YAVNE, Israel, Dec. 14, 2020 (GLOBE NEWSWIRE) -- MediWound Ltd. (Nasdaq: MDWD), a fully-integrated biopharmaceutical company bringing innovative therapies to address unmet needs in severe burn and wound management, today announced it has further expanded its NexoBrid® global presence and entered the Middle Eastern markets with the signing of a distribution agreement granting Ghassan Aboud Group (GAG), an international conglomerate based in the United Arab Emirates (UAE), an exclusive right to market and distribute NexoBrid in UAE for the treatment of severe burns. Commercialization of NexoBrid in the UAE will commence upon securing regulatory approval, which is expected within a year.

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MediWound Continues Global Expansion with Distribution Agreement for NexoBrid in United Arab Emirates with Ghassan Aboud Group

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LYON, France and CAMBRIDGE, Mass., Dec. 14, 2020 (GLOBE NEWSWIRE) -- ERYTECH Pharma (Nasdaq & Euronext: ERYP), a clinical-stage biopharmaceutical company developing innovative therapies by encapsulating therapeutic drug substances inside red blood cells, today announced the completion of enrollment in the TRYbeCA-1 Phase 3 trial in second-line pancreatic cancer.

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ERYTECH Completes Enrollment in TRYbeCA-1 Phase 3 Trial in Second-Line Pancreatic Cancer

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NEW YORK, Dec. 14, 2020 (GLOBE NEWSWIRE) -- Mesoblast Limited (Nasdaq:MESO; ASX:MSB), global leader in allogeneic cellular medicines for inflammatory diseases, will host a webcast today to provide a corporate update.

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Mesoblast Corporate Update

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NEW YORK, Dec. 14, 2020 (GLOBE NEWSWIRE) -- Mesoblast Limited (Nasdaq:MESO; ASX:MSB), global leader in allogeneic cellular medicines for inflammatory diseases, today announced top-line results from the landmark DREAM-HF Phase 3 randomized controlled trial of its allogeneic cell therapy rexlemestrocel-L (REVASCOR®) in 537 patients with advanced chronic heart failure1.

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Mesoblast Provides Topline Results From Phase 3 Trial of Rexlemestrocel-L for Advanced Chronic Heart Failure

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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

Alypn Beauty's Wildhuckleberry 8 Acid Polishing Peel is a multitasking skin treat that works as a ... [+] mask, skin peel and polish.

After a refreshing facial wash, double cleanse with an ultra gentle, multitasking mask, peel and scrub and polish in a jar. ALPYN BEAUTYs Wild Huckleberry 8-Acid Polishing Peel has sold out several times since it launched in Octoberand for good reason. This skin treat harnesses the powers of a natural salicylic, citric, malic, tartaric, glycolic, azelac, ferulic and lactic acids to exfoliate skin and dissolve dead skin cells. Wild crafted huckleberry protects skin barrier, while bamboo power combined with blueberry seed paste effect natural microdermabrasion of skin. This light and delicious skin peel is so gentle you can use is daily, day and night. Ingredients used for this heaven-sent are all sustainably sourced and wild-crafted so you know that it does your skin, soul and planet good. This peel is also ideal for prepping skin before makeup application.

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

Wipe away deep seated dirt and impurities with M-61s PowerGlow Toner. Formulated with Glycolic Acid and Salicylic Acid, this gentle skin toner exfoliates impurities, refines pores and smoothens skin texture to prevent blemishes and existing acne. Unlike other harsh anti-acne toners that leave skin dry, extra sensitive and flaky, the PowerGlow brings out soft, hydrated and glowing skin. It is also ideal for sensitive skin types that need an extra anti-acne boost.

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.

Cutting-edge Graphene Technology is used in the sheet mask, which emits infrared rays into the skin, offering enhanced absorption of each ingredient. There are five sheets in every box of Press Refresh. Applied during evenings on alternating days, Press Fresh does a remarkable job in reducing redness and inflammation. By the time the last sheet was used, skin texture had dramatically improved. Pores also appeared smaller. Post acne, Press Refresh is highly recommended as a routine mask for preventing future breakouts. ZITSTICKA is a brand dedicated to creating innovative skin solution that help to fight acne breakouts at all stages.

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

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

bu/bg

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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

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

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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Exploiting the diphtheria toxin internalization receptor enhances delivery of proteins to lysosomes for enzyme replacement therapy - Science Advances

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NEW YORK, Dec. 11, 2020 (GLOBE NEWSWIRE) -- Prevail Therapeutics Inc. (Nasdaq: PRVL), a biotechnology company developing potentially disease-modifying AAV-based gene therapies for patients with neurodegenerative diseases, today announced that the first patient has been dosed in the Phase 1/2 PROCLAIM clinical trial evaluating PR006, an investigational AAV9 gene therapy delivering the GRN gene, for the treatment of frontotemporal dementia patients with GRN mutations (FTD-GRN).

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Prevail Therapeutics Announces First Patient Dosed in Phase 1/2 PROCLAIM Clinical Trial Evaluating PR006 for the Treatment of Frontotemporal Dementia...

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Signals momentum in addressing social equity, tax and banking reform, and the widespread adoption of cannabis in the U.S. Signals momentum in addressing social equity, tax and banking reform, and the widespread adoption of cannabis in the U.S.

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Golden Leaf Holdings Applauds the Historic Passing of the MORE Act

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SUNNY ISLES, Fla., Dec. 11, 2020 (GLOBE NEWSWIRE) -- Bespoke Extracts, Inc. (OTC Pink: BSPK), producer of high quality, hemp-derived CBD products, today formally congratulated professional Mixed Martial Artist Cody Law on his big TKO win in the third round against opponent Kenny Champion at Bellator 254 held last night in Uncasville, Connecticut. As previously announced, Bespoke Extracts was Law’s corporate sponsor at the event.

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Bespoke Extracts Congratulates Pro MMA Fighter Cody Law on TKO Win at Bellator 254

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CAMBRIDGE, Mass., Dec. 11, 2020 (GLOBE NEWSWIRE) -- TCR² Therapeutics Inc. (Nasdaq: TCRR), a clinical-stage immunotherapy company developing a pipeline of novel T cell therapies for patients suffering from cancer, today announced that the Company plans to discuss interim data from the Phase 1 portion of the TC-210 Phase 1/2 clinical trial for patients with mesothelin-expressing solid tumors in a premarket press release and webcast to be held on Monday, December 14th, 2020.

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TCR² Therapeutics to Announce Interim Data from Phase 1/2 Clinical Trial of TC-210 in Mesothelin-Expressing Solid Tumors

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– Event to be webcast live on Monday, December 14, 2020 at 8:00 a.m. ET –

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Arvinas to Host Webcast Presentation of New Clinical Data from ARV-471 and ARV-110 PROTAC® Protein Degrader Development Programs

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BOSTON, Dec. 11, 2020 (GLOBE NEWSWIRE) -- Albireo Pharma, Inc. (Nasdaq: ALBO), a clinical-stage orphan pediatric liver disease company developing novel bile acid modulators, today announced the grant of inducement stock options exercisable for an aggregate of 18,500 shares of Albireo’s common stock. The stock options are exercisable at a price of $39.77 per share, the closing price of Albireo’s common stock on December 9, 2020, the grant date, and were granted as inducements material to the employee’s acceptance of employment with Albireo in accordance with Nasdaq Listing Rule 5635(c)(4). Each stock option has a 10-year term and vests over a four-year period, subject to the employee’s continued service with Albireo through the applicable vesting dates. The vesting schedule for each stock option is 25 percent on the one-year anniversary of the employee’s start date with Albireo and 75 percent in 12 equal quarterly installments thereafter. The stock options are subject to the terms and conditions of Albireo’s 2020 Inducement Equity Incentive Plan.

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Albireo Reports Inducement Grant Under Nasdaq Listing Rule 5635(c)(4)

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CAMPBELL, Calif., Dec. 11, 2020 (GLOBE NEWSWIRE) -- VIVUS, Inc. (the “Company”), a biopharmaceutical company, today announced that it has received approval from the United States Bankruptcy Court for the District of Delaware (the “Bankruptcy Court”) on its Second Amended Joint Prepackaged Chapter 11 Plan of Reorganization of VIVUS, Inc. and Its Affiliated Debtors [Docket No. 339] (the “Plan”).

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VIVUS Receives Court Approval of Joint Chapter 11 Plan of Reorganization

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Primary endpoint achieved with twice-daily dose group at 28 day time point versus latanoprost control group.

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ANN ARBOR, Mich., Dec. 11, 2020 (GLOBE NEWSWIRE) -- Kraig Biocraft Laboratories, Inc. (OTCQB: KBLB) (“Company” or “Kraig Labs”), announced today that it has secured $950,000 in bridge financing and simultaneously filed with the SEC to become a mandatory, fully reporting company. Today the Company filed a Form 8-A to become fully reporting issuer. This financing will also make a significant contribution to that effort.

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Kraig Biocraft Laboratories Secures Bridge Funding and Files to Become Fully Reporting Company

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NEW YORK and MAINZ, GERMANY, December 11, 2020 — Pfizer Inc. (NYSE: PFE) and BioNTech SE (Nasdaq: BNTX) announced today that the U.S. Food and Drug Administration (FDA) has authorized the emergency use of the mRNA vaccine, BNT162b2, against COVID-19 in individuals 16 years of age or older. The vaccine is now authorized under an Emergency Use Authorization (EUA) while Pfizer and BioNTech gather additional data and prepare to file a planned Biologics License Application (BLA) with the FDA for a possible full regulatory approval in 2021.

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Demonstrates modularity of Intellia’s in vivo liver insertion technology to durably restore protein, compared to traditional gene therapy

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Intellia Therapeutics Achieves Normal Human Alpha-1 Antitrypsin Protein Levels in Non-Human Primates Through Targeted Gene Insertion for the Treatment...

Global News Feed Comments Off on Intellia Therapeutics Achieves Normal Human Alpha-1 Antitrypsin Protein Levels in Non-Human Primates Through Targeted Gene Insertion for the Treatment… | December 13th, 2020

VASCEPA COVID-19 CardioLink-9 Randomized Trial suggests improvement in patient-reported COVID-19 symptoms while achieving its primary endpoint by demonstrating a 25% reduction in high-sensitivity C-reactive protein (hsCRP) with encouraging short-term safety and tolerability data using VASCEPA loading dose

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Amarin Reports Encouraging Efficacy and Safety Results from Pilot Study Treating COVID-19 Infected Outpatients with VASCEPA® (Icosapent Ethyl) in...

Global News Feed Comments Off on Amarin Reports Encouraging Efficacy and Safety Results from Pilot Study Treating COVID-19 Infected Outpatients with VASCEPA® (Icosapent Ethyl) in… | December 13th, 2020