Ex Vivo Hepatocyte Reprograming Promotes Homology-Directed DNA Repair to Correct Metabolic Disease in Mice After Transplantation

Abstract

Ex vivo CRISPR/Cas9-mediated gene editing in hepatocytes using homology-directed repair (HDR) is a potential alternative curative therapy to organ transplantation for metabolic liver disease. However, a major limitation of this approach in quiescent adult primary hepatocytes is that nonhomologous end-joining is the predominant DNA repair pathway for double-strand breaks (DSBs). This study explored the hypothesis that ex vivo hepatocyte culture could reprogram hepatocytes to favor HDR after CRISPR/Cas9-mediated DNA DSBs. Quantitative PCR (qPCR), RNA sequencing, and flow cytometry demonstrated that within 24 hours, primary mouse hepatocytes in ex vivo monolayer culture decreased metabolic functions and increased expression of genes related to mitosis progression and HDR. Despite the down-regulation of hepatocyte function genes, hepatocytes cultured for up to 72 hours could robustly engraft in vivo. To assess functionality long-term, primary hepatocytes from a mouse model of hereditary tyrosinemia type 1 bearing a single-point mutation were transduced ex vivo with two adeno-associated viral vectors to deliver the Cas9 nuclease, target guide RNAs, and a 1.2-kb homology template. Adeno-associated viral Cas9 induced robust cutting at the target locus, and, after delivery of the repair template, precise correction of the point mutation occurred by HDR. Edited hepatocytes were transplanted into recipient fumarylacetoacetate hydrolase knockout mice, resulting in engraftment, robust proliferation, and prevention of liver failure. Weight gain and biochemical assessment revealed normalization of metabolic function. Conclusion: The results of this study demonstrate the potential therapeutic effect of ex vivo hepatocyte-directed gene editing after reprogramming to cure metabolic disease in a preclinical model of hereditary tyrosinemia type 1.

Figure 1

Ex vivo reprogramming of cultured hepatocytes up‐regulates DNA repair pathways. (A) Averaged relative quantification values showing expression levels of five genes involved in homologous recombination (Brca1, Brca2, Rad51, Rad52, and Pcna) relative to the length of culture as determined by comparative qPCR on RNA isolated from primary hepatocytes. Samples are standardized to B2m expression. (B) Deconvolution of these five genes, showing consistent up‐regulation over time. (C) Representative histogram of Ki‐67 intensity in primary hepatocytes after increasing time in culture, with peak heights normalized to mode of sample. (D) Quantification of mean fluorescence intensity in biological replicates (n = 2). (E) Hierarchical clustering of six RNA‐Seq samples at three different time points (0 hours, 24 hours, and 48 hours). The 24‐hour and 48‐hour time points are more similar to one another than either is to 0 hours. (F) Heat map of gene expression in all samples for differentially expressed genes between times 0 hours and 48 hours (n = 5,254). (G) Enrichment analysis of highly affected gene sets clustered by biologic functions between cells in culture for 48 hours relative to 0 hours. (H) Detailed enrichment summary illustrating differences in the regulation of two selected Gene Ontology function annotations of interest—DNA repair (horizontal black bars on the left) and cellular amino acid catabolic process (horizontal green bars on the left)—among the three time points collected (presented by column). The heat map includes the top and bottom 500 differentially expressed genes ranked by fold change between times 0 hours and 48 hours. Individual DNA repair genes are up‐regulated, whereas individual cellular and amino acid catabolic process genes are down‐regulated in the 24‐hour and 48‐hour groups. Abbreviations: FDR, false discovery rate; and ns, not significant at α = 0.05.

Figure 2

Hepatocytes cultured ex vivo can engraft in the liver after at least 72 hours in culture. (A) In vivo imaging of hepatocyte transplant (Tx) recipients 2 hours and 26 hours following Tx. Hepatocytes were cultured for t = 24 hours, 48 hours, or 72 hours and transplanted through splenic injection (n = 3 for each Tx time point). Hepatocyte engraftment in the liver can be seen 26 hours after Tx, even in the group that received cells after 72 hours in culture. (B) All livers imaged ex vivo at the 26‐hour time point showed luciferase activity from engrafted hepatocytes. Flux was standardized and quantified, showing no significant difference among the three groups.

Figure 3

AAV‐Cas9 and AAV‐HT can disrupt and correct the Fah locus in primary Fah^‐/‐hepatocytes transduced ex vivo. (A) Schematic illustration of the position of the two Cas9 gRNAs and protospacer adjacent motif sequences (in green) that were used relative to the Fah locus and the HT1 mutation (in red). Also shown are the two AAV vectors used in the subsequent experiments. AAV‐HT contains the corrected mutation, additional silent sequence modifications introduced for selective PCR (in blue), and Cas9 Guide 1. AAV‐Cas9 carries the Staphylococcus aureus Cas9 gene under the cytomegalovirus promotor, as well as Cas9 Guide 2. (B) PCR amplicon deep sequencing of the Fah locus from primary Fah^‐/‐hepatocytes transduced with AAV‐Cas9 and AAV‐HT showed that low gene‐editing rates in hepatocytes are detectable after 72 hours in culture. Sequence distribution and top 10 hits are shown. Both complete and partial homologous recombination were classified as HDR. (C) Selective PCR amplification of the corrected sequence showed that correction was occurring in primary hepatocytes transduced with both AAV‐Cas9 and AAV‐HT, but not in hepatocytes transduced with AAV‐HT alone. (D) Primary hepatocytes were transduced with AAV‐Cas9 and AAV‐HT, and were harvested at 24 hours, 48 hours, and 72 hours, respectively. The cells only control is primary hepatocytes that were not transduced and were collected at 72 hours. Abbreviations: CMV, cytomegalovirus; ITR, inverted terminal repeat.

Figure 4

Primary hepatocytes transduced with AAV‐Cas9 and AAV‐HT and cultured ex vivo can phenotypically correct a mouse model of hereditary tyrosinemia type I. (A) Workflow of ex vivo transplant experiments. Hepatocytes are isolated from a Fah^‐/‐ donor mouse, transduced with AAV‐Cas9 and AAV‐HT, cultured for 48 hours, and transplanted into a Fah^‐/‐recipient mouse through splenic injection. (B) Weight charts for female recipients (green; n = 3), male recipients (red; n = 3), and control mice (black; n = 4). Gray bars indicate time on the protective drug, NTBC. ^*Euthanization of control mice. Females phenotypically rescued from liver failure with one fewer NTBC cycle than did the males. (C) Blood data for Fah^‐/‐mice sacrificed after 20 days of NTBC withdrawal (black; n = 5), Fah^‐/‐mice that were never withdrawn from NTBC (dark gray; n = 5), experimental mice treated with AAV‐Cas9 and AAV‐HT (green/red; n = 6), and wild‐type control mice (light gray; n = 4). **P < 0.01; ***P < 0.001; and ****P < 0.0001.

Figure 5

An ex vivo protocol using AAV‐Cas9 and AAV‐HT can molecularly correct a mouse model of hereditary tyrosinemia type I. (A) Immunohistochemistry for fumarylacetoacetate hydrolase in liver sections of all six experimental mice. Black scale bar is 4 mm. (B) Untreated Fah^‐/‐mouse liver stained for FAH. (C) PCR amplicon deep sequencing of seven predicted off‐target sites, showing percentage of non‐wild‐type alleles from the livers of both treated and untreated control mice. (D) NGS data of the Fah locus from liver samples from all six experimental mice showed that 86% of alleles were unmodified. Green letters indicate HDR‐corrected bases; red letters indicate other changed bases; and bolded letters indicate bases targeted for change in the homology template. Perfect correction at this locus was seen in 2.6% of alleles, although the point mutation change was seen in over 12.6% of alleles.

Figure 6

HDR‐mediated correction of the Fah single‐nucleotide polymorphism is repeatable and durable. (A) Weight curves for two hepatocyte transplant donors that were cured following the dual AAV treatment, as well as weight curves for seven serial recipient mice transplanted with hepatocytes from the first two mice after cycling and rescue. The dark gray bar indicates time on NTBC for every animal, and the light gray bar indicates time on NTBC for only the starred red animal. (B) Serum data for Fah^‐/‐mice sacrificed after 20 days of NTBC withdrawal (black; n = 5), Fah^‐/‐mice who were never withdrawn from NTBC (gray; n = 5), experimental serial transplant recipient mice (red, 1 × 10⁶ hepatocytes, n = 4; blue, 5 × 10⁵ hepatocytes, n = 3), and wild‐type control mice (green; n = 4). (C) Immunohistochemistry staining for FAH in treated mice and an untreated control. (D) Quantification of the levels of FAH expression in the livers of both treated groups and their untreated controls at time of sacrifice. *P < 0.05; **P < 0.01; and ****P < 0.0001.