Muscle-specific, liver-detargeted adeno-associated virus gene therapy rescues Pompe phenotype in adult and neonate Gaa-/- mice.

Pompe disease (PD) is a neuromuscular disorder caused by acid α-glucosidase (GAA) deficiency. Reduced GAA activity leads to pathological glycogen accumulation in cardiac and skeletal muscles responsible for severe heart impairment, respiratory defects, and muscle weakness. Enzyme replacement therapy with recombinant human GAA (rhGAA) is the standard-of-care treatment for PD, however, its efficacy is limited due to poor uptake in muscle and the development of an immune response. Multiple clinical trials are ongoing in PD with adeno-associated virus (AAV) vectors based on liver- and muscle-targeting. Current gene therapy approaches are limited by liver proliferation, poor muscle targeting, and the potential immune response to the hGAA transgene. To generate a treatment tailored to infantile-onset PD, we took advantage of a novel AAV capsid able to increase skeletal muscle targeting compared to AAV9 while reducing liver overload. When combined with a liver-muscle tandem promoter (LiMP), and despite the extensive liver-detargeting, this vector had a limited immune response to the hGAA transgene. This combination of capsid and promoter with improved muscle expression and specificity allowed for glycogen clearance in cardiac and skeletal muscles of Gaa-/- adult mice. In neonate Gaa-/- , complete rescue of glycogen content and muscle strength was observed 6 months after AAV vector injection. Our work highlights the importance of residual liver expression to control the immune response toward a potentially immunogenic transgene expressed in muscle. In conclusion, the demonstration of the efficacy of a muscle-specific AAV capsid-promoter combination for the full rescue of PD manifestation in both neonate and adult Gaa-/- provides a potential therapeutic avenue for the infantile-onset form of this devastating disease.

Intramuscular plasmid DNA electrotransfer: biodistribution and degradation.

We have studied radiolabelled plasmid DNA biodistribution and degradation in the muscle at different times after injection, with or without electrotransfer using previously defined conditions. Radiolabelled plasmid progressively left the muscle and was degraded as soon as 5 min after plasmid injection, with or without electrotransfer. Autoradiography showed that the major part of injected radioactivity was detected in the interfibrilar space of a large proportion of the muscle. Large zones of accumulation of radioactivity, which seems to be contained in some fibres (more than 20 microm), were identified as soon as 5 min after electrotransfer. Such structures were never observed on slices of non-electrotransferred muscles. However, these structures were not frequent and probably lesional. The surprising fact is that despite the amount of intact plasmid having been greatly reduced between 5 min and 3 h after injection, the level of transfection remains unchanged whether electric pulses were delivered 20 s or 3 h after injection. Such a behavior was similarly observed when injecting 0.3, 3 or 30 microg of plasmid DNA. Moreover, the transfection level was correlated to the amount of plasmid DNA injected. These results suggest that as soon as it is injected, plasmid DNA is proportionally partitioned between at least two compartments. While a major part of plasmid DNA is rapidly cleared and degraded, the electrotransferable pool of plasmid DNA represents a very small part of the amount injected and belongs to another compartment where it is protected from endogenous DNAses.