Volume 6, Issue 2, Pages 199-209 (August 2002) Highly Efficient Lentiviral Vector-Mediated Transduction of Nondividing, Fully Reimplantable Primary Hepatocytes  Tuan Huy Nguyen, José Oberholzer, Jacques Birraux, Pietro Majno, Philippe Morel, Didier Trono  Molecular Therapy  Volume 6, Issue 2, Pages 199-209 (August 2002) DOI: 10.1006/mthe.2002.0653 Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 1 Phenotypic analysis and transduction of primary human hepatocytes. (A) Expression of Albumin. Primary hepatocytes were trypsinized on day 7 post-plating, permeabilized and stained with anti-human albumin-FITC conjugated antibodies (dark) or IgG isotypic FITC-conjugated antibody (light) as control and analyzed by FACS. Result was similar on day 2 post-plating. (B) BrdU labeling of hepatocytes. Hepatocytes were labeled for 16 hours with BrdU on day 2 post-plating and incorporation was detected using a BrdU-specific mouse monoclonal antibody and TRITC-conjugated anti-mouse IgG. Nuclei were counterstained with DAPI (left). Nuclei that have incorporated BrdU can be identified by their red fluorescence (right, ×200). (C) Efficient transduction of human primary hepatocytes. FACS analysis of hepatocytes transduced on day 2 post-plating through a 16-hour exposure to 30 HTU per cell of a CLL-GFP-CLL. Control infections were done in the presence of the RT inhibitor Nevirapine or with a VSV G-pseudotyped MLV-based retroviral vector (MLV-GFP), or with medium only (mock) as indicated. The percentage of GFP-positive cells is indicated in the lower right corner of each plot. (D) BrdU incorporation and GFP expression in target cells. Hela cells (upper panels) and human primary hepatocytes on day 2 post-plating (lower panels) were incubated with (left) or without (right) CLL-GFP-CLL lentivectors for 16 hours in the presence of BrdU before immunostaining 3 days later. Cells were stained for both BrdU incorporation (brown nucleus) and GFP expression (red cytoplasm). The short arrow points to a BrdU-positive, transduced HeLa cell, and the long arrows to transduced nonproliferating cells (×200). Molecular Therapy 2002 6, 199-209DOI: (10.1006/mthe.2002.0653) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 2 Detection of integrated vector provirus. Genomic DNA isolated from primary human hepatocytes, either mock-treated or transduced with CLL-GFP-CLL vector at the indicated MOI was subjected to nested Alu-HIV PCR. Amplification products were resolved on 1% agarose gel containing ethidium bromide and detected by UV transillumination. PCR1 and PCR2 correspond respectively to the first and second rounds of PCR. Molecular Therapy 2002 6, 199-209DOI: (10.1006/mthe.2002.0653) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 3 Dose-response of human hepatocytes to lentiviral transduction. (A) Human hepatocytes and Hela cells were exposed during 16 hours to increasing MOI (equals the number of HTU per cell) of HIV-derived vector and GFP-positive cells were quantified by FACS. The values represent the mean of three measurements with standard deviation. (B) FACS analysis of human hepatocytes transduced on day 2 by a 16-hour exposure to 5 × 103 and 5 × 104 cpm of RT activity equivalent of GFP vectors with cPPT-CTS in sense (TRIP-GFP) or antisense (TRIPinv-GFP) orientations. Percentage of GFP-positive cells and mean fluorescence intensity (MFI) are indicated. Molecular Therapy 2002 6, 199-209DOI: (10.1006/mthe.2002.0653) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 4 Decreased sensitivity of rodent hepatocytes to lentiviral transduction. (A) Human and rodent hepatocytes were transduced on day 2 post-plating with a GFP-expressing HIV-derived vector at a low MOI to stay in the linear range of the assay and GFP positive cells were scored by FACS. (B) Same experiment with increasingly high MOIs. (C) Same experiment, transducing human and rodent hepatocytes at an MOI of 30 HTU/cell at different times after plating. (D) PCR analysis of reverse transcription. Lysates of hepatocytes infected at different MOIs (0, untransduced; 1, MOI = 1; 10, MOI = 10) prepared at the indicated times post-infection were amplified by PCR with LTR-, GFP- and β-actin-specific primers. LTR5/LTR6 primers amplify a 140-bp fragment from strong stop DNA (early reverse transcript) and GFP primers a 417-bp fragment from elongated reverse transcription products. As a control, human cells were infected at an MOI of 10 in the presence of nevirapine (N). Molecular Therapy 2002 6, 199-209DOI: (10.1006/mthe.2002.0653) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 5 Optimized ex vivo liver gene therapy protocol. (A) Representative FACS analysis of bulk population of hepatocytes placed in culture and trypsinized; viable cells are gated in R1. (B) Vitamin E allows for an accelerated transduction protocol. Human hepatocytes were transduced on day 2 post-plating through a 4-hour exposure to increasing amounts of vectors in the presence (lozenges) or absence (squares) of vitamin E. Hepatocytes were then washed, cultured in regular hepatocyte medium, and analyzed by FACS to determine the percent of viable (closed lozenge and square) and GFP-positive cells (open lozenge and open square). (C) Transduction of freshly isolated human hepatocytes in suspension. Hepatocytes were transduced in suspension immediately after purification, by a 4-hour exposure to vector at indicated MOIs in the presence (open lozenge) or absence (closed square) of vitamin E. Cells were then washed, plated, and cultured without vitamin E. The percentage of GFP-positive and viable cells were determined by FACS 5 days later. (D) Transduction of human primary hepatocytes with residual vector from the wash medium of hepatocytes transduced in suspension. Hepatocytes were either infected in suspension for 4 hours (left and middle panel) or plated immediately after harvest. The latter hepatocytes were then incubated for 16 hours with the medium from the last wash of the suspension-transduced hepatocytes (right panel). We determined the percentage of GFP-positive cells by FACS 5 days later. Molecular Therapy 2002 6, 199-209DOI: (10.1006/mthe.2002.0653) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 6 Transplantation of lentivector-transduced hepatocytes. (A) Vitamin Emediated stimulation of rat hepatocyte transduction. Freshly isolated rat hepatocytes were transduced in suspension at an MOI of 10 for 4 hours with (open bar) or without (black bar) vitamin E, placed in culture and analyzed 5 days later for expression of the GFP transgenethen washed. Combined results of two experiments are shown, with error bar indicative of mean ± SD (B) Primary hepatocytes (2 × 107 cells) transduced in the presence of vitamin E at an MOI of 10 (transduction efficiency, 31%; data not shown) were immediately transplanted into the spleen of rats that had been subjected to a two-thirds partial hepatoctomy 24 hours earlier (n = 2). Livers were examined by immunohistochemistry with a GFP-specific monoclonal antibody at 2 (first row) and 5 (second row) weeks post-transplantation. Animal examined at 5 weeks had undergone an additional hemi-hepatectomy at 2 weeks. Nontransplanted controls are shown in the third row. Original magnification: ×100 (left column), ×200 (right column). (C) Low magnification view (×40) of the liver section shown in the first row of (B). Molecular Therapy 2002 6, 199-209DOI: (10.1006/mthe.2002.0653) Copyright © 2002 American Society for Gene Therapy Terms and Conditions