733. Somatically Repairing Compound Heterozygous Recessive Mutations by Chromosomal Cut-and-Paste for In Vivo Gene Therapy

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Patients affected by monogenic recessive genetic disorders often carry two different mutated alleles of the same gene, which is known as compound heterozygous. Theoretically, exchanging the genetic material between the two mutated alleles will reconstitute a mutation-free allele that can be therapeutic (FigureFigure). We hypothesized that generating DNA double-stranded breaks at the same location on both mutant alleles can induce allele exchange, reconstitute a mutation-free allele, and therefore yield therapeutic benefit. We first tested this hypothesis in a targeted knock-in mouse model that carries GFPN-term-intron-tdTomatoC-term and tdTomatoN-term-intron-GFPC-term expression cassettes, respectively, at the same genomic location on each copy of Chr 11. Therefore, allele exchange at the intronic region will reconstitute the full-length GFP and tdTomato, serving as a reporter system. We injected recombinant AAV (rAAV) vectors expressing SpCas9 and sgRNA targeting the intron into adult mice by tail vein injection. Five weeks later, we observed GFP and tdTomato fluorescence in cryosections of peripheral tissues including liver and heart, whereas there was no such fluorescence observed in the tissue samples from untreated mice, demonstrating that allele exchange occurred, and that the reconstituted alleles yielded protein expression. Furthermore, we generated mice that carry two different mutations of the Aspa gene as a compound heterozygous mouse model of Canavan disease. We treated these mice with rAAV vectors expressing SpCas9 and sgRNA targeting an intron between the two mutation sites. Three weeks after treatment, we detected reconstituted, mutation-free Aspa DNA sequence by allele-specific PCR and single-molecule, high-throughput DNA sequencing in the liver. The reconstituted Aspa allele carried insertion at the predicted SpCas9 cleavage site, indicating that the DNA allele exchange was mediated by the non-homologous end joining DNA repair pathway. We also observed allele exchange in mouse liver using the SaCas9 system. Evaluation of the therapeutic benefit following Cas9/sgRNA-mediated allele exchange in various compound heterozygous mouse models and patient cell lines is underway. The gene repairing strategy described here is a novel approach to tackling a broad range of autosomal recessive genetic disorders.View Large Image | Download PowerPoint Slide