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

Vectors in stealth mode

Gene therapy using adeno-associated viral vectors (AAVs) has shown safety and efficacy in patients with hemophilia. However, AAVs have limitations hindering their efficacy in a subgroup of patients. The use of lentiviral vectors (LVs) has been explored as possible alternative; however, preclinical data reported low transduction efficacy possibly due to fast clearance by phagocytes. Now, Milani et al. developed a shielded LV able to escape phagocytosis by increasing the content of the phagocytosis inhibitor CD47 on their surface. Upon intravenous administration in monkeys, the LVs showed high transduction efficacy without signs of toxicity. The results suggest that LV-mediated gene therapy might be an effective strategy for treating hemophilia and possibly other disorders.


Liver-directed gene therapy for the coagulation disorder hemophilia showed safe and effective results in clinical trials using adeno-associated viral vectors to replace a functional coagulation factor, although some unmet needs remain. Lentiviral vectors (LVs) may address some of these hurdles because of their potential for stable expression and the low prevalence of preexisting viral immunity in humans. However, systemic LV administration to hemophilic dogs was associated to mild acute toxicity and low efficacy at the administered doses. Here, exploiting intravital microscopy and LV surface engineering, we report a major role of the human phagocytosis inhibitor CD47, incorporated into LV cell membrane, in protecting LVs from uptake by professional phagocytes and innate immune sensing, thus favoring biodistribution to hepatocytes after systemic administration. By enforcing high CD47 surface content, we generated phagocytosis-shielded LVs which, upon intravenous administration to nonhuman primates, showed selective liver and spleen targeting and enhanced hepatocyte gene transfer compared to parental LV, reaching supraphysiological activity of human coagulation factor IX, the protein encoded by the transgene, without signs of toxicity or clonal expansion of transduced cells.


Liver-directed gene therapy for the treatment of the inherited coagulation disorder hemophilia is among the most successful application of gene therapy (1). Protein replacement therapy (PRT) with recombinant coagulation factor VIII or IX (FIX), whose activity is lacking in hemophilia A or B, respectively, is the standard of care. Gene therapy, however, may establish a stable tissue source of functional factor after a single administration, bypassing the lifelong requirement of frequent intravenous PRT infusion and potentially providing a definitive cure of the disease. A single intravenous administration of recombinant adeno-associated virus (AAV)–derived vectors delivering a functional copy of a clotting factor gene to the liver, its natural site of production, has shown safety and efficacy in patients with hemophilia and is poised to become a clinically available treatment option (24). However, because AAV vectors do not actively integrate into the host cell genome and the anti-AAV immune responses limit vector readministration (1, 5), it may be difficult to apply this type of gene therapy to pediatric patients. Moreover, a sizable fraction of adult patients is immunized against AAV; thus, they either are not eligible to receive AAV vector administration, because of neutralizing anti-AAV antibodies (Abs), or necessitate immune suppression for a period of time to maintain AAV-transduced hepatocytes, because of cellular immunity against AAV capsids (1, 5). On the contrary, HIV-derived lentiviral vectors (LVs) integrate into the target cell chromatin and are maintained as cells divide, a potential advantage for establishing long-term expression if not associated with a significant risk of insertional mutagenesis (6, 7). Furthermore, the lower prevalence of HIV compared to AAV infection in humans ( makes LVs attractive gene delivery vehicles to complement the reach and broaden the scope of AAV vector–based gene therapy for liver diseases. We have developed LVs that achieve stable transgene expression in the liver and reconstitute FIX activity in mouse models of hemophilia B, without detectable evidence of genotoxicity (8, 9). These outcomes are dependent on stringent targeting of expression to hepatocytes by combining transcriptional and posttranscriptional microRNA-mediated regulation (10). However, systemic LV administration to dogs was associated to a mild acute toxicity, and efficacy was low at the administered LV doses (9). These limitations might reflect poor biodistribution of intravenously administered LV to hepatocytes, possibly due to fast clearance from the circulation by hepatic and splenic professional phagocytes, which, in turn, triggers innate immune activation upon sensing the taken up viral particles. For these reasons, we set out to investigate LV biodistribution to different liver cell types after intravenous administration of escalating LV doses and counteract their capture by professional phagocytes exploiting a natural inhibitor, CD47. By this strategy, we generated phagocytosis-shielded LVs with higher efficiency of hepatocyte gene transfer and reduced activation of acute inflammatory response, after intravenous administration, and evaluated their safety and efficacy in nonhuman primates (NHPs).


Professional phagocytes in the liver uptake most intravenously administered LVs

We first evaluated the correlation between the administered dose of LVs bearing a hepatocyte-specific FIX expression cassette (LV-FIX) (8) and transgene expression in C57BL/6 adult mice (n = 48). The previously described hepatocyte-specific expression cassette contains an engineered hepatocyte-specific promoter (enhanced transthyretin) and target sequences for the hematopoietic lineage–specific microRNA 142, abrogating off-target transgene expression in antigen-presenting cells (APCs) in the liver and spleen (11). We observed a nonlinear dose response and a rapid increase in FIX output above a threshold dose, achieving about 1 μg/ml (corresponding to 20% of normal) at an LV dose of 3 × 1010 transducing units (TU)/kg (Fig. 1A). Professional phagocytes in the liver and spleen provide a major clearance mechanism for blood-born particles, including viral vectors (12, 13). We thus investigated the biodistribution of intravenously administered LVs to the spleen and liver cell subpopulations. We administered LV expressing green fluorescent protein [GFP; 1, 2, or 4 × 1010 TU/kg (n = 10 per dose)] to C57BL/6 adult mice and measured transgene expression and vector copies per diploid genome [vector copy number (VCN)] 2 months thereafter. Whereas both the percentages of GFP-positive hepatocytes and VCN in total liver DNA nearly doubled from 1 to 2 × 1010 TU/kg, there was a disproportionately higher increase in GFP-positive hepatocytes when further doubling the vector dose (Fig. 1B), in line with the nonlinear dose response observed for LV-FIX. We then separated cellular fractions enriched in hepatocytes [parenchymal cells (PCs)] from non-PCs (nPCs) in freshly dissociated livers. We further purified hepatocytes from the PC fraction and liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs), and plasmacytoid dendritic cells (pDCs) from total nPCs by fluorescence-activated cell sorting (FACS; fig. S1) and measured the VCN in these subpopulations. We observed very high VCN in KCs at all tested doses, reaching 27. This high VCN in nPCs and KCs plateaued at increasing LV doses (Fig. 1C). It is reported that the liver comprises about 70% PCs and 30% nPCs, of which 50% are LSECs (15% of total), 20% are KCs (6% of total), and the remaining 30% are biliary ducts cells, hepatic stellate cells, and other cells (14). We thus calculated the relative contribution to the total liver vector content of each subpopulation, considering their reported abundance, and estimated that only about 30% of the liver LV DNA is found in PCs at low doses, while it becomes >50% at the highest dose. Conversely, almost 70% of the LV DNA is found in nPCs at the low doses and about 40% at the highest dose (fig. S2A). There was a linear correlation between the VCN measured in total liver samples at each LV dose and that calculated summing up the contributions of each subpopulation (fig. S2, B to D). Whereas the best-fit correlation between the percentages of GFP-positive hepatocytes and total liver VCN was exponential, a perfectly linear correlation was found between GFP-positive hepatocytes and the VCN in PCs (fig. S2, E and F). These data indicate that the VCN measured in total liver does not reflect the percentage of hepatocyte transduction due to the different distribution of LV DNA observed within the liver cell subpopulations at different LV doses. Moreover, the results suggest that, as the LV input reaches a threshold dose, LV uptake by KCs is saturated and more LVs are available to transduce hepatocytes, thus giving rise to a nonlinear dose response for hepatocyte gene transfer and transgene expression.

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.

Recognition of human CD47 on LV particles inhibits phagocytosis and increases hepatocyte gene transfer

Phagocytosis is physiologically inhibited by CD47, a ubiquitously expressed species-specific ligand of signal regulatory protein α (SIRP-α) receptor expressed by professional phagocytes (15). Human CD47 is incorporated into LVs when they bud from producer cells. It has been shown that SIRP-α of NOD mice has high affinity for human CD47 (16). We thus compared the outcome of LV-FIX administration to NOD mice and C57BL/6 F9 knockout mice, a mouse model of hemophilia B. We observed a significantly longer half-life of LV particles in the first hour upon bolus intravenous injection (P = 0.0001) and >10-fold higher FIX expression in the blood (P = 0.0046) in NOD compared to C57BL/6 mice (Fig. 1, D and E). Consistent with the increased FIX output, we found fourfold higher LV VCN in sorted hepatocytes and 30- and 5-fold lower VCN in KCs and spleen, respectively, in NOD versus C57BL/6 mice (Fig. 1F). LV copies were also >10-fold lower in NOD pDCs, which are known sensors of viral nucleic acid and were reported to release type I interferon after exposure to LV particles (17). We confirmed a strong correlation between the VCN in sorted hepatocytes and FIX expression (fig. S2G). On the basis of the VCN in the liver cell subpopulations, we estimated that only about 5% of the liver LV DNA was in sorted hepatocytes in C57BL/6 mice, whereas it was about 50% in NOD mice (fig. S2H). Despite the higher VCN in sorted hepatocytes found in NOD compared to C57BL/6 mice, the VCN measured in total liver samples was lower in the former compared to the latter strain (fig. S2I), further confirming that VCN in bulk liver does not reflect the extent of transduction of hepatocytes. When we calculated the expected liver VCN on the basis of the relative contribution of the VCN measured in sorted hepatocytes and KCs (from Fig. 1F), we obtained a higher total liver VCN in C57BL/6 compared to NOD mice (fig. S2I). To confirm the role of human CD47 in the observed outcome, we repeated the experiment with LVs produced by cells in which we disrupted the CD47 gene by CRISPR-Cas9 (CD47-free LVs; Fig. 1, G to I, and figs. S2J and S3). The interstrain differences in LV half-life, transgene expression, and biodistribution among liver cell types, observed when we administered LVs, were almost completely abrogated when we administered CD47-free LVs, at both lower and higher doses, indicating that the interaction between the NOD SIRP-α and the human CD47 molecule on LV particles was primarily responsible for the differences observed between the two strains. These findings underline a major role of CD47 in inhibiting LV uptake by phagocytes and innate immune sensors, substantially affecting LV in vivo biodistribution.

CD47 overexpression in producer cells protects LVs from uptake by human macrophages

Because the density of CD47 molecules is differentially regulated among distinct cell types and can determine their susceptibility to phagocytosis (1821), we exposed a human macrophage cell line or human primary macrophages to LVs and found a higher content of LV genome than in reference 293T cells, suggesting that LVs are actively phagocytized by these cells and that the amount of CD47 incorporated in the LV particles might be rate-limiting for inhibiting phagocytosis by human cells (Fig. 2, A and B). Note that the high content of LV genome did not correspond to high transgene expression, consistent with the reported postentry restriction of LV transduction in human mature macrophages (22, 23). We thus engineered both LV stable producer cell lines previously reported in (24) and 293T cells used for LV production by transient transfection to overexpress CD47. To this end, we transduced these cells with CD47-expressing self-inactivating γ-retroviral vectors (SIN RVs), which cannot be cross-packaged by LVs (25). We achieved stable 10- to 30-fold overexpression of the CD47 protein on the cell surface of both 293T cells and LV producer cell lines (CD47hi cells; fig. S4, A to E). LVs produced by CD47hi cells had comparable titer and infectivity on reference 293T cells as vectors produced by the parental cells (fig. S4, F to K) and showed significantly increased (P < 0.0001) immunostaining for CD47 by electron microscopy (Fig. 2, C and D). The amount of CD47 on the LV surface did not affect the presence of the envelope protein, vesicular stomatitis virus protein G (VSV.G), consistent with unaltered infectivity of CD47hi LV (Fig. 2E). When matched input of CD47hi and control LVs was incubated with a macrophage cell line or primary human macrophages, we found a significantly reduced uptake of CD47hi LV (P = 0.0303), which reached the basal level observed in the reference 293T cells (see Fig. 2B). Conversely, we found a significantly higher uptake of CD47-free than control LVs (P = 0.0083) by human macrophages (Fig. 2F). These data indicate that modulating the quantity of CD47 on LV particles affects their uptake by human macrophages.

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.

CD47hi LVs show reduced susceptibility to phagocytosis and innate immune activation in NOD mice

We then evaluated whether CD47 amount affected LV biodistribution in vivo, using the NOD mouse strain, which recognizes the human CD47. The half-life in the blood was significantly higher (P = 0.0005) for CD47hi than control LVs (Fig. 2G), with a dose-dependent change in biodistribution. At lower vector doses, KC uptake of control LVs was significantly higher (P = 0.0157) than uptake of CD47hi LVs (Fig. 2H). At higher vector doses, KC uptake was reduced for both vector types, compared to the lower vector doses (Fig. 2I and fig. S5A). These data suggest that the density of CD47 on the control LV surface is limiting and inhibits phagocytosis by liver KCs and other APCs only at high LV doses, when the total particle input may act by bulk action (nonparticle autonomous), whereas CD47hi LVs are consistently protected at the individual particle level even at low input. There was no difference in hepatocyte transduction or FIX expression between CD47hi LVs and control LVs in these experimental settings (Fig 2, H to J). Differential surface display of CD47 affected the acute cytokine and chemokine release, after intravenous LV administration. Specifically, macrophage inflammatory protein 1α (MIP-1α; P = 0.0363), MIP-1β (P < 0.0001), monocyte chemoattractant protein-1 (MCP-1; P = 0.0007), the chemokine (C-X-C motif) ligand 1 (CXCL-1; P = 0.0002), and granulocyte colony-stimulating factor (G-CSF; P < 0.0001) significantly increased in LV-treated compared to untreated NOD mice 3 hours after LV administration and then returned to baseline 2 to 7 days after LV treatment, depending on the cytokine, whereas their serum concentration was not different in CD47hi LV–treated compared to untreated NOD mice (Fig. 2, K to O, and fig. S5, B to F). Conversely, administration of CD47-free LV to NOD mice triggered the strongest increase in these macrophage-related cytokines (Fig. 2, K to O). These data are in line with the observed modulation of APC uptake by the CD47 content of the LV particles.

Intravital imaging shows that CD47 regulates the rate and extent of LV phagocytosis by KCs

To investigate the kinetics of LV phagocytosis in the liver in real time upon intravenous administration, we performed intravital two-photon microscopy (IV2PM). To visualize LVs, we produced them in control 293T cells, CD47hi 293T cells, or CD47-negative 293T cells expressing GFP fused to the membrane-targeting domain of pp60Src, a chimeric protein previously shown to be effectively incorporated in the budding HIV envelope (26). LV uptake was recorded live in the surgically exposed liver of anesthetized mice (27). We observed that administration of GFP-labeled LVs in C57BL/6 mice resulted in rapid and widespread uptake by KCs (visualized by red fluorescent anti-F4/80 Ab infusion before LV administration (28)), which became 90% LV positive in the examined fields within 4 to 8 min upon administration (Fig. 3 and movie S1). By contrast, we observed that administration of the same LVs into NOD mice reached 90% LV-positive KCs only after 15 to 28 min after LV treatment. When CD47hi LVs were administered to NOD mice, only 12 to 52% of KCs became LV positive at the end of recording (38 min after LV treatment). Note that the GFP signal is lost once LV envelope fuses with endosome membrane and the LV core escapes into the cytoplasm. According to this design, we could follow LV accumulation in KC endophagosomes (yellow signal), but not hepatocyte transduction, because of overall lower LV entry per cell. The CD47-free LV uptake by KCs observed in NOD mice appeared overlapping with that observed in control LVs injected in C57BL/6 mice, reaching 90% LV-positive KCs 6 min after LV treatment. These data provide visual evidence that the recognition and content of CD47 on the LV surface affect timing and extent of LV uptake by KCs and, together with the results shown above, indicate a major role of this molecule in shielding LVs from phagocytosis in vivo.

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).

Intravenous administration of phagocytosis-shielded LVs to NHPs is safe and well tolerated

Because the sequence homology of SIRP-α and CD47 between NHPs and humans is 94 and 99%, respectively, whereas the murine SIRP-α and CD47 are only 70 to 75% homologous to the human genes, we predicted that the protection afforded by CD47 to LVs may be even more effective in NHPs, which represent the closest model to humans. We chose Macaca nemestrina as recipient because of the lower restriction to HIV infection compared to other NHP species (29, 30). We produced large-scale batches of control or CD47hi LVs using the previously described major histocompatibility complex (MHC)–free 293T cells (24), purified and qualified for potency, purity, and sterility according to the protocol and specifications used for clinical grade LVs (table S1 and fig. S6A). We administered these LVs via a peripheral vein to six NHPs at the target dose of 7.5 × 109 TU/kg (three for each LV version). The infusion was well tolerated, with only a minor elevation of aspartate aminotransferase 1 day after administration (twofold the preadministration value) and with serum alanine aminotransferases and body temperature remaining within the means ± 3 SDs of pretreatment values (Fig. 4, A to C, and tables S2 to S8). We observed a transient self-limiting leukopenia, mostly noted in lymphocytes, 1 day after LV administration, which might be explained by migration of these cells into the liver (Fig. 4, D and E). We measured a panel of 23 cytokines in the serum of treated NHPs before and after LV administration and observed a transient increase in interleukin-2 (IL-2), IL-1 receptor antagonist, IL-18, IL-10, and the macrophage-related cytokines MIP-1α, MIP-1β, and MCP-1, compared to the vehicle-treated animal, suggesting mild self-limiting inflammation (fig. S6, B to H). In line with the data shown above in NOD mice, we observed a milder rise in MIP-1α, MIP-1β, and MCP-1 in CD47hi LVs compared to LV-treated NHPs, and the concentrations of these chemokines in CD47hi LV–treated animals were nearly overlapping with those of the vehicle-treated individuals (fig. S6, B to H). Overall, these data show that in vivo administration of LVs to NHPs is safe and well tolerated and suggests reduced activation of the innate immune system by CD47hi LVs.

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.

Intravenous administration of LVs to NHPs results in robust hepatocyte gene transfer further increased by CD47hi LVs

We measured human-specific FIX antigen and activity in the plasma of treated NHPs and found an average of 1 μg/ml and 1 U/ml (corresponding to 20 and 100% of normal, respectively) in LV-treated animals, which was stable for 90 days after LV treatment, when the study was terminated (Fig. 4, F and G). Instead, FIX antigen and activity were 2.9 μg/ml and 2.42 U/ml (corresponding to 58 and 242% of normal, respectively) on average in CD47hi LV–treated animals, nearly threefold higher than those of LV-treated animals [P = 0.0002 (Fig. 4F) and P < 0.0001 (Fig. 4G)], in the first month after LV treatment. Note that FIX activity was four- to fivefold higher than antigen, as expected by the use of the hyperfunctional FIX Padua variant (8, 31). After the first month of follow-up, the three NHPs expressing higher FIX developed antihuman FIX Abs at increasing titer with increasing FIX level (Fig. 4, H and I), in line with other studies administering high doses of recombinant human FIX or vectors expressing human FIX to NHPs (32, 33). The emergence of low-titer non-neutralizing anti-FIX Abs in CD47hi LV2 and LV3 was associated with the formation of detectable circulating immune complexes (Fig. 4J), which likely caused a concomitant overestimation of human-specific FIX antigen and activity by the immune capture assays toward the end of the study (see Fig. 4, F and G). Instead, the higher titer, neutralizing anti-FIX Abs developed by CD47hi LV1 caused human FIX antigen and activity to become undetectable (Fig 4I). By measuring p24 in the serum of treated NHPs, we observed almost overlapping clearance of the two LV versions, with a 4-log decrease in serum p24 the day after LV administration and becoming undetectable 1 week after LV administration (fig. S7A). The serum concentration of C3a increased above the mean ± 3 SDs of pretreatment values shortly after LV administration, suggesting that a part of circulating LV particles may be lysed by the complement system (fig. S7B). As expected, all the animals developed anti-VSV.G Abs (fig. S7C). After necropsy, we measured LV DNA in the liver, spleen, and major organs of treated animals. We found between 0.5 and 1.8 LV VCN in the liver, accounting for 80 to 90% of all the retrieved LV DNA, showing selective targeting of the liver by LVs in NHPs, with almost a 3-log difference in VCN measured in the liver and the highest VCN measured in the other organs (Fig. 5A). In line with the mouse data, the measured FIX output, but not the total liver VCN, was higher in CD47hi LV–treated compared to LV-treated NHPs. These findings likely reflect a different vector distribution between liver cell subpopulations, favoring hepatocyte transduction and decreasing KC uptake for CD47hi LVs. Increased hepatocyte transduction in CD47hi LV–treated NHPs was further indicated by measuring LV RNA expression, which is selectively targeted to hepatocytes, in liver samples and performing RNA in situ hybridization (ISH) on liver slices with a probe targeting the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) present on the transgene RNA. We found higher LV RNA content (P = 0.0358) and more LV-expressing cells (P < 0.0001) in the liver of CD47hi LV– than LV-treated NHPs, except in CD47hi LV1, who showed hardly any LV RNA and very few LV-expressing cells, suggesting delayed clearance of transduced hepatocytes accompanying the development of neutralizing antihuman FIX Abs (Fig. 5, B to D). We estimated the difference in LV VCN of KCs compared to hepatocytes of CD47hi LV– and LV-treated NHPs on the basis of a mathematical model built on experimental data obtained in mice (fig. S7, D and E). Pathology analysis of the liver, spleen, and major organs was performed by two independent veterinary pathologists at the end of the study in all NHPs, including the vehicle-treated control and no macroscopic or microscopic lesions were reported, except for splenic follicular hyperplasia and minimal liver inflammatory foci found in vector-treated NHPs, which could not be conclusively attributed to vector treatment because of the single control animal analyzed and whose features were considered within the range of normal for NHPs. Blood hematology and clinical biochemistry parameters were within the range of pretreatment values in the follow-up of vector administration, except for minor fluctuations (see tables S2 to S8).

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.

Quantitative high-throughput vector integration site (IS) analysis performed at the end of the study on genomic DNA from multiple liver samples and one spleen sample from all NHPs showed high diversity, with thousands of distinct ISs contributing by few occurrences to the total (Fig. 5E, fig. S8A, and table S9). There were no dominant clones in the liver or spleen of all NHPs analyzed, and >93% ISs were represented by one to four genomes, whereas the remaining ISs were represented by a maximum abundance of 32 genomes (Fig. 5F and fig. S8B). The frequency of IS mapping near or within cancer genes, defined by the oncogenes and tumor suppressor genes listed in UniProt for both human and murine annotations, was about 2% on average, similar to the percentage of cancer genes in the whole genome, and did not show enrichment even in the few ISs with an abundance of more than four genomes (table S10). The genomic distribution of ISs across the genome showed the appearance of few hotspots (common IS; table S11), which, with few exceptions, clustered in regions syntenic to human genomic regions known to be frequently targeted by LV insertions in human hematopoietic cells as the result of an intrinsic integration bias of the parental virus not associated with genotoxicity (3436). IS was more enriched in the liver than in spleen (P < 0.0001) when comparing the CD47hi LV versus control LV groups (see table S9), likely reflecting the CD47 action.

Overall, we describe efficient liver gene transfer in NHPs, which is further and substantially increased by CD47hi LVs, likely because of skewed vector biodistribution within the liver, favoring transduction of hepatocytes at the expense of KCs and leading to human-specific FIX activity in the plasma up to supraphysiological, without molecular evidence of clonal expansion of transduced cells or enrichment of LV ISs at cancer genes.


Here, we show tolerability, selectivity, and efficiency of liver gene transfer after systemic intravenous LV administration to NHPs. We observed a more favorable LV dose response for FIX expression in NHPs than in dogs and mice, which required a fourfold higher dose to reach the same FIX output. This favorable outcome may be due, at least in part, to better recognition of the human CD47 present on LVs by the NHP SIRP-α receptor as compared to its murine or canine counterpart. In addition, HIV has evolved mechanisms to evade intracellular sensing and innate immune triggering in humans (37), which may further explain the limited acute reaction to intravenous LV administration observed in M. nemestrina in our study. The preferential gene transfer to the liver and spleen by VSV.G-pseudotyped LVs, despite systemic administration and the well-known VSV.G pantropism, may be due to the abundant blood supply to these filter organs, the facilitated access to perisinusoidal space by the discontinuous microvascular endothelium, and the engagement of low-density lipoprotein (LDL) receptors on hepatocytes by VSV.G on vector particles (38), mimicking LDL uptake. This LV biodistribution, combined with tailored choices of transcriptional and posttranscriptional regulation (39, 40), provides a platform for stringently targeting transgene expression to specific cell types within the liver or spleen, as shown here for hepatocytes.

The major role of the producer cell–derived CD47 molecule in modulating LV biodistribution among liver and spleen cell types shown here is consistent with previous observations that incorporation of CD47 increases the half-life and decreases phagocytosis of intravenously administered nanoparticles and LV particles in a SIRP-α–dependent manner (20, 41). Here, we extend these findings as we compared mice permissive or refractory to human CD47 signaling, and LV with or without CD47, and show that decreased clearance by professional phagocytes favors LV transduction of hepatocytes and alleviates the innate immune response to systemic administration. Furthermore, intravital imaging of the liver during LV administration provided direct evidence of the rapid scavenging of vector particles from the sinusoids by mouse KCs and the rate-limiting function of CD47 on the vector surface in modulating this clearance mechanism. LVs with high surface content of CD47, besides being more resistant to phagocytosis, were also less prone to innate immune sensing by APCs and showed improved efficiency of gene transfer to hepatocytes in NHPs, compared to LVs with basal content of CD47. Such increase in hepatocyte transduction and FIX output by CD47hi LVs was not observed in NOD mice. The difference between mice and NHPs may be due, at least in part, to the different rates of intravenous administration, which was performed by bolus injection in mice and by slow infusion (10 ml/kg per hour) in NHPs. In the former setting, a peak of LV blood concentration at the beginning of infusion may have allowed CD47 to act by total mass action (nonparticle autonomous) on professional phagocytes, thus masking the benefit provided by increasing the CD47 density per LV particle, which might instead be more relevant at lower LV blood concentrations, as upon the slow LV infusion performed in NHPs. This explanation is also consistent with our interpretation of the different extent of KC uptake and its modulation by CD47 at high versus low input vector doses in NOD mice. Different liver histological features, such as the size of LSEC fenestrations, may also contribute to the different LV dose response observed for hepatocyte gene transfer in different animal models (42). Note, however, that CD47hi LV did not show delayed clearance from the circulation as compared to control LVs in NHPs, suggesting that additional complement-mediated mechanisms might contribute to clear nonphagocytized CD47hi LVs from the circulation by direct lysis or opsonization of complement-bound particles that have lost transduction capacity. In support of this hypothesis, we observed a higher peak of complement activation in CD47hi LV– than LV-treated NHPs.

Antihuman FIX immune responses were observed in this, as well as previous gene therapy studies performed in NHPs with different vectors (33, 43), and may raise concerns for clinical translation. The association of immune response with high human FIX concentration in the blood and the delayed time of onset suggest that these responses may be mediated by cross-presentation in MHC classes I and II of FIX antigen taken up from the circulation by APCs and thus were not fully prevented by shielding LVs from phagocytosis at the time of administration. Such immune responses may not necessarily imply a risk when translating the treatment in previously exposed Abs-negative patients.

Whereas genome-wide LV integration necessarily implies a concern for delayed oncogenesis, our findings show no signs of expansion of LV-transduced hepatocytes or of liver tumors in NHPs, albeit within the inherent limitations of number of treated individuals and length of observation. LV IS analysis in NHP liver and spleen showed diverse composition with nearly all unique ISs being represented by one to four genomes, indicating that no clonal expansions have occurred and consistent with the low to null proliferation rate expected for most liver cells in the treated NHPs. Moreover, to the best of our knowledge, no evidence of insertional oncogenesis has been reported until now in hundreds of patients treated by LV-transduced hematopoietic stem cells (HSCs) or T cells in several clinical trials, with a follow-up of >10 years for the earliest treated patients (1, 6). These findings are reassuring, given the sensitivity of HSCs to oncogenic transformation, which is likely to be higher than hepatocytes, and support the claim that LV design, IS selection, and polyclonal reconstitution all contribute to alleviate such risk.

The main limitations of this work are the small sample size of the NHP study due to feasibility and ethical reasons; a relatively short window of observation of LV-treated NHPs, which cannot exclude long-term adverse effects due to insertional mutagenesis; and the single vector dose evaluated when comparing CD47hi LVs and parental LVs in NHPs, which calls for additional preclinical studies at lowered vector doses to further evaluate the safety, efficacy, and immunogenicity of in vivo gene therapy with CD47hi LVs. Overall, the enhanced therapeutic index of phagocytosis-shielded CD47hi LVs should allow reducing the effective LV dose, thus easing manufacturing demand, alleviating concerns for any residual dose-dependent LV toxicity, and paving the way to future clinical testing with the aim to broaden applicability of liver gene therapy to more hemophilia patients and, possibly, other diseases.


Study design

Sample size in experiments with mice was chosen according to previous experience with experimental models and assays. Mouse studies were designed to evaluate susceptibility of LVs to phagocytosis after intravenous administration. LV-derived transgene expression was measured in the plasma of treated animals, and LV transduction of different cell types was determined at end point. NHP study was designed to evaluate acute toxicity, transduction efficiency, and biodistribution of intravenously administered LVs by measuring transgene expression in the plasma, blood chemistry, histological, and molecular analyses. Sample size in the NHP study was limited by ethical and feasibility reasons. No sample or animal was excluded from the analyses. Mice and NHPs were randomly assigned to each experimental group. Investigators were not blinded.

Vector production

Large-scale purified LV batches used for the NHP study were produced by MolMed S.p.A., as previously described (9), and formulated in phosphate-buffered saline (PBS) with 5% dimethyl sulfoxide (DMSO). Results of selected quality control assays performed on these batches are reported in table S1. A sterile “vehicle” solution (PBS with 5% DMSO) was prepared in parallel. Laboratory-grade VSV.G-pseudotyped third-generation SIN LVs were produced by calcium phosphate transient transfection into 293T cells or by LV stable producer cell lines (24). 293T cells were transfected with a solution containing a mix of the selected LV genome transfer plasmid, the packaging plasmids pMDLg/pRRE and pCMV.REV, pMD2.G, and pAdvantage, as previously described (9). Medium was changed 14 to 16 hours after transfection, and supernatant was collected 30 hours after medium change. Alternatively, LV production was induced when LV producer cells were in a subconfluent state by replacing the culture medium with medium containing doxycycline (1 μg/ml; Sigma-Aldrich), and supernatant was collected 3 days after induction. LV-containing supernatants were sterilized through a 0.22-μm filter (Millipore) and, when needed, transferred into sterile polyallomer tubes (Beckman) and centrifuged at 20,000g for 120 min at 20°C (Beckman Optima XL-100 K Ultracentrifuge). LV pellet was dissolved in the appropriate volume of PBS to allow 500× to 1000× concentrations. VSV.G-pseudotyped SIN RVs were produced by calcium phosphate transient transfection into 293T cells. 293T cells were transfected with pRT43.3.PGK.CD47, the packaging plasmid pCMV-Gag/Pol (Moloney Leukemia Virus), and pMD2.G, as described (44). Medium was changed 14 to 16 hours after transfection, and RV-containing supernatant was collected 30 hours after medium change and concentrated 1000× by ultracentrifugation as above.

Mice experiments

Founder C57BL/6 F9 knockout mice were originally obtained from the laboratory of I. Verma at the Salk Institute (45). NOD and wild-type C57BL/6 mice were purchased from Charles River Laboratories. All mice were maintained in specific pathogen-free conditions. Vector administration was carried out in adult (7 to 10 weeks old) mice by tail-vein injection. Mice were bled from the retro-orbital plexus using capillary tubes, and blood was collected into 0.38% sodium citrate buffer (pH 7.4). Mice were deeply anesthetized with tribromoethanol (Avertin) and euthanized by CO2 inhalation at the scheduled times. All animal procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee.

Intravital imaging

C57BL/6 or NOD mice were surgically prepared for liver IV2PM, as described (28). Mice were intravenously injected with PE-conjugated anti-F4/80 Ab (clone BM8, BioLegend) 20 min before imaging. GFP-labeled LVs, CD47hi LVs, or CD47-free LVs were intravenously injected 2 min after the start of video recording. Images (TrimScope II) were obtained with a Nikon Ti-U fluorescence inverted microscope and a 25× objective (numerical aperture, 0.95). For four-dimensional analysis, 8 to 12 z stacks (spacing, 4 μm) of xy sections (300 to 400 μm2) were acquired every 20 s for 40 min. Liver sinusoids were visualized by injecting intravenous nontargeted Quantum Dots 655 (Invitrogen) immediately before imaging. Sequences of image stacks were transformed into volume-rendered four-dimensional videos using Imaris software (Bitplane).

NHP study

Seven adult (body weight, 3 to 5 kg) male M. nemestrina were purchased from BioPRIM (Baziège, France). Macaques were housed in an enriched environment with access to toys, fresh fruits, and vegetables at the Boisbonne Center (Nantes, France), under protocol APAFIS no. 4302-2015122314563838 that was approved by the Institutional Animal Care and Use Committee of the Pays de la Loire. For LV administration, animals were anesthetized with dexmedetomidine (30 μg/kg; Domitor) and ketamine (7 mg/kg) and maintained under gas anesthesia, 1 to 2% isoflurane (Vetflurane). The LV-containing solution or vehicle solution (PBS with 5% DMSO) was administered using a syringe with controlled flow rate fixed at 10 ml/kg per hour via a catheter in the saphenous vein for an approximate time of 2 hours. The administered LV dose was verified by sampling and titering the dosing solution at the end of infusion. Whereas 5 of 6 animals received the target dose, between 6.9 and 7.7 × 109 TU/kg, one animal (CD47hi LV2) received a higher dose of CD47hi LVs (1.0 × 1010 TU/kg) because of a mistake in pooling LV aliquots of different volume. Note, however, that this animal showed the lowest transgene expression among its group, thus suggesting that the slightly higher administered dose was not instrumental in driving the outcome of higher transgene expression for the CD47hi LV group. An anti-inflammatory and antihistamine premedication regimen was administered: dexamethasone (0.3 mg/kg; Dexadreson) intravenously 24 hours before LV administration and just before LV administration and dexchlorpheniramine (4 mg/kg; Polaramine) intravenously 30 min before LV administration. Blood samples were taken at different time points from the femoral vein upon anesthesia with ketamine (10 mg/kg; Imalgene) intramuscularly. For hematology, 1 ml of total blood samples was collected on EDTA-coated tubes. For clinical biochemistry, 2 ml of total blood samples was collected on heparin-coated tubes, and for hemostasis, 1.8 ml of total blood was collected in citrate-coated tubes. Blood tests were performed on fresh samples at the Veterinary School of Nantes (LDHvet, Oniris). Biochemistry parameters were analyzed by automatons and analyzers on the basis of spectrometry, reflectometry, potentiometry, and enzyme immunoassays. Hemostasis was analyzed by a hemostasis analyzer on the basis of electromechanical clot detection (viscosity-based detection system). Tissue samples were collected after euthanasia, performed by overdose pentobarbital sodium (Dolethal) intravenous injection.

FIX assays

The concentration of human FIX was determined in mouse plasma by an enzyme-linked immunosorbent assay (ELISA) specific for human FIX antigen (Asserachrom IX:Ag, Stago) following the manufacturer’s instructions. Absorbance of each sample was determined spectrophotometrically using a Multiskan GO microplate reader (Thermo Fisher Scientific) and normalized to antigen standard curves. The concentration of human FIX was determined in NHP plasma by an ELISA specific for human FIX antigen (AHIX-5041, Haematologic Technologies). Human FIX activity was quantified in NHP plasma by a modified FIX chromogenic assay, human FIX in plasma samples was first captured by a human FIX–specific Ab immobilized onto a 96-well plate (AHIX-5041, Haematologic Technologies), and then chromogenic activity of human FIX was measured using a Hyphen Biophen Factor IX kit (221806-RUO , Aniara Corp.) following the manufacturer’s instructions. Standard curves were obtained by diluting recombinant human FIX (BeneFIX, Pfizer) into untreated NHP plasma. Total anti–human FIX Abs were quantified in heat-inactivated NHP serum by ELISA, coating recombinant human FIX (BeneFIX, Pfizer) and developing with a horseradish peroxidase (HRP)–conjugated mouse anti-monkey immunoglobulin G (IgG) Ab (1:8000; SouthernBiotech). Antihuman FIX/human FIX complexes were quantified in NHP serum by ELISA, coating with a human FIX–specific Ab (AHIX-5041, Haematologic Technologies) and developing with a HRP-conjugated mouse anti-monkey IgG Ab (1:8000; SouthernBiotech). Absorbance of each sample was determined spectrophotometrically using a Multiskan GO microplate reader (Thermo Fisher Scientific) and normalized to coated monkey IgG standard curves ( Neutralizing anti–human FIX Abs were determined by Bethesda assay. The test sample was incubated for 2 hours at 37°C with recombinant human FIX (1 U/ml; BeneFIX, Pfizer) with FIX activity of 100%. Residual FIX activity was measured using a Hyphen Biophen Factor IX kit (221806-RUO, Aniara Corp.) and converted into Bethesda units per milliliters, where one Bethesda unit is defined as the inverse of the dilution factor of the test sample that yields 50% residual FIX activity.

Statistical analysis

Statistical analyses were performed upon consulting with professional statisticians at the San Raffaele University Center for Statistics in the Biomedical Sciences (CUSSB). When normality assumptions were not met, nonparametric statistical tests were performed. Mann-Whitney or Kruskal-Wallis tests with Dunn’s multiple comparison posttest were performed when comparing two or more experimental groups, respectively. For repeated measures over time, two-way ANOVA was performed. Comparison between the proportions of ISs retrieved in the liver and spleen in NHPs was performed by the Fisher’s exact test. When sample size was lower than five and no repeated measurements were available for the same finding, inferential statistical analysis was not performed. To test differences in FIX expression or activity over time in NHPs, we applied a nonparametric two-way ANOVA (robust rank-based method for factorial designs).


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)


Acknowledgments: We thank A. Nonis, C. Brombin, and C. Di Serio of CUSSB for statistical consulting, A. Lombardo and A. Migliara for help with genetic disruption of CD47, the ALEMBIC facility at the San Raffaele Scientific Institute for help with electron microscopy analysis, MolMed S.p.A. for large-scale production and purification of the LV batches used in the NHP study, and all members of the Naldini laboratory for helpful discussions. M.M. conducted this study as partial fulfillment of her international PhD course in Molecular Medicine at San Raffaele University, Milan. Funding: This work was supported by Telethon (SR-Tiget Core Grant 2011–2016) and Bioverativ sponsored research agreement. Author contributions: M.M. and A.A. designed and performed experiments, analyzed and interpreted data, and wrote the manuscript. F.M. designed and performed IV2PM experiments, analyzed data, and edited the manuscript. T.L. set up and performed FIX and Ab assays, analyzed data, and edited the manuscript. D.C. and A. Calabria performed IS analysis and analyzed data. S.B., M.B., and F.R. performed experiments and analyzed data. I.V. set up and performed VCN assays on NHP samples. A.R. performed electron microscopy experiments. S.P.-W. and D.D. performed FIX and Ab assays. P.C. supervised I.V.’s research. E.A. coordinated experiments with NHPs, interpreted data, and edited the manuscript. E.M. supervised D.C. and A. Calabria for IS analysis and interpretation and edited the manuscript. R.P. supervised T.L.’s research. M.I. supervised F.M.’s research and edited the manuscript. L.N. and A. Cantore conceived the project, supervised the research, interpreted data, and wrote the manuscript. L.N. provided overall coordination. Competing interests: L.N., A. Cantore, A.A., M.M., R.P., T.L., and S.P.-W. are inventors on patent applications (P105283GB and P114659GB, Vector Production) submitted by Foundation Telethon (FT) and San Raffaele Scientific Institute (SRSI) or Bioverativ on LV technology related to the work presented in this manuscript. FT and SRSI, through SR-Tiget, have established a research collaboration on liver-directed lentiviral gene therapy of hemophilia with Bioverativ. All other authors declare that they have no competing interests. Data and materials availability: The LVs and reagents described in the manuscript are available to interested scientists upon signing a material transfer agreement with standard provisions. There are some restrictions on the use of the provided materials in research involving LV-based gene therapy of hemophilia, except for research aimed at reproducing the findings reported in the manuscript, according to the collaboration agreement between FT, SRSI, and Bioverativ. All data associated with this study are present in the paper or the Supplementary Materials.

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