Updates from ASGCT 2020

The 23rd Annual Meeting of the American Society of Gene & Cell Therapy (ASGCT) kicked off on Tuesday, May 12 to a large audience, despite COVID-19 concerns causing the meeting to be held virtually.  The first morning started off with a strong focus on Duchenne, with a session titled “Successes in Gene Therapy for Musculoskeletal Disease” featuring talks from prominent leaders in dystrophin gene therapy.

  • Xiao Xiao, of the University of North Carolina at Chapel Hill and co-founder of Bamboo Therapeutics, gave an overview of Duchenne muscular dystrophy, Duchenne therapeutic strategies in general, and micro-dystrophin gene therapy programs specifically. 
  • Jeff Chamberlain of the University of Washington School of Medicine gave a detailed presentation on the strategy and data that helped develop and optimize the micro-dystrophins used in Duchenne gene therapy.
  • Jerry Mendell of Nationwide Children’s Hospital took us from his first clinical neuromuscular gene therapy studies and the evolution of approaches to get us to the present day, sharing videos from some boys who have received micro-dystrophin gene therapy for Duchenne. 

Poster 1074 – Targeted Delivery of Oligonucleotide Therapeutics to Muscle Demonstrates Potential to Treat Duchenne Muscular Dystrophy.

Dyne Therapeutics disclosed a novel approach to enhance the delivery of antisense oligonucleotides (ASOs), therapeutic molecules to skeletal, cardiac and smooth muscles as a treatment for Duchenne muscular dystrophy.

The company’s FORCE platform utilizes a selective Transferrin receptor-1 (TFR-1) antibody conjugated with an exon-skipping oligonucleotide. TFR-1 is highly expressed on the surface of muscle cells, and this allows for a targeted delivery of a disease-modifying therapeutic payload directly to skeletal, cardiac and smooth muscle tissue.

Preclinical results in the DMD mdx mouse model were disclosed showing that treatment with a single dose of FORCE-M23D resulted in efficient exon skipping and dystrophin protein restoration in both skeletal and cardiac muscle, and was accompanied with improvements in functional activity and a reduction in creatine kinase levels. https://www.dyne-tx.com/wp-content/uploads/2020-ASGCT-DyneTx-Poster-FINAL-2020-05-11.pdf

These data support the therapeutic potential of Dyne’s muscle-targeted antibody-ASO conjugates for the treatment of Duchenne.

These data support the therapeutic potential of Dyne’s muscle-targeted antibody-ASO conjugates for the treatment of Duchenne.

Poster 237 – Development of a Minimized Exons 45-55 Skipping Cocktail for the Treatment of Duchenne Muscular Dystrophy.

Kenji Rowel Q. Lim and colleagues from the University of Alberta () reported the design of phosphorodiamidate morpholino oligomer (PMO) antisense oligos (ASOs) targeting exons 45-55 of the human DMD gene. This region is known as a “mutation hotspot,” and deletion of these 10 exons is associated with a mild phenotype in Becker muscular dystrophy patients. If successful, this approach could treat approximately 50% of all Duchenne patients.

They demonstrated that a 12-PMO cocktail restored up to ~15.9% of exons 45-55 skipped dystrophin compared to normal levels in immortalized DMD patient-derived muscle cells with various mutations.

More recent studies have found that the number of PMOs in this cocktail can be reduced and yet retain appreciable levels of exons 45-55 skipping. Immortalized patient-derived myotubes carrying an exon 52 deletion were transfected with a 5-PMO cocktail at 3 days post-differentiation. At 2 days post-treatment, 34% exons 45-55 skipping (on average) was observed by RT-PCR in the treated cells. Surprisingly, this result was not significantly different from the 25% skipping obtained using the full 12-PMO cocktail.

Current studies are aimed at identifying PMO cell-penetrating peptide conjugates to enhance efficacy. DG9-PMO was identified as a promising peptide-conjugate, and demonstrated enhanced exon skipping in vivo in the hDMDdel52;mdx mouse model.

Future work will test the efficacy of the DG9-conjugated version of this cocktail in vivo.

During the second day of the ASGCT 2020 conference, both Sarepta Therapeutics and Solid Biosciences presented their respective micro-dystrophin gene therapy programs, although no new data was revealed. 

In a symposium Sarepta sponsored, Louise Rodino-Klapac reviewed the design of Sarepta’s SRP-9001 gene therapy product, which uses an AAVrh74 vector, MCHCK74 promoter, and a micro-dystrophin transgene.  Sarepta’s Phase 2 placebo-controlled trial of SRP-9001 (with 41 participants total) completed dosing in January 2020, and we are awaiting results.  In the meantime, Rodino-Klapac indicated an additional 60-subject trial is being planned to start later this year. 

J Patrick Gonzales from Solid Biosciences also presented Solid’s SGT-001 micro-dystrophin gene therapy, which utilizes an AAV9 vector, CK8 promoter, and a micro-dystrophin construct that includes the NOS (Nitric Oxide Synthase) binding domain.  As Solid reported last week, their Phase1/2 trial is on clinical hold, and they are working to respond to the FDA’s requests for further data and analyses related to their manufacturing processes. In the meantime, all study-related activities with all enrolled subjects is continuing.  

Poster 1065 – Extracellular Vesicles for Monitoring Therapeutic Antisense Oligonucleotide Drug Activity in Duchenne Muscular Dystrophy

Lauren Sullivan and colleagues from Massachusetts General Hospital and Boston Children’s Hospital reported on their work developing a non-invasive method for detecting the effectiveness of drugs targeting dystrophin.

One therapeutic strategy in Duchenne involves the use of antisense oligonucleotides (ASOs) to induce removal, or “skipping” of certain exons to restore the reading frame and produce a partially functional dystrophin protein. In clinical trials of ASOs in Duchenne, muscle tissue biopsies are used to detect exon skipping.  However, tissue biopsies are invasive, impractical for long term monitoring of therapeutic response, and require general anesthesia in children.

In order to develop a non-invasive method, the authors focused on extracellular vesicles (EVs), which are small amounts of cell membrane (and cell contents) that are naturally released by cells.  Some of these EVs circulate through the body and end up in urine, where they can be collected and analyzed.  RNA contained within urine EVs have been shown to reveal mutation-specific deletions or exon-skipping of dystrophin. But the analytic and clinical validity of EV RNA for monitoring ASO drug activity is unknown.

Therefore, the authors validated their method in mdx animals.  Urine and various tissue samples were collected from mdx mice treated with an exon-skipping ASO.  Using droplet digital PCR for sensitive and quantitative analysis, they determined that the degree of exon skipping could be reliably and accurately measured even from the low copy number of dystrophin RNA transcripts obtained from urine.

Extending these methods to humans, urine samples from four DMD patients on weekly eteplirsen treatment were collected, to measure the efficiency of exon 51 skipping over time.  Interestingly, high levels of exon skipping (60-80%) were observed at earlier timepoints in 3 individuals, but this effect diminished over time.  Given the small sample size, it’s hard to draw overarching conclusions about this pattern until more individuals are studied. 

In general, however, a non-invasive measurement such as this may enable long-term monitoring of exon skipping ASO activity in larger number of individuals, reduce the need for invasive muscle biopsies, enable finding more efficacious doses, and even facilitate the development of other, more effective exon-skipping ASOs. 

Poster 1144 – A Single AAV Micro-Dystrophin Therapy in 3-Month-Old Mdx Mice Results in Persistent Improvement in Whole Body Performance and Heart Function for 22-Months

Clinical trials with AAV delivered micro-dystrophin are currently in progress. While the initial results are extremely encouraging, the durability of the response (i.e. to provide long-term mini-dystrophin expression and muscle protection in young patients) following a single injection is currently unknown.

To help address this, scientists from the University of Missouri and Pusan National University have examined the duration of the effect of a single systemic AAV micro-dystrophin treatment on whole body performance and heart function in mdx mice.

Three month old mdx mice were injected with AAV micro-dystrophin vector (1e13 vg) and evaluated at two time points – 12 and 22 months of age. Immunostaining and western blot showed robust micro-dystrophin expression both in the heart and skeletal muscle up to the end of the study.

Performance was evaluated by forelimb grip strength, treadmill running and serum CK levels. Treated mice significantly outperformed untreated mice in grip force and running distance at both time points, and treatment significantly reduced the CK level.

Significant improvements were also observed in ECG and cardiac hemodynamics. Several ECG parameters (e.g. heart rate, PR interval, QRS duration, and QTc interval) and hemodynamic parameters (e.g. end-diastolic/systolic volume, dP/dt max and min, max pressure, and ejection fraction) were completely normalized in treated mice at 22 months of age.

These results provide direct evidence that a single systemic AAV micro-dystrophin therapy has the potential to provide long-lasting benefits in mice.

In the middle of the third science-packed day of the ASGCT 2020 conference, the organization took a moment to unveil a new, modern logo to take them forward in this time of enormous progress in gene and cell therapies. 

PTC Therapeutics also sponsored a symposium and presented an overview of their gene therapy programs.  PTC has a long-standing commitment to Duchenne community, including commercialization of Emflaza and Ataluren (EU only), but is not pursuing gene therapy in Duchenne. 

Poster 188 – Lack of Toxicity in Non-Human Primates (NHPs) Receiving Clinically Relevant Doses of an AAV9.U7snRNA Vector Designed to Induce DMD Exon 2 Skipping

Dystrophinopathies are caused by a myriad of mutations of the DMD gene, and exon duplications are common and affect approximately 11% of all patients. Duplication of exon 2 (Dup2) is the most common single-exon duplication seen in Duchenne patients.

scAAV9.U7.ACCA was designed to contain four copies of the non-coding U7 small nuclear RNA targeting the splice sites of exon 2, and previously preclinical studies established its efficacy in vivo in restoring full-length dystrophin expression in the Dup2 mouse model of DMD

Researchers from Audentes Therapeutics and Nationwide Children’s Hospital (Columbus Ohio) have now reported on the non-clinical development studies with the AAV9.U7snRNA vector., This toxicology work was an important part of the preclinical development plan that led to the initiation of the ongoing gene therapy trial (https://clinicaltrials.gov/ct2/show/NCT04240314) to treat boys with DMD exon 2 duplications.

The toxicity of AAV9.U7snRNA was evaluated in male juvenile non-human primates (NHPs), where each received a single intravenous infusion of scAAV9.U7.ACCA vector of either 3e13 vg/kg (the minimal efficacious dose for a planned clinical trial), or 8e13 vg/kg, along with a cohort with vehicle control in a study performed in accordance with good laboratory practice.

Dose-dependent exon 2 skipping was confirmed in all muscle tissues (heart, quadriceps, diaphragm) and results showed that systemic delivery of AAV9.U7.ACCA to NHPs was safe and well-tolerated at clinically relevant doses. These findings supported initiating the first in human study of this vector for the treatment of exon 2 duplication Duchenne patients.

Poster 216 – Gene Editing with CRISPR/Cas9 to Correct DMD Exon Duplication Mutations in Patient-Derived Cells and Mice

In another body of work from researchers at Nationwide Children’s Hospital and also targeting duplication of DMD exon 2 (Dup2), a CRISPR/Cas9 system was used to eliminate the duplicated exon as a different potential therapeutic strategy.  This research is at very early stages but supports further exploration and development of CRISPR-mediated gene editing approaches for DMD duplication mutations. 

By targeting a small region shared by most Dup2 individuals, researchers designed CRISPR guide RNAs (gRNAs) that bind to this region in both copies of exon 2 and facilitate the cleavage of the DNA in between (thus leaving only one copy of exon 2).  This approach was successful in removing the duplicated exon in cells derived from human Dup2 individuals.  A similar strategy was also successful in targeting a larger duplication in exons 2 thru 7 (Dup2-7) in vitro.

The researchers also showed the CRISPR gene editing approach worked in vivo in Dup2 mice, resulting in partial restoration of muscle dystrophin expression.   Future studies will expand on these findings, determining the dose and timing of treatment that gives the largest effect when the CRISPR/Cas9 components are delivered to Dup2 mice via AAV9. 

1069 – AAV.U7 Induced Exon Skipping for a Mutational Hotspot (~6%) of the DMD Gene Results in Efficient Exon Skipping, Protein Restoration and Force Improvement

This collaboration between researchers at Nationwide Children’s Hospital (Columbus Ohio) and UCLA focused on exon 44 in the DMD gene coding for dystrophin.  Exon 44 is within a mutation hotspot in the gene and skipping of exon 44 could be potentially beneficial to approximately 6% of patients. 

AAV with a U7 promoter and various constructs targeting exon 44 were first tested in myotubes derived from skin biopsies from 3 DMD patients, to select for the constructs which most effectively induced skipping of exon 44. 

The top 3 were then tested in a new humanized DMD mouse model lacking exon 45, called hDMDdel45/mdx.  The absence of exon 45 in this mouse disrupts the reading frame of the DMD gene, and the mouse does not express any dystrophin.  The goal of skipping exon 44 in these mice already lacking exon 45 is to restore the reading frame and allow for expression of a truncated dystrophin (which lacks only exons 44 and 45). 

Intramuscular injection of AAV.U7-ex44 in hDMDdel45/mdx resulted in efficient exon 44 skipping and expression of a truncated dystrophin at 1- and 3- months post injection.  Muscle strength (tibialis anterior specific force and eccentric contraction) was significantly improved when tested 3- months post injection (compared to untreated animals).

With these promising results, the next steps are to find the minimal efficacious dose (MED) and move to systemic injections to determine the effect at the whole-body level.   

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