Intraosseous delivery of lentiviral vectors targeting factor VIII expression in platelets corrects murine hemophilia A

Abstract

Intraosseous (IO) infusion of lentiviral vectors (LVs) for in situ gene transfer into bone marrow may avoid specific challenges posed by ex vivo gene delivery, including, in particular, the requirement of preconditioning. We utilized IO delivery of LVs encoding a GFP or factor VIII (FVIII) transgene directed by ubiquitous promoters (a MND or EF-1α-short element; M-GFP-LV, E-F8-LV) or a platelet-specific, glycoprotein-1bα promoter (G-GFP-LV, G-F8-LV). A single IO infusion of M-GFP-LV or G-GFP-LV achieved long-term and efficient GFP expression in Lineage(-)Sca1(+)c-Kit(+) hematopoietic stem cells and platelets, respectively. While E-F8-LV produced initially high-level FVIII expression, robust anti-FVIII immune responses eliminated functional FVIII in circulation. In contrast, IO delivery of G-F8-LV achieved long-term platelet-specific expression of FVIII, resulting in partial correction of hemophilia A. Furthermore, similar clinical benefit with G-F8-LV was achieved in animals with pre-existing anti-FVIII inhibitors. These findings further support platelets as an ideal FVIII delivery vehicle, as FVIII, stored in α-granules, is protected from neutralizing antibodies and, during bleeding, activated platelets locally excrete FVIII to promote clot formation. Overall, a single IO infusion of G-F8-LV was sufficient to correct hemophilia phenotype for long term, indicating that this approach may provide an effective means to permanently treat FVIII deficiency.

Figure 1

GFP expression in BM cells following IO infusion of M-GFP-LV. C57BL/6 mice were intraosseously delivered with M-GFP-LV (1.1 × 10⁸ ifu/animal, n = 6). (a) Schematic of self-inactivating LV genome encoding GFP under the control of the modified myeloid proliferative sarcoma virus promoter (MND) (M-GFP-LV). (b) Schematic of IO infusion of vectors into the mice with an infusion speed of 10 µl/minute, which was precisely controlled by a programmable microfluidics syringe pump. (c) GFP expression in BM cells of LV-treated mice detected by a fluorescent microscope. Top panel, bright field; bottom panel, green fluorescent signals. (d,f) Flow cytometry analysis of total BM cells isolated from naive or LV-treated mice. Top panel, naive mice; bottom panel, mice treated with LVs. (e,g) GFP expression in Lin^-Sca1⁺c-Kit⁺ HSCs. Top panels, Lin^- cells were gated; middle panels, Lin^-Scal⁺c-Kit⁺ HSCs were gated; bottom panels, GFP⁺Lin^-Sca1⁺c-Kit⁺ HSCs were gated. d and e shown were representative plots of control and treated mice. Data were expressed as mean ± SD. Differences were considered significant at P < 0.01 (**).

Figure 2

In vivo GFP expression in transduced BM cells and platelets following IO infusion of G-GFP-LV. (a) Schematic of self-inactivating LV genomes encoding GFP under the control of platelet-specific glycoprotein 1bα promoter (Gp1bα) (G-GFP-LV). (b) C57BL/6 mice were intraosseously infused with G-GFP-LV (5 × 10⁷ ifu/animal, n = 3) or PBS (20 µl/animal, mock, n = 3) on day 0. The experimental mice were subsequently sacrificed, their BM cells were isolated, and GFP expression levels in Lin^-Sca1⁺c-Kit⁺ HSCs (left panel), B220⁺ (middle panel) and CD11c⁺ (right panel) cells were detected by flow cytometry on day 31. (c) For long-term follow up, C57/BL6 mice were treated with G-GFP-LV (5 × 10⁷ ifu/animal, n = 5) or PBS (20 µl/animal, mock, n = 3) on day 0. Platelets were isolated from peripheral blood and marked with CD42d⁺, and their GFP expression levels over 5 months were measured by flow cytometry. Left panel, representative flow data at 5 months after infusion; right panel, summary plot over time. Figures shown were representative of two independent experiments.

Figure 3

Circulatory FVIII in plasma of HemA mice following IO infusion of E-F8-LV. (a) Schematic of a self-inactivating LV genome encoding hFVIII variant with the proximal 226 amino acid region of the B-domain (FVIII/N6) under the control of EF-1α promoter (E-F8-LV). (b) E-F8-LV (1 × 10⁸ ifu/animal, n = 3) or PBS (20 µl/animal, mock, n = 2) was intraosseously infused into HemA mice on day 0. hFVIII expression in Lin^-Sca1⁺c-Kit⁺ HSCs was checked by flow cytometry on day 12 after infusion. Figures shown were representative of two independent experiments. (c) HemA mice were intraosseously infused with E-F8-LV (5 × 10⁷ ifu/animal, n = 4) or PBS (20 µl/animal, mock, n = 3) on day 0. Plasma samples were collected and hFVIII activity and anti-FVIII antibodies were measured by aPTT and Bethesda assay, respectively. In mock mice, the level of hFVIIII activity was corrected as 0% of Normal and the level of anti-FVIII antibodies was 0 Bethesda Unit. Each line represents an individual LV-treated animal. Data shown were representative of four independent experiments.

Figure 4

hFVIII specific expression in CD42d⁺ platelets. (a) Schematic of a self-inactivating LV genome encoding hFVIII variant with the proximal 226 amino acid region of the B-domain (FVIII/N6) under the control of Gp1bα promoter (G-F8-LV). HemA mice were intraosseously infused with G-F8-LV (2.2 × 10⁷ ifu/animal, n = 6) or PBS (20 µl/animal, mock, n = 3) on day 0. (b) BM cells, (c) white blood cells and (d) platelets from peripheral blood were isolated. hFVIII expression was detected in Lin^-Sca1⁺c-Kit⁺ HSCs on day 8 (b), in CD3ɛ⁺, CD11c⁺, B220⁺, CD11b⁺ blood cells on day 35 (c), and in CD42d⁺ platelets on day 91 (d) by flow cytometry. Representative flow data of two independent experiments were shown in the figures.

Figure 5

Long-term stable hFVIII levels in platelets were obtained in HemA mice after a single IO infusion of G-F8-LVs and their phenotype was corrected. HemA mice were given IO infusion of G-F8-LV (2.2 × 10⁷ ifu/animal or 2.2 × 10⁶ ifu/animal) or PBS (20 µl/animal, mock) on day 0. (a) Platelets were isolated from peripheral blood of high- (n = 8) or low- (n = 5) titer G-F8-LV treated or mock (n = 3) mice. hFVIII expression levels in CD42d⁺ platelets were evaluated by flow cytometry on day 27, 62, 84, 112 and 160. (b) hFVIII levels in platelet lysates of high (n = 5) or low (n = 5) titer LV-treated mice or mock (n = 3) were measured by ELISA on day 112. (c) HemA phenotype correction of G-F8-LV treated mice was monitored by tail clip assay on day 35, 118, and 160 (n = 4–7/group). The average blood loss of untreated HemA mice was set as 100%. Wild-type C57BL/6 mice were used as positive controls. (d) Plasma samples were collected from high-titer G-F8-LV treated (n = 10) or mock (n = 3) mice, and hFVIII activity and anti-FVIII antibodies were measured by aPTT and Bethesda assay, respectively. For mock mice, the level of hFVIIII activity was corrected as 0% of normal and the level of anti-FVIII antibodies was 0 Bethesda unit. Each symbol represented an individual animal. Data were expressed as mean ± SD. Differences were considered significant at P < 0.05 (*) and P < 0.01 (**). n.s. was an abbreviation for non-significant. Data shown were from two independent experiments.

Figure 6

hFVIII expression in platelets of G-F8-LV treated inhibitor HemA mice corrected their hemophilia A phenotype. (a) Inhibitor HemA mice were established by repeated intraperitoneal injection (3x/week for 2 weeks) of 3U rhFVIII into 10- to 12-week-old HemA mice. These inhibitor HemA mice were then intraosseously infused with G-F8-LV (2.2 × 10⁷ ifu/animal) or PBS (20 µl/animal, mock) on day 0. (b) Platelets were isolated from peripheral blood of LV-treated (n = 5) and mock (n = 3) mice and lysed. The resulting lysate was examined for hFVIII expression level by ELISA on day 27 after infusion. (c) The phenotypic correction of G-F8-LV treated HemA inhibitor mice (n = 7) was examined by tail clip assays on day 160 after infusion. The average blood loss of untreated HemA (n = 10) mice was set as 100%. Wild-type C57BL/6 mice (n = 8) were used as positive controls. Data shown were from two independent experiments. Data were expressed as mean ± SD. Differences were considered significant at P < 0.05 (*) and P < 0.01 (**).