Full-length dystrophin restoration via targeted exon integration by AAV-CRISPR in a humanized mouse model of Duchenne muscular dystrophy

Abstract

Targeted gene-editing strategies have emerged as promising therapeutic approaches for the permanent treatment of inherited genetic diseases. However, precise gene correction and insertion approaches using homology-directed repair are still limited by low efficiencies. Consequently, many gene-editing strategies have focused on removal or disruption, rather than repair, of genomic DNA. In contrast, homology-independent targeted integration (HITI) has been reported to effectively insert DNA sequences at targeted genomic loci. This approach could be particularly useful for restoring full-length sequences of genes affected by a spectrum of mutations that are also too large to deliver by conventional adeno-associated virus (AAV) vectors. Here, we utilize an AAV-based, HITImediated approach for correction of full-length dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy (DMD). We co-deliver CRISPR-Cas9 and a donor DNA sequence to insert the missing human exon 52 into its corresponding position within the DMD gene and achieve full-length dystrophin correction in skeletal and cardiac muscle. Additionally, as a proof-of-concept strategy to correct genetic mutations characterized by diverse patient mutations, we deliver a superexon donor encoding the last 28 exons of the DMD gene as a therapeutic strategy to restore full-length dystrophin in >20% of the DMD patient population. This work highlights the potential of HITI-mediated gene correction for diverse DMD mutations and advances genome editing toward realizing the promise of full-length gene restoration to treat genetic disease.

Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing

“…16,18). While lower count numbers were detected in some samples, they are nonetheless in accordance to published literature 49,51,52 . CIRCLE-seq predicted off-target sites were PCR amplified and subjected to NGS analysis, wherein no difference in off-target CRIPSR activity was seen in CCExo compared to Cas9 treated cells, even in some K562 cell samples, where higher off-target activity was predicted by CIRCLE-seq analysis (Fig.…”

CRISPR-mediated optogene expression from a cell-specific endogenous promoter in retinal ON-bipolar cells to restore vision

“…For HITI, we compared the most known and widely used SpCas9 (~4.1 kb) with the smaller SaCas9 (~3.1 kb), which would allow us to drive nuclease expression by ON-BPC-specific promoters (Hulliger et al, 2020), thus potentially reducing offtarget expression and increasing the safety of the system. However, unlike observations by others (Pickar-Oliver et al, 2021), SaCas9 turned out to be less efficient compared to SpCas9. For MITI, we compared LbCpf1 and AsCpf1, which are similar in size (~3.9 kb) and recognize the same PAM sequence.…”

CRISPR applications for Duchenne muscular dystrophy: From animal models to potential therapies

“…The first hDMDTg KO model was the hDMDTgEx52∆/ mdx model, generated using TALEN‐mediated gene targeting (homologous recombination) in hDMDTg/ mdx mouse ES cells (Veltrop et al, 2018 ). Another hDMDTgEx52∆/ mdx model has also been independently generated by CRISPR microinjection of hDMDTg/ mdx zygotes to remove exon 52 of hDMDTg (Pickar‐Oliver et al, 2021 ). CRISPR was also used to generate hDMDTgEx45∆/ mdx and hDMDTgEx45∆/ mdxD2 mice by first inducing the deletion of exon 45, by zygotic microinjection of Cas9 mRNA with two pairs of gRNAs targeting the introns flanking exon 45 (Young et al, 2017 ).…”