Category Archives: Gene therapy

California-based Kriya Therapeutics announced on Tuesday that it concluded an $80.5 million Series A financing round, led by QVT, Dexcel Pharma, Foresite Capital, Bluebird Ventures, Narya Capital, Amplo, Paul Manning, and Asia Alpha. The company, which was founded in 2019, has a pipeline that includes multiple AAV-based gene therapies for the treatment of type 1 and type 2 diabetes, as well as obesity.

"There have been numerous successful gene therapies focused on rare monogenic diseases in recent years," said Shankar Ramaswamy, M.D., Co-Founder, Chairman, and CEO of Kriya Therapeutics. "We see tremendous potential to expand the field and apply gene therapy to highly prevalent serious diseases. We are focused on designing gene therapies using algorithmic tools, scalable infrastructure, and proprietary technology to optimize the efficacy and durability of our treatments. We look forward to accelerating the development of our pipeline, platform technologies, and internal GMP manufacturing capability with the funds raised in this Series A financing."

Kriya focuses on developing gene therapies for conditions that impact millions of patients. Its goal is to design one-time gene therapies to express therapeutic proteins within specific human tissues. Kriyas pipeline includes KT-A112, KT-A522, and KT-A832, all of which are investigational gene therapies.

Kriyas leadership team is composed of experts who have experience designing, developing and manufacturing successful gene therapies as well.

"Kriya is building a leading team and cutting-edge infrastructure to engineer best-in-class gene therapies for severe chronic conditions and accelerate their advancement into human clinical trials," said Roger Jeffs, Ph.D., Co-Founder and Vice Chairman of Kriya. "The company is committed to incorporating the latest advancements in the field into the design and development of its therapeutic constructs. Through its R&D laboratory capabilities in the Bay Area and in-house process development and manufacturing infrastructure in Research Triangle Park, I believe that Kriya will be uniquely positioned to become a leader in the gene therapy field."

Another company with a focus on gene therapy that recently came out of stealth mode is Dyno Therapeutics. On Monday, the Massachusetts-based organization announced that it is now eligible for more than $2 billion in upfront payments, research support and various milestones and options fees through its research-and-development and collaboration deals.

Dyno, which launched in 2018 with approximately $9 million in financing, has a technology platform built on the intellectual property that came from the laboratory of George Church. Church, who is the Robert Winthrop Professor of Genetics at Harvard Medical School, is a cofounder of Dyno.

At Dyno, we see a vast opportunity to expand the treatment landscape for gene therapies, said Eric D. Kelsic, co-founder and chief executive officer of Dyno. The success of gene therapy relies on the ability of vectors to safely and precisely deliver a gene to the intended target cells and tissues. Our approach addresses the major limitations of naturally occurring AAV vectors and creates optimized, disease-specific vectors for gene therapies with great curative potential. Our portfolio of R&D programs and newly-announced collaborations with leading gene therapy developers reflect the applicability of our AI-powered approach to improve treatments for patients and expand the number of treatable diseases with gene therapies.

Dyno has announced partnerships with Novartis and Sarepta Therapeutics thus far. With Sarepta, Dyno will design and discover novel AAV capsids to improve gene therapy for muscle diseases. Along with Novartis, Dyno will focus on developing improved AAV vectors for gene therapies for ocular diseases.

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Kriya Therapeutics to Focus on Gene Therapy with $80.5M in Funding - BioSpace

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Like other CDMOs, Thermo sees a massive opportunity in the field of gene therapy.

Thermo paid $1.7 billion last year to buy viral vector contract manufacturer Brammer Bio. The company later opened a $90 million manufacturing facility in Lexington, Massachusetts, and it's expanded capacity at sites in Cambridge, Massachusetts, and Alachua, Florida, as well.

Thermo's new space in Massachusetts will rival a 300,000-square-foot plant operated by Swiss drug manufacturer Lonza in Pearland, Texas, outside of Houston. Lonza called the plant the world's largest dedicated to cell and gene therapy production when it opened in 2018.

Lonza has also been steadily making deals in an effort to support biotech and pharma customers through every step of the process in gene therapy. As of April 2019, the company had worked with more than 45 viral vector customers and expected that number to keep climbing.

Other companies are expanding, too. Fujifilm in November announced plans to spend about $120 million in the gene therapy field, including a new innovation center in Texas. Catalent last year paid $1.2 billion for Paragon Bioservices to bolster its manufacturing capacity for gene therapies.

Thermo said its new plant will take advantage of the latest technology, with digital connectivity and advanced operator training. The company said it chose to construct the site in Plainville to take advantage of nearby Thermo facilities and draw on the Boston-area talent pool

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Thermo Fisher to invest $180M in new gene therapy plant - BioPharma Dive

Japan's national health insurance will cover a gene therapy drug for a rare childhood genetic disorder that costs 167 million yen ($1.56 million) per treatment, making it the most expensive medication funded by the public system, government officials said Wednesday.

An advisory panel to the health minister approved provision of Swiss pharmaceutical giant Novartis AG's drug Zolgensma for spinal amyotrophy patients under the age of 2 starting as early as May 20, the officials said.

The drug, which costs over 200 million yen in the United States, is known as the world's most expensive medication.

Supplied photo shows samples of Swiss pharmaceutical giant Novartis AG's gene therapy drug "Zolgensma" for spinal amyotrophy patients. (Photo courtesy of Novartis)

It is a one-time therapy for the genetic disorder, which causes motor neuron loss and muscle wasting, and its coverage by insurance will offer hope for patients of the disease and their families.

With Japan's social security expenses ballooning amid the rapid aging of its population, some experts have expressed concerns over the burden on the insurance system of inclusion of the treatment. They also note that new drugs have tended to be highly expensive in recent years.

But a senior health ministry official said that given the small number of patients of the rare illness, "the fiscal impact is going to be limited."

The previous most expensive drug in Japan was Kymriah, approved last May to treat leukemia and other hematologic cancers, at 33.49 million yen.

Novartis Pharma K.K., a Tokyo-based unit of the Swiss pharmaceutical company, produces and sells both Kymriah and Zolgensma.

Spinal amyotrophy affects one or two infants out of every 100,000 and can lead to severe respiratory problems and early death. Without use of an artificial respirator, it is said that most die within 18 months.

Zolgensma will be given as a one-time infusion into the vein, which can introduce normal genes into human cells to recover motor function.

Novartis expects that the drug will be administered to about 25 patients per year in Japan, estimating annual sales of 4.2 billion yen.

Under the Japanese insurance system, out-of-pocket medical expenses for those under 2 are set at 20 percent of the total. But the real payment is minimal as the central and municipal governments cover almost all the expense under subsidy programs.

Related coverage:

Japan health insurance to cover new leukemia therapy worth 33 mil. yen

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Japan's health insurance to cover $1.5 million gene therapy drug - Kyodo News Plus

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Cutting-edge research on rewriting the genes responsible for Tay-Sachs disease, sickle-cell anemia, and other disorders will be presented at this weeks online annual meeting of the American Society for Gene and Cell Therapy. Originally planned as a Boston gathering, the scientific groups get-together became a virtual one because of the Covid-19 pandemic.

From Tuesday through Friday, academic researchers will be presenting their latest data online, along with updates from firms like Bluebird Bio (ticker: BLUE), Voyager Therapeutics (VYGR), Fate Therapeutics (FATE), Beam Therapeutics (BEAM), Axovant Gene Therapies (AXGT), and many others. Patients and their families have found their way to clinical trials through the societys website.

Bluebird plans presentations on its cell therapy against the blood cancer known as multiple myeloma. Using a technology known as CAR-T, the company creates supercharged versions of a patients immune cells that have halted disease progression in some of the 18 patients enrolled in a continuing Phase 1 trial.

Featured on Friday will be reports on the first babies treated with gene therapy for the debilitating neurodegenerative disorder Tay-Sachs. The treatment is being developed by the London-based Axovant under license from the University of Massachusetts Medical School.

Voyager will discuss its preclinical mouse studies on treating neurological disorders like amyotrophic lateral sclerosis and Huntingtons disease by using techniques that block the rogue signals generated by defective genes.

Fate Therapuetics is scheduled to show a new off-the-shelf CAR-T technology that it hopes will allow the immune system to target a broad range of solid tumors as well as multiple myeloma. The approach is licensed from Harvard Universitys Dana-Farber Cancer Institute.

Beam, meanwhile, will detail success it has shown in preclinical editing of the genetic defect that causes sickle-cell anemia. The company is developing a sharper-edged way of rewriting faulty genes than the widely used Crispr technology that Beam licensed from researchers at the Broad institute of Harvard and MIT. Beam founder and Crispr pioneer Feng Zhang will give a featured lecture as part of the online meeting on Thursday.

Write to Bill Alpert at william.alpert@barrons.com

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Gene-Therapy Treatments for Tay-Sachs, Sickle Cell to Be Featured in Online Gathering - Barron's

Sarepta Therapeutics Inc. andDyno Therapeutics Inc., have announced an agreement to develop next-generation Adeno-Associated Virus (AAV) vectors for muscle diseases, using Dynos CapsidMap platform. Dynos proprietary CapsidMap platform opens up new ways to identify novel capsids the cell-targeting protein shell of viral vectors that could offer improved muscle targeting and immune-evading properties, in addition to advantages in packaging and manufacturing.Our agreement with Dyno provides us with another valuable tool to develop next-generation capsids for gene therapies to treat rare diseases, saidDoug Ingram, Sareptas president and CEO. By leveraging Dynos AI platform and Sareptas deep expertise in gene therapy development, our goal is to advance next-generation treatments with improved muscle-targeting capabilities.Under the terms of the agreement, Dyno will be responsible for the design and discovery of novel AAV capsids with improved functional properties for gene therapy and Sarepta will be responsible for conducting preclinical, clinical and commercialization activities for gene therapy product candidates using the novel capsids. If successful, Dyno could receive over$40 millionin upfront, option and license payments during the research phase of the collaboration. Additionally, if Sarepta develops and commercializes multiple candidates for multiple muscle diseases, Dyno will be eligible for additional significant future milestone payments. Dyno will also receive royalties on worldwide net sales of any commercial products developed through the collaboration.This agreement is a major step forward in our plan to realize the potential of Dynos AI platform for gene therapies to improve patient health. We are excited to work with Sarepta to create gene therapies with improved properties to address a range of muscle-related diseases, stated Dynos CEO and co-founderEric D. Kelsic, Ph.D. The success of the gene therapies developed through this collaboration with Sarepta will rely on AI-powered vectors that allow gene therapies to be safely and precisely targeted to the muscle tissue.

By designing capsids that confer improved functional properties to Adeno-Associated Virus (AAV)vectors, Dynos proprietary CapsidMap platform overcomes the limitations of todays gene therapies on the market and in development. CapsidMap uses artificial intelligence (AI) technology for the design of novel capsids, the cell-targeting protein shell of viral vectors. The CapsidMap platform applies DNA library synthesis and next-generation DNA sequencing to measureinvivogene delivery properties in high throughput. At the core of CapsidMap are advanced search algorithms leveraging machine learning and Dynos massive quantities of experimental data, that together build a comprehensive map of sequence space and thereby accelerate the discovery and optimization of synthetic AAV capsids.

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Sarepta And Dyno Therapeutics Will Develop Next-Gen Gene Therapy Vectors - Contract Pharma

PALO ALTO, Calif.andDURHAM Flush with cash, Kriya Therapeutics has big plans.

The biotech startup, with headquarters in Durham and Palo Alto, California, has secured $80.5 million in Series A financing to fund the development of its gene therapies for highly serious diseases.

Among them: type 1 and type 2 diabetes, severe obesity and other indications affecting millions of patients.

Series A investors include QVT, Dexcel Pharma, Foresite Capital, Bluebird Ventures (associated with Sutter Hill Ventures), Narya Capital, Amplo,Paul Manning, andAsia Alpha. This Series A round follows an initial seed financing completed by the company in the fourth quarter of 2019 led by Transhuman Capital, who also participated in the Series A round.

Kriya said financing proceeds would go towards supporting the development of the companys pipeline, internal discovery engine, and proprietary GMP manufacturing infrastructure.

There have been numerous successful gene therapies focused on rare monogenic diseases in recent years, said Shankar Ramaswamy, M.D., Co-Founder, Chairman, and CEO of Kriya Therapeutics, in a statement.

We see tremendous potential to expand the field and apply gene therapy to highly prevalent serious diseases. We are focused on designing gene therapies using algorithmic tools, scalable infrastructure, and proprietary technology to optimize the efficacy and durability of our treatments. We look forward to accelerating the development of our pipeline, platform technologies, and internal GMP manufacturing capability with the funds raised in this Series A financing.

Founded in 2019, the companys team includesformer senior leadership from Spark Therapeutics, AveXis, Sangamo Therapeutics, and other gene therapy companies.

Kriyas initial pipeline includes:

Kriya is building a leading team and cutting-edge infrastructure to engineer best-in-class gene therapies for severe chronic conditions and accelerate their advancement into human clinical trials, saidRoger Jeffs, Ph.D., Co-Founder and Vice Chairman of Kriya, in a statement.

The company is committed to incorporating the latest advancements in the field into the design and development of its therapeutic constructs. Through its R&D laboratory capabilities in the Bay Area and in-house process development and manufacturing infrastructure inResearch Triangle Park, I believe that Kriya will be uniquely positioned to become a leader in the gene therapy field.

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Durham's Kriya Therapuetics lands $80M to advance gene therapies for diabetes, severe obesity - WRAL Tech Wire

People who are overweight and suffering from joint pain caused by osteoarthritis are often reluctant to exercise, even though physical activity can boost muscle strength and relieve pain. A new study suggests gene therapy may someday be a good option for those peopleand it may help them shed pounds, too.

Researchers at the Washington University School of Medicine gave young mice a single injection of the gene that makes follistatin, a protein that normally blocks another protein called myostatin, which modulates muscle growth. The therapy caused a significant buildup of muscle mass in the mice while also preventing obesity, the team reported in the journal Science Advances.

We've identified here a way to use gene therapy to build muscle quickly, said senior investigator Farshid Guilak, Ph.D., professor of orthopaedic surgery and director of research at Shriners Hospitals for Children St. Louis, in a statement. It had a profound effect in the mice and kept their weight in check, suggesting a similar approach may be effective against arthritis, particularly in cases of morbid obesity."

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In fact, the mice didnt just build muscle, they also nearly doubled their strength without exercising any more than they usually did. Despite being fed a high-fat diet, they had fewer metabolic issues and stronger hearts than did animals that did not receive the follistatin gene. Their joints were healthier, with less cartilage damage and inflammatory markers than their untreated counterparts, the researchers reported.

Whats more, the Washington University team discovered that the gene therapy promoted the beiging of white fat, meaning it turned some unhealthy white adipose tissue into brown fat, which positively correlates with increased triglyceride clearance, normalized glucose level, and reduced inflammation, the researchers wrote in the study. Therefore, delivering the follistatin gene could serve as a very promising approach to induce beiging of [white adipose tissue] in obesity, they wrote.

RELATED: Could gene therapy be the solution to obesity and diabetes?

This is not the first time gene therapy has been proposed as a potential treatment for obesity and other metabolic diseases. Australian researchers demonstrated that removing the gene RCAN1 from mice, for example, helped turn white fat into brown fat. And a team in South Korea used the gene editing system CRISPR to remove the FABP4 gene from mice that had been fed a high-fat diet, resulting in a 20% loss of body weight and a reduction in insulin resistance.

The Washington University teams approach is distinctive in that it focuses on building muscle. But the researchers noted theyll have to do further studies to determine whether the gene therapy has any negative effect on heart muscle. Even though heart health improved in the mice, any thickening of the hearts walls could be dangerous over time.

Still, Guilak and his colleagues believe that follistatin gene therapy could be a promising approach to treating several conditions, including muscular dystrophy and other diseases that cause muscle wasting, they said in the study.

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Gene therapy cuts fat and builds muscle in sedentary mice on unhealthy diets - FierceBiotech

- Molecular Methods Quantified Precision and Efficiency of Nuclease-Free Gene Editingfor PKU -

- Manufacturing Enhancements Led to Improved Productivity, Quality and Scalability of Commercial Process, Confirmed in 2,000L Bioreactor -

- Data Highlight Unique Characteristics of AAVHSC Genetic Medicines Platform -

BEDFORD, Mass., May 12, 2020 (GLOBE NEWSWIRE) -- Homology Medicines, Inc. (Nasdaq: FIXX), a genetic medicines company, announced today the presentation of data at the American Society of Gene & Cell Therapy (ASGCT) 23rd Annual Meeting. Among Homologys seven presentations are data from its in vivo nuclease-free gene editing program for phenylketonuria (PKU) and in vivo gene therapy program for metachromatic leukodystrophy (MLD), both of which are in IND-enabling studies. Presentations also focus on the Companys commercial manufacturing platform, as well as data on the differentiating characteristics of Homologys family of AAVHSC vectors, particularly when compared to other AAVs, which highlight the potential of the Companys dual gene therapy and editing platform.

Homology has made substantial progress in understanding the unique properties of our AAVHSC-based technology and this enables us to move our dual genetic medicines platform forward to develop potential treatments, or cures, for patients, stated Albert Seymour, Ph.D., Chief Scientific Officer of Homology Medicines. We are pleased to share data here that describe the molecular methods we have developed to characterize in vivo, nuclease-free gene editing efficiency and precision at the DNA level. Additional data from our in vivo MLD gene therapy program demonstrates the impact on key biomarkers in two species, as well as the durability of effect in the murine model of disease with data out to 52 weeks. Underpinning all our programs is our internal GMP process and manufacturing capabilities, where we have now confirmed our commercial HEK293 suspension platform at the 2,000-liter scale, bringing our total internal capacity to 3,500 liters. Additionally, we are presenting data showing improved AAVHSC packaging as compared to the non-Clade F vector AAV5.

Highlights from Homologys 2020 ASGCT Presentations

The presentation, Molecular Characterization of Precise In Vivo Targeted Gene Editing in Human Cells using AAVHSC15, a New AAV Derived from Hematopoietic Stem Cells (AAVHSC), describes quantitative molecular methods to measure efficiency and precision of nuclease-free, homologous recombination-based gene editing. The studies, which used a single I.V. administration of a gene editing construct to insert the human PAH gene, which is mutated in people with phenylketonuria (PKU), in a humanized liver murine model, show:

Two posters related to Homologys internal commercial manufacturing platform will be presented.In Molecular Design and Characterization of Packaging Plasmid Sequences for Improved Production of Novel Clade F AAVHSCs, the data demonstrate:

In Development and Scalability of Transfection-Based Production and Purification of Novel Clade F Adeno-Associated Viruses Isolated from Human Hematopoietic Stem Cells (AAVHSCs), Homology describes high-quality productivity and scalability of its mammalian, suspension-based manufacturing, including:

Related to Homologys HMI-202 investigational gene therapy for MLD, the presentation, Gene Therapy for Metachromatic Leukodystrophy (MLD) That Crosses the Blood-Nerve and Blood-Brain Barriers in Mice and Non-Human Primates, details that a single I.V. administration:

In collaboration with Childrens Hospital of Philadelphia (CHOP), Homology also presents, In Vivo Transduction of Murine Hematopoietic Stem Cells after Intravenous Injection of AAVHSC15 and AAVHSC17, which shows:

As Homology has advanced its AAVHSC technology, it is presenting mechanistic data on the platform, including the following two presentations.In Role of Terminal Galactose in Cellular Uptake, Intracellular Trafficking, and Tissue Tropism Using Adeno-Associated Viruses Isolated from Human Stem Cells (AAVHSCs), the data show:

In AAVHSCs Transduction Does Not Significantly Elicit p53-Mediated Apoptosis or Alter Cell Cycle in Human iPSCs and Primary Cells When Compared to Non-Clade F AAV Vectors, the studies demonstrate that AAVHSCs:

For more information about the presentations, visit Homologys website at http://www.homologymedicines.com/publications.

About Homology Medicines, Inc. Homology Medicines, Inc. is a genetic medicines company dedicated to transforming the lives of patients suffering from rare genetic diseases with significant unmet medical needs by curing the underlying cause of the disease. Homologys proprietary platform is designed to utilize its human hematopoietic stem cell-derived adeno-associated virus vectors (AAVHSCs) to precisely and efficiently deliver genetic medicinesin vivoeither through a gene therapy or nuclease-free gene editing modality across a broad range of genetic disorders. Homology has a management team with a successful track record of discovering, developing and commercializing therapeutics with a particular focus on rare diseases, and intellectual property covering its suite of 15 AAVHSCs. Homology believes that its compelling preclinical data, scientific expertise, product development strategy, manufacturing capabilities and intellectual property position it as a leader in the development of genetic medicines. For more information, please visitwww.homologymedicines.com.

Forward-Looking Statements This press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. All statements contained in this press release that do not relate to matters of historical fact should be considered forward-looking statements, including without limitation statements regarding our expectations surrounding the potential, safety, efficacy, and regulatory and clinical progress of our product candidates; our beliefs regarding our manufacturing capabilities; our position as a leader in the development of genetic medicines; and our participation in upcoming presentations and conferences. These statements are neither promises nor guarantees, but involve known and unknown risks, uncertainties and other important factors that may cause our actual results, performance or achievements to be materially different from any future results, performance or achievements expressed or implied by the forward-looking statements, including, but not limited to, the following: the impact of the COVID-19 pandemic on our business and operations, including our preclinical studies and clinical trials, and on general economic conditions; we have and expect to continue to incur significant losses; our need for additional funding, which may not be available; failure to identify additional product candidates and develop or commercialize marketable products; the early stage of our development efforts; potential unforeseen events during clinical trials could cause delays or other adverse consequences; risks relating to the capabilities of our manufacturing facility; risks relating to the regulatory approval process; our product candidates may cause serious adverse side effects; inability to maintain our collaborations, or the failure of these collaborations; our reliance on third parties; failure to obtain U.S. or international marketing approval; ongoing regulatory obligations; effects of significant competition; unfavorable pricing regulations, third-party reimbursement practices or healthcare reform initiatives; product liability lawsuits; failure to attract, retain and motivate qualified personnel; the possibility of system failures or security breaches; risks relating to intellectual property and significant costs as a result of operating as a public company. These and other important factors discussed under the caption Risk Factors in our Quarterly Report on Form 10-Q for the quarterly period ended March 31, 2020 and our other filings with the SEC could cause actual results to differ materially from those indicated by the forward-looking statements made in this press release. Any such forward-looking statements represent managements estimates as of the date of this press release. While we may elect to update such forward-looking statements at some point in the future, we disclaim any obligation to do so, even if subsequent events cause our views to change.

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Homology Medicines Announces Presentations on its In Vivo Gene Therapy and Gene Editing Programs and Commercial Manufacturing Platform at the American...

Genespire and SR-Tiget announce strategic alliance for the development of transformative gene therapies for genetic diseases and disclose collaboration focus

Pre-clinical data from SR-Tiget, included in the alliance with Genespire, to be presented at ASGCT 23rd Annual Meeting

Italy, Milan, 13 May 2020: The San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), one of the worlds leading gene therapy research institutes jointly managed by Fondazione Telethon and Ospedale San Raffaele and Genespire, a gene therapy company developing transformative therapies for genetic diseases, and spin-out of SR-Tiget, announced today their alliance on the research and development of candidate therapeutic products for people affected by primary immunodeficiencies and metabolic diseases based on novel gene editing and lentiviral vector technologies developed by SR-Tiget.

Genespire was co-founded in March 2020 by SR-Tiget director and gene therapy pioneer Prof. Luigi Naldini and Dr. Alessio Cantore, Fondazione Telethon and Ospedale San Raffaele. Genespire recently raised 16 million in a Series A financing round from Sofinnova Partners.

Under the terms of the alliance, Genespire and SR-Tiget will study and further develop novel gene therapies, which have the unique potential to address severe unmet medical need and exploit gene editing and lentiviral vector technologies developed by SR-Tiget.

Genespire was granted an exclusive global license for the research, development and commercialization of gene therapies for metabolic diseases based on SR-Tigets alloantigen free, microRNA-regulated lentiviral vectors, which allow for stable liver gene therapy even for diseases with early onset, requiring administration at a young age.

Genespire was also granted exclusive licenses and options to the results of a joint research and development program with SR-Tiget in the T-cell and Hematopoietic Stem Cells field to address genetic diseases, in particular primary immunodeficiencies, exploiting the ex vivo gene editing technology. SR-Tiget and Genespire will first collaborate to bring an ex-vivo autologous edited T-cell gene therapy for X-linked Hyper IgM syndrome (HIGM1) to the clinic, which becomes Genespires lead candidate product. HIGM1 is caused by inherited mutations of the CD40 ligand gene (CD40L), resulting in impaired antibody response and innate immunity, meaning that people find it difficult to fight off infections and eventually succumb to them. The treatment objective is to correct the defective gene through targeted editing of the endogenous locus, thereby maintaining physiological regulation of the CD40L gene, with the aim of improving the immune response of the patients.

Preclinical results of SR-Tiget on HIGM1 will be disclosed in an oral presentation at the American Society for Cell and Gene Therapy (ASGCT) 23rd Annual Meeting, taking place virtually from 12-15 May 2020 by SR-Tiget (details of Presentation 1 below). The presentation will outline the technology and its preclinical validation in the disease model and patient derived cells and discuss the potential of the gene edited T-cell treatment approach for patients with Hyper IgM.

Dr. Alessio Cantore will also present novel data related to the potential of the lentiviral vector platform for liver gene therapy in an oral presentation at ASGCT (details of Presentation 2 below). The presentation will focus on investigating the stability of lentiviral vector genetically modified liver cells following post-natal liver growth in mice, in view of its potential application to pediatric patients.

Luigi Naldini, Director of SR-Tiget and scientific co-founder of Genespire said: We are excited to have secured a path for bringing forward some of the gene therapy work pioneered at SR-Tiget to eventually help individuals affected by severe metabolic and immunodeficiency disorders. SR-Tigets alliance with Genespire will provide the means to progress effectively to clinical trials, with a strong view to develop efficacious and safe medicines ready for market access.

Julia Berretta, Chief Executive Officer of Genespire commented: SR-Tiget brings outstanding expertise and significant experience in developing gene therapies from bench to bedside. We believe that our strong partnership with SR-Tiget, led by internationally recognized experts Prof. Luigi Naldini and Dr. Alessio Cantore will be fundamental for Genespire to carry out its goal of translating pioneering science into transformative therapeutic solutionsfor patients.

-ENDS-

Oral presentation 1 details: Title: Modeling, Optimization and Comparative Efficacy of HSC- and T-cell Based Editing Strategies for Treating Hyper IgM Syndrome Authors: Valentina Vavassori, Elisabetta Mercuri, Genni Marcovecchio, Maria Carmina Castiello, Giulia Schiroli , Luisa Albano, Elena Fontana, Andrea Annoni, Valentina Capo, Carrie Margulies, Frank Buquicchio, Joseph Kovacs, Eugenio Scanziani, Cecilia Cotta-Ramusino, Anna Villa, Luigi Naldini, Pietro Genovese Date and time: May 14th 2020, 3:45 PM EDT Session: 354 Gene Therapies for Hemophilia and Immune Disorders Abstract #937 Oral Presentation 2 Details Title: Investigating the stability of lentiviral vector targeted liver cells during post-natal growth for in vivo gene therapy applications Authors: Michela Milani, Francesco Starinieri, Cesare Canepari, Tongyao Liu, Federica Moalli, Gioia Ambrosi, Tiziana Plati, Mauro Biffi, Cesare Covino, Timothy Nichols, Matteo Iannacone, Robert Peters, Luigi Naldini, Alessio Cantore Date and time: May 14th 2020, 4:15 pm EDT Session: 350 RNA Virus Vectors Abstract #911

Notes to Editors

About Hyper IgM Syndrome (HIGM)

Hyper IgM is a Primary Immune Deficiency affecting 1:250,000-500,000 patients. The disease is linked to mutations in the CD40L gene, which is expressed in activated CD4 T cells, and results in impaired antibody production and innate immunity. The current standard of care is constituted by continuous Ig replacement, and antibiotic-antifungal prophylaxis, but the disease is still linked to high morbidity and reduced life expectancy. Allogeneic hematopoietic stem cell transplant (HSCT) is potentially curative, but is limited by matched donor availability and is associated with high risk of graft versus host disease, infections and death. Thus, improved therapeutic alternatives are strongly needed.

About Genespire

Genespire is a biotechnology company focused on the development of transformative gene therapies for patients affected by genetic diseases, particularly primary immunodeficiencies and inherited metabolic diseases. Based in Milan, Italy, Genespire was founded in March 2020 by the gene therapy pioneer Prof. Luigi Naldini and Dr. Alessio Cantore, Fondazione Telethon and Ospedale San Raffaele. It is a spin-off of SR-Tiget, a world leading cell and gene therapy research institute and is backed by Sofinnova Partners. http://www.genespire.com

About SR-Tiget

Based in Milan, Italy, the San Raffaele-Telethon Institute for Gene Therapy (SR-Tiget) is a joint venture between the Ospedale San Raffaele and Fondazione Telethon. SR-Tiget was established in 1995 to perform research on gene transfer and cell transplantation and translate its results into clinical applications of gene and cell therapies for different genetic diseases. Over the years, the Institute has given a pioneering contribution to the field with relevant discoveries in vector design, gene transfer strategies, stem cell biology, identity and mechanism of action of innate immune cells. SR-Tiget has also established the resources and framework for translating these advances into novel experimental therapies and has implemented several successful gene therapy clinical trials for inherited immunodeficiencies, blood and storage disorders, which have already treated >115 patients and have led through collaboration with industrial partners to the filing and approval of novel advanced gene therapy medicines.

About Fondazione Telethon

Fondazione Telethon is a non-profit organisation created in 1990 as a response to the appeals of a patient association group of stakeholders, who saw scientific research as the only real opportunity to effectively fight genetic diseases. Thanks to the funds raised through the television marathon, along with other initiatives and a network of partners and volunteers, Telethon finances the bestscientific research on rare genetic diseases, evaluated and selected by independent internationally renowned experts, with the ultimate objective of making the treatments developed available to everyone who needs them. Throughout its 30 years of activity, Fondazione Telethon has invested more than 528 million in funding more than 2.630 projects to study more than 570 diseases, involving over 1.600 scientists. Fondazione Telethon has made a significant contribution to the worldwide advancement of knowledge regarding rare genetic diseases and of academic research and drug development with a view to developing treatments. For more information, please visit:www.telethon.it

About Ospedale San Raffaele

Ospedale San Raffaele (OSR) is a clinical-research-university hospital established in 1971 to provide international-level specialised care for the most complex and difficult health conditions. OSR is part ofGruppo San Donato, the leading hospital group in Italy. The hospital is a multi-specialty center with over 60 clinical specialties; it is accredited by the Italian National Health System to provide care to both public and private, national and international patients. Research at OSR focuses on integrating basic, translational and clinical activities to provide the most advanced care to our patients. The institute is recognized as a global authority in molecular medicine and gene therapy, and is at the forefront of research in many other fields. Ospedale San Raffaele is a first-class institute which treats many diseases and stands out for the deep interaction between clinical and scientific area. This makes the transfer of scientific results from the laboratories to the patients bed easier. Its mission is to improve knowledge of diseases, identify new therapies and encourage young scientists and doctor to grow professionally. For more information, please visit:www.hsr.it

Enquiries: Genespire Julia Berretta, CEOTel: +39 02 83991300info@genespire.com Consilium Strategic Communications Amber Fennell / Matthew Neal Tel: +44 (0) 20 3709 5700genespire@consilium-comms.com

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Genespire and SR-Tiget announce strategic alliance for the development of transformative gene therapies for genetic diseases and disclose...

Abstract

Obesity-associated inflammation and loss of muscle function play critical roles in the development of osteoarthritis (OA); thus, therapies that target muscle tissue may provide novel approaches to restoring metabolic and biomechanical dysfunction associated with obesity. Follistatin (FST), a protein that binds myostatin and activin, may have the potential to enhance muscle formation while inhibiting inflammation. Here, we hypothesized that adeno-associated virus 9 (AAV9) delivery of FST enhances muscle formation and mitigates metabolic inflammation and knee OA caused by a high-fat diet in mice. AAV-mediated FST delivery exhibited decreased obesity-induced inflammatory adipokines and cytokines systemically and in the joint synovial fluid. Regardless of diet, mice receiving FST gene therapy were protected from post-traumatic OA and bone remodeling induced by joint injury. Together, these findings suggest that FST gene therapy may provide a multifactorial therapeutic approach for injury-induced OA and metabolic inflammation in obesity.

Osteoarthritis (OA) is a multifactorial family of diseases, characterized by cartilage degeneration, joint inflammation, and bone remodeling. Despite the broad impact of this condition, there are currently no disease-modifying drugs available for OA. Previous studies demonstrate that obesity and dietary fatty acids (FAs) play a critical role in the development of OA, and metabolic dysfunction secondary to obesity is likely to be a primary risk factor for OA (1), particularly following joint injury (2, 3). Furthermore, both obesity and OA are associated with a rapid loss of muscle integrity and strength (4), which may contribute directly and indirectly to the onset and progression of OA (5). However, the mechanisms linking obesity, muscle, and OA are not fully understood and appear to involve interactions among biomechanical, inflammatory, and metabolic factors (6). Therefore, strategies that focus on protecting muscle and mitigating metabolic inflammation may provide an attractive target for OA therapies in this context.

A few potential interventions, such as weight loss and exercise, have been proposed to reverse the metabolic dysfunction associated with obesity by improving the quantity or quality of skeletal muscle (7). Skeletal muscle mass is modulated by myostatin, a member of the transforming growth factor (TGF-) superfamily and a potent negative regulator of muscle growth (8), and myostatin is up-regulated in obesity and down-regulated by exercise (9). While exercise and weight loss are the first line of therapy for obesity and OA, several studies have shown difficulty in achieving long-term maintenance of weight loss or strength gain, particularly in frail or aging populations (10). Thus, targeted pharmacologic or genetic inhibition of muscle-regulatory molecules such as myostatin provides a promising approach to improving muscle metabolic health by increasing glucose tolerance and enhancing muscle mass in rodents and humans (8).

Follistatin (FST), a myostatin- and activin-binding protein, has been used as a therapy for several degenerative muscle diseases (11, 12), and loss of FST is associated with reduced muscle mass and prenatal death (13). In the context of OA, we hypothesize that FST delivery using a gene therapy approach has multifactorial therapeutic potential through its influence on muscle growth via inhibition of myostatin activity (14) as well as other members of the TGF- family. Moreover, FST has been reported to reduce the infiltration of inflammatory cells in the synovial membrane (15) and affect bone development (16), and pretreatment with FST has been shown to reduce the severity of carrageenan-induced arthritis (15). However, the potential for FST as an OA therapy has not been investigated, especially in exacerbating pathological conditions such as obesity. We hypothesized that overexpression of FST using a gene therapy approach will increase muscle mass and mitigate obesity-associated metabolic inflammation, as well as the progression of OA, in high-fat diet (HFD)induced obese mice. Mice fed an HFD were treated with a single dose of adeno-associated virus 9 (AAV9) to deliver FST or a green fluorescent protein (GFP) control, and the effects on systemic metabolic inflammation and post-traumatic OA were studied (fig. S1).

Dual-energy x-ray absorptiometry (DXA) imaging of mice at 26 weeks of age (Fig. 1A) showed significant effects of FST treatment on body composition. Control-diet, FST-treated mice (i.e., Control-FST mice) exhibited significantly lower body fat percentages, but were significantly heavier than mice treated with a GFP control vector (Control-GFP mice) (Fig. 1B), indicating that increased muscle mass rather than fat was developed with FST. With an HFD, control mice (HFD-GFP mice) showed significant increases in weight and body fat percentage that were ameliorated by FST overexpression (HFD-FST mice).

(A) DXA images of mice at 26 weeks of age. (B) DXA measurements of body fat percentage and bone mineral density (BMD; 26 weeks) and body weight measurements over time. (C) Serum levels for adipokines (insulin, leptin, resistin, and C-peptide) at 28 weeks. (D) Metabolite levels for glucose, triglycerides, cholesterol, and FFAs at 28 weeks. (E) Serum levels for cytokines (IL-1, IL-1, MCP-1, and VEGF) at 28 weeks. (F) Fluorescence microscopy images of visceral adipose tissue with CD11b:Alexa Fluor 488 (green), CD11c:phycoerythrin (PE) (red), and 4,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 100 m. Data are presented as mean SEM; n = 8 to 10; two-way analysis of variance (ANOVA), P < 0.05. Groups not sharing the same letter are significantly different with Tukey post hoc analysis. For IL-1 and VEGF, P < 0.05 for diet effect and AAV effect. For MCP-1, P < 0.05 for diet effect.

In the HFD group, overexpression of FST significantly decreased serum levels of several adipokines including insulin, leptin, resistin, and C-peptide as compared to GFP-treated mice (Fig. 1C). HFD-FST mice also had significantly lower serum levels of glucose, triglycerides, cholesterol, and free FAs (FFAs) (Fig. 1D), as well as the inflammatory cytokine interleukin-1 (IL-1) (Fig. 1E) when compared to HFD-GFP mice. For both dietary groups, AAV-FST delivery significantly increased circulating levels of vascular endothelial growth factor (VEGF) while significantly decreasing IL-1 levels. Furthermore, obesity-induced inflammation in adipose tissue was verified by the presence of CD11b+CD11c+ M1 pro-inflammatory macrophages or dendritic cells (Fig. 1F).

To determine whether FST gene therapy can mitigate injury-induced OA, mice underwent surgery for destabilization of the medial meniscus (DMM) and were sacrificed 12 weeks after surgery. Cartilage degeneration was significantly reduced in DMM joints of the mice receiving FST gene therapy in both dietary groups (Fig. 2, A and C) when compared to GFP controls. FST overexpression also significantly decreased joint synovitis (Fig. 2, B and D) when compared to GFP controls. To evaluate the local influence of pro-inflammatory cytokines to joint degeneration and inflammation, synovial fluid (SF) was harvested from surgical and ipsilateral nonsurgical limbs and analyzed using a multiplexed array. The DMM joints from mice with FST overexpression exhibited a trend toward lower levels of pro-inflammatory cytokines, including IL-1, IL-1, and IL-6, and a higher level of interferon- (IFN-)induced protein (IP-10) in the SF of DMM joints as compared to contralateral controls (Fig. 2E).

(A) Histologic analysis of OA severity via Safranin O (glycosaminoglycans) and fast green (bone and tendon) staining of DMM-operated joints. (B) Histology [hematoxylin and eosin (H&E) staining] of the medial femoral condyle of DMM-operated joints. Thickened synovium (S) from HFD mice with a high density of infiltrated cells was observed (arrows). (C) Modified Mankin scores compared within the diet. (D) Synovitis scores compared within the diet. (E) Levels of proinflammatory cytokines in the SF compared within the diet. (F) Hot plate latency time and sensitivity to cold plate exposure, as measured using the number of jumps in 30 s, both for non-operated algometry measurements of pain sensitivity compared within the diet. Data are presented as mean SEM; n = 5 to 10 mice per group; two-way ANOVA, P < 0.05. Groups not sharing the same letter are significantly different with Tukey post hoc analysis.

To investigate the effect of FST on pain sensitivity in OA, animals were subjected to a variety of pain measurements including hot plate, cold plate, and algometry. Obesity increased heat withdrawal latency, which was rescued by FST overexpression (Fig. 2F). Cold sensitivity trended lower with obesity, and because no significant differences in heat withdrawal latency were found with surgery (fig. S2), no cold sensitivity was measured after surgery. We found that FST treatment protected HFD animals from mechanical algesia at the knee receiving DMM surgery, while Control-diet DMM groups demonstrated increased pain sensitivity following joint injury.

A bilinear regression model was used to elucidate the relationship among OA severity, biomechanical factors, and metabolic factors (table S1). Factors significantly correlated with OA were then selected for multivariate regression (Table 1). Both multivariate regression models revealed serum tumor necrosis factor- (TNF-) levels as a major predictor of OA severity.

, standardized coefficient. ***P < 0.001.

We analyzed the effects of FST treatment on muscle structure and mass, and performance measures were conducted on mice in both dietary groups. Both Control-FST and HFD-FST limbs exhibited visibly larger muscles compared to both AAV-GFP groups (Fig. 3A). In addition, the muscle masses of tibialis anterior (TA), gastrocnemius, and quadriceps increased significantly with FST treatment (Fig. 3B). Western blot analysis confirmed an increase in FST expression in the muscle at the protein level in FST-treated groups compared to GFP-treated animals in Control and HFD groups (Fig. 3C). Immunofluorescence labeling showed increased expression of FST in muscle (Fig. 3D) and adipose tissue (Fig. 3E) of the AAV-FST mice, with little or no expression of FST in control groups.

(A) Photographic images and (B) measured mass of tibialis anterior (TA), gastrocnemius (GAS), and quadriceps (QUAD) muscles; n = 8, diet and AAV effects both P < 0.05. (C) Western blot showing positive bands of FST protein only in FST-treated muscles, with -actin as a loading control. Immunolabeling of (D) GAS muscle and (E) adipose tissue showing increased expression of FST, particularly in skeletal muscle. (F) H&E-stained sections of GAS muscles were measured for (G) mean myofiber diameter; n = 100 from four mice per group, diet, and AAV effects; both P < 0.05. (H) Oil Red O staining was analyzed for (I) optical density values of FAs; n = 6. (J) Second-harmonic generation imaging of collagen in TA sections was quantified for intensity; n = 6. (K) Western blotting showing the level of phosphorylation markers of protein synthesis in GAS muscle. (L) Functional analysis of grip strength and treadmill time to exhaustion; n = 10. Data are presented as mean SEM; two-way ANOVA, P < 0.05. Groups not sharing the same letter are significantly different with Tukey post hoc analysis. Photo credit: Ruhang Tang, Washington University.

To determine whether the increases in muscle mass reflected muscle hypertrophy, gastrocnemius muscle fiber diameter was measured in H&E-stained sections (Fig. 3F) at 28 weeks of age. Mice with FST overexpression exhibited increased fiber diameter (i.e., increased muscle hypertrophy) relative to the GFP-expressing mice in both diet treatments (Fig. 3G). Oil Red O staining was used to determine the accumulation of neutral lipids in muscle (Fig. 3H). We found that HFD-FST mice were protected from lipid accumulation in muscles compared to HFD-GFP mice (Fig. 3I). Second-harmonic generation imaging confirmed the presence of increased collagen content in the muscles of HFD mice, which was prevented by FST gene therapy (Fig. 3J). We also examined the expression and phosphorylation levels of the key proteins responsible for insulin signaling in muscles. We observed increased phosphorylation of AktS473, S6KT389, and S6RP-S235/2369 and higher expression of peroxisome proliferatoractivated receptor coactivator 1- (Pgc1-) in muscles from FST mice compared to GFP mice, regardless of diet (Fig. 3K). In addition to the improvements in muscle structure with HFD, FST-overexpressing mice also showed improved function, including higher grip strength and increased treadmill running endurance (Fig. 3L), compared to GFP mice.

Because FST has the potential to influence cardiac muscle and skeletal muscle, we performed a detailed evaluation on the effect of FST overexpression on cardiac function. Echocardiography and short-axis images were collected to visualize the left ventricle (LV) movement during diastole and systole (fig. S3A). While the Control-FST mice had comparable LV mass (LVM) and left ventricular posterior wall dimensions (LVPWD) with Control-GFP mice (fig. S3, B and C), the HFD-FST mice have significantly decreased LVM and trend toward decreased LVPWD compared to HFD-GFP. Regardless of the diet treatments, FST overexpression enhanced the rate of heart weight/body weight (fig. S3D). Although Control-FST mice had slightly increased dimensions of the interventricular septum at diastole (IVSd) compared to Control-GFP (fig. S3E), there was significantly lower IVSd in HFD-FST compared to HFD-GFP. In addition, we found no difference in fractional shortening among all groups (fig. S3F). Last, transmitral blood flow was investigated using pulse Doppler. While there was no difference in iso-volumetric relaxation time (IVRT) in Control groups, HFD-FST mice had a moderate decrease in IVRT compared to HFD-GFP (fig. S3G). Overall, FST treatment mitigated the changes in diastolic dysfunction and improved the cardiac relaxation caused by HFD.

DXA demonstrated that FST gene therapy improved bone mineral density (BMD) in HFD compared to other groups (Fig. 1B). To determine the effects of injury, diet intervention, and overexpression of FST on bone morphology, knee joints were evaluated by microcomputed tomography (microCT) (Fig. 4A). The presence of heterotopic ossification was observed throughout the GFP knee joints, whereas FST groups demonstrated a reduction or an absence of heterotopic ossification. FST overexpression significantly increased the ratio of bone volume to total volume (BV/TV), BMD, and trabecular number (Tb.N) of the tibial plateau in animals, regardless of diet treatment (Fig. 4B). Joint injury generally decreased bone parameters in the tibial plateau, particularly in Control-diet mice. In the femoral condyle, BV/TV and Tb.N were significantly increased in mice with FST overexpression in both diet types, while BMD was significantly higher in HFD-FST compared to HFD-GFP mice (Fig. 4B). Furthermore, AAV-FST delivery significantly increased trabecular thickness (Tb.Th) and decreased trabecular space (Tb.Sp) in the femoral condyle of HFD-FST compared to HFD-GFP animals (fig. S4).

(A) Three-dimensional (3D) reconstruction of microCT images of non-operated and DMM-operated knees. (B) Tibial plateau (TP) and femoral condyle (FC) regional analyses of trabecular bone fraction bone volume (BV/TV), BMD, and trabecular number (Tb.N). Data are presented as mean SEM; n = 8 to 19 mice per group; two-way ANOVA. (C) 3D microCT reconstruction of metaphysis region of DMM-operated joints. (D) Analysis of metaphysis BV/TV, Tb.N, and BMD. (E) 3D microCT reconstruction of cortical region of DMM-operated joints. (F) Analysis of cortical cross-sectional thickness (Ct.Cs.Th), polar moment of inertia (MMI), and tissue mineral density (TMD). (D and F) Data are presented as mean SEM; n = 8 to 19 mice per group; Mann-Whitney U test, *P < 0.05.

Further microCT analysis was conducted on the trabecular (Fig. 4C) and cortical (Fig. 4E) areas of the metaphyses. FST gene therapy significantly increased BV/TV, Tb.N, and BMD in the metaphyses regardless of the diet (Fig. 4D). Furthermore, FST delivery significantly increased the cortical cross-sectional thickness (Ct.Cs.Th) and polar moment of inertia (MMI) of mice on both diet types, as well as tissue mineral density (TMD) of cortical bones of mice fed control diet (Fig. 4F).

To elucidate the possible mechanisms by which FST mitigates inflammation, we examined the browning/beiging process in subcutaneous adipose tissue (SAT) with immunohistochemistry (Fig. 5A). Here, we found that key proteins expressed mainly in brown adipose tissue (BAT) (PGC-1, PRDM16, thermogenesis marker UCP-1, and beige adipocyte marker CD137) were up-regulated in SAT of the mice with FST overexpression (Fig. 5B). Increasing evidence suggests that an impaired mitochondrial oxidative phosphorylation (OXPHOS) system in white adipocytes is a hallmark of obesity-associated inflammation (17). Therefore, we further examined the mitochondrial respiratory system in SAT. HFD reduced the amount of OXPHOS complex subunits (Fig. 5C). We found that proteins involved in OXPHOS, including subunits of complexes I, II, and III of mitochondria OXPHOS complex, were significantly up-regulated in AAV-FSToverexpressing animals compared to AAV-GFP mice (Fig. 5D).

(A) Immunohistochemistry of UCP-1 expression in SAT. Scale bar, 50 m. (B) Western blotting of SAT for key proteins expressed in BAT, with -actin as a loading control. (C) Western blot analysis of mitochondria lysates from SAT for OXPHOS proteins using antibodies against subunits of complexes I, II, III, and IV and adenosine triphosphate (ATP) synthase. (D) Change of densitometry quantification normalized to the average FST level of each OXPHOS subunit. Data are presented as mean SEM; n = 3. *P < 0.05, t test comparison within each pair.

Our findings demonstrate that a single injection of AAV-mediated FST gene therapy ameliorated systemic metabolic dysfunction and mitigated OA-associated cartilage degeneration, synovial inflammation, and bone remodeling occurring with joint injury and an HFD. Of note, the beneficial effects were observed across multiple tissues of the joint organ system, underscoring the value of this potential treatment strategy. The mechanisms by which obesity and an HFD increase OA severity are complex and multifactorial, involving increased systemic metabolic inflammation, joint instability and loss of muscle strength, and synergistic interactions between local and systemic cytokines (4, 6). In this regard, the therapeutic consequences of FST gene therapy also appear to be multifactorial, involving both direct and indirect effects such as increased muscle mass and metabolic activity to counter caloric intake and metabolic dysfunction resulting from an HFD while also promoting adipose tissue browning. Furthermore, FST may also serve as a direct inhibitor of growth factors in the TGF- family that may be involved in joint degeneration (18).

FST gene therapy showed a myriad of notable beneficial effects on joint degeneration following joint injury while mitigating HFD-induced obesity. These data also indirectly implicate the critical role of muscle integrity in the onset and progression of post-traumatic OA in this model. It is important to note that FST gene therapy mitigated many of the key negative phenotypic changes previously associated with obesity and OA, including cartilage structural changes as well as bone remodeling, synovitis, muscle fibrosis, and increased pain, as compared to GFP controls. To minimize the number of animals used, we did not perform additional controls with no AAV delivery; however, our GFP controls showed similar OA changes as observed in our previous studies, which did not involve any gene delivery (2). Mechanistically, FST restored to control levels a number of OA-associated cytokines and adipokines in the serum and the SF. While the direct effects of FST on chondrocytes remains to be determined, FST has been shown to serve as a regulator of the endochondral ossification process during development (19), which may also play a role in OA (20). Furthermore, previous studies have shown that a 2-week FST treatment of mouse joints is beneficial in reducing infiltration of inflammatory cells into the synovial membrane (15). Our findings suggest that FST delivery in skeletally mature mice, preceding obesity-induced OA changes, substantially reduces the probability of tissue damage.

It is well recognized that FST can inhibit the activity of myostatin and activin, both of which are up-regulated in obesity-related modalities and are involved in muscle atrophy, tissue fibrosis, and inflammation (21). Consistent with previous studies, our results show that FST antagonizes the negative regulation of myostatin in muscle growth, reducing adipose tissue content in animals. Our observation that FST overexpression decreased inflammation at both serum systemic and local joint inflammation may provide mechanistic insights into our findings of mitigated OA severity in HFD-fed mice. Our statistical analysis implicated serum TNF- levels as a major factor in OA severity, consistent with previous studies linking obesity and OA in mice (22). Although the precise molecular mechanisms of FST in modulating inflammation remain unclear, some studies postulate that FST may act like acute-phase protein in lipopolysaccharide-induced inflammation (23).

In addition to these effects of skeletal muscle, we found that FST gene therapy normalized many of the deleterious changes of an HFD on cardiac function without causing hypertrophy. These findings are consistent with previous studies showing that, during the process of aging, mice with myostatin knockout had an enhanced cardiac stress response (24). Furthermore, FST has been shown to regulate activin-induced cardiomyocyte apoptosis (1). In the context of this study, it is also important to note that OA has been shown to be a serious risk factor for progression of cardiovascular disease (25), and severity of OA disability is associated with significant increases in all-cause mortality and cardiovascular events (26).

FST gene therapy also rescued diet- and injury-induced bone remodeling in the femoral condyle, as well as the tibial plateau, metaphysis, and cortical bone of the tibia, suggesting a protective effect of FST on bone homeostasis of mice receiving an HFD. FST is a known inhibitor of bone morphogenetic proteins (BMPs), and thus, the interaction between the two proteins plays an essential role during bone development and remodeling. For example, mice grown with FST overexpression via global knock-in exhibited an impaired bone structure (27). However, in adult diabetic mice, FST was shown to accelerate bone regeneration by inhibiting myostatin-induced osteoclastogenesis (28). Furthermore, it has been reported that FST down-regulates BMP2-driven osteoclast activation (29). Therefore, the protective role of FST on obesity-associated bone remodeling, at least in part, may result from the neutralizing capacity of FST on myostatin in obesity. In addition, improvement in bone quality in FST mice may be explained by their enhanced muscle mass and strength, as muscle mass can dominate the process of skeletal adaptation, and conversely, muscle loss correlates with reduced bone quality (30).

Our results show that FST delivery mitigated pain sensitivity in OA joints, a critical aspect of clinical OA. Obesity and OA are associated with both chronic pain and pain sensitization (31), but it is important to note that structure and pain can be uncoupled in OA (32), necessitating the measurement of both behavioral and structural outcomes. Of note, FST treatment protected only HFD animals from mechanical algesia at the knee post-DMM surgery and also rescued animals from pain sensitization induced by HFD in both the DMM and nonsurgical limb. The mitigation in pain sensitivity observed here with FST treatment may also be partially attributed to the antagonistic effect of FST on activin signaling. In addition to its role in promoting tissue fibrosis, activin A has been shown to regulate nociception in a manner dependent on the route of injection (33, 34). It has been shown that activin can sensitize the transient receptor potential vanilloid 1 (TRPV1) channel, leading to acute thermal hyperalgesia (33). However, it is also possible that activin may induce pain indirectly, for example, by triggering neuroinflammation (35), which could lead to sensitization of nociceptors.

The earliest detectable abnormalities in subjects at risk for developing obesity and type 2 diabetes are muscle loss and accumulation of excess lipids in skeletal muscles (4, 36), accompanied by impairments in nuclear-encoded mitochondrial gene expression and OXPHOS capacity of muscle and adipose tissues (17). PGC-1 activates mitochondrial biogenesis and increases OXPHOS by increasing the expression of the transcription factors necessary for mitochondrial DNA replication (37). We demonstrated that FST delivery can rescue low levels of OXPHOS in HFD mice by increasing expression PGC-1 (Fig. 3H). It has been reported that high-fat feeding results in decreased PGC-1 and mitochondrial gene expression in skeletal muscles, while exercise increases the expression of PGC-1 in both human and rodent muscles (38, 39). Although the precise molecular mechanism by which FST promotes PGC-1 expression has not been established, the infusion of lipids decreases expression of PGC-1 and nuclear-encoded mitochondrial genes in muscles (40). Thus, decreased lipid accumulation in muscle by FST overexpression may provide a plausible explanation for the restored PGC-1 in the FST mice. These findings were further confirmed by the metabolic profile, showing reduced serum levels of triglycerides, glucose, FFAs, and cholesterol (Fig. 1D), and are consistent with previous studies, demonstrating that muscles with high numbers of mitochondria and oxidative capacity (i.e., type 1 muscles with high levels of PGC-1 expression) are protected from damage due to an HFD (4).

In addition, we found increased phosphorylation of protein kinase B (Akt) on Ser473 in the skeletal muscle of FST-treated mice as compared to untreated HFD counterparts (Fig. 3K), consistent with restoration of a normal insulin response. A number of studies have demonstrated that the serine-threonine protein kinase Akt1 is a critical regulator of cellular hypertrophy, organ size, and insulin signaling (41). Muscle hypertrophy is stimulated both in vitro and in vivo by the expression of constitutively active Akt1 (42, 43). Furthermore, it has been demonstrated that constitutively active Akt1 also promotes the production of VEGF (44).

BAT is thought to be involved in thermogenesis rather than energy storage. BAT is characterized by a number of small multilocular adipocytes containing a large number of mitochondria. The process in which white adipose tissue (WAT) becomes BAT, called beiging or browning, is postulated to be protective in obesity-related inflammation, as an increase in BAT content positively correlates with increased triglyceride clearance, normalized glucose level, and reduced inflammation. Our study shows that AAV-mediated FST delivery serves as a very promising approach to induce beiging of WAT in obesity. A recent study demonstrated that transgenic mice overexpressing FST exhibited an increasing amount of BAT and beiging in subcutaneous WAT with increased expression of key BAT-related markers including UCP-1 and PRDM16 (45). In agreement with previous reports, our data show that Ucp1, Prdm16, Pgc1a, and Cd167 are significantly up-regulated in SAT of mice overexpressing FST in both dietary interventions. FST has been recently demonstrated to play a crucial role in modulating obesity-induced WAT expansion by inhibiting TGF-/myostatin signaling and thus promoting overexpression of these key thermogenesis-related genes. Together, these findings suggest that the observed reduction in systemic inflammation in our model may be partially explained by FST-mediated increased process of browning/beiging.

In conclusion, we show that a single injection of AAV-mediated FST, administered after several weeks of HFD feeding, mitigated the severity of OA following joint injury, and improved muscle performance as well as induced beiging of WAT, which together appeared to decrease obesity-associated metabolic inflammation. These findings provide a controlled model for further examining the differential contributions of biomechanical and metabolic factors to the progression of OA with obesity or HFD. As AAV gene therapy shows an excellent safety profile and is currently in clinical trials for a number of conditions, such an approach may allow the development of therapeutic strategies not only for OA but also, more broadly, for obesity and associated metabolic conditions, including diseases of muscle wasting.

All experimental procedures were approved by and conducted in accordance with the Institutional Animal Care and Use Committee guidelines of Washington University in Saint Louis. The overall timeline of the study is shown in fig. S1A. Beginning at 5 weeks of age, C57BL/6J mice (The Jackson Laboratory) were fed either Control or 60% HFD (Research Diets, D12492). At 9 week of age, mice received AAV9-mediated FST or GFP gene delivery via tail vein injection. A total of 64 mice with 16 mice per dietary group per AAV group were used. DMM was used to induce knee OA in the left hind limbs of the mice at the age of 16 weeks. The non-operated right knees were used as contralateral controls. Several behavioral activities were measured during the course of the study. Mice were sacrificed at 28 weeks of age to evaluate OA severity, joint inflammation, and joint bone remodeling.

Mice were weighed biweekly. The body fat content and BMD of the mice were measured using a DXA (Lunar PIXImus) at 14 and 26 weeks of age, respectively.

Complementary DNA synthesis for mouse FST was performed by reverse transcriptase in a reverse transcription quantitative polymerase chain reaction (RT-qPCR) ( Invitrogen) mixed with mRNAs isolated from the ovary tissues of C57BL/6J mouse. The PCR product was cloned into the AAV9-vector plasmid (pTR-UF-12.1) under the transcriptional control of the chicken -actin (CAG) promoter including cytomegalovirus (CMV) enhancers and a large synthetic intron (fig. S1B). Recombinant viral vector stocks were produced at Hope Center Viral Vectors Core (Washington University, St. Louis) according to the plasmid cotransfection method and suspension culture. Viral particles were purified and concentrated. The purity of AAV-FST and AAV-GFP was evaluated by SDSpolyacrylamide gel electrophoresis (PAGE) and stained by Coomassie blue. The results showed that the AAV protein components in 5 1011 vector genomes (vg) are only stained in three major protein bands: VR1, 82 kDa; VR2, 72 kDa; and VR3, 62 kDa. Vector titers were determined by the DNA dot-blot and PCR methods and were in the range of 5 1012 to 1.5 1013 vector copies/ml. AAV was delivered at a final dose of 5 1011 vg per mouse by intravenous tail injection under red light illumination at 9 weeks of age. This dose was determined on the basis of our previous studies showing that AAV9-FST gene delivery by this route resulted in a doubling of muscle mass at a dose of 2.5 1011 vg in 4-week-old mice or at 5 1011 vg in 8-week-old mice (46).

At 16 weeks of age, mice underwent surgery for the DMM to induce knee OA in the left hindlimb as previously described (2). Briefly, anesthetized mice were placed on a custom-designed device, which positioned their hindlimbs in 90 flexion. The medial side of the joint capsule was opened, and the medial meniscotibial ligament was transected. The joint capsule and subcutaneous layer of the skin were closed with resorbable sutures.

Mice were sacrificed at 28 weeks of age, and changes in joint structure and morphology were assessed using histology. Both hindlimbs were harvested and fixed in 10% neutral-buffered formalin (NBF). Limbs were then decalcified in Cal-Ex solution (Fisher Scientific, Pittsburgh, PA, USA), dehydrated, and embedded in paraffin. The joint was sectioned in the coronal plane at a thickness of 8 m. Joint sections were stained with hematoxylin, fast green, and Safranin O to determine OA severity. Three blinded graders then assessed sections for degenerative changes of the joint using a modified Mankin scoring system (2). Briefly, this scoring system measures several aspects of OA progression (cartilage structure, cell distribution, integrity of tidemark, and subchondral bone) in four joint compartments (medial tibial plateau, medial femoral condyle, lateral tibial plateau, and lateral femoral condyle), which are summed to provide a semiquantitative measure of the severity of joint damage. To assess the extent of synovitis, sections were stained with H&E to analyze infiltrated cells and synovial structure. Three independent blinded graders scored joint sections for synovitis by evaluating synovial cell hyperplasia, thickness of synovial membrane, and inflammation in subsynovial regions in four joint compartments, which were summed to provide a semiquantitative measure of the severity of joint synovitis (2). Scores for the whole joint were averaged among graders.

Serum and SF from the DMM and contralateral control limbs were collected, as described previously (2). For cytokine and adipokine levels in the serum and SF fluid, a multiplexed bead assay (Discovery Luminex 31-Plex, Eve Technologies, Calgary, AB, Canada) was used to determine the concentration of Eotaxin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), IFN-, IL-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IP-10, keratinocyte chemoattractant (KC), leukemia inhibitory factor (LIF), liposaccharide-induced (LIX), monocyte chemoattractant protein-1 (MCP-1), M-CSF, monokine induced by gamma interferon (MIG), macrophage inflammatory protein1 (MIP-1), MIP-1, MIP-2, RANTES, TNF-, and VEGF. A different kit (Mouse Metabolic Array) was used to measure levels for amylin, C-peptide, insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), ghrelin, glucagon, insulin, leptin, protein phosphatase (PP), peptide yy (PYY), and resistin. Missing values were imputed using the lowest detectable value for each analyte.

Muscles were cryopreserved by incubation with 2-methylbutane in a steel beaker using liquid nitrogen for 30 s, cryoembedded, and cryosectioned at 8 m thickness. Tissue sections were stained following standard H&E protocol. Photomicrographs of skeletal muscle fiber were imaged under brightfield (VS120, Olympus). Muscle slides fixed in 3.7% formaldehyde were stained with 0.3% Oil Red O (in 36% triethyl phosphate) for 30 min. Images were taken in brightfield (VS120, Olympus). The relative concentration of lipid was determined by extracting the Oil Red O with isopropanol in equally sized muscle sections and quantifying the OD500 (optical density at 500 nm) in a 96-well plate.

To determine spatial expression of FST in different tissues, cryosections of gastrocnemius muscles and adipose tissue were immunolabeled for FST. Tissue sections were fixed in 1.5% paraformaldehyde solution, and primary anti-FST antibody (R&D Systems, AF-669, 1:50) was incubated overnight at 4C after blocking with 2.5% horse serum (Vector Laboratories), followed by labeling with a secondary antibody (Alexa Fluor 488, Invitrogen, A11055) and with 4,6-diamidino-2-phenylindole (DAPI) for cell nuclei. Sections were imaged using fluorescence microscopy.

Second-harmonic generation images of TA were obtained from unstained slices using backscatter signal from an LSM 880 confocal microscope (Zeiss) with Ti:sapphire laser tuned to 820 nm (Coherent). The resulting image intensity was analyzed using ImageJ software.

To measure bone structural and morphological changes, intact hindlimbs were scanned by microCT (SkyScan 1176, Bruker) with an 18-m isotropic voxel resolution (455 A, 700-ms integration time, and four-frame averaging). A 0.5-mm aluminum filter was used to reduce the effects of beam hardening. Images were reconstructed using NRecon software (with 10% beam hardening and 20 ring artifact corrections). Subchondral/trabecular and cortical bone regions were segmented using CTAn automatic thresholding software. Tibial epiphysis was selected using the subchondral plate and growth plate as references. Tibial metaphysis was defined as the 1-mm region directly below the growth plate. The cortical bone analysis was performed in the mid-shaft (4 mm below the growth plate with a height of 1 mm). Hydroxyapatite calibration phantoms were used to calibrate bone density values (mg/cm3).

Fresh visceral adipose tissues were collected, frozen in optimal cutting temperature compound (OCT), and cryosectioned at 5-m thickness. Tissue slides were then acetone-fixed followed by incubation with Fc receptor blocking in 2.5% goat serum (Vector Laboratories) and incubation with primary antibodies cocktail containing anti-CD11b:Alexa Fluor 488 and CD11c:phycoerythrin (PE) (BioLegend). Nuclei were stained with DAPI. Samples were imaged using fluorescence microscopy (VS120, Olympus).

Adipose tissues were fixed in 10% NBF, paraffin-embedded, and cut into 5-m sections. Sections were deparaffinized, rehydrated, and stained with H&E. Immunohistochemistry was performed by incubating sections (n = 5 per each group) with the primary antibody (antimUCP-1, U6382, Sigma), followed by a secondary antibody conjugated with horseradish peroxidase (HRP). Chromogenic substrate 3,3-diaminobenzidine (DAB) was used to develop color. Counterstaining was performed with Harris hematoxylin. Sections were examined under brightfield (VS120, Olympus).

Proteins of the muscle or fat tissue were extracted using lysis buffer containing 1% Triton X-100, 20 mM tris-HCl (pH 7.5), 150 mM NaCl, 1 mm EDTA, 5 mM NaF, 2.5 mM sodium pyrophosphate, 1 mM -glycerophosphate, 1 mM Na3VO4, leupeptin (1 g ml1), 0.1 mM phenylmethylsulfonyl fluoride, and a cocktail of protease inhibitors (Sigma, St. Louis, MO, USA, catalog no. P0044). Protein concentrations were measured with Quick Start Bradford Dye Reagent (Bio-Rad). Twenty micrograms of each sample was separated in SDS-PAGE gels with prestained molecular weight markers (Bio-Rad). Proteins were wet-transferred to polyvinylidene fluoride membranes. After incubating for 1.5 hours with a buffer containing 5% nonfat milk (Bio-Rad #170-6404) at room temperature in 10 mM tris-HCl (pH 7.5), 100 mM NaCl, and 0.1% Tween 20 (TBST), membranes were further incubated overnight at 4C with antiUCP-1 rabbit polyclonal antibody (1:500, Sigma, U6382), anti-PRDM16 rabbit antibody (Abcam, ab106410), anti-CD137 rabbit polyclonal antibody (1:1000, Abcam, ab203391), total OXPHOS rodent western blot (WB) antibodies (Abcam, ab110413), anti-actin (Cell Signaling Technology, 13E5) rabbit monoclonal antibody (Cell Signaling Technology, 4970), followed by HRP-conjugated secondary antibody incubation for 30 min. A chemiluminescent detection substrate (Clarity, Western ECL) was applied, and the membranes were developed (iBrightCL1000).

The effects of HFD and FST gene therapy on thermal hyperalgesia were examined at 15 weeks of age. Mice were acclimatized to all equipment 1 day before the onset of testing, as well as a minimum of 30 min before conducting each test. Thermal pain tests were measured in a room set to 25C. Peripheral thermal sensitivity was determined using a hot/cold analgesia meter (Harvard Apparatus, Holliston, MA, USA). For hot plate testing, the analgesia meter was set to 55C. To prevent tissue damage, a maximum cutoff time of 20 s was established a priori, at which time an animal would be removed from the plate in the absence of pain response, defined as paw withdrawal or licking. Animals were tested in the same order three times, allowing each animal to have a minimum of 30 min between tests. The analgesia meter was cleaned with 70% ethanol between trials. The average of the three tests was reported per animal. To evaluate tolerance to cold, the analgesia meter was set to 0C. After 1-hour rest, animals were tested for sensitivity to cold over a single 30-s exposure. The number of jumps counted per animal was averaged within each group and compared between groups.

Pressure-pain tests were conducted at the knee using a Small Animal Algometer (SMALGO, Bioseb, Pinellas Park, FL, USA). Surgical and nonsurgical animals were evaluated over serial trials on the lateral aspect of the experimental and contralateral knee joints. The average of three trials per limb was calculated for each limb. Within each group, the pain threshold of the DMM limb versus non-operated limb was compared using a t test run on absolute values of mechanical pain sensitivity for each limb, P 0.05.

To assess the effect of HFD and AAV-FST treatments on neuromuscular function, treadmill running to exhaustion (EXER3, Columbus Instruments) was performed at 15 m/min, with 5 inclination angle on the mice 4 months after gene delivery. Treadmill times were averaged within groups and compared between groups.

Forelimb grip strength was measured using Chatillon DFE Digital Force Gauge (Johnson Scale Co.) for front limb strength of the animals. Each mouse was tested five times, with a resting period of 90 s between each test. Grip strength measurements were averaged within groups and compared between groups.

Cardiac function of the mice was examined at 6 months of age (n = 3) using echocardiography (Vevo 2100 High-Resolution In Vivo Imaging System, VisualSonics). Short-axis images were taken to view the LV movement during diastole and systole. Transmitral blood flow was observed with pulse Doppler. All data and images were performed by a blinded examiner and analyzed with an Advanced Cardiovascular Package Software (VisualSonics).

Detailed statistical analyses are described in methods of each measurement and its corresponding figure captions. Analyses were performed using GraphPad Prism, with significance reported at the 95% confidence level.

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

Acknowledgments: Funding: This study was supported, in part, by NIH grants AR50245, AR48852, AG15768, AR48182, AG46927, AR073752, OD10707, AR060719, AR074992, and AR75899; the Arthritis Foundation; and the Nancy Taylor Foundation for Chronic Diseases. Author contributions: R.T. and F.G. developed the concept of the study; R.T., N.S.H., C.-L.W., K.H.C., and Y.-R.C. collected and analyzed data; S.J.O. analyzed data; and all authors contributed to the writing of the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Gene therapy for follistatin mitigates systemic metabolic inflammation and post-traumatic arthritis in high-fat dietinduced obesity - Science Advances