Long-Term Efficacy of AAV9-U7snRNA-Mediated Exon 51 Skipping in mdx52 Mice.

Gene therapy and antisense approaches hold promise for the treatment of Duchenne muscular dystrophy (DMD). The advantages of both therapeutic strategies can be combined by vectorizing antisense sequences into an adeno-associated virus (AAV) vector. We previously reported the efficacy of AAV-U7 small nuclear RNA (U7snRNA)-mediated exon skipping in the mdx mouse, the dys - /utr - mouse, and the golden retriever muscular dystrophy (GRMD) dog model. In this study, we examined the therapeutic potential of an AAV-U7snRNA targeting the human DMD exon 51, which could be applicable to 13% of DMD patients. A single injection of AAV9-U7 exon 51 (U7ex51) induces widespread and sustained levels of exon 51 skipping, leading to significant restoration of dystrophin and improvement of the dystrophic phenotype in the mdx52 mouse. However, levels of dystrophin re-expression are lower than the skipping levels, in contrast with previously reported results in the mdx mouse, suggesting that efficacy of exon skipping may vary depending on the targeted exon. Additionally, while low levels of exon skipping were measured in the brain, the dystrophin protein could not be detected, in line with a lack of improvement of their abnormal behavioral fear response. These results thus confirm the high therapeutic potential of the AAV-mediated exon-skipping approach, yet the apparent discrepancies between exon skipping and protein restoration levels suggest some limitations of this experimental model.

Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system.

Recombinant adeno-associated viruses (rAAV) are largely used for gene transfer in research, preclinical developments, and clinical trials. Their broad in vivo biodistribution and long-term efficacy in postmitotic tissues make them good candidates for numerous gene transfer applications. Upstream processes able to produce large amounts of rAAV were developed, particularly those using baculovirus expression vector system. In parallel, downstream processes present a large panel of purification methods, often including multiple and time consuming steps. Here, we show that simple tangential flow filtration, coupled with an optimized iodixanol-based isopycnic density gradient, is sufficient to purify several liters of crude lysate produced by baculovirus expression vector system in only one working day, leading to high titers and good purity of rAAV products. Moreover, we show that the viral vectors retain their in vitro and in vivo functionalities. Our results demonstrate that simple, rapid, and relatively low-cost methods can easily be implemented for obtaining a high-quality grade of gene therapy products based on rAAV technology.

Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors.

Recombinant adeno-associated viruses (rAAV) are promising candidates for gene therapy approaches. The last two decades were particularly fruitful in terms of processes applied in the production and purification of this type of gene transfer vectors. This rapid technological evolution led to better yields and higher levels of vector purity. Recently, some reports showed that rAAV produced by transient tri-transfection method in adherent human embryonic kidney 293 cells can be harvested directly from supernatant, leading to easier and faster purification compared to classical virus extraction from cell pellets. Here, we compare these approaches with new vector recovery method using small quantity of detergent at the initial clarification step to treat the whole transfected cell culture. Coupled with tangential flow filtration and iodixanol-based isopycnic density gradient, this new method significantly increases rAAV yields and conserves high vector purity. Moreover, this approach leads to the reduction of the total process duration. Finally, the vectors maintain their functionality, showing unexpected higher in vitro and in vivo transduction efficacies. This new development in rAAV downstream process once more demonstrates the great capacity of these vectors to easily accommodate to large panel of methods, able to furthermore ameliorate their safety, functionality, and scalability.

Actin scaffolding by clathrin heavy chain is required for skeletal muscle sarcomere organization.

The ubiquitous clathrin heavy chain (CHC), the main component of clathrin-coated vesicles, is well characterized for its role in intracellular membrane traffic and endocytosis from the plasma membrane (PM). Here, we demonstrate that in skeletal muscle CHC regulates the formation and maintenance of PM-sarcomere attachment sites also known as costameres. We show that clathrin forms large coated lattices associated with actin filaments and the muscle-specific isoform of α-actinin at the PM of differentiated myotubes. Depletion of CHC in myotubes induced a loss of actin and α-actinin sarcomeric organization, whereas CHC depletion in vivo induced a loss of contractile force due to the detachment of sarcomeres from the PM. Our results suggest that CHC contributes to the formation and maintenance of the contractile apparatus through interactions with costameric proteins and highlight an unconventional role for CHC in skeletal muscle that may be relevant to pathophysiology of neuromuscular disorders.