Recombinant adeno-associated viruses (rAAVs) are promising for use in liver targeted human gene therapy. However, for rAAV vectors to be useful for therapies of inborn errors affecting the liver, these vectors must be able to have continuous long-term and regulated expression of the transgene. Currently, standard rAAV vectors have strong promoters that can lead to hepatocellular carcinoma if random integration occurs in proximity to an oncogene. rAAVs have a limited payload capacity and would be unsuitable for gene therapy of mutations occurring in large genes. Additionally, rAAVs are mainly non-integrating and function therapeutically by episomal expression which can be lost during cell replication. One method to address these limitations is to directly repair the mutation in hepatocytes. However, efficiency of in vivo gene repair with currently available methods is low. Selection of hepatocytes with the corrected mutation is one way to increase the efficiency of in vivo gene correction. One way to achieve this is to use the selectable Gene Ride vector that has been described by our lab. The selectable Gene Ride vector provides a selectable advantage to correctly target hepatocytes, allowing their expansion over untargeted hepatocytes. In this proposal, we aim to develop the promoterless selectable Gene Ride vector to have broader clinical potential. To allow the Gene Ride vector to be functional in any gene, we propose to utilize the CRISPR/Cas9 gene editing system in place of shRNAs to overcome the limitations of shRNAs. shRNAs typically knock down but do not knock out a gene, while mutations caused by gRNAs can lead to gene knockout. Furthermore, high expression levels of shRNAs are required for gene knockdown. We hypothesize that lower levels of gRNAs will be sufficient for gene knockouts. In order to allow the gRNA to be expressed from an endogenous polymerase 2 promoter, we will flank the gRNA with ribozymes. These ribozymes are self-cleaving and allow release of the active gRNA. The current selectable Gene Ride vector utilizes the small molecule inhibitor CEHPOBA for selection. CEHPOBA is not an FDA approved drug, making translation into the clinic difficult. We propose to examine more clinically relevant gene knockout and drug selection combinations. This includes NTCP, the main bile acid transporter in hepatocytes. In combination with a cholic acid diet, knockout of NTCP can be used to select for correctly targeted hepatocytes. Cytochrome p450 reductase (Cypor), is a cofactor for all cytochrome p450s. A knockout of Cypor in combination with the hepatotoxic drug retrorsine is an alternative method for selection. Development of the self-cleaving ribozyme gRNA-selectable Gene Ride vector provides a potential gene therapy approach for treating genetically inherited disorders. !
The selectable Gene Ride vector is a novel rAAV based gene therapy vector, which allows for the in vivo selection of hepatocytes that have undergone targeting the highly expressed albumin gene. In this study we will test novel Gene Ride vector designs and selection regimens. In aggregate, this work will enhance the feasibility of therapeutic gene editing in the liver. !