Research ArticleGene Therapy

A universal system to select gene-modified hepatocytes in vivo

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Science Translational Medicine  08 Jun 2016:
Vol. 8, Issue 342, pp. 342ra79
DOI: 10.1126/scitranslmed.aad8166
  • Fig. 1. Identification of an shRNA that rescues Fah deficiency.

    (A) The tyrosine catabolic pathway. Genetic deficiency of Fah causes hereditary tyrosinemia type 1 (HT1) due to accumulation of FAA in hepatocytes. The disease can be treated pharmacologically (NTBC) or by shRNA knockdown of the genes required for making FAA. CEHPOBA (4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate) inhibits FAH and causes accumulation of FAA. TAT, tyrosine aminotransferase; MAI, maleylacetoacetate isomerase. (B) Lentiviral construct. shRNAs targeting Hpd, Hgd, or Tat were expressed from a U6 promoter. Vector expresses a green fluorescent protein (GFP) reporter. LTR, long terminal repeat; UbC, ubiquitin promoter. (C) Experimental timeline. Neonatal Fah−/− mice were injected with shTat, shHpd, and shHgd lentiviruses or a vector devoid of any shRNA (control) and kept on NTBC until weaning. NTBC was then withdrawn to permit liver injury and selection of resistant hepatocytes. (D) Mouse weights during selection starting at 5 weeks of age. Gray bars represent periods of intermittent NTBC therapy. Only the control and shHgd and shTat cohorts were given NTBC after week 6. Data are means − SD (downward tick) (n = 4 to 6). (E) Mice were injected with a nonselectable vector (control) or shHpd. Liver tissues were stained for the reporter GFP and for α-fetoprotein (AFP), which is highly expressed in mutant Fah−/− hepatocytes. Yellow arrow denotes the absence of AFP within a selected nodule compared with AFP-positive surrounding tissue (black arrows). Scale bars, 100 μm. (F) Polymerase chain reaction (PCR) amplification of genomic liver DNA with primers flanking the lentiviral-shRNA sequence (two primer sets).

  • Fig. 2. Selection of integrating rAAV vectors.

    (A) Ribosomal rAAV constructs capable of chromosomal integration. Top: Standard vector control. Bottom: Selectable Hpd shRNA vector. Both vectors contained rDNA homology arms to enhance chromosomal integration, and both expressed hF9. ITR, inverted terminal repeat. (B) Experimental timeline. Selection: Periods of NTBC withdrawal were different between cohorts and are indicated in (C). (C) Weights and NTBC administration in animals receiving control or shHpd vector. Black rectangles indicate periods of NTBC treatment in control mice, whereas gray bar is NTBC for mice receiving shHpd. Data are means ± SD (n = 3 to 5). (D) Plasma hF9 measured by enzyme-linked immunosorbent assay (ELISA). Data are means ± SD (n = 4 to 5). *P < 0.05, **P = 0.01 versus controls, by Student’s two-tailed t test assuming equal variance.

  • Fig. 3. Selection of targeted integrations in the albumin locus.

    (A) GeneRide rAAV constructs designed for chromosomal integration. Top: The selectable shHpd construct is driven by the pol3 U6 promoter. Below: The shHpd is embedded within a miRNA and controlled by the endogenous albumin promoter. Both albumin-targeted GeneRide vectors encoded hF9 complementary DNA (cDNA) flanked by mouse albumin homology arms. The structures of the wild-type and gene-targeted albumin locus are also shown. Homologous recombination led to generation of a fused mRNA transcript. RNA processing liberated the shHpd and ribosome skipping at the 2A peptide coding sequence generated separate mouse albumin (Alb) and hF9 proteins. (B) Experimental timeline. Selection cycles starting at week 5: off NTBC for 3 weeks, then on for 5 days, until week 20. (C) Plasma hF9 measured by ELISA in mice treated with the selectable or control GeneRide rAAV. The 5 and 100% levels of normal hF9 blood levels are shown with a dashed line. Data are means ± SD (n = 4). **P < 0.01, ***P < 0.001 versus controls, by Student’s two-tailed t test assuming equal variance. (D) hF9 liver immunohistochemistry showing representative nodules from mice with plasma F9 levels of 38,000 and 27,000 ng/ml (high), 800 ng/ml (low), and a control (no selection). Scale bars, 100 μm. (E) Plasma hF9 levels in mice treated with the selectable GeneRide rAAV and subjected to NTBC withdrawal from 6 to 9 weeks of age followed by reintroduction of NTBC thereafter. The dashed line indicates the therapeutic level of hF9 (250 ng/ml or 5%). Data are single measurements from individual mice (n = 3).

  • Fig. 4. Histology of shHpd selected hF9-positive nodules.

    (A) Serial sections of representative fields from three separate Fah−/− mice after multiple cycles of selection (NTBC withdrawal). Mice were given either the shHpd vector or saline control. The adjacent sections were stained for expression of hF9; glutamine synthase, a marker for hepatocytes adjacent to the central vein of the hepatic lobule (zone 3); hematoxylin and eosin (H&E); and the proliferation marker Ki67. The “x” and “o” symbols mark vessels as landmarks for lining up the serial sections. For shHpd mouse 1 (shHpd 1), serial sections are the same area in low magnification (×62); for shHpd 2 and the control mouse, serial sections are in high magnification (×150). Black arrows indicate nodules expressing hF9. Scale bars, 100 μm. (B) In vivo selection after gene transfer in adults. Four adult male Fah−/− mice treated from birth with NTBC were injected with 8 × 1011 vg of the GeneRide vector (Fig. 3A) at day 52. NTBC therapy was stopped 10 days later. hF9 levels were measured. Data are averages ± SD (n = 4). Statistical differences between hF9 levels at different time points were evaluated by Student’s two-tailed t test assuming equal variance.

  • Fig. 5. Selection of gene-targeted hepatocytes in wild-type mice using pharmacologic FAH inhibition.

    (A) Experimental timeline. Beginning at 4 weeks of age, the mice were given daily intraperitoneal injections of CEHPOBA (1 μmol/g) or saline until 8 weeks of age. (B) Plasma hF9 measured by ELISA in mice treated with CEHPOBA or saline. Data are individual mice (n = 3). Dashed line denotes 5% of normal F9 levels, which is considered therapeutic. The gray rectangle indicates CEHPOBA treatment periods. A second period of CEHPOBA treatment was administered to only one CEHPOBA mouse (inverted triangle). (C) hF9 immunohistochemistry showing representative liver nodules from two separate CEHPOBA-treated mice and two saline-treated controls. Arrows denote hF9-positive hepatocytes. Scale bars, 100 μm. (D) H&E staining of liver and kidney tissues from a representative CEHPOBA-treated animal and saline-treated control. Scale bars, 100 μm.

  • Fig. 6. Histology of CEHPOBA-treated livers.

    (A) Liver histology of a representative CEHPOBA-treated animal and a saline control 1 month after stopping the drug. An untreated age-matched Fah−/− off NTBC (a positive control from a separate experiment) is shown at the right side for comparison. Serial sections were stained with H&E, followed by antibody stains for Ki67, HNF4α, and CYPE1. Black arrows show portal bile ducts. The “*” marks central veins, and “o” delineates portal veins. Pleiocytosis is marked by yellow arrows. (B) Relative abundance of fused transcripts/mouse albumin transcripts from saline-treated C57Bl6 mice, CEHPOBA-treated C57Bl6 mice (8 weeks after completing selection), and NTBC-cycled Fah−/− mice injected with either control vector or the GeneRide vector harboring the shHpd selection cassette (20 weeks of age; see Fig. 3C). Data are individual animals (n = 3) with means ± SD.

  • Table 1. Liver function tests after CEHPOBA selection.

    Liver function parameters 1 month after the end of CEHPOBA or saline selection. Data are means ± SD (n = 3 animals per group). P values were determined by Student’s two-tailed t test assuming equal variance. ALT, alanine aminotransferase; AST, aspartate aminotransferase.

    ALT (U/liter)17.3 ± 2.115.3 ± 1.50.13
    AST (U/liter)68 ± 3239 ± 80.10
    Alkaline phosphatase (U/liter)111 ± 30114 ± 470.46
    Total bilirubin (mg/dl)0.2 ± 0.010.13 ± 0.060.06
    Albumin (g/dl)2.9 ± 0.22.9 ± 0.20.50
    Total protein (g/dl)5.4 ± 0.65.4 ± 0.20.50

Supplementary Materials

  • Supplementary Material for:

    A universal system to select gene-modified hepatocytes in vivo

    Sean Nygaard, Adi Barzel, Annelise Haft, Angela Major, Milton Finegold, Mark A. Kay, Markus Grompe*

    *Corresponding author. Email: grompem{at}

    Published 8 June 2016, Sci. Transl. Med. 8, 342ra79 (2016)
    DOI: 10.1126/scitranslmed.aad8166

    This PDF file includes:

    • Fig. S1. Integration site analysis after selection in gene-targeted mouse liver DNA.
    • Table S1. shRNA sequences tested in lentiviral vectors.
    • Data values in tabular format

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