In Vivo Selection of Transplanted Hepatocytes by Pharmacological Inhibition of Fumarylacetoacetate Hydrolase in Wild-type Mice

Abstract

Genetic fumarylacetoacetate hydrolase (Fah) deficiency is unique in that healthy gene-corrected hepatocytes have a strong growth advantage and can repopulate the diseased liver. Unfortunately, similar positive selection of gene-corrected cells is absent in most inborn errors of liver metabolism and it is difficult to reach the cell replacement index required for therapeutic benefit. Therefore, methods to transiently create a growth advantage for genetically modified hepatocytes in any genetic background would be advantageous. To mimic the selective pressure of Fah deficiency in normal animals, an efficient in vivo small molecule inhibitor of FAH, 4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate (CEHPOBA) was developed. Microarray analysis demonstrated that pharmacological inhibition of FAH produced highly similar gene expression changes to genetic deficiency. As proof of principle, hepatocytes lacking homogentisic acid dioxygenase (Hgd) and hence resistant to FAH inhibition were transplanted into sex-mismatched wild-type recipients. Time course analyses of 4-6 weeks of CEHPOBA administration after transplantation showed a linear relationship between treatment length and replacement index. Compared to controls, recipients treated with the FAH-inhibitor had 20-100-fold increases in liver repopulation. We conclude that pharmacological inhibition of FAH is a promising approach to in vivo selection of hepatocytes.

A universal system to select gene-modified hepatocytes in vivo

“…In order to apply this system in any genetic liver disease, selection must work in animals that not deficient in FAH. We previously described a small-molecule inhibitor of FAH that was capable of inducing FAA accumulation and exerting selective pressure in vivo in mice (22, 23). In order to demonstrate that the Hpd shRNA could be used in any genetic background, neonatal wild-type C57Bl6 mice were injected with 2×10 11 vg of the promoterless F9 generide construct.…”

“…We have previously shown that transplanted hepatocytes can be selected with CEHPOBA in vivo if they carry a genetic mutation in tyrosine catabolic enzyme (23). However, patients with such mutations are rare leaving the vast majority of clinically relevant metabolic liver diseases unamenable to this powerful selection.…”

“…1A) could be strongly selected in wild-type mice treated with an FAH-inhibitor 4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate (CEHPOBA) (23). Based on this finding we reasoned that shRNA-mediated knockdown of enzymes upstream of FAH would make hepatocytes resistant to CEHPOBA and achieve their in vivo selection (Fig.…”

Therapeutic hepatocyte transplant for inherited metabolic disorders: functional considerations, recent outcomes and future prospects

“…Mice with genetically-engineered deletion of the fah gene represent perhaps the first instance of a genetic disorder in which the feasibility of HTx was documented (Karnezis et al 2001); (Erker et al 2010); (Paulk et al 2012). Grompe and colleagues successfully exploited fah deficiency in the distal portion of the tyrosine metabolic pathway to provide an engraftment advantage for exogenously administered fah +/+ cells to fah -deficient mice.…”

Adeno-associated Virus as a Mammalian DNA Vector

“…tissue-restricted promoters); however, as these sequences are not specific to AAV vectors they will not be discussed here. Regarding AAV vector-specific enhancements for gene addition applications, a pivotal advancement was spurred by the observation that transgenic cassettes less than half the size of the AAV genome (≤2.3 kb) packaged both monomer and dimer single-strand DNA species that had the capability to form a duplex vector genome through antiparallel single strand complementarity (158, 174, 176). Shortly thereafter, two reports demonstrated that these dimer species could be intentionally induced using a plasmid containing a wild-type ITR sequence, transgenic DNA (≤2.3 kb) and a truncated ITR in which the trs is deleted and therefore, is deficient for Rep-induced resolution of the typical double-stranded AAV genome intermediate (Figure 2) (161, 162, 177).…”