[Background] Recombinant adeno-associated viruses (AAVs) have been used to manipulate transgene expression for scientific investigations and therapeutic applications in mice and humans. AAVs have various serotypes, and the efficiency of organ-specific gene transfection varies among serotypes. AAV serotype 1 (AAV 1), the most conventional serotype, has been widely applied based on its broad organ specificity. Tabebordbar et al. recently showed improved transgene expression in skeletal muscles using a modified AAV (MyoAAV 1A). The comparison of transfection efficiency between AAV 1 and MyoAAV 1A in undifferentiated or differentiated C2C12 myocytes and mouse skeletal muscles via intravenous injection has not been thoroughly investigated.

[Material and Methods] AAV was produced by transfection of three plasmid vectors into HEK293T cells to produce infectious viral particles: pAAV-CMV-GFP, pHelper, and pAAV-RC (rep/cap expression). Plasmid pAAV-RC encodes serum-specific capsid protein for either AAV 1 or MyoAAV 1A. The produced AAV was purified, and viral yield was quantified by real-time PCR targeting the common region of the AAV viral genome. Undifferentiated C2C12 myoblasts were allowed to differentiate into myocytes under the 2% horse serum condition. C2C12 myoblasts and myocytes were infected by adding AAV 1 and MyoAAV 1A to the medium at progressively higher titers (10^7, 10^8, and 10^9 viral genomes per μL, vg/μL) and incubated for 48 hours. Gene delivery efficiency was evaluated by GFP expression at 48 and 96 hours after the start of AAV incubation. For in vivo study, mice were injected intravenously with 2x10^12 vg/kg of AAV 1 or MyoAAV 1A or the same volume of saline for control. Gastrocnemius muscle was harvested seven days after injection. GFP expression was assessed by immunohistochemical staining and Western blotting.

[Results] In C2C12 myoblast, greater GFP expression was observed in AAV 1 compared to MyoAAV 1A. The ratio of GFP-positive cells was 80.4 ± 5.0% and 5.6 ± 1.3% for AAV 1 and MyoAAV 1A, respectively. In contrast, the efficiency of gene transduction on differentiated myocytes was superior with MyoAAV 1A; 13.7 ± 2.7% and 86.4 ± 2.8% (Fig. 1). GFP expression was confirmed in gastrocnemius muscle injected intravenously with MyoAAV 1A as analyzed by Western blotting and immunostaining, while that with AAV 1 was marginal compared to the control.

[Discussion] Skeletal muscle also functions as an endocrine organ. Cytokines released from skeletal muscle are called myokines and have systemic effects. This study suggests that skeletal muscle can serve as a transgene expression site for cytokines/myokines and deliver them systemically. Transgenic delivery to skeletal muscle can be potentially beneficial to multiple organs in a variety of pathological conditions. The high transfection efficiency of MyoAAV 1A, both in vitro and in vivo, especially that in differentiated myocytes, demonstrated in this study, is expected to contribute to the development of gene therapy.

[Conclusion] MyoAAV 1A is more efficient for gene transduction in skeletal muscles than AAV 1, exerting more specific expression in differentiated myocytes, and will be thus helpful in AAV-based myocyte gene therapy.