Key Words: Hepatocyte transplantation, Stem cells, gene-therapy
During the past decade, liver-directed cell therapy and gene therapy for inherited metabolic disorders has progressed to a point where successful clinical application is in sight. Our current preclinical targets are inherited hyperbilirubinemia (Crigler-Najjar syndrome, CN-1), mucopolysaccharidosis VII (MPS-VII) and alpha-1 antitrypsin (AAT) deficiency. We have been pursuing several approaches for liver-directed gene therapy as follows.
Subproject 1. Hepatocyte-based therapies for genetic liver diseases. The conventional source for human hepatocytes is livers from deceased allogeneic donors. Hurdles to broad clinical application of this highly promising approach include the scarcity of usable donor organs and the need for prolonged immunosuppression. To overcome these limitations, we are developing methods to induce preferential proliferation of the transplanted hepatocytes by preparative irradiation of the host liver, so that a small number of donor hepatocytes can repopulate the liver. Our work has been translated into the first successful hepatocyte allotransplantation in a patient with CN-1. Classic AAT deficiency disease arises from the inheritance of the ATZ variant that is misfolded, and is prone to polymerization and aggregation within the hepatocyte endoplasmic reticulum (ER). Inefficient secretion of tne mutant protein leads to reduced plasma levels, causing pulmonary emphysema due to loss of AAT function, whereas in some patients, accumulation of the misfolded protein within hepatocytes causes liver disease as a "gain of function". Recently, we have shown that normal hepatocytes transplanted in the livers of mice expressing human AAT-Z spontaneously proliferate, competitively replacing hepatocytes containing globules of PiZ. The mechanism of hepatic repopulation is being investigated in the above models of genetic liver diseases.
Subproject 2. Human embryonic and pluripotent stem cells as sources of hepatocytes: As a novel source of hepatocytes, we are generating differentiated hepatocytes by manipulating human embryonic stem cells and induced pluripotential stem cells (iPSCs) in culture from normal individuals and patients. We have developed cellular models of several inherited disorders, based on hepatocyte-like cells derived from patient-specific hepatocytes. Partial liver repolulation with normal iPSC-derived hepatocytes has been achieved in both immunodeficient mice and immunosuppressed rodent models. Transplantation of normal iPSC-derived human hepatocytes into UGT1A1-deficient jaundiced Gunn rats (model of Crigler-Najjar syndrome, type 1 resulted in amelioration of hyperbilirubinemia. Current studies are focused on (1) developing cellular models of inherited liver-based disorders, e.g. Crigler-Najjar syndrome, alpha-1 antitrypsin deficiency and primary hyperoxaluria, (2) improvement of differentiation of iPSCs to hepatocytes and (3) evaluation of metabolic function after transplantation into animal models of human metabolic diseases.
Subproject 3. Gene therapy via recombinant viral vectors and non-viral vehicles: We have developed novel adenoviral and lentiviral vectors that permit ex vivo, as well as systemic gene therapy. Site-specific gene insertion and repair of mutation based on zinc finger-nuclease, TALEN and CRISPR technologies are also being pursued.
Basma H, Soto-Gutiérrez A, Yannam GR, Liu L, Ito R, Yamamoto T, Ellis E, Carson SD, Sato S, Chen Y, Muirhead D, Navarro-Álvarez N, Wong R, Roy-Chowdhury J, Platt JL, Mercer DF, Miller JD, Strom SC, Kobayashi N, Fox IJ. Differentiation and Transplantation of Human Embryonic Stem Cell-Derived Hepatocytes. Gastroenterology 136:990-9, 2009.
Louboutin J-P, Chemasova AA, Marusich E, Roy-Chowdhury J, Strayer DS. Efficient CNS gene delivery by intravenous injection. Nature Methods, 7:905-7, 2010. Pubmed PMID 20953176.
Ding J, Yannam GR, Roy-Chowdhury N, Hidvegi T, Basma H, Rennard SI, Wong RJ, Avsar Y, Guha C, Perlmutter DH, Fox IJ, Roy-Chowdhury J. Spontaneous hepatic repopulation in transgenic mice expressing mutant human alpha 1-anti-trypsin by wildtype donor hepatocytes. J. Clin. Invest. 121(5):1930-4, 2011. Pubmed PMID 21505264.
Zhou H, Dong X, Kabarriti R, Chen Y, Avsar A, Wang X, Ding J, Liu L, Fox IJ, Roy-Chowdhury J, Roy-Chowdhury N*, Guha C*. Single liver lobe repopulation with wildtype hepatocytes using regional hepatic irradiation cures jaundice in Gunn rats. PLoS One, 2012, 7(10): e46775. doi:10.1371/journal.pone.0046775. (*Co-communicating authors).
Sauer V, Roy-Chowdhury N, Guha C, Roy-Chowdhury J. Induced pluripotent stem cells as a source of hepatocytes. Curr Pathobiol Rep 2:11–20, 2014. (DOI 10.1007/s40139-013-0039-2). NIHMS555298
Chen Y, Li Y, Wang X, Zhang W, Sauer V, Chang C-J, Han B, Tchaikovskya T, Avsar Y, Tafaleng EN, Sanal MG, Tar K, Polgar Z, Strom S, Bouhassira EE, Guha C, Fox IJ, Roy-Chowdhury J, Roy-Chowdhury N. Amelioration of hyperbilirubinemia in Gunn rats after transplantation of human induced pluripotent stem cell-derived hepatocytes. Stem Cell Reports 5:22-30, 2015.
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