Research ArticleGene Therapy

Phagocytosis-shielded lentiviral vectors improve liver gene therapy in nonhuman primates

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Science Translational Medicine  22 May 2019:
Vol. 11, Issue 493, eaav7325
DOI: 10.1126/scitranslmed.aav7325
  • Fig. 1 Role of CD47 in LV biodistribution within the liver of intravenously injected mice.

    (A) Means with SEM of human FIX (hFIX) expression measured in the plasma of C57BL/6 mice treated at the indicated LV doses (n = 48, from eight independent experiments, performed with three different LV batches; the n of mice per dose is reported on the top of each point). (B) Means with SEM of the percentage of GFP-positive PCs (green line) in liver sections (5 to 10 optical fields scored from six to eight nonconsecutive sections per mouse), and VCN measured in genomic DNA extracted from whole liver (black line) of mice treated with the indicated dose of LV with hepatocyte-specific expression 2 months after administration (n = 5 per dose cohort). (C) Means with SEM of VCN measured in fractionated liver PCs (brown line) or nPCs (light blue line), FACS-sorted LSECs (red line), or KCs (purple line) of mice 2 months after LV administration (n = 4 per dose cohort). (D and E) Means with SEM of (D) LV particles (in ng HIV Gag p24/ml) measured in serum, and (E) hFIX expression (in ng/ml) measured in plasma of C57BL/6 hemophilia B mice (n = 6, black line) or nonobese diabetic mice (NOD mice; n = 6, dark red line), treated with LV-FIX at the indicated time after administration. Two-way analysis of variance (ANOVA) for repeated measures. (F) Single values and means with SEM of VCN measured in FACS-sorted hepatocytes (Hep), LSECs, KCs, or pDCs, and whole spleen (spleen), as indicated, of C57BL/6 hemophilia B mice (n = 5 to 6, black circles) or NOD mice (n = 5 to 6, dark red circles) 2 months after administration (1.2 ×1010 TU/kg). Mann-Whitney test. (G and H) Means with SEM of (G) LV particles or (H) hFIX expression as in (D and E) measured in C57BL/6 hemophilia B mice or NOD mice, treated with LV-FIX produced by CD47-negative 293T cells at 1.2 × 1010 TU/kg [C57-HemB, n = 11 (black line); NOD, n = 11 (dark red line)] or 2 × 1010 TU/kg [C57-HemB, n = 6 (gray line); NOD, n = 6 (light red line)]. Two-way ANOVA for repeated measures. (I) Single values and means with SEM of VCN measured as in (F) of C57BL/6 hemophilia B mice (n = 9) or NOD mice (n = 7 to 11) 2 months after CD47-free LV administration (2 × 1010 TU/kg). Mann-Whitney test.

  • Fig. 2 Generation and evaluation of CD47hi LV.

    (A) Single values and means with SEM of the percentage of GFP-positive–differentiated THP-1 cells transduced with LV (n = 3, black circles) or CD47hi LVs (n = 6, yellow circles), at the indicated multiplicity of infection (MOI) analyzed by flow cytometry, 3 days after transduction (two independent experiments performed with two different CD47hi LV batches). (B) Single values and means with SEM of VCN in 293T cells and primary human macrophages transduced with LVs (293T cells, n = 4; macrophages, n = 6) or CD47hi LVs (293T cells, n = 4; macrophages, n = 5) at MOI 10 and analyzed 3 days after transduction (two independent experiments performed with two different CD47hi LV batches produced by transfection into CD47-overexpressing 293T cells or by CD47-overexpressing LV-GFP stable producer cell line and two different healthy blood donors). Mann-Whitney test. (C to E) Representative photomicrographs (C) and quantitative analysis (D and E) of LV batches produced by control (LVs, black circles), CD47-overexpressing (CD47hi LVs, yellow circles), or CD47-negative 293T cells (CD47-free LVs, light blue circles), immunostained with anti-CD47 (D) or anti-VSV.G (E) Abs, as indicated, or as staining control without the primary Ab (ctrl, black triangles) and analyzed by electron microscopy (n = 41 to 70 virions per sample). Kruskal-Wallis test with Dunn’s multiple comparison tests. Scale bar, 100 nm. (F) Single values and means with SEM of VCN in 293T cells and primary human macrophages (293T cells, n = 6; macrophages, n = 15) transduced with LVs (black circles) or CD47-free LVs (light blue circles) at MOI 10 and analyzed 3 days after transduction (two independent experiments with five different healthy blood donors). Mann-Whitney test. Note that VCN denotes integrated or nonintegrated reverse-transcribed LV genome. (G) Single values and means with SEM of percentages of HIV Gag p24 recovered at 24 hours compared to 10 min after LV (n = 25, black circles) or CD47hi LV (n = 23, yellow circles) administration to NOD mice. Mann-Whitney test. (H and I) Single values and means with SEM of VCN in FACS-sorted hepatocytes, LSECs, KCs, or pDCs, and whole spleen, as indicated, of NOD mice (H) injected with LVs [n = 13 to 19; pDCs, n = 4 (black circles)] or CD47hi LVs [n = 11 to 16; pDCs, n = 4 (yellow circles)] at 1.2 to 2 × 1010 TU/kg (three independent experiments) or (I) injected with LVs (n = 6 to 12) or CD47hi LVs (n = 7 to 14) at 4 to 8 × 109 TU/kg (three independent experiments). VCN measured 2 months after LV administration. Mann-Whitney test. (J) Single values and means with SEM of hFIX expression (in ng/ml) measured in plasma of NOD mice injected with LVs (n = 12) or CD47hi LVs (n = 8 to 11) at the indicated vector dose. Mann-Whitney test. (K to O) Means with SEM of the concentration of (K) MIP-1α, (L) MIP-1β, (M) MCP1, (N) CXCL1, and (O) G-CSF in the serum of NOD mice at the indicated time after administration of LVs [n = 29 for (K) to (M); n = 14 for (N) and (O)], CD47hi LVs [n = 12 for (K) to (M); n = 7 for (N) and (O)], CD47-free LVs [n = 11 for (K) to (M); n = 7 for (N) and (O)], or left untreated [n = 17 for (K) to (M); n = 12 for (N) and (O)]. The dashed lines show the mean concentration in untreated cohorts. Kruskal-Wallis test with Dunn’s multiple comparison tests. Reported statistics refer to comparison between LV-treated (red line) or CD47-free LV–treated (blue line) and untreated (dashed line) mice 3 hours after LV administration. *P < 0.05; ***P < 0.001; ****P < 0.0001. Complete statistical analysis of data in (K) to (O) is in fig. S5.

  • Fig. 3 Intravital imaging of LV, CD47hi LV, or CD47-free LV uptake by liver KCs in mice.

    (A) IV2PM images from 8 to 12 z stacks spacing 4 μm of liver of C57BL/6 or NOD mice treated with GFP-labeled LVs, CD47hi LVs, or CD47-free LVs as indicated, at the indicated time (min; note that LV intravenous injection starts at 2 min). Sinusoids are labeled in white, and KCs are labeled in red. Scale bars, 30 μm. Separate channels (white and red or white and green) are also shown for the 30-min time point. (B) Single values of the percentage of LV-positive KCs (yellow stained) over time in C57BL/6 or NOD mice treated with LVs, CD47hi LVs, or CD47-free LVs, as indicated (analyzed KCs per mouse, n = 43 to 130).

  • Fig. 4 Tolerability and efficacy of intravenous LV gene therapy in NHPs.

    (A to E) Means with SEM of the concentration of (A) alanine transaminase (ALT), (B) aspartate transaminase (AST), (C) body temperature, (D) counts of white blood cell (WBC), and (E) lymphocytes of vehicle-treated (n = 1, red circles), LV-treated (n = 3, black squares), or CD47hi LV–treated (n = 3, yellow squares) NHPs at the indicated time after administration. The black dashed lines show the means ± 3 SDs calculated on 14 pre-LV samples taken from the same animals; the blue dashed lines show the normal reference values for M. fascicularis. (F to J) Concentration of (F) hFIX antigen or (G) hFIX activity measured in the plasma or (H) total anti–hFIX Abs, or (I) neutralizing anti–hFIX Abs, or (J) anti-FIX/hFIX immune complexes measured in the serum of vehicle-treated (n = 1, red circles), LV-treated (n = 3, black symbols), or CD47hi LV–treated (n = 3, yellow symbols) NHPs at the indicated time after administration. Nonparametric two-way ANOVA on the first 30 days after LV. BU, Bethesda units.

  • Fig. 5 Selectivity of intravenous LV gene therapy in NHPs and IS analysis.

    (A) Single values of VCN in the indicated organs of vehicle-, LV- or CD47hi LV–treated NHPs at necropsy (90 days after LV treatment). The dashed lines defining the gray area represent the lower limit of detection (0.0004) and the lower limit of quantification (0.006); thus, values in the gray area can be detected (different from the negative control) but not reliably quantified (see Materials and Methods section). PBMCs, peripheral blood mononuclear cells. (B) Expression analysis by quantitative real-time polymerase chain reaction of WPRE normalized on the endogenous TAF7 gene (2ΔCt) on RNA extracted from different liver lobes of LV- or CD47hi LV–treated NHPs, as indicated. Mann-Whitney test. (C and D) Counts of LV-RNA–positive cells [(D) LV-expressing cells] by ISH on liver tissue slices of the indicated NHPs (n = 5 random fields taken from five nonconsecutive slides per NHP); representative images are shown in (C). Scale bar, 100 μm. Mann-Whitney test. (E and F) Stacked bar plots representing the abundance of each LV IS retrieved from the liver of LV- or CD47hi LV–treated NHPs. (E) Each LV IS is represented by a different color with the height in relative proportion with the number of retrieved genomes (frequency) over the total. (F) The frequency by which individual LV integrations are found in one or more genomes is plotted in groups of increasing number of genomes.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/493/eaav7325/DC1

    Materials and Methods

    Fig. S1. Fractionation and sorting of liver cell subpopulations.

    Fig. S2. LV biodistribution within the liver cell subpopulations.

    Fig. S3. Generation of CD47-negative cells.

    Fig. S4. Generation of CD47-overexpressing cells.

    Fig. S5. Cytokine and chemokine response to LV, CD47hi LV, or CD47-free LV administration in NOD mice.

    Fig. S6. Cytokine and chemokine response to LV or CD47hi LV administration in NHPs.

    Fig. S7. LV gene therapy in NHPs.

    Fig. S8. IS analysis in CD47hi LV– or LV-treated NHP spleen.

    Table S1. Large-scale LV batches used in NHP study.

    Table S2. Clinical biochemistry, hematology, and hemostasis of vehicle NHP.

    Table S3. Clinical biochemistry, hematology, and hemostasis of LV1.

    Table S4. Clinical biochemistry, hematology, and hemostasis of LV2.

    Table S5. Clinical biochemistry, hematology, and hemostasis of LV3.

    Table S6. Clinical biochemistry, hematology, and hemostasis of CD47hi LV1.

    Table S7. Clinical biochemistry, hematology, and hemostasis of CD47hi LV2.

    Table S8. Clinical biochemistry, hematology, and hemostasis of CD47hi LV3.

    Table S9. LV IS in NHPs.

    Table S10. LV IS in cancer genes.

    Table S11. LV CIS.

    Movie S1. IV2PM of LVs, CD47hi LVs, or CD47-free LVs upon administration.

    References (4653)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Fractionation and sorting of liver cell subpopulations.
    • Fig. S2. LV biodistribution within the liver cell subpopulations.
    • Fig. S3. Generation of CD47-negative cells.
    • Fig. S4. Generation of CD47-overexpressing cells.
    • Fig. S5. Cytokine and chemokine response to LV, CD47hi LV, or CD47-free LV administration in NOD mice.
    • Fig. S6. Cytokine and chemokine response to LV or CD47hi LV administration in NHPs.
    • Fig. S7. LV gene therapy in NHPs.
    • Fig. S8. IS analysis in CD47hi LV– or LV-treated NHP spleen.
    • Table S1. Large-scale LV batches used in NHP study.
    • Table S2. Clinical biochemistry, hematology, and hemostasis of vehicle NHP.
    • Table S3. Clinical biochemistry, hematology, and hemostasis of LV1.
    • Table S4. Clinical biochemistry, hematology, and hemostasis of LV2.
    • Table S5. Clinical biochemistry, hematology, and hemostasis of LV3.
    • Table S6. Clinical biochemistry, hematology, and hemostasis of CD47hi LV1.
    • Table S7. Clinical biochemistry, hematology, and hemostasis of CD47hi LV2.
    • Table S8. Clinical biochemistry, hematology, and hemostasis of CD47hi LV3.
    • Table S9. LV IS in NHPs.
    • Table S10. LV IS in cancer genes.
    • Table S11. LV CIS.
    • Legend for movie S1
    • References (4653)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). IV2PM of LVs, CD47hi LVs, or CD47-free LVs upon administration.

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