GP64-pseudotyped lentiviral vectors target liver endothelial cells and correct hemophilia A mice

Abstract

Lentiviral vectors (LV) are efficient vehicles for in vivo gene delivery to the liver. LV integration into the chromatin of target cells ensures their transmission upon proliferation, thus allowing potentially life-long gene therapy following a single administration, even to young individuals. The glycoprotein of the vesicular stomatitis virus (VSV.G) is widely used to pseudotype LV, as it confers broad tropism and high stability. The baculovirus-derived GP64 envelope protein has been proposed as an alternative for in vivo liver-directed gene therapy. Here, we perform a detailed comparison of VSV.G- and GP64-pseudotyped LV in vitro and in vivo. We report that VSV.G-LV transduced hepatocytes better than GP64-LV, however the latter showed improved transduction of liver sinusoidal endothelial cells (LSEC). Combining GP64-pseudotyping with the high surface content of the phagocytosis inhibitor CD47 further enhanced LSEC transduction. Coagulation factor VIII (FVIII), the gene mutated in hemophilia A, is naturally expressed by LSEC, thus we exploited GP64-LV to deliver a FVIII transgene under the control of the endogenous FVIII promoter and achieved therapeutic amounts of FVIII and correction of hemophilia A mice.

Keywords: Envelope Engineering; Hemophilia A; In Vivo Gene Therapy; Lentiviral Vectors; Liver Endothelial Cells.

Figure 1. In vitro characterization of GP64-pseudotyped LV.

(A–C) Single values, mean, and standard deviation of the mean (SEM) of LV-infectious titer (A), physical particles (B) or specific infectivity (C) of VSV.G-LV or GP64-LV measured by transducing 293T or Huh7 cell lines, as indicated (n = 6 biological replicates). TU Transducing Units. (A, C) Wilcoxon matched-pairs signed rank test; (B) Mann–Whitney test. (D) GP64-LV-infectious titer measured on Huh7 is 20-fold higher compared to that measured on 293 T cells (n = 6), while VSV.G-LV transduce both cell lines similarly (n = 6). Single values, mean and SEM. Mann–Whitney test. (E). Percentage of titer recovered, compared to the no-serum control on Huh7 cell line (2 independent assays performed at the indicated LV concentration, n = 1 per dose per LV, except for n = 2 for GP64-LV at 9e4 TU/mL, mean and SEM) of GP64-LV or VSV.G-LV, incubated for 1 h with heat-inactivated (H.I., empty symbols) or fresh complement-preserved (filled symbols) human sera. (F–I) Recovered titer on Huh7 (y axis), of VSV.G-LV (F, G) or GP64-LV (H, I) as indicated, at the indicated concentration (x axis), incubated for 1 h in medium only (IMDM) or in H.I. (F, H) or fresh (G, I) serum. Data used to calculate percentages of titer recovered shown in (E). nd not detectable. Source data are available online for this figure.

Figure 2. Evaluation of phagocytosis of complement-opsonized LV.

(A) Representative ImageStream images of macrophages left untreated (top panel) or incubated 6 h with fluorescent LV particles (bottom panel). Ch1: Hoechst nuclear staining; Ch2: GFP-positive LV clusters; Ch3: Brightfield. (B) Mean and SEM with single values of total LV spots per 100 primary human macrophages or 293T cells, as indicated, analyzed by ImageStream after incubation with VSV.G-LV, LV lacking envelope protein (Bald-LV) or left untreated (three independent experiments performed with macrophages derived from six different normal donors). Mann–Whitney test. (C–F) Mean and SEM with single values of phagocytosed VSV.G-LV (C, D) or GP64-LV (E, F) opsonized with fresh complement-preserved human sera (Fresh serum) compared to no-serum control (IMDM) by primary human macrophages analyzed by ImageStream. LV tested in these assays have been produced in standard 293 T cells (C, n = 20 normal donors; E, n = 6), or in CD47hi 293 T cells (CD47hi LV, D, n = 17; F, n = 6). Multiplicity of infection (MOI) 10. Wilcoxon matched-pairs signed rank test. Source data are available online for this figure.

Figure 3. GP64-LV efficiently transduce liver sinusoidal endothelial cells in vivo.

(A, B) Mean and SEM with single values of the VCN (A) or GFP-positive tissue (B) measured in the total liver of 8-week-old mice 1 week after i.v. administration of VSV.G-LV or GP64-LV at the indicated doses (n = 5 mice per group). Mann–Whitney test. (C) Mean and SEM of the distribution of the GFP-positive tissue among the major liver cell types of mice shown in (A, B) at the indicated doses (KC Kupffer cells, LSEC liver sinusoidal endothelial cells, Hep hepatocytes). (D) Representative images and merge of immunofluorescent staining of liver analyzed in B and C at 4e10 TU/kg (n = 5 mice). Red: F4/80-positive cells (KC); white: Lyve1-positive cells (LSEC); green: GFP (transduced cells); blue: Hoechst (nuclei). Orange arrows indicate GFP-positive LSEC. Blue arrows indicate GFP-positive KC. Scale bar: 250 µm. Source data are available online for this figure.

Figure 4. In vivo evaluation of CD47hi GP64-LV.

(A) Mean with SEM of human FIX (hFIX) concentration measured in the plasma of C57BL/6 mice or NOD mice, as indicated, treated at the indicated LV doses with VSV.G-LV (n = 116, historical dataset from twenty-four independent experiments) or GP64-LV (n = 22 C57BL/6, n = 35 NOD, from four independent experiments, performed with four different LV batches). (B) Mean with SEM with single values of VCN in measured in non-parenchymal cells (nPC), FACS-sorted hepatocytes (Hep), LSEC, KC, and plasmacytoid dendritic cells (pDC) as indicated, of C57BL/6 mice (n = 5) or NOD mice (n = 5), 2 months after administration of the indicated doses of LV. Kruskal–Wallis test with Dunn’s multiple comparisons test (compared to C57BL/6 mice). (C) Single values of human FIX (hFIX) concentration measured over time in the plasma of NOD mice treated at the indicated LV doses with CD47hi GP64-LV (n = 5). Two-way ANOVA. (D) Mean with SEM with single values of VCN measured in liver subpopulations as indicated, of mice shown in (A), 2 months after administration. Mann–Whitney test. (E) Mean with SEM with single values of VCN measured in the indicated liver subpopulations of mice shown in Fig. 3F (LV) and 4B (CD47hi LV), reported here for direct comparison. Mann–Whitney test. Source data are available online for this figure.

Figure 5. Ex vivo transduction of primary human hepatocytes and immortalized LSEC.

(A, B) Mean and SEM with single values of the % (A) or mean fluorescent intensity (MFI, B) of GFP-positive primary human hepatocytes (n = 3 technical replicates) transduced with VSV.G-LV or GP64-LV at the indicated multiplicity of infections (MOI). Mann–Whitney test. (C) Mean and SEM with single values of the amount of hFIX measured in the culture supernatant of primary human hepatocytes (n = 3 technical replicates) transduced with VSV.G-LV or GP64-LV at the indicated MOI, or left untreated. (D). Mean and SEM with single values of the vector copy numbers (VCN) of reversed transcribed LV genome of the primary human hepatocytes (shown in (A–C), n = 3 technical replicates) transduced with VSV.G-LV or GP64-LV encoding for GFP or hFIX at the indicated MOI, or left untreated. (E, F) Mean and SEM with single values of the % (E) or MFI (F) of GFP-positive primary human immortalized LSEC derived from a healthy donor (n = 4 independent experiments) transduced with VSV.G-LV or GP64-LV at the indicated MOI. (G) Mean and SEM with single values of the amount of hFVIII measured in the culture supernatant of primary human immortalized LSEC derived from a healthy donor (n = 4 independent experiments) transduced with VSV.G-LV or GP64-LV at the indicated MOI, or left untreated. (H) Mean and SEM with single values of the VCN of reversed transcribed LV genome of the primary human LSEC (shown in (E–G)) transduced with VSV.G-LV or GP64-LV encoding for GFP or hFVIII at the indicated MOI, or left untreated. Source data are available online for this figure.

Figure 6. Evaluation of GP64-LV mediated gene therapy in hemophilia A mice.

(A–C) Single values of hFVIII antigen (A), activity (B), or anti-hFVIII antibodies (Abs, C) measured over time in the plasma of immune-competent HemoA-R593C mice treated at 7 weeks of age by i.v. injection of 5 × 10¹⁰ TU/kg of VSV.G-LV (n = 6) or GP64-LV (n = 5) of LV-F8.coFVIII, or left untreated (n = 5). (D) Single values and mean with SEM of anti-hFVIII inhibitors of mice shown in (A–C) at the indicated time after LV administration. BU Bethesda Units. (E) Survival of mice shown in (A–C) 24 h after hemostatic challenge. Note that mice treated with VSV.G-LV were not tested (NT) since they do not display measurable hFVIII in the circulation. WT wild type. (F) Mean with SEM with single values of VCN measured in nPC, FACS-sorted Hep, LSEC, KC, pDC and whole liver, as indicated, of mice shown in (A–C), 36 weeks after LV administration. Mann–Whitney test. (G) Mean with SEM with single values of LV expression measured in FACS-sorted Hep or LSEC, as indicated, of mice shown in (A–C), 36 weeks after LV administration. WPRE: woodchuck hepatitis B virus post-transcriptional regulatory element, present in the transgene mRNA. (H–J) Single values of hFVIII antigen (H), activity (I) or anti-hFVIII Abs (J) measured over time in the plasma of immune-competent HemoA mice treated as newborns by temporal vein injection of 3 × 10¹⁰ TU/kg of VSV.G-LV (n = 6) or GP64-LV (n = 5) of LV-F8.coFVIII. (K) Single values and mean with SEM of anti-hFVIII inhibitors of mice shown in (H–J) at the indicated time after LV administration. (L) Survival of mice shown in (F–H) 24 h after hemostatic challenge. WT wild type. (M) Mean with SEM with single values of VCN measured in the total liver of mice shown in (F–H), 36 weeks after LV administration. Mann–Whitney test. (N–P) Single values of hFVIII antigen (N), activity (O) or anti-hFVIII Abs (P) measured over time in the plasma of immune-competent HemoA mice treated as newborns by temporal vein injection of 9 × 10¹⁰ TU/kg of VSV.G-LV (n = 4) or GP64-LV (n = 4) of LV-F8.coFVIII. Source data are available online for this figure.

Figure EV1. Electron microscopy analysis of CD47 content on LV particles.

(A) Single values, mean and SEM of gold particles per virion of LV batches produced by control (GP64-LV), CD47-overexpressing (CD47hi GP64-LV or VSV.G-LV), or CD47-negative 293T cells (CD47free GP64-LV), immunostained with anti-CD47 antibody, or as staining control without the primary antibody (Ctrl) and analyzed by electron microscopy (n = 57–114 virions per sample). Kruskal–Wallis test with Dunn’s multiple comparison test (compared to GP64-LV).

Figure EV2. Evaluation of GP64-LV liver transduction in vivo in mice.

(A–C) Single values, mean and SEM of the % of KC (A), hepatocytes (B) or LSEC (C) in GFP-positive cells shown in Fig. 3C, reported here separately for statistical analysis. n = 5 per group. Mann–Whitney test. (D). Representative merge images of immunofluorescent staining of liver sections analyzed in Fig. 3B, C at the indicated doses. Red: F4/80-positive cells (KC); White: Lyve1-positive cells (LSEC); Green: GFP (transduced cells); Blue: Hoechst (nuclei). Scale bar: 250 µm. Images of the 4e10 TU/kg LV dose are also presented in Fig. 3D. (E) Representative images of immunofluorescent staining of a liver section from an untreated control mouse. Red: F4/80-positive cells (KC); White: Lyve1-positive cells (LSEC); Green: GFP (transduced cells); Blue: Hoechst (nuclei). Scale bar: 250 µm.

Figure EV3. Biodistribution analysis of GP64-LV after systemic administration in mice.

(A–E) Single values, mean and SEM of GFP-positive tissue measured by immunohistochemistry in the liver (A), lung (B), spleen (C), heart (D) or kidney (E) of 8-week-old mice analyzed 10 days after i.v. administration of VSV.G-LV or GP64-LV at 4e10 TU/kg (n = 6 mice per group), or left untreated for background signal evaluation (n = 3). Kruskal–Wallis test with Dunn’s multiple comparison test. (F) Representative anti-GFP immunohistochemistry images of liver, brain, heart, kidney, lungs, spleen of mice treated with GP64-LV or VSV.G-LV, as indicated, analyzed in (A–E). Scale bar: 300 µm. (G) Higher magnification of liver and spleen sections shown in (F). Orange arrows indicate GFP-positive endothelial cells, identified by morphology. Scale bar: 100 µm.

Figure EV4. GP64-LV half-life after systemic administration in mice.

(A) Percentage of the serum concentrations of LV particles (measured as HIV Gag p24) recovered at the indicated time (minutes) after administration of GP64-LV or CD47hi GP64-LV relative to the total amount of administered LV particles to C57BL/6 or NOD mice, as indicated (8e10 TU/kg). Kruskal–Wallis test with Dunn’s multiple comparison test (compared to C57 LV). (B) Single values, mean, and SEM of the percentage of GFP-positive Huh7 cells, transduced with GP64-LV or CD47hi GP64-LV, at the indicated MOI (n = 3).

Figure EV5. Ex vivo transduction of primary human blood outgrown endothelial cells.

(A, B) Mean and SEM with single values of the % (C) or MFI (D) of GFP-positive primary blood outgrowth endothelial cells (BOEC) derived from a hemophilia A donor (n = 2 independent experiments) transduced with VSV.G-LV or GP64-LV at the indicated MOI. (C) Mean and SEM with single values of the amount of hFVIII measured in the culture supernatant of primary BOEC derived from a hemophilia A donor (n = 2 independent experiments) transduced with VSV.G-LV or GP64-LV at the indicated MOI, or left untreated. hFVIII concentration reported is obtained from two supernatant collection. (D) Mean and SEM with single values of the VCN of reversed transcribed LV genome of the primary BOEC (shown in (A–C)) transduced with VSV.G-LV or GP64-LV encoding for GFP or hFVIII at the indicated MOI, or left untreated.