Systemic Delivery of Dysferlin Overlap Vectors Provides Long-Term Gene Expression and Functional Improvement for Dysferlinopathy

Abstract

Dysferlinopathies comprise a family of disorders caused by mutations in the dysferlin (DYSF) gene, leading to a progressive dystrophy characterized by chronic muscle fiber loss, fat replacement, and fibrosis. To correct the underlying histopathology and function, expression of full-length DYSF is required. Dual adeno-associated virus vectors have been developed, defined by a region of homology, to serve as a substrate for reconstitution of the full 6.5 kb dysferlin cDNA. Previous work studied the efficacy of this treatment through intramuscular and regional delivery routes. To maximize clinical efficacy, dysferlin-deficient mice were treated systemically to target all muscles through the vasculature for efficacy and safety studies. Mice were evaluated at multiple time points between 4 and 13 months post treatment for dysferlin expression and functional improvement using magnetic resonance imaging and magnetic resonance spectroscopy and membrane repair. A systemic dose of 6 × 1012 vector genomes resulted in widespread gene expression in the muscles. Treated muscles showed a significant decrease in central nucleation, collagen deposition, and improvement of membrane repair to wild-type levels. Treated gluteus muscles were significantly improved compared to placebo-treated muscles and were equivalent to wild type in volume, intra- and extramyocellular lipid accumulation, and fat percentage using magnetic resonance imaging and magnetic resonance spectroscopy. Dual-vector treatment allows for production of full-length functional dysferlin with no toxicity. This confirms previous safety data and validates translation of systemic gene delivery for dysferlinopathy patients.

Emerging Issues in AAV-Mediated In Vivo Gene Therapy

“… 31 The re-assembly of the full-length transgene expression cassette is then achieved upon co-infection of the same cell by both dual AAV vectors followed by: (1) homologous recombination (HR) between 5′ and 3′ genomes (dual AAV overlapping vectors; Figure 1 B); (2) ITR-mediated tail-to-head concatemerization of 5′ and 3′ genomes (dual AAV trans -splicing vectors; Figure 1 C); or (3) a combination of these two mechanisms (dual AAV hybrid vectors; Figure 1 D). 30 Remarkably, the use of dual AAV vectors in vivo results in the expression of full-length proteins and therapeutic efficacy, as demonstrated in several animal models of disease, 31 , 37 , 38 , 39 , 40 , 41 , 42 although the efficiency of these systems is still lower when compared with canonical single AAV vectors, thus requiring higher vector doses. 30 , 38 Nevertheless, for some applications, as seen in gene transfer directed to confined tissues such as the eye, the use of the dual AAV vector platform may represent an efficient and viable gene transfer strategy for transgenes of >4.7 kb in size.…”

Clinical development on the frontier: gene therapy for duchenne muscular dystrophy

“…Furthermore, because muscle cells are non-dividing and long-lived, there is potential that a one-time treatment could have long-term and perhaps even life-long benefits. In fact, preclinical and clinical studies in neuromuscular diseases to date indicate no loss in durability [25,[52][53][54][55]. The objective of gene therapy for DMD is to deliver a gene encoding a functional version of dystrophin systemically to all target tissues involved in DMD pathology and thereby ameliorate the progression of the disease.…”

“…Subsequent calculation of the number of transgenes per nucleus will determine if most cells are receiving the transgene, keeping in mind that muscle cells are multi-nucleated with up to a few hundred nuclei per cell [83]. Specifically, the rAAVrh74 capsid induces high-level transduction of both cardiac and skeletal muscle [54,84].…”

Emerging Concepts and Challenges in Rheumatoid Arthritis Gene Therapy

“…Dysferlin-deficient mice were treated systemically by two AAV vectors to provide reconstitution of the full 6.5 kb dysferlin cDNA. The treatment provided long-term gene expression and functional improvement for dysferlinopathy [144]. However, the clinical usability of such systems remains questionable.…”