335. Toward a Cellular Therapy for Metabolic Liver Disease: Gene Targeting of the Ornithine Transcarbamylase (Otc) Locus in Bipotential Murine Oval Liver Cells (BMOL) Using Adeno-Associated Virus

Liver progenitor cells (LPC) possess significant potential as a cellular therapy as they are easily isolated, can differentiate into hepatocytes and cholangiocytes and maintain stable phenotype in long-term culture. However, their use to correct recessive genetic disease in an autologous context will require repair of mutant loci. Adeno-associated virus induced homologous recombination (AAV-HR) is a highly effective tool for this task as it is u003e1000-fold more efficient than alternative approaches such as plasmid DNA and adenovirus. As with other HR systems, the frequency of AAV-HR is increased by DNA strand breaks (DSB) at the target locus. To develop a model of gene targeting in LPC, an AAV vector was constructed to target the Otc locus in Spfash mice which would both correct the mutant base and insert Otc exons 5-10 as well as a polyadenylation signal to support transcription and translation of the targeted gene. A neomycin phosphotransferase expression cassette was also included to permit G418-selection of transduced cells. The capacity of the construct (OTCe4-neo) to correctly target was confirmed by restoration of OTC expression in hepatocytes of male Spfash mice injected as neonates with 2.2 × 1011 vg packaged in hepatotropic serotype 8 capsids. Using an enzyme activity assay, clusters of OTC positive cells were observed in liver tissue sections consistent with locus repair and subsequent replication of corrected cells. The vector was also used to target the Otc locus in BMOL-TAT-LacZ, a murine LPC line derived from a transgenic mouse after three weeks on a choline-deficient, ethionine-supplemented (CDE) diet to increase LPC frequency. In vitro targeting was detected by nested PCR analysis of G418 selected populations. Based on this promising data, cell lines were isolated from male Spfash mice maintained on the CDE diet and validated to be BMOL by (i) expression of LPC markers (E-Cad, A6, EpCAM, CD24 and CK19) using immuno-histochemistry and (ii) their differentiation into hepatoblasts or ductal epithelial cells when maintained in appropriate in vitro conditions. Ongoing experiments will test the capacity of OTCe4-neo to target the Otc locus in the BMOL-Spfash lines. We have also developed CRISPR technology to induce DSB in exon 4 of Otc and functionally validated the constructs in BMOL-Spfash. While not essential for success of the project, application of CRISPR should increase the basal frequency of AAV-HR in BMOL- Spfash and expedite downstream experiments designed to test the therapeutic efficacy of LPC.