the Texas Medical Centers great stories which enabled durable repair of artery walls weakened by aneurysms. He invented the technique on his wifes sewing machine using the then new material, bought at Foleys in downtown Houston when they were out of nylon and vinyon, the fabrics he preferred. He soon determined Dacron was superior because it didnt degenerate over time.

The role of Providence in human endeavor is speculative, but I like to think that in a personal case it was purposeful, DeBakey wrote in the American Surgeon in 2008. Obviously, because of my good fortune, I was ahead of everyone else in the field.

The invention, one of more than 50 he devised to repair hearts and arteries, won DeBakey the 1963 Lasker Award, the top American award in medicine.

The following year, while attempting a surgery that proved too difficult to complete, Dr. DeBakey improvised a coronary bypass procedure only previously performed successfully in dogs. In so doing, he became the first surgeon to perform a successful coronary bypass on a human patient.

In 1968, DeBakey was credited with the first simultaneous, multi-organ transplant, overseeing a team that removed the heart, lobe of one lung and both kidneys from a 20-year-old victim of a gunshot wound. The organs were transplanted into four patients: a 50-year-old man got the heart; a 39-year man got the partial lung; and two men, 41 and 22, each received a kidney.

Meanwhile, Cooley focused on hearts, performing an estimated 65,000 over four decades, more than any other surgeon. At one time, his surgical team was performing one-tenth of all open heart surgeries in the U.S.

Cooley stood above all others because of his speed and dexterity, a combination that produced what was described at the time as Woolworth volume and Tiffany qualtiy. He was quoted saying he always wanted to be known as the Sam Walton of heart surgery, in reference to the founder of Walmart.

MORNING REPORT: Get the top stories on HoustonChronicle.com sent directly to your inbox

But Cooley also pushed the boundaries of heart surgery. Dr. Christian Baarnard in South Africa beat him to the first heart transplant in December 1967, but Cooley matched the achievement five months later and his patient went on to live 204 days, compared to 18 for Baarnards patient.

In 1969, Cooley stunned the world by implanting a mechanical heart into the chest of Haskell Karp, a printing estimator in the last stages of heart failure. The device worked long enough to replace it with a donor heart when one became available three days later, although Karp died 32 hours later of pneumonia and kidney failure.

For all the attention it generated, the event didnt set off a wave of implants across the nation, the technology considered premature, rejection issues not yet well understood. Instead, it focused attention on alternatives known as left ventricular assist devices (LVADs), which assist the chamber that pumps blood throughout the body replace the heart. The approach was pioneered by DeBakey after he abandoned research into the total artificial heart.

Also pioneered in Houston: a minimally invasive procedure to replace a failing heart valve. The surgery, which entails threading the new valve to the heart through a blood vessel in the patients groin rather than open-heart surgery, was approved first for patients too sick and frail for open-heart surgery, then for patients at intermediate risk. More recently, studies showed it proved better than open surgery in young, healthy patients.

Houston doctors are at forefront of the next great hope for cardiovascular care too: regenerative medicine. The field is based on the idea that stem cells found in early stage embryos and adults, prized for their ability to easily divide and develop into various types of cells may be able to repair injuries and degeneration to heart tissue, an idea first tested at Texas Heart Institute around 2000. Though still a work in progress, the idea is considered the next frontier.

todd.ackerman@chron.com

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Houston healthcare in 1945 was 'mediocre.' The rivalry between DeBakey and Cooley changed it forever - Houston Chronicle

Cardiac Stem Cells Comments Off on Houston healthcare in 1945 was ‘mediocre.’ The rivalry between DeBakey and Cooley changed it forever – Houston Chronicle | December 13th, 2020

New York, Dec 12 (IANS) Researchers have revealed the appealing array of fruit and candy flavours that entice millions of young people to take up vaping are cardiotoxic and disrupt the heart's normal electrical activity.

Mounting studies indicate that the nicotine and other chemicals delivered by vaping, while generally less toxic than conventional cigarettes, can damage the lungs and heart.

"But so far there has been no clear understanding about what happens when the vaporized flavouring molecules in flavoured vaping products, after being inhaled, enter the bloodstream and reach the heart," said study author Sami Noujaim from the University of South Florida in the US.

In the study, published in the American Journal of Physiology-Heart and Circulatory Physiology, the research team reported on a series of experiments assessing the toxicity of vape flavourings in cardiac cells and in young mice.

The flavoured electronic nicotine delivery systems widely popular among teens and young adults are not harm-free.

"Altogether, our findings in the cells and mice indicate that vaping does interfere with the normal functioning of the heart and can potentially lead to cardiac rhythm disturbances," Noujaim said.

In mouse cardiac muscle cells (HL-1 cells), the researchers tested the toxicity of three different popular flavours of e-liquid: fruit flavour, cinnamon, and vanilla custard.

All three were toxic to HL-1 cells exposed to e-vapour bubbled into the laboratory dish where the cells were cultured.

Cardiac cells derived from human pluripotent stem cells were exposed to three distinct e-vapours.

The first e-vapour containing the only solvent interfered with the electrical activity and beating rate of cardiac cells in the dish. A second e-vapour with nicotine added to the solvent increased the toxic effects on these cells.

The third e-vapour comprised of nicotine, solvent, and vanilla custard flavouring (the flavour previously identified as most toxic) augmented damage to the spontaneously beating cells even more.

"This experiment told us that the flavouring chemicals added to vaping devices can increase harm beyond what the nicotine alone can do," Noujaim said.

The findings showed that mice exposed to vaping were more prone to an abnormal and dangerous heart rhythm disturbance known as ventricular tachycardia compared to control mice.

"Our research matters because regulation of the vaping industry is a work in progress," Noujaim noted.

--IANS

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Flavours added to vaping devices can damage the heart: Study - Sify News

Cardiac Stem Cells Comments Off on Flavours added to vaping devices can damage the heart: Study – Sify News | December 13th, 2020

Abstract

Enzyme replacement therapy, in which a functional copy of an enzyme is injected either systemically or directly into the brain of affected individuals, has proven to be an effective strategy for treating certain lysosomal storage diseases. The inefficient uptake of recombinant enzymes via the mannose-6-phosphate receptor, however, prohibits the broad utility of replacement therapy. Here, to improve the efficiency and efficacy of lysosomal enzyme uptake, we exploited the strategy used by diphtheria toxin to enter into the endolysosomal network of cells by creating a chimera between the receptor-binding fragment of diphtheria toxin and the lysosomal hydrolase TPP1. We show that chimeric TPP1 binds with high affinity to target cells and is efficiently delivered into lysosomes. Further, we show superior uptake of chimeric TPP1 over TPP1 alone in brain tissue following intracerebroventricular injection in mice lacking TPP1, demonstrating the potential of this strategy for enhancing lysosomal storage disease therapy.

Lysosomal storage diseases (LSDs) are a group of more than 70 inherited childhood diseases characterized by an accumulation of cellular metabolites arising from deficiencies in a specific protein, typically a lysosomal hydrolase. Although each individual disease is considered rare, LSDs have a combined incidence of between 1/5000 and 1/8000 live births, and together, they account for a substantial proportion of the neurodegenerative diseases in children (1). The particular age of onset for a given LSD varies depending on the affected protein and the percentage of enzymatic activity still present; however, in most cases, symptoms manifest early in life and progress insidiously, affecting multiple tissues and organs (2). In all but the mildest of cases, disease progression results in severe physical disability, possible intellectual disability, and a shortened life expectancy, with death occurring in late childhood or early adolescence.

As they are monogenic diseases, reintroducing a functional form of the defective enzyme into lysosomes is in principle a viable strategy for treating LSDs. Enzyme replacement therapy (ERT) is now approved for the treatment of seven LSDs, and clinical trials are ongoing for five others (3). However, delivering curative doses of recombinant lysosomal enzymes into lysosomes remains a major challenge in practice. ERT typically takes advantage of a specific N-glycan posttranslational modification, mannose-6-phosphorylation (M6P), which controls trafficking of endogenous lysosomal enzymes, as well as exogenous uptake of lysosomal enzymes from circulation by cells having the cation-independent M6P receptor (CIMPR) (4). Hence, a combination of factors including (i) the abundance of the M6P receptor in the liver, (ii) poor levels of CIMPR expression in several key target tissue types such as bone and skeletal muscle, (iii) incomplete and unpredictable M6P labeling of recombinant enzymes, and (iv) the highly variable affinity of recombinant lysosomal enzymes for CIMPR [viz., Kds (dissociation constants) ranging from low to mid micromolar (5, 6)] all contribute to diminishing the overall effectiveness of therapies using CIMPR for cell entry (3).

To improve the delivery of therapeutic lysosomal enzymes, we drew inspiration from bacterial toxins, which, as part of their mechanism, hijack specific host cellsurface receptors to gain entry into the endolysosomal pathway. While we and others have explored exploiting this pathway to deliver cargo into the cytosol (7, 8), here we asked whether this same approach could be used to enhance the delivery of lysosomal enzymes into lysosomes. We choose the diphtheria toxin (DT)diphtheria toxin receptor (DTR) system owing to the ubiquitous nature of the DTR, in particular its high expression levels on neurons.

Corynebacterium diphtheriae secretes DT exotoxin, which is spread to distant organs by the circulatory system, where it affects the lungs, heart, liver, kidneys, and the nervous system (9). It is estimated that 75% of individuals with acute disease also develop some form of peripheral or cranial neuropathy. This multiorgan targeting results from the fact that the DTR, heparin-binding EGF (epidermal growth factor)like growth factor (HBEGF), is ubiquitously expressed. The extent to which DT specifically targets difficult-to-access tissues such as muscle and bone, however, is not currently known.

DT is a three-domain protein that consists of an N-terminal ADP (adenosine diphosphate)ribosyl transferase enzyme (DTC), a central translocation domain (DTT), and a C-terminal receptorbinding domain (DTR). The latter is responsible for both binding cell surface HBEGF with high affinity [viz., Kd = 27 nM (10)] and triggering endocytosis into early endosomes (Fig. 1A). Within endosomes, DTT forms membrane-spanning pores that serve as conduits for DTC to enter the cytosol where it inactivates the host protein synthesis machinery. The remaining portions of the toxin remain in the endosomes and continue to lysosomes where they are degraded (11, 12). We hypothesized that the receptor-binding domain, lacking any means to escape endosomes, would proceed with any attached cargo to lysosomes and, thus, serve as a means to deliver cargo specifically into lysosomes following high-affinity binding to HBEGF.

(A) DT intoxication pathway (left), DT domain architecture, and LTM structure (right). (B and C) DTK51E/E148K, LTM, mCherry-LTM, and LTM-mCherry compete with wild-type DT for binding and inhibit its activity in a dose-dependent manner with IC50 (median inhibitory concentration) values of 46.9, 10.1, 52.7, and 76.1 nM, respectively (means SD; n = 3). (D and E) C-terminal and N-terminal fusions of LTM to mCherry were immunostained (red) and observed to colocalize with the lysosomal marker LAMP1 (39). (F) Fractional co-occurrence of the red channel with the green channel (Manders coefficient M2) were calculated for mCherry-LTM and LTM-mCherry and were found to be 0.61 0.10 and 0.52 0.11, respectively (means SD; n = 6).

In this study, we generated a series of chimeric proteins containing the DTR-binding domain, DTR, with the goal of demonstrating the feasibility of delivering therapeutic enzymes into lysosomes through the DT-HBEGF internalization pathway. We showed that DTR serves as a highly effective and versatile lysosome-targeting moiety (LTM). It can be placed at either the N or C terminus of the cargo, where it retains its high-affinity binding to HBEGF and the ability to promote trafficking into lysosomes both in vitro and in vivo. On the basis of its advantages, over M6P-mediated mechanisms, we further investigated the utility of LTM for the lysosomal delivery of human tripeptidyl peptidase-1 (TPP1) with the long-term goal of treating Batten disease.

To evaluate whether the DTR-binding fragment could function autonomously to traffic cargo into lysosomes, we first asked whether the isolated 17-kDa DTR fragment could be expressed independently from DT holotoxin and retain its affinity for HBEGF. We cloned, expressed, and purified the receptor-binding fragment and evaluated its ability to compete with full-length DT for the DTR, HBEGF. Before treating cells with a fixed dose of wild-type DT that completely inhibits protein synthesis, cells were incubated with a range of concentrations of LTM or a full-length, nontoxic mutant of DT (DTK51E/E148K). LTM-mediated inhibition of wild-type DT-mediated toxicity was equivalent to nontoxic DT (Fig. 1B), demonstrating that the receptor-binding fragment can be isolated from the holotoxin without affecting its ability to fold and bind cell surface HBEGF. Next, we evaluated whether LTM had a positional bias (i.e., was able to bind HBEGF with a fusion partner when positioned at either terminus). To this end, we generated N- and C-terminal fusions of LTM to the model fluorescent protein mCherry (i.e., mCherry-LTM and LTM-mCherry). To determine binding of each chimera to HBEGF, we quantified the ability of each chimera to compete with wild-type DT on cells in the intoxication assay. Both constructs competed with wild-type DT to the same extent as LTM alone and DTK51E/E148K (Fig. 1C), demonstrating that LTM is versatile and autonomously folds in different contexts.

To evaluate intracellular trafficking, HeLa cells were treated with either LTM-mCherry or mCherry-LTM and then fixed and stained 4 hours later with an antibody against the lysosomal marker LAMP1. In both cases, we observed significant uptake of the fusion protein (Fig. 1, D and E). We calculated Manders coefficients (M2) to quantify the extent to which signal in the red channel (LTM-mCherry and mCherry-LTM) was localizing with signal in the green channel (LAMP1). The fraction of red/green co-occurrence was calculated to be 0.61 for mCherry-LTM and 0.52 for LTM-mCherry, indicating trafficking to the lysosomal compartments of the cells and no significant difference (P = 0.196) between the two orientations of chimera (Fig. 1F). Together, these results confirm that the LTM is capable of binding HBEGF and trafficking associated cargo into cells and that the LTM can function in this manner at either terminus of a fusion construct.

With minimal positional bias observed in the mCherry fusion proteins, we next screened LTM fusions to TPP1 to identify a design that maximizes expression, stability, activity, and, ultimately, delivery. TPP1 is a 60-kDa lysosomal serine peptidase encoded by the CLN2 gene, implicated in neuronal ceroid lipofuscinosis type 2 or Batten disease. Loss of function results in the accumulation of lipofuscin, a proteinaceous, autofluorescent storage material (13). Exposure to the low-pH environment of the lysosome triggers autoproteolytic activation of TPP1 and release of a 20-kDa propeptide that occludes its active site. From a design perspective, we favored an orientation in which the LTM was N terminal to TPP1, as autoprocessing of TPP1 would result in the release of the upstream LTM-TPP1 propeptide, liberating active, mature TPP1 enzyme in the lysosome (Fig. 2A). Given the need for mammalian expression of lysosomal enzymes, we generated synthetic genetic fusions of the LTM to TPP1, in which we converted the codons from bacterially derived DT into the corresponding mammalian codons. Human embryonic kidney (HEK) 293F suspension cells stably expressing recombinant TPP1 (rTPP1) and TPP1 with an N-terminal LTM fusion (LTM-TPP1) were generated using the piggyBac transposon system (14). A C-terminal construct (TPP1-LTM) was also produced; however, expression of this chimera was poor in comparison with rTPP1 and LTM-TPP1 (~0.4 mg/liter, cf. 10 to 15 mg/liter).

(A) Design of LTM-TPP1 fusion protein and delivery schematic. (B) Enzyme kinetics of rTPP1 and LTM-TPP1 against the synthetic substrate AAF-AMC are indistinguishable. Michaelis-Menten plots were generated by varying [AAF-AMC] at a constant concentration of 10 nM enzyme (means SD; n = 3). Plots and kinetic parameters were calculated with GraphPad Prism 7.04. (C) Maturation of TPP1 is unaffected by the N-terminal fusion of LTM. (D) LTM-TPP1 inhibits wild-type DT activity in a dose-dependent manner (IC50 of 17.2 nM), while rTPP1 has no effect on protein synthesis inhibition by DT (means SD; n = 3). (E) LTM and DTR-TPP1 bind HBEGF with apparent Kds of 13.3 and 19.1 nM, respectively. (F) LTM-TPP1 (39) colocalizes with LAMP1 staining (red).

The activity of rTPP1 and LTM-TPP1 against the tripeptide substrate Ala-Ala-Phe-AMC (AAF-AMC) was assessed to determine any effects of the LTM on TPP1 activity. The enzyme activities of rTPP1 and LTM-TPP1 were determined to be equivalent, as evidenced through measurements of their catalytic efficiency (Fig. 2B), demonstrating that there is no inference by LTM on the peptidase activity of TPP1. Maturation of LTM-TPP1 through autocatalytic cleavage of the N-terminal propeptide was analyzed by SDSpolyacrylamide gel electrophoresis (PAGE) (Fig. 2C). Complete processing of the zymogen at pH 3.5 and 37C occurred between 5 and 10 min, which is consistent with what has been observed for the native recombinant enzyme (15).

The ability of LTM-TPP1 to compete with DT for binding to extracellular HBEGF was first assessed with the protein synthesis competition assay. Similar to LTM, mCherry-LTM, and LTM-mCherry, LTM-TPP1 prevents protein synthesis inhibition by 10 pM DT with an IC50 (median inhibitory concentration) of 17.2 nM (Fig. 2D). As expected, rTPP1 alone was unable to inhibit DT-mediated entry and cytotoxicity. To further characterize this interaction, we measured the interaction between LTM and LTM-TPP1 and recombinant HBEGF using surface plasmon resonance (SPR) binding analysis (Fig. 2E). By SPR, LTM and LTM-TPP1 were calculated to have apparent Kds of 13.3 and 19.1 nM, respectively, values closely corresponding to the IC50 values obtained from the competition experiments (10.1 and 17.2 nM, respectively). Consistent with these results, LTM-TPP1 colocalizes with LAMP1 by immunofluorescence (Fig. 2F).

To study uptake of chimeric fusion proteins in cell culture, we generated a cell line deficient in TPP1 activity. A CRISPR RNA (crRNA) was designed to target the signal peptide region of TPP1 in exon 2 of CLN2. Human HeLa Kyoto cells were reverse transfected with a Cas9 ribonucleoprotein complex and then seeded at low density into a 10-cm dish. Single cells were expanded to colonies, which were picked and screened for TPP1 activity. A single clone deficient in TPP1 activity was isolated and expanded, which was determined to have ~4% TPP1 activity relative to wild-type HeLa Kyoto cells plated at the same density (Fig. 3A). The small residual activity observed is likely the result of another cellular enzyme processing the AAFAMC (7-amido-4-methlycoumarin) substrate used in this assay, as there is no apparent TPP1 protein being produced (Fig. 3B). Sanger sequencing of the individual alleles confirmed complete disruption of the CLN2 gene (fig. S1). In total, three unique mutations were identified within exon 2 of CLN2: a single base insertion resulting in a frameshift mutation and two deletions of 24 and 33 base pairs (bp), respectively.

(A) CLN2 knockout cells exhibit ~4% TPP1 activity relative to wild-type HeLa Kyoto cells (means SD; n = 3). (B) Western blotting against TPP1 reveals no detectable protein in the knockout cells. (C) (Left) In vitro maturation of pro-rTPP1 and LTM-TPP1 (16 ng) was analyzed by Western blot. (Right) TPP1 present in wild-type (WT) and TPP1/ cells, and TPP1/ cells treated with 100 nM rTPP1 and LTM-TPP1. (D) Uptake of rTPP1 and LTM-TPP1 into HeLa Kyoto TPP1/ cells was monitored by TPP1 activity (means SD; n = 4). (E) TPP1 activity present in HeLa Kyoto TPP1/ cells following a single treatment with 50 nM LTM-TPP1 (means SD; n = 3).

Next, we compared the delivery and activation of rTPP1 and LTM-TPP1 into lysosomes by treating TPP1/ cells with a fixed concentration of the enzymes (100 nM) and by analyzing entry and processing by Western blot (Fig. 3C). In both cases, most enzymes were present in the mature form, indicating successful delivery to the lysosome; however, the uptake of LTM-TPP1 greatly exceeded the uptake of rTPP1. As both rTPP1 and LTM-TPP1 receive the same M6P posttranslational modifications promoting their uptake by CIMPR, differences in their respective uptake should be directly attributable to uptake by HBEGF. To quantify the difference in uptake and lysosomal delivery, cells were treated overnight with varying amounts of each enzyme, washed, lysed, and assayed for TPP1 activity. The activity assays were performed without a preactivation step, so signal represents protein that has been activated in the lysosome. For both constructs, we observed a dose-dependent increase in delivery of TPP1 to the lysosome (Fig. 3D). Delivery of LTM-TPP1 was significantly enhanced compared with TPP1 alone at all doses, further demonstrating that uptake by HBEGF is more efficient than that by CIMPR alone. TPP1 activity in cells treated with LTM-TPP1 was consistently ~10 greater than that of cells treated with rTPP1, with the relative difference increasing at the highest concentrations tested. This may speak to differences in abundance, replenishment, and/or recycling of HBEGF versus CIMPR, in addition to differences in receptor-ligand affinity. Uptake of LTM-TPP1 and rTPP1 into several other cell types yielded similar results (fig. S2). To assess the lifetime of the delivered enzyme, cells were treated with LTM-TPP1 (50 nM) and incubated overnight. Cells were washed and incubated with fresh media, and TPP1 activity was assayed over the course of several days. Cells treated with LTM-TPP1 still retained measurable TPP1 activity at 1 week after treatment (Fig. 3E).

While the DT competition experiment demonstrated that HBEGF is involved in the uptake of LTM-TPP1 but not rTPP1 (Fig. 2D), it does not account for the contribution of CIMPR to uptake. Endoglycosidase H (EndoH) cleaves between the core N-acetylglucosamine residues of high-mannose N-linked glycans, leaving behind only the asparagine-linked N-acetylglucosamine moiety. Both rTPP1 and LTM-TPP1 were treated with EndoH to remove any M6P moieties, and delivery into Hela TPP1/ was subsequently assessed. While rTPP1 uptake is completely abrogated by treatment with EndoH, LTM-TPP1 uptake is only partially decreased (Fig. 4), indicating that while HBEGF-mediated endocytosis is the principal means by which LTM-TPP1 is taken up into cells, uptake via CIMPR still occurs. The fact that CIMPR uptake is still possible in the LTM-TPP1 fusion means that the fusion is targeted to two receptors simultaneously, increasing its total uptake and, potentially, its biodistribution.

Uptake of LTM-TPP1 via the combination of HBEGF and CIMPR was shown to be 3 to 20 more efficient than CIMPR alone in cellulo (fig. S2). To interrogate this effect in vivo, TPP1-deficient mice (TPP1tm1pLob or TPP1/) were obtained as a gift from P. Lobel at Rutgers University. Targeted disruption of the CLN2 gene was achieved by insertion of a neo cassette into intron 11 in combination with a point mutation (R446H), rendering these mice TPP1 null by both Western blot and enzyme activity assay (16). Prior studies have demonstrated that direct administration of rTPP1 into the cerebrospinal fluid (CSF) via intracerebroventricular or intrathecal injection results in amelioration of disease phenotype (17) and even extension of life span in the disease mouse (18). To compare the uptake of LTM-TPP1 and rTPP1 in vivo, the enzymes were injected into the left ventricle of 6-week-old TPP1/ mice. Mice were euthanized 24 hours after injection, and brain homogenates of wild-type littermates, untreated, and treated mice were assayed for TPP1 activity (Fig. 5A). Assays were performed without preactivation, and therefore, the results report on enzyme that has been taken up into cells, trafficked to the lysosome, and processed to the mature form.

(A) Assay schematic. (B) TPP1 activity in brain homogenates of 6-week-old mice injected with two doses (5 and 25 g) of either rTPP1 or LTM-TPP1 (5 g, P = 0.01; 25 g, P = 0.002). (C) TPP1 activity in brain homogenates following a single 25-g dose of LTM-TPP1, 1, 7, and 14 days postinjection. Data are presented as box and whisker plots, with whiskers representing minimum and maximum values from n 4 mice per group. Statistical significance was calculated using paired t tests with GraphPad Prism 7.04.

While both enzymes resulted in a dose-dependent increase in TPP1 activity, low (5 g) and high (25 g) doses of rTPP1 resulted in only modest increases of activity, representing ~6 and ~26% of the wild-type levels of activity, respectively (Fig. 5B). At the same doses, LTM-TPP1 restored ~31 and ~103% of the wild-type activity. To assess the lifetime of enzyme in the brain, mice were injected intracerebroventricularly with 25 g of LTM-TPP1 and euthanized either 1 or 2 weeks postinjection. Remarkably, at 1 week postinjection, ~68% of TPP1 activity was retained (compared with 1 day postinjection), and after 2 weeks, activity was reduced to ~31% (Fig. 5C).

ERT is a lifesaving therapy that is a principal method of treatment in non-neurological LSDs. Uptake of M6P-labeled enzymes by CIMPR is relatively ineffective due to variable receptor affinity (5, 6), heterogeneous expression of the receptor, and incomplete labeling of recombinantly produced enzymes (19). Despite its inefficiencies and high cost (~200,000 USD per patient per year) (20), it remains the standard of care for several LSDs, as alternative treatment modalities (substrate reduction therapy, gene therapy, and hematopoietic stem cell transplantation) are not effective, not as well developed, or inherently riskier (2125). Improving the efficiency and distribution of recombinant enzyme uptake may help address some of the current shortcomings in traditional ERT.

Several strategies have been used to increase the extent of M6P labeling on recombinantly produced lysosomal enzymes: engineering mammalian and yeast cell lines to produce more specific/uniform N-glycan modification (19, 26, 27), chemical or enzymatic modification of N-glycans posttranslationally (28), and covalent coupling of M6P (29). M6P-independent uptake of a lysosomal hydrolase by CIMPR has been demonstrated for both -glucuronidase (28) and acid -glucosidase (30, 31). In the latter work, a peptide tag (GILT) targeting insulin-like growth factor II receptor (IGF2R) was fused to recombinant alpha glucosidase, which enabled receptor-mediated entry into cells. CIMPR is a ~300-kDa, 15-domain membrane protein with 3 M6P-binding domains and 1 IGF2R domain. By targeting the IGF2R domain with a high-affinity (low nanomolar) peptide rather than the low-affinity M6P-binding domain, the authors were able to demonstrate a >20-fold increase in the uptake of a GAA-peptide fusion protein in cell culture and a ~5-fold increase in the ability to clear built-up muscle glycogen in GAA-deficient mice.

In this study, we have demonstrated efficient uptake and lysosomal trafficking of a model lysosomal enzyme, TPP1, via a CIMPR-independent route, using the receptor-binding domain of a bacterial toxin. HBEGF is a member of the EGF family of growth factors, and DT is its only known ligand. Notably, it plays roles in cardiac development, wound healing, muscle contraction, and neurogenesis; however, it does not act as a receptor in any of these physiological processes (32). Intracellular intoxication by DT is the only known process in which HBEGF acts as a receptor, making it an excellent candidate receptor for ERT, as there is no natural ligand with which to compete. Upon binding, DT is internalized via clathrin-mediated endocytosis and then trafficked toward lysosomes for degradation (33, 34). Acidification of endosomal vesicles by vacuolar ATPases (adenosine triphosphatases) promotes insertion of DTT into the endosomal membrane and subsequent translocation of the catalytic DTC domain into the cytosol. In the absence of an escape mechanism, the majority of internalized LTM should be trafficked to the lysosome, as we have demonstrated with our chimera (Figs. 2F and 3C). Uptake of LTM-TPP1 in vitro is robustly relative to rTPP1 (Fig. 3D and fig. S2), and TPP1 activity is sustained in the lysosome for a substantial length of time (Fig. 3E). We have also demonstrated that the increase in uptake efficiency that we observed in cell culture persists in vivo. TPP1 activity in the brains of CLN2-null mice was significantly greater in animals treated with intracerebroventricularly injected LTM-TPP1, as compared with those treated with TPP1 at two different doses (Fig. 5B), and, remarkably, this activity persists with an apparent half-life of ~8 days (Fig. 5C).

An important consideration for further development of the LTM platform for clinical development is the potential immunogenicity of using a bacterial fragment in this context. Previously, we demonstrated that the receptor-binding fragment of DT could be replaced with a human scFv (single-chain fragment variable) targeting HBEGF (8). With our demonstration of the potential for targeting HBEGF for LSDs, future efforts will focus on increasing the affinity and specificity of these first-generation humanized LTMs to develop high-affinity chimeras with greatly reduced immunogenicity for further development.

While the ability of LTM-TPP1 to affect disease progression has yet to be determined, recent positive clinical trial results (35) and the subsequent approval of rTPP1 (cerliponase alfa) for treatment of neuronal ceroid lipofuscinosis 2 (NCL2) provide support for this approach. In that clinical trial, 300 mg of rTPP1 was administered by biweekly intracerebroventricular injection to 24 affected children, and this was able to prevent disease progression. While this dose is of the same order of magnitude as other approved ERTs (<1 to 40 mg/kg) (36, 37), it represents a substantial dose, especially considering that it was delivered to a single organ. Improving the efficiency of uptake by targeting an additional receptor as we have done here, is expected to greatly decrease the dose required to improve symptoms, while at the same time decreasing costs and the chances of dose-dependent side effects.

DTK51E/E148K, LTM, LTM-mCherry, mCherry-LTM, and HBEGF constructs were cloned using the In-Fusion HD cloning kit (Clontech) into the Champion pET SUMO expression system (Invitrogen). Recombinant proteins were expressed as 6His-SUMO fusion proteins in Escherichia coli BL21(DE3)pLysS cells. Cultures were grown at 37C until an OD600 (optical density at 600 nm) of 0.5, induced with 1 mM IPTG (isopropyl--d-thiogalactopyranoside) for 4 hours at 25C. Cell pellets harvested by centrifugation were resuspended in lysis buffer [20 mM tris (pH 8.0), 160 mM NaCl, 10 mM imidazole, lysozyme, benzonase, and protease inhibitor cocktail] and lysed by three passages through an EmulsiFlex C3 microfluidizer (Avestin). Following clarification by centrifugation at 18,000g for 20 min and syringe filtration (0.2 m), soluble lysate was loaded over a 5-ml His-trap FF column (GE Healthcare) using an AKTA FPLC. Bound protein was washed and eluted over an imidazole gradient (20 to 150 mM). Fractions were assessed for purity by SDS-PAGE, pooled, concentrated, and frozen on dry ice in 25% glycerol for storage at 80C.

TPP1 cDNA was obtained from the SPARC BioCentre (The Hospital for Sick Children) and cloned into the piggyBac plasmid pB-T-PAF (J.M.R., University of Toronto) using Not I and Asc I restriction sites to generate two expression constructs (pB-T-PAF-ProteinA-TEV-LTM-TPP1 and pB-T-PAF-ProteinA-TEV-TPP1). Stably transformed expression cell lines (HEK293F) were then generated using the piggyBac transposon system, as described (14). Protein expression was induced with doxycycline, and secreted fusion protein was separated from expression media using immunoglobulin G (IgG) Sepharose 6 fast flow resin (GE Healthcare) in a 10-ml Poly-Prep chromatography column (Bio-Rad). Resin was washed with 50 column volumes of wash buffer [10 mM tris (pH 7.5) and 150 mM NaCl] and then incubated overnight at 4C with TEV (Tobacco Etch Virus) protease to release the recombinant enzyme from the Protein A tag. Purified protein was then concentrated and frozen on dry ice in 50% glycerol for storage at 80C.

Cellular intoxication by DT was measured using a nanoluciferase reporter strain of Vero cells (Vero NlucP), as described previously (8). Briefly, Vero NlucP cells were treated with a fixed dose of DT at EC99 (10 pM) and a serial dilution of LTM, LTM-mCherry, mCherry-LTM, DTK51E/E148K, LTM-TPP1, or rTPP1 and incubated overnight (17 hours) at 37C. Cell media was then replaced with a 1:1 mixture of fresh media and Nano-Glo luciferase reagent (Promega), and luminescence was measured using a SpectraMax M5e (Molecular Devices). Results were analyzed with GraphPad Prism 7.04.

SPR analysis was performed on a Biacore X100 system (GE Healthcare) using a CM5 sensor chip. Recombinant HBEGF was immobilized to the chip using standard amine coupling at a concentration of 25 g/ml in 10 mM sodium acetate (pH 6.0) with a final response of 1000 to 2500 resonance units (RU). LTM and LTM-TPP1 were diluted in running buffer [200 mM NaCl, 0.02% Tween 20, and 20 mM tris (pH 7.5)] at concentrations of 6.25 to 100 nM and injected in the multicycle analysis mode with a contact time of 180 s and a dissociation time of 600 s. The chip was regenerated between cycles with 10 mM glycine (pH 1.8). Experiments were performed in duplicate using two different chips. Binding data were analyzed with Biacore X100 Evaluation Software version 2.0.2, with apparent dissociation constants calculated using the 1:1 steady-state affinity model.

HeLa cells were incubated with LTM-mCherry (0.5 M), mCherry-LTM (0.5 M), or LTM-TPP1 (2 M) for 2 hours. Cells were washed with ice-cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100. mCherry constructs were visualized with a rabbit polyclonal antibody against mCherry (Abcam, ab16745) and anti-rabbit Alexa Fluor 568 (Thermo Fisher Scientific). LAMP1 was stained with a mouse primary antibody (DSHB 1D4B) and anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific).

Colocalization was quantified using the Volocity (PerkinElmer) software package to measure Manders coefficients of mCherry signal with LAMP1 signal. The minimal threshold for the 488- and 568-nm channels was adjusted to correct the background signal. The same threshold for both channels was used for all the cells examined.

CLN2/ fibroblast 19494 were incubated with LTM-TPP1 (2 M) for 2 hours. Cells were washed with ice-cold PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100. LTM-TPP1 was visualized with a mouse monoclonal against TPP1 (Abcam, ab54685) and anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific). LAMP1 was stained with rabbit anti-LAMP1 and anti-rabbit Alexa Fluor 568 (Thermo Fisher Scientific).

TPP1 protease activity was measured using the synthetic substrate AAF-AMC using a protocol adapted from Vines and Warburton (38). Briefly, enzyme was preactivated in 25 l of activation buffer [50 mM NaOAc (pH 3.5) and 100 mM NaCl] for 1 hour at 37C. Assay buffer [50 mM NaOAc (pH 5.0) and 100 mM NaCl] and substrate (200 M AAF-AMC) were then added to a final volume of 100 l. Fluorescence (380 nm excitation/460 nm emission) arising from the release of AMC was monitored in real time using a SpectraMax M5e (Molecular Devices). TPP1 activity in cellulo was measured similarly, without the activation step. Cells in a 96-well plate were incubated with 25 l of 0.5% Triton X-100 in PBS, which was then transferred to a black 96-well plate containing 75 l of assay buffer with substrate in each well.

crRNA targeting the signal peptide sequence in exon 2 of CLN2 was designed using the Integrated DNA Technologies (www.idtdna.com) design tool. The gRNA:Cas9 ribonucleoprotein complex was assembled according to the manufacturers protocol (Integrated DNA Technologies) and reverse transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific) into HeLa Kyoto cells (40,000 cells in a 96-well plate). Following 48 hours of incubation, 5000 cells were seeded into a 10-cm dish. Clonal colonies were picked after 14 days and transferred to a 96-well plate. Clones were screened for successful CLN2 knockout by assaying TPP1 activity and confirmed by Sanger sequencing and Western blot against TPP1 antibody (Abcam, ab54385).

The pro-form of TPP1 was matured in vitro to the active form in 50 mM NaOAc (pH 3.5) and 100 mM NaCl for 1 to 30 min at 37C. The autoactivation reaction was halted by the addition of 2 Laemmli SDS sample buffer containing 10% 2-mercaptoethanol and boiled for 5 min. Pro and mature TPP1 were separated by SDS-PAGE and imaged on a ChemiDoc gel imaging system (Bio-Rad).

Proteins or cellular lysate were separated by 4 to 20% gradient SDS-PAGE before being transferred to a nitrocellulose membrane using the iBlot (Invitrogen) dry transfer system. Membranes were then blocked for 1 hour with a 5% milktris-buffered saline (TBS) solution and incubated overnight at room temperature with a 1:100 dilution of mouse monoclonal antibody against TPP1 (Abcam, ab54685) in 5% milk-TBS. Membranes were washed 3 5 min with 0.1% Tween 20 (Sigma-Aldrich) in TBS before a 1-hour incubation with a 1:5000 dilution of sheep anti-mouse IgG horseradish peroxidase secondary antibody (GE Healthcare) in 5% milk-TBS. Chemiluminescent signal was developed with Clarity Western ECL substrate (Bio-Rad) and visualized on a ChemiDoc gel imaging system (Bio-Rad).

rTTP1 and LTM-TPP1 were treated with EndoH (New England Biolabs) to remove N-glycan modifications. Enzymes were incubated at 1 mg/ml with 2500 U of EndoH for 48 hours at room temperature in 20 mM tris (pH 8.0) and 150 mM NaCl in a total reaction volume of 20 l. Cleavage of N-glycans was assessed by SDS-PAGE, and concentrations were normalized to native enzyme-specific activities.

Cryopreserved TPP1+/ embryos were obtained from P. Lobel at Rutgers University and rederived in a C57/BL6 background at The Centre for Phenogenomics in Toronto. Animal maintenance and all procedures were approved by The Centre for Phenogenomics Animal Care Committee and are in compliance with the CCAC (Canadian Council on Animal Care) guidelines and the OMAFRA (Ontario Ministry of Agriculture, Food, and Rural Affairs) Animals for Research Act.

TPP1/ mice (60 days old) were anesthetized with isoflurane (inhaled) and injected subcutaneously with sterile saline (1 ml) and meloxicam (2 mg/kg). Mice were secured to a stereotactic system, a small area of the head was shaved, and a single incision was made to expose the skull. A high-speed burr was used to drill a hole at stereotaxic coordinates: anteroposterior (A/P), 1.0 mm; mediolateral (M/L), 0.3 mm; and dorsoventral (D/V), 3.0 mm relative to the bregma, and a 33-gauge needle attached to a 10-l Hamilton syringe was used to perform the intracerebroventricular injection into the left ventricle. Animals received either 1 or 5 l of enzyme (5 g/l), injected at a constant rate. Isoflurane-anesthetized animals were euthanized by transcardial perfusion with PBS. Brains were harvested and frozen immediately, then thawed and homogenized in lysis buffer [500 mM NaCl, 0.5% Triton X-100, 0.1% SDS, and 50 mM Tris (pH 8.0)] using 5-mm stainless steel beads in TissueLyser II (Qiagen). In vitro TPP1 assay was performed, as described, minus the activation step.

Acknowledgments: We thank P. Lobel at Rutgers University for providing the TPP1-deficient mice. Funding: We are grateful to the Canadian Institutes of Health Research for funding. Author contributions: S.N.S.-M. devised and performed experiments and drafted the initial manuscript. G.L.B. provided materials and assisted in conceptualization and experimental design. X.Z., D.Z., and R.H. contributed to the experimental design and performed experiments. P.K.K. and B.A.M. contributed to the experimental design. J.M.R. contributed to the experimental design and revised the manuscript. R.A.M. assisted in conceptualization, contributed to the experimental design, and assisted in writing the manuscript. Competing interests: B.A.M. is a chief medical advisor at Taysha Gene Therapies. The authors declare that they have no other 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. Additional data related to this paper may be requested from the authors.

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