Efficient engraftment and viral transduction of human hepatocytes in an FRG rat liver humanization model

Abstract

Humanized liver rodent models, in which the host liver parenchyma is repopulated by human hepatocytes, have been increasingly used for drug development and disease research. Unlike the leading humanized liver mouse model in which Fumarylacetoacetate Hydrolase (Fah), Recombination Activating Gene (Rag)-2 and Interleukin-2 Receptor Gamma (Il2rg) genes were inactivated simultaneously, generation of similar recipient rats has been challenging. Here, using Velocigene and 1-cell-embryo-targeting technologies, we generated a rat model deficient in Fah, Rag1/2 and Il2rg genes, similar to humanized liver mice. These rats were efficiently engrafted with Fah-expressing hepatocytes from rat, mouse and human. Humanized liver rats expressed human albumin and complement proteins in serum and showed a normal liver zonation pattern. Further, approaches were developed for gene delivery through viral transduction of human hepatocytes either in vivo, or in vitro prior to engraftment, providing a novel platform to study liver disease and hepatocyte-targeted therapies.

IL-6–GP130 signaling protects human hepatocytes against lipid droplet accumulation in humanized liver models

“…FSRG and FSRG-Il6 HumIn mice were generated with VelociGene and VelociMouse technologies as described previously ( 41 , 42 ). FRG rats were generated as previously described ( 6 ). All studies were done in accordance with the Institutional Animal Care and Use Committee guidelines at Regeneron Pharmaceuticals Inc.…”

“…Hepatocyte number and viability was determined using a ViaStain AOPI staining solution (Nexcelom, CS2-0106), using the Nexcelom Cellometer Auto 2000. Mouse and rat transplantations were performed as described previously ( 3 , 6 ). In general, transplantations were done in female FSRG mice or mix-gendered FSRG-Il6 HumIn mice at ages of 6 to 9 weeks or male FRG rats at ages of about 9 to 12 weeks.…”

“…Lentiviral vector production, titration, and infection were performed as previously described with slight modifications ( 6 ). Lentiviral particles were produced following standard Lipofectamine-mediated cotransfection of HEK 293 T cells with the transfer plasmid encoding mIl6r , rIl6r , or human GP130 Y186-Y190del under the cytomegalovirus (CMV) promoter ( pLVX-CMV -, subcloned from pLVX-EF1a-IRES-puro plasmid, Takara), a second-generation packaging plasmid encoding the gag , pol , and rev genes ( psPAX2 , obtained from the Tronolab at Ecole Polytechnique Fédérale de Lausanne, Switzerland) and a plasmid encoding the vesicular stomatitis virus envelope glycoprotein G (VSV-G) as an envelope plasmid ( pMD2-G , Tronolab).…”

“…Humanized liver mouse and rat models, in which donor human hepatocytes repopulate recipient rodent livers, have been widely used to study human liver biology, diseases, and therapeutics (1)(2)(3)(4)(5)(6). However, it has been widely observed that engrafted human hepatocytes in both humanized liver mice and rats show defects, including increased lipid droplet accumulation (5)(6)(7).…”

“…Humanized liver mouse and rat models, in which donor human hepatocytes repopulate recipient rodent livers, have been widely used to study human liver biology, diseases, and therapeutics (1)(2)(3)(4)(5)(6). However, it has been widely observed that engrafted human hepatocytes in both humanized liver mice and rats show defects, including increased lipid droplet accumulation (5)(6)(7). Ameliorating such defects would improve the accuracy of the models to recapitulate normal human liver biology and shed light on some of the underlying mechanisms that regulate lipid accumulation in hepatocytes.…”