Ex vivo gene therapy for metabolic liver disease

? DESCRIPTION (provided by applicant): The applicant for the K01 Mentored Career Development Award, Dr. Raymond Hickey, is currently a postdoctoral fellow under the mentorship of Dr. Stephen Russell and Dr. Scott Nyberg at Mayo Clinic. Dr. Hickey's long-term career goal is to establish an independent research program focused on liver regenerative medicine as a tenure-track investigator at an academic institution. Liver transplantation remains the only curative intervention for inherited metabolic liver disorders. A potential alternative strategy is hepatocyte transplantation. However, treatment is severely restricted by a shortage of donor hepatocytes, limited cell engraftment, and elicitation of an immune response against the transplanted allogeneic cells. Autologous cell transplantation, in which the patient's own hepatocytes undergo ex vivo gene therapy prior to being retransplanted, is a potential curative therapy for these disorders. The objective of this proposal is to prevent liver failure in a mouse model of metabolic liver disease using a novel gene repair strategy in hepatocytes. The central hypothesis is that the efficacy of ex vivo gene repair and cell engraftment can be improved through the use of integration-impaired lentiviral vectors and three-dimensional hepatocyte spheroids. The hypothesis has been formulated on the basis of preliminary data that demonstrates (a) robust lentiviral vector transduction of primary hepatocytes using novel conditions, (b) induction of targeted modifications at site-specific loci using genome-editing nucleases, (c) functional engraftment of short-term cultured hepatocyte spheroids in vivo, and (d) non-invasive imaging of transplanted hepatocytes in vivo using the sodium-iodide-symporter reporter gene and nuclear imaging. Three specific aims are proposed in this study: (1) Determine the effect of vector episomal structure on gene repair in hepatocytes in vitro; (2) Determine whether engraftment of hepatocyte spheroids is impacted by ex vivo culture and in vivo sinusoidal vasodilation; and (3) Determine whether ex vivo gene-corrected hepatocytes can regenerate functional liver in vivo. With respect to expected outcomes, the work proposed here will demonstrate for the first time that ex vivo gene correction in hepatocytes can prevent liver failure in a mouse model of metabolic disease. The training period provided by the K01 Mentored Career Development Award will provide Dr. Hickey with the necessary skills needed for his independent career investigating novel mechanisms of gene and cell therapy for metabolic liver disorders.