Improved viral vector design for homology-independent targeted integration (HITI)

Gene therapies for recessive disorders are becoming commonplace, but treatments for dominant gain-of-function disorders are under-researched due to the need to achieve both knock down of the dominant mutant allele and replacement of normal function. This project aims to design high-efficiency AAV vectors with the additional complexity required to tackle dominant disorders. Cas9-mediated Homology-Independent Targeted Integration (HITI) facilitates insertion of an arbitrary sequence at any selected gene editing nuclease cleaved PAM site (Suzuki 2016 PMID:27851729). HITI utilises the NHEJ-pathway, which is substantially more efficient than conventional HDR pathways, and therefore offers more realistic in vivo gene editing strategies. We have a 'HITI reporter' mouse model to allow in vivo HITI to be assessed in any organ. We have preliminary data with AAV Cas9 HITI vectors showing controlled, error-free, PAM-targeted, DNA insertion into mouse liver cells in vivo. As a disease exemplar, we plan to use HITI for the dominant liver disorder, alpha-1 antitrypsin (AAT) deficiency. Specifically, we will target the common, dominant AAT genetic variant (PiZ) that leads to AAT protein precipitation in hepatocytes causing deficient disease-causing levels in the lung. Depending on project progression there is also the opportunity to develop the HITI vector platform for other organs.