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CRISPR editing of muscle stem cells (MuSCs) with adeno-associated virus serotype-9 (AAV9) holds promise for sustained gene repair therapy for muscular dystrophies. However, conflicting evidence exists on whether AAV9 transduces MuSCs. To rigorously address this question, we used a muscle graft model. The grafted muscle underwent complete necrosis before regenerating from its MuSCs. We injected AAV9.Cre into Ai14 mice. These mice express tdTomato upon Cre-mediated removal of a floxed stop codon. About 28%–47% and 24%–89% of Pax7(+) MuSCs expressed tdTomato in pre-grafts and regenerated grafts (p > 0.05), respectively, suggesting AAV9 efficiently transduced MuSCs, and AAV9-edited MuSCs renewed successfully. Robust MuSC transduction was further confirmed by delivering AAV9.Cre to Pax7-ZsGreen-Ai14 mice in which Pax7(+) MuSCs are genetically labeled by ZsGreen. Next, we co-injected AAV9.Cas9 and AAV9.gRNA to dystrophic mdx mice to repair the mutated dystrophin gene. CRISPR-treated and untreated muscles were grafted to immune-deficient, dystrophin-null NSG.mdx4cv mice. Grafts regenerated from CRISPR-treated muscle contained the edited genome and yielded 2.7-fold more dystrophin(+) cells (p = 0.015). Importantly, increased dystrophin expression was not due to enhanced formation of revertant fibers or de novo transduction by residual CRISPR vectors in the graft. We conclude that AAV9 effectively transduces MuSCs. AAV9 CRISPR editing of MuSCs may provide enduring therapy.

Recombinant adeno-associated virus (AAV)-based gene delivery is a promising approach to treat muscle diseases. However, body-wide muscle delivery and pre-existing immune responses pose significant challenges to AAV muscle gene therapy. While the determinants of tissue tropism and immunogenicity of AAV are amenable to traditional molecular engineering, the development of a muscle-specific, immunosilent AAV vector has remained elusive. Recent advances in understanding the relationship between capsid structural motifs and functional domains have created exciting developments in the search for a muscle-specific AAV. Novel approaches to generate unique AAV properties through forced evolution have resulted in capsids with improved immune properties and/or muscle-targeting efficiency. Optimization of the gene cassette to restrict expression to mature muscle fibers provides another level of control. These reengineered AAV vectors have the potential to greatly increase efficacy and reduce off-target effects for body-wide muscle gene therapy. In this chapter, we discuss recent advances in the development of a next-generation, muscle-specific AAV vector.

Duchenne muscular dystrophy (DMD) is a X-linked, progressive childhood myopathy caused by mutations in the dystrophin gene, one of the largest genes in the genome. It is characterized by skeletal and cardiac muscle degeneration and dysfunction leading to cardiac and/or respiratory failure. Adeno-associated virus (AAV) is a highly promising gene therapy vector. AAV gene therapy has resulted in unprecedented clinical success for treating several inherited diseases. However, AAV gene therapy for DMD remains a significant challenge. Hurdles for AAV-mediated DMD gene therapy include the difficulty to package the full-length dystrophin coding sequence in an AAV vector, the necessity for whole-body gene delivery, the immune response to dystrophin and AAV capsid, and the species-specific barriers to translate from animal models to human patients. Capsid engineering aims at improving viral vector properties by rational design and/or forced evolution. In this review, we discuss how to use the state-of-the-art AAV capsid engineering technologies to overcome hurdles in AAV-based DMD gene therapy.

Gene therapy is on the verge of producing viable treatments for numerous diseases. Millions of years of viral evolution has made virus an ideal system to deliver foreign genes to host cells. Here we discuss the most common viral vectors used in gene therapy such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, and poxvirus. For each viral vector, we review the virus biology, viral vector development and relevant clinical applications.