Abstract/Summary

The use of Adeno-associated viral (AAV) vectors for gene therapy has been most promising because of their safety profile; however, current production methods limit desirable amounts of AAV vectors. Significant efforts have been made to improve AAV production systems, including the optimisation of vector expression cassettes and the regulation of producer cell factors. In this study, we endeavoured to alter the host cell gene expression using CRISPR/Cas9 technology to improve AAV production. We generated knockouts of Y-Box protein (YB)1 in 293T, and the putatively identified homologue Spodoptera frugiperda Y-Box protein (SfYB) in Sf9 cells. We present the first example in which CRISPR/Cas9 genome editing can be utilised to regulate cell-intrinsic factors that may be implicated in AAV vector production; although, the disruption of YB1 or SfYB did not generate enhanced AAV vector producer cell lines. YB1 knockout cells presented with heightened sensitivity to chloroquine and limited its use for AAV vector production. The protective function of YB1 to chloroquine-induced cytotoxicity was demonstrated, and analysis of YB1 mutants suggested that its cold shock domain was the principle mediator of this resistance. We also identified YB1 associations with AAV serotype 2 (AAV2) inverted terminal repeat (ITR) in vitro, and a rather distinct colocalisation between YB1 mutant – encompassing YB1’s C-terminal domain (CTD) and AAV2 intact particle and AAV2 capsid to the nucleolar compartment. Therefore, there may be associations between YB1 and its CTD in AAV2 vector production. We also present the advantages of using a repertoire of assays to characterise CRISPR/Cas9-edited cell lines. This included the advantage of establishing clonal populations that were homozygous for their knockout mutation(s), and utilising target-specific antibodies for screening knockouts. Regardless, CRISPR/Cas9 has become a mainstream technology allowing for specific and efficient genome editing, and is revolutionising human gene therapy with the potential of giving rise to an entirely new class of therapeutics.