Gene Editing in Hemophilia: A “CRISPR” Choice?

The use of CRISPR/Cas9 gene editing can have a potentially positive impact on patients with hemophilia, according to the authors of a review that was published in the journal Blood.

Hemophilia B (HemB) stands as an attractive target for gene therapy trials, and specifically, studies implementing adeno-associated viral (AAV) vectors, without any genome editing, have shown efficacy in the phenotype that causes severe bleeding in adults with HemB, thus eradicating the need for preventative FIX replacement therapy. Although the durability of this treatment in table FIX expression has recently been demonstrated, according to the authors, “these long-lasting therapeutic effects may not be reproducible in pediatric patients, those with the most to gain from effective bleed control, because of high hepatocyte cellular turnover, resultant dilution of transduced cells, and eventual loss of transgene expression.

Recently, gene correction approaches have emerged as viable alternatives to nongenomic editing therapies to treat diseases such as HemB, and these techniques include zinc-finger nuclease (ZFN)-based HDR, transcription activator-like effector nuclease-based HDR, and CRISPR/Cas9 technology, which appears the most viable option. A previous study conducted by Wang et al showed that AAV- and CRISPR/Cas9-mediated gene targeting resulted in clinically valuable expression of human FIX when used in a HemB mouse model, and because this gene-editing approach was not mutation position specific, it could be applied to most HemB patients.

In the study, a dual AAV8 vector system that includes a donor vector that contained partial human FIX complementary DNA (exons 2 to 8) carrying the hyperactive FIX Padua mutation and a single guide RNA vector was used to target exon 2 of murine FIX. By pinpointing this region of the FIX gene, the researchers were able to impact applicability because most (up to 95%) of HemB mutations are nearest to exon 1. This approach has been described in previous studies however, the researcher increased the Fix-specific activity 10-fold. Moreover, they displayed that the treated mice survived a partial hepatectomy 8 weeks subsequent to vector injection endured for an additional 24 weeks, suggesting stable gene targeting.

Although the Wang et al study reported promising results to support the use of CRISPR in hemophilia, this review notes that there are still unresolved issues that require careful attention before applying this modality in a clinical setting. For one, elevated vector dose used in this approach highlights concerns about AAV-related toxicity, and moreover, the strategy of gene targeting the FIX exon 2 will not serve HemB patients with mutations within specific FIX promoter regions or the signal peptide. The review says that the possibility of off-target edits “is particularly relevant and needs careful assessment not just by in silico techniques, but also by experimental analyses described recently.”

The authors concluded by writing that to “supplant current therapies with gene therapy in general, and gene editing in particular, careful consideration needs to be given to identifying and eliminating possible off-target edits, choosing an appropriate and safe vehicle for in vivo delivery without any associated toxicity, and assessment of the risk-to-benefit ratio for each individual.”

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