Key Words:  Liver, Inherited liver diseases; Cell and Gene Therapy

Our current focus is on developing cell and gene-based therapies for monogenic liver diseases, such as inherited hyperbilirubinemia (Crigler-Najjar syndrome, CN-1), a₁ antitrypsin (AAT) deficiency, dyslipidemias and hemophilias A and B.

 Subproject 1.  Hepatocyte-based therapies for genetic liver diseases.  To develop a minimally invasive alternative to liver transplantation, we are developing strategies to repopulate the liver by transplanted hepatocytes. To overcome the hurdles of inefficient hepatocyte engraftment and failure of transplanted hepatocytes to proliferate, we are evaluating targeted hepatic irradiation and mitotic stimulation of the transplanted hepatocytes. Regiospecific conformal hepatic irradiation (HIR) is being used to transiently disrupt the sinusoidal endothelial barrier, thereby enhancing initial engraftment. HIR makes the host hepatocytes in the irradiated region less mitotically competent. We are exploring different types of mitotic agents to stimulate the proliferation of the engrafted hepatocytes, which can competitively repopulate the host liver. We are also evaluating whether co-transplantation of liver sinusoidal endothelial cells can augment liver repopulation by hepatocytes. Our work was translated into the first successful hepatocyte transplantation in a CN-1 patient, and more recently in two patients with urea cycle disorders and one with phenyl ketonuria. 

AAT deficiency (ATD) is one of the most common potentially lethal monogenic liver disorders in the West. In classic ATD, a mutant misfolded AAT (ATZ) tis secreted inefficiently and is retained within hepatocytes. Circulatory AAT deficiency leads to unrestrained neutrophil elastase activity in the lung, causing pulmonary emphysema, whereas ATZ accumulation within hepatocytes results liver disease. We showed that wildtype hepatocytes transplanted into transgenic mice expressing human ATZ competitively replace the host hepatocytes. Our current focus is to disrupt ATZ expression in a fraction of the hepatocyte mass by DNA break-enhanced homologous recombination in vivo, so that the gene-edited hepatocytes can repopulate the liver, thereby providing normal AAT and correcting the liver disease.

Subproject 2.  Transplantation of endothelial cells to repopulate mutant liver endothelial cells (LSEC): LSECs are highly specialized endothelial cells that are important in maintaining liver architecture, as well as hepatocyte regeneration and function. In addition, these cells express coagulation factor VIII (the deficiency of which causes hemophilia A) and Von Willebrand factor. In ongoing work, we have found that intravenous infusion of LSECs  following regional HIR results in engraftment of LSECs in the liver. Subsequent pharmacological stimulation of the LSECs results in regional liver repopulation by the transplanted cells. This procedure has cured the bleeding disorder in Factor VIII-deficient hemophilic mice.

In ongoing studies we are correcting the genetic lesions in mutant hepatocytes and LSECs, using CRISPR-cas or zinc finger nuclease-enhanced homologous recombination to generate phenotypically corrected for transplantation into animal models of inherited human liver diseases.

Recent Publications:

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 expressingmutant human alpha 1-anti-trypsin by wildtype donor hepatocytes. J. Clin. Invest. 121:1930-4, 2011.

Chen Y, Li Y, Wang X,  Zhang W, Sauer V, Chang CJ, Han B, Tchaikovskaya T, Avsar Y, Tafaleng E, Madhusudana Girija S, Tar K, Stephen S, Bouhassira E, Guha C, Fox IJ, Roy-Chowdhury J and Roy-Chowdhury N. Amelioration of hyperbilirubinemia in Gunn rats after transplantation of hepatocytes derived from human induced pluripotent stem cells. Stem Cell Reports 5:1-9, 2015.

Sauer V, Tchaikovskaya T, Wang X, Li Y, Zhang W Tar K, Polgar Z, Ding J, Guha C, Fox IJ, Roy-Chowdhury N, Roy-Chowdhury J. Human urinary epithelial cells as a source of engraftable hepatocyte-like cells using stem cell technology. Cell transplant, 2016, 25:2221-2243.

Soltys KA, Setoyama K, Tafaleng EN, et al. Host conditioning and rejection monitoring in hepatocyte transplantation in humans. J Hepatol. 66:987-1000, 2017. doi: 10.1016/j.jhep.2016.12.017.  PMID: 28027971.

Peterson EA, Polgar Z, Devakanmalai GS, Li Y, Jaber FL, Zhang W, Wang X, Iqbal NJ, Murray JW, Roy-Chowdhury N, Quispe Tintaya W, Maslov AY, Tchaikovskaya TL, Sharma Y, Rogler LE, Gupta S, Zhu L, Roy-Chowdhury J, Shafritz DA. Genes and pathways promoting long-term liver repopulation by ex vivo hYAP-ERT2 transduced hepatcytes and treatment of jaundice Gunn rats. Hepatology Communications 2019; 3:129-146

Barahman M, Zhang W, Harris HY, Aiyer A, Kabarriti R, Kinkhabwala M, Roy-Chowdhury N, Beck AP, Scanlan TS, Roy-Chowdhury J, Asp P,  Guha C. Radiation-primed hepatocyte transplantation in murine monogeneic dyslipidemia normalizes cholesterol and prevents atherosclerosis. J Hepatol. 2019 70:1170-1179.

Li Y, Guha C, Asp P, Wang X, Tchaikovskya TL, Kim K, Mendel M, Cost GJ, Perlmutter DH, Roy-Chowdhury N, Fox IJ, Conway A, Roy-Chowdhury J. Resolution of hepatic fibrosis after ZFN-mediated gene editing in the PiZ mouse model of human α1-antitrypsin deficiency. Hepatol Commun. 2023 Feb 27;7(3):e0070. PMCID: PMC9974076.