1: Gene Therapy and Regulation of Gene Expression, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona    2: Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona    3: Bioinformatics Core, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona    4: Grousbeck Gene Therapy Center, Schepens Eye Research Institute, Mass Eye and Ear, Boston, MA    5: Ocular Genomics Institute, Mass Eye and Ear, Harvard Medical School, Boston, MA

Targeted gene disruption mediated by programmable nuclease generates double-strand breaks (DSB) that are prevalently repaired by the error-prone non-homologous end-joining system (NHEJ). NHEJ introduces unpredictable deletions or insertions (indels) resulting in frameshift events or the introduction of early stop codon leading to the disruption of the gene function.

To minimize the variability of NHEJ in vivo, we co-injected two AAV-Cas9 nucleases carrying two different guides that target close regions in the Hao1 gene in a mouse model of primary hyperoxaluria type I (PH1). NGS analysis showed an overall deletion of the sequence in between the two guide-targeted sequences, resulting in the complete elimination of the translated protein, as measured by western blot. However, this strategy might duplicate the chance of off-target events, a rising safety concern for clinical applications.

Thus, to generate a safer gene disruption, paired gRNAs were combined with the nickase variant of Cas9. Nickase Cas9 creates nicks instead of DSB, which are sensed by high-fidelity single-strand break repair pathways. Therefore, simultaneous nicks, appropriately spaced and oriented may lead to site-specific DSB and gene knockout.

Recently, we demonstrated that the simultaneous administration of two AAV-nickase Cas9, carrying the guides described above, mediated highly efficient disruption of Hao1 in vivo. By NGS analysis on-target, we determined that paired nickase Cas9 system do not lose the editing efficiency of paired nuclease Cas9. Additionally, in animals treated with only one guide we observed that individual nicks were faithfully repaired, as detected by NGS and normal target gene expression, thus minimizing potential off-target.

Moreover, we proved that the minimal AAV dose for targeted gene knockout can be reduced by combining nickase Cas9 and the two gRNAs in a single AAV vector (AAV-all-in-one). Additionally, we observed that the all-in-one system allowed the reduction of the vector dose to achieve a similar effect. More importantly, we established that Hao1 disruption, mediated by AAV-all-in-one, at the minimal tested dose, is therapeutically relevant, in a mouse model of PH1.

Finally, CAST-seq quantitative analysis of eventual chromosomal rearrangements in vivo will further investigate the safety and clinical relevance of paired nickase system, for diseases that could benefit from permanent gene disruption like PH1.