Kim Shontz

A Phase 1/2a Follistatin Gene Therapy Trial for Becker Muscular Dystrophy

Becker muscular dystrophy (BMD) is a variant of dystrophin deficiency resulting from DMD gene mutations. Phenotype is variable with loss of ambulation in late teenage or late mid-life years. There is currently no treatment for this condition. In this BMD proof-of-principle clinical trial, a potent myostatin antagonist, follistatin (FS), was used to inhibit the myostatin pathway. Extensive preclinical studies, using adeno-associated virus (AAV) to deliver follistatin, demonstrated an increase in strength. For this trial, we used the alternatively spliced FS344 to avoid potential binding to off target sites. AAV1.CMV.FS344 was delivered to six BMD patients by direct bilateral intramuscular quadriceps injections. Cohort 1 included three subjects receiving 3 × 10(11) vg/kg/leg. The distance walked on the 6MWT was the primary outcome measure. Patients 01 and 02 improved 58 meters (m) and 125 m, respectively. Patient 03 showed no change. In Cohort 2, Patients 05 and 06 received 6 × 10(11) vg/kg/leg with improved 6MWT by 108 m and 29 m, whereas, Patient 04 showed no improvement. No adverse effects were encountered. Histological changes corroborated benefit showing reduced endomysial fibrosis, reduced central nucleation, more normal fiber size distribution with muscle hypertrophy, especially at high dose. The results are encouraging for treatment of dystrophin-deficient muscle diseases.

Overexpression of Galgt2 in skeletal muscle prevents injury resulting from eccentric contractions in both mdx and wild-type mice

The cytotoxic T cell (CT) GalNAc transferase, or Galgt2, is a UDP-GalNAc:beta1,4-N-acetylgalactosaminyltransferase that is localized to the neuromuscular synapse in adult skeletal muscle, where it creates the synaptic CT carbohydrate antigen {GalNAcbeta1,4[NeuAc(orGc)alpha2, 3]Galbeta1,4GlcNAcbeta-}. Overexpression of Galgt2 in the skeletal muscles of transgenic mice inhibits the development of muscular dystrophy in mdx mice, a model for Duchenne muscular dystrophy. Here, we provide physiological evidence as to how Galgt2 may inhibit the development of muscle pathology in mdx animals. Both Galgt2 transgenic wild-type and mdx skeletal muscles showed a marked improvement in normalized isometric force during repetitive eccentric contractions relative to nontransgenic littermates, even using a paradigm where nontransgenic muscles had force reductions of 95% or more. Muscles from Galgt2 transgenic mice, however, showed a significant decrement in normalized specific force and in hindlimb and forelimb grip strength at some ages. Overexpression of Galgt2 in muscles of young adult mdx mice, where Galgt2 has no effect on muscle size, also caused a significant decrease in force drop during eccentric contractions and increased normalized specific force. A comparison of Galgt2 and microdystrophin overexpression using a therapeutically relevant intravascular gene delivery protocol showed Galgt2 was as effective as microdystrophin at preventing loss of force during eccentric contractions. These experiments provide a mechanism to explain why Galgt2 overexpression inhibits muscular dystrophy in mdx muscles. That overexpression also prevents loss of force in nondystrophic muscles suggests that Galgt2 is a therapeutic target with broad potential applications.

AAV.Dysferlin Overlap Vectors Restore Function in Dysferlinopathy Animal Models

Dysferlinopathies are a family of untreatable muscle disorders caused by mutations in the dysferlin gene. Lack of dysferlin protein results in progressive dystrophy with chronic muscle fiber loss, inflammation, fat replacement, and fibrosis; leading to deteriorating muscle weakness. The objective of this work is to demonstrate efficient and safe restoration of dysferlin expression following gene therapy treatment.

Safety and Efficacy of AAV Retrograde Pancreatic Ductal Gene Delivery in Normal and Pancreatic Cancer Mice

Recombinant adeno-associated virus (rAAV)-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9) expressing GFP in a self-complementary (sc) AAV vector under an EF1α promoter (scAAV.GFP) following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 1012 viral genomes (vg). Intraductal delivery of 1 × 1011 vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, ∼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 1011 vg. In a KrasG12D-driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.

609. Systemic Delivery of Dysferlin Overlap Vectors Mediates Functional Recovery of Dysferlin Deficiency

Dysferlinopathies comprise a family of disorders caused by mutations in the dysferlin (DYSF) gene leading to absent or mutant protein. Dysferlin protein has been implicated in multiple functional roles specifically in membrane stabilization/repair, t-tubule formation and vesicle trafficking. The loss of dysferlin causes a progressive dystrophy characterized by chronic muscle fiber loss, fat replacements and fibrosis resulting in deteriorating muscle weakness. Efforts made in the gene therapy arena with dysferlin or surrogate gene replacement have shown some efficacy in restoring membrane repair, however, only delivery of full-length DYSF has been able to correct the underlying histopathology. Therefore, there is a strong rationale to develop therapies that deliver the entire DYSF cDNA. A potential issue with this is the size of the DYSF gene (6.5 kb) which is too large for canonical AAV packaging. To circumvent this, we have developed and previously shown efficacy with a unique dual vector system using AAV to deliver and express DYSF specifically in muscle cells. This two vector system (AAV.DYSF.DV) packaged in the rh. 74 serotype is defined by a 1 kb region of homology between the two vectors. Following delivery to muscle, this overlap serves as a substrate for recombination/repair to generate the full-length gene. Our previous work studied the efficacy of this treatment strategy through intramuscular and regional vascular delivery routes. However, as generalized muscle weakness is common in dysferlinopathies, therapies targeting all muscle groups are warranted to maximize clinical efficacy. In this update, we have treated dysferlin-deficient mice systemically by intravenous injection to target all muscles through the vasculature for efficacy and safety studies. Mice were evaluated at 3 and 6 months post-treatment for dysferlin expression, restoration of membrane repair capability, diaphragm specific force measurements and muscle histology. Additional animals are awaiting MRI analysis following 1 year of treatment. A single systemic dose of 6×1012 vector genomes (3×1012 vg of each vector) resulted in widespread gene expression exceeding 30% of muscle fibers. Treated muscles showed a significant decrease in central nucleation and collagen deposition. Membrane repair ability was improved toward wild-type and force deficits in the diaphragm were restored to wildtype levels. The mice showed no evidence of local or systemic toxicity, further confirming previous safety data. This study, in conjunction with our previous work, lays the foundation for clinical trial.

C-2. Eteplirsen, a Phosphorodiamidate Morpholino Oligomer (PMO) for the Treatment of Duchenne Muscular Dystrophy (DMD): 168 Week Update on Six-Minute Walk Test (6MWT), Pulmonary Function Testing (PFT), and Safety

DMD is a rare, degenerative, X-linked recessive genetic disease that results in progressive muscle loss and premature death. DMD is caused by mutations in the dystrophin gene that lead to a reading frame shift and premature translation termination. Exon skipping, a promising disease-modifying approach for DMD, can be induced by eteplirsen, a charge neutral PMO that selectively binds to exon 51 of dystrophin pre-mRNA, restoring the open reading frame and enabling production of an internally truncated yet functional dystrophin protein as found in the less severe dystrophinopathy, Becker muscular dystrophy (BMD).

G.O.24

DMD, a rare, degenerative, genetic disease that results in progressive muscle loss and premature death affects 1:5000 male births. It is caused by deletions in the dystrophin gene, which prevents production of the dystrophin protein. There are no approved treatments available, although corticosteroids have shown some benefit. Eteplirsen is an investigational drug designed to enable functional dystrophin production in boys who are amenable to skipping exon 51. Twelve boys aged 7–13years with eligible genotypes were randomized 1:1:1 to eteplirsen 30mg/kg/wk, 50mg/kg/wk, or placebo IV for 24-weeks. All patients transitioned into an ongoing open-label extension with 30 or 50mg/kg eteplirsen. Clinical efficacy endpoints included the 6-min walk test (6MWT) and pulmonary function testing. Safety assessments included adverse event recording, EKG, ECHO, hematology, blood chemistry and urinalysis. After 120weeks of treatment, a significant clinical benefit of 65m was observed ( p =0.006) on the 6MWT for ambulatory-evaluable patients in the combined eteplirsen-treated cohorts ( n =6) versus the placebo/delayed-treatment cohort ( n =4). The eteplirsen-treated cohorts showed a decline of less than 14m in walking ability from baseline, which was not statistically significant. After a substantial decline in the first 36weeks of the study, the placebo/delayed-treatment cohort demonstrated stabilization in walking ability at week 48, i.e., 12weeks after initiation of treatment with eteplirsen when meaningful levels of dystrophin were likely produced, with