Molecular and Biochemical Assessment of Gene Therapy in the Canine Model of Duchenne Muscular Dystrophy.

Mutations in the dystrophin gene result in Duchenne muscular dystrophy (DMD), a progressive muscle-wasting disease. Adeno-associated virus (AAV) mediated gene replacement, and CRISPR/Cas9-mediated genome editing hold the potential to treat DMD. Molecular and biochemical analyses are essential to determine gene transfer efficiency and therapeutic efficacy. In this chapter, we present a series of methods routinely used in our laboratory to extract and quantify DNA, RNA, and protein in gene therapy studies performed in the canine DMD model.

Assessment of the Gene Therapy Immune Response in the Canine Muscular Dystrophy Model.

The immune response is a primary hurdle in the development of gene therapy for neuromuscular diseases. Both innate and adaptive immune responses have been observed in human trials. The canine model is an excellent platform to understand immunological consequences of gene therapy. Over the last several decades, we have conducted gene replacement and gene repair therapies in the canine model of Duchenne muscular dystrophy (DMD) using adeno-associated virus (AAV)-mediated expression of the highly abbreviated micro-dystrophin gene, the larger mini-dystrophin gene, and the Cas9-based CRISPR genome editing machinery. We have evaluated the innate, humoral, and cellular immune responses to the AAV vector and the transgene product. In this chapter, we share our experience in collecting and processing of the dog blood samples for immunological assays, and our protocols for quantitative evaluation of cytokines and chemokines, antibodies, and T-cell responses.

Cas9-specific immune responses compromise local and systemic AAV CRISPR therapy in multiple dystrophic canine models.

Adeno-associated virus (AAV)-mediated CRISPR-Cas9 editing holds promise to treat many diseases. The immune response to bacterial-derived Cas9 has been speculated as a hurdle for AAV-CRISPR therapy. However, immunological consequences of AAV-mediated Cas9 expression have thus far not been thoroughly investigated in large mammals. We evaluate Cas9-specific immune responses in canine models of Duchenne muscular dystrophy (DMD) following intramuscular and intravenous AAV-CRISPR therapy. Treatment results initially in robust dystrophin restoration in affected dogs but also induces muscle inflammation, and Cas9-specific humoral and cytotoxic T-lymphocyte (CTL) responses that are not prevented by the muscle-specific promoter and transient prednisolone immune suppression. In normal dogs, AAV-mediated Cas9 expression induces similar, though milder, immune responses. In contrast, other therapeutic (micro-dystrophin and SERCA2a) and reporter (alkaline phosphatase, AP) vectors result in persistent expression without inducing muscle inflammation. Our results suggest Cas9 immunity may represent a critical barrier for AAV-CRISPR therapy in large mammals.