(F-206) Evaluation of precision and efficacy of CRISPR-mediated transgene integration at muscle-specific safe-harbor loci.

Introduction:: In vivo genome engineering has shown promise in treating inherited disease in preclinical models and in clinical trials of inherited blindness and amyloidosis. Yet significant challenges remain to creating precise DNA corrections and gene replacement in vivo. An approach that enabled precise gene integration could address heterogenous patient mutations and correct larger deactivating mutations. Targeted integration of therapeutic transgenes has advanced considerably with advantages over the semi-random integration pattern of gammaretroviral vectors and lentiviral vectors. CRISPR-based integration has emerged as a potential route for long-term therapeutic transgene delivery. Despite major efforts, there has been limited research on the safety and efficacy of targeted gene integration in skeletal muscle. In this study, we identified new potential safe harbor sites in skeletal muscle that can provide safe and stable integration and replacement gene expression. The selection of skeletal muscle as a gene therapy integration site can decrease the invasiveness of the procedure, with minimal local or systemic toxicity associated with intramuscular injection.


Materials and Methods:: We validated two high-expressing skeletal-muscle loci as safe-harbor sites by integrating a promoterless reporter gene (green fluorescent protein, GFP) and therapeutic gene (human factor 9, hFIX) with homology-independent targeted integration (HITI). HITI promotes transgene integration by simultaneous nuclease activity at the target locus and the donor DNA. We co-delivered CRISPR-Cas9 specific for the intronic region of the target loci and a HITI donor insert plasmid with a panel of candidate splice sites to hijack the endogenous promoter of muscle-specific loci in myoblasts. The promoter of the endogenous gene is leveraged to achieve high-level and constitutive gene expression of the transgene with limited possibility of silencing, which is a major concern when expressing the transgene with exogenous promoter. We evaluated gene expression with qRT-PCR and protein expression with ELISA and western blot. Precision was evaluated with unbiased deep sequencing methods and specificity was determined with RNA sequencing of modified myoblasts.


Results, Conclusions, and Discussions:: Targeted integration of green fluorescent protein (GFP) demonstrated precise in-frame integration of GFP transgene in mouse DNA and mRNA by deep sequencing at both 5’ and 3’ end junctions between the target locus integration site and the GFP insert. Using PCR-enriched Illumina short-read sequencing, we confirmed that the precision of integration of GFP is 60% at the DNA level and up to 96% at the mRNA level. This suggests that integrating in the intronic region reduced unintended transcription modifications. To further investigate the efficiency and precision of integration, we also performed high-throughput short- and long-read sequencing using unbiased tagmentation-based library construction or CRISPR-based enrichment strategies to eliminate the bias of PCR-enriched quantification efficiency. We then applied this approach to integrate a promoterless hFIX therapeutic gene in mouse myoblasts. At the RNA level, we analyzed the integration of hFIX in the muscle-specific locus in mouse myotubes by qRT-PCR analysis. This integration resulted in a significant increase in relative hFIX gene expression up to 2,500-fold higher than that observed in a control group treated with scrambled gRNA group (Fig. 1A). We determined a dramatic increase in hFIX protein production in mouse myotubes that were treated, as determined by an ELISA. The concentration of the excreted hFIX protein was found to be between 0.5-1.9 ng/mL (Fig. 1B). Furthermore, we also assessed the alteration in global transcriptome after hFIX integration in mouse genome in vitro by RNA sequencing to evaluate and determine the safety of the loci. The study's findings present a potentially valuable approach for creating a reliable and stable gene therapy through intramuscular injection. Additionally, the targeted integration technique employed in this study may be adaptable to hundreds of genetic disorders that involve the loss of serum proteins and serve as a platform for in vivo synthetic biology.


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