Ex Vivo Cell Therapy by Ectopic Hepatocyte Transplantation Treats the Porcine Tyrosinemia Model of Acute Liver Failure

Abstract

The effectiveness of cell-based therapies to treat liver failure is often limited by the diseased liver environment. Here, we provide preclinical proof of concept for hepatocyte transplantation into lymph nodes as a cure for liver failure in a large-animal model with hereditary tyrosinemia type 1 (HT1), a metabolic liver disease caused by deficiency of fumarylacetoacetate hydrolase (FAH) enzyme. Autologous porcine hepatocytes were transduced ex vivo with a lentiviral vector carrying the pig Fah gene and transplanted into mesenteric lymph nodes. Hepatocytes showed early (6 h) and durable (8 months) engraftment in lymph nodes, with reproduction of vascular and hepatic microarchitecture. Subsequently, hepatocytes migrated to and repopulated the native diseased liver. The corrected cells generated sufficient liver mass to clinically ameliorate the acute liver failure and HT1 disease as early as 97 days post-transplantation. Integration site analysis defined the corrected hepatocytes in the liver as a subpopulation of hepatocytes from lymph nodes, indicating that the lymph nodes served as a source for healthy hepatocytes to repopulate a diseased liver. Therefore, ectopic transplantation of healthy hepatocytes cures this pig model of liver failure and presents a promising approach for the development of cures for liver disease in patients.

Fat‐associated lymphoid clusters as expandable niches for ectopic liver development

“…The native tyrosinemic liver was still highly involved in bilirubin and bile acid metabolism and transport. Given that the ectopic livers did not have a biliary system connected to the intestine [5,6] and no accumulation of bile, [7,8] the evidence suggested that hepatocytes in the ectopic livers were actively transporting bile to the intestine, possibly through the bloodstream using the remnant native tyrosinemic liver. Our previous work in the mouse [5,6] and our two recent reports in the pig on the ectopic liver [7,8] concurred with the notion that bile acids and bilirubin are transported outside of the ectopic livers.…”

“…[2][3][4] However, most patients with severe liver disease have fibrotic and cirrhotic liver obstructing blood flow, which constitutes an adverse environment for liver regeneration. Our group has previously demonstrated that lymph nodes (LNs) are excellent extrahepatic sites for ectopic liver development [5][6][7][8] as well as for other tissues, [9][10][11][12] and direct injection of hepatocytes into LNs resulted in the formation of a functional ectopic liver. [6] When transplanted into the peritoneal cavity, hepatocytes migrated to the abdominal LNs to generate ectopic livers.…”

Fumarylacetoacetate hydrolase gene as a knockout target for hepatic chimerism and donor liver production

“…Tbx3 , OC1/2 , Mab21l2 , and UbC have never been tested as targets for blastocyst complementation. Fah is an appealing target for blastocyst complementation for several reasons, including the well-described ability of Fah −/− livers to host the development of WT hepatocytes, whether the WT cells are introduced through genetic reversion or via deliberate transplantation ( Nicolas et al., 2020 ; Overturf et al., 1996 ; Zhang et al., 2016 ).…”

“…In the 1990s, it was observed that children with HT1 commonly had spontaneous reversion of the FAH mutation in hepatocytes, causing overgrown nodules of normal tissue in their livers as the reverted hepatocytes out-competed those damaged by FAA accumulation ( Kvittingen et al., 1994 ). Subsequent experiments demonstrated that exogenously introduced WT hepatocytes can repopulate Fah −/− livers in mice, rats, and pigs ( Hickey et al., 2016 ; Nicolas et al., 2020 ; Overturf et al., 1996 ; Zhang et al., 2016 ). Human hepatocytes and hepatocyte-like cells can be infused into immunodeficient Fah −/− mice, repopulating the liver with human hepatocytes ( Azuma et al., 2007 ; Bissig et al., 2010 ; Bissig-Choisat et al., 2016 ; Yuan et al., 2018 ).…”

mRNA-based therapy proves superior to the standard of care for treating hereditary tyrosinemia 1 in a mouse model

“…To date, experimental approaches to treat tyrosinemia type 1 involve adenovirus- and AAV-mediated gene transfer, 13 , 14 gene delivery of naked DNA constructs, 15 , 16 genome editing, 17 , 18 , 19 and ex vivo therapy. 20 , 21 , 22 The approach presented herein focuses on mRNA-based therapy, a powerful tool with tremendous potential to treat a variety of indications; e.g., infectious diseases, personalized cancer therapy, protein replacement therapy, and gene editing. 23 , 24 , 25 …”