119. Engineering a Self-Inactivating CRISPR System for AAV Vectors

Significant strides in nuclease engineering have enabled a broad range of biomedical applications. However, numerous challenges remain, and in particular the long term expression mediated by AAV delivery of nuclease-encoding constructs to post-mitotic cells raises concerns with specificity and immunogenicity. That is, off-target nuclease activity can induce genotoxicity, and expression of an exogenous nuclease has the potential to elicit an immune response against transduced cells. Thus, it would be advantageous to limit the duration of nuclease expression following delivery. We have engineered self-inactivating nuclease constructs using the CRISPR/Cas9 system, which consist of a Cas9 nuclease such as that from Streptococcus pyogenes (SpCas9), a chimeric single guide RNA (sgRNA) molecule for targeting, and flanking sites targeted by that sgRNA. For example, we modified an SpCas9/sgRNA construct targeted at the VEGFA locus by introducing copies of the target site flanking the nuclease construct. The result is a negative feedback loop where Cas9 cuts both the target genomic locus and its own coding construct and thereby self-limits its expression. We demonstrate that this construct can eliminate >90% of its expression within 72 hours, and tuning of different parameters enables retention of up to 65% on-target efficiency and reduction of off target-cutting by up to 80%. We further show that by engineering the flanking target sites to contain mismatches to the sgRNA, the Cas9 expression duration can be modulated and the on-target/off-target cutting ratios improved. By retaining strong activity while minimizing off-target effects and by eliminating potentially immunogenic, long-term expression of foreign protein, these self-inactivating constructs have the potential to address two substantial concerns with therapeutic application of engineered nucleases.