In vivo Gene Editing of Muscles and Muscle Stem Cells using AAV-CRISPR

Abstract

Recent advances in targeted gene editing offer researchers unprecedented access to modify mammalian genomes for experimental and therapeutic purposes. Particularly, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system directs an RNA guided endonuclease (RGEN)-Cas9 to virtually any genomic location specified by nucleobase complementarity with a short guide RNA (gRNA) and generates a DNA double strand break (DBS) there. Cells mend such endonuclease-generated DSBs via non-homologous end joining (NHEJ) or homology-directed repair (HDR), which are the major cellular pathways that protect against genomic lesions and cell death. While NHEJ is active throughout the cell cycle and in non-dividing cells, it is relatively error-prone and produces variable sequence outcomes due to highly unpredictable nucleotide insertions and deletions at the DBS site. In contrast, HDR offers more precise gene-editing outcomes, as well as the unique capacity to introduce entirely new sequence elements; however, HDR is generally believed to be inefficient in post-mitotic organs and additionally requires homologous DNA present on either endogenous chromosomes or exogenous templates. Both CRISPR-mediated NHEJ and HDR have been extensively studied and utilized in vitro and ex vivo for basic research, genetic screening, and cell/gene therapy applications. Yet, the promise of these systems for in vivo gene editing of tissues and tissue stem cells in their native environment remains underexplored due to challenges of efficient delivery of genome modification enzymes to cells in vivo and lack of sensitive reporters that can detect and track successful gene editing outcomes at single cell level. In this thesis, I sought to address this significant gap in application of CRISPR editing in vivo. First, I describe a system for in vivo delivery of CRISPR-Cas9 endonucleases, coupled with paired gRNAs, via adeno-associated virus (AAV), and show that this system is effective for therapeutic gene modification in a mouse model of Duchenne’s muscular dystrophy (DMD). Specifically, in mdx mice carrying a nonsense mutation in the Dmd allele, AAV-CRISPR was shown to transduce affected cardiac and muscle cell lineages, and to genomically excise the mutated Dmd exon23 via NHEJ, correcting the Dmd reading frame and recovering Dystrophin expression and muscle function. AAV-CRISPR further transduced muscle satellite cells in situ and targeted genome modification in these regenerative muscle stem cells within their native niche. Finally, I used the AAV-CRISPR system to test the feasibility of achieving multi-organ HDR in vivo in postnatal mammals, and particularly, in postnatal stem cells, which provide a reservoir of regenerative cells to support ongoing tissue turnover and repair. For these efforts, I developed a GFP-BFP color-switching reporter system to track genome-editing outcomes in vivo at the single cell level, and showed that postnatal cardiac muscle, skeletal muscle, and muscle stem cells can be modified by AAV-CRISPR induced templated HDR at discrete developmental time points in mice. Together, this work provides a proof-of-principle for therapeutic gene editing of diseased muscle, documents robust AAV targeting of muscle stem cells in their native niche, and demonstrates sequence-directed, systemically disseminated, in vivo AAV-CRISPR-mediated HDR in heart, muscle, and muscle stem cells, providing new opportunities for therapeutic and experimental applications.