Hemophilia A gene therapy via intraosseous delivery of factor VIII-lentiviral vectors

Abstract

Current treatment of hemophilia A (HemA) patients with repeated infusions of factor VIII (FVIII; abbreviated as F8 in constructs) is costly, inconvenient, and incompletely effective. In addition, approximately 25 % of treated patients develop anti-factor VIII immune responses. Gene therapy that can achieve long-term phenotypic correction without the complication of anti-factor VIII antibody formation is highly desired. Lentiviral vector (LV)-mediated gene transfer into hematopoietic stem cells (HSCs) results in stable integration of FVIII gene into the host genome, leading to persistent therapeutic effect. However, ex vivo HSC gene therapy requires pre-conditioning which is highly undesirable for hemophilia patients. The recently developed novel methodology of direct intraosseous (IO) delivery of LVs can efficiently transduce bone marrow cells, generating high levels of transgene expression in HSCs. IO delivery of E-F8-LV utilizing a ubiquitous EF1α promoter generated initially therapeutic levels of FVIII, however, robust anti-FVIII antibody responses ensued neutralized functional FVIII activity in the circulation. In contrast, a single IO delivery of G-FVIII-LV utilizing a megakaryocytic-specific GP1bα promoter achieved platelet-specific FVIII expression, leading to persistent, partial correction of HemA in treated animals. Most interestingly, comparable therapeutic benefit with G-F8-LV was obtained in HemA mice with pre-existing anti-FVIII inhibitors. Platelets is an ideal IO delivery vehicle since FVIII stored in α-granules of platelets is protected from high-titer anti-FVIII antibodies; and that even relatively small numbers of activated platelets that locally excrete FVIII may be sufficient to promote efficient clot formation during bleeding. Additionally, combination of pharmacological agents improved transduction of LVs and persistence of transduced cells and transgene expression. Overall, a single IO infusion of G-F8-LV can generate long-term stable expression of hFVIII in platelets and correct hemophilia phenotype for long term. This approach has high potential to permanently treat FVIII deficiency with and without pre-existing anti-FVIII antibodies.

Keywords: Anti-FVIII inhibitory antibodies; Factor VIII; Gene therapy; Hemophilia A; Intraosseous delivery; Lentiviral vectors; Megakaryocyte-specific gene expression; Stem cell gene therapy.

Fig. 1

GFP expression in BM cells following IO infusion of M-GFP-LV. a Schematic of IO infusion of vectors into the mice with an infusion speed of 10 μl/min, which was precisely controlled by a programmable microfluidics syringe pump. b C57BL/6 mice were intraosseously delivered with M-GFP-LV (1.1 × 10⁸ ifu/animal, n = 6) on day 0. BM cells were isolated from treated or untreated legs and GFP expression in Lin⁻Sca1⁺c-Kit⁺ HSCs were examined on day 7 by flow cytometry. c C57BL/6 mice were given IO infusion of M-GFP-LV (1.1 × 10⁸ ifu; n = 8) on day 0. GFP expression in Lin⁻Sca1⁺c-Kit⁺ HSCs of treated and untreated legs was evaluated on day 124. d C57BL/6 mice were given IO infusion of M-GFP-LV (8.8 × 10⁸ ifu/animal; n = 10) or PBS (20 μl/animal, mock; n = 5) on day 0. Long-term GFP expression in Lin⁻Sca1⁺c-Kit⁺ HSCs was detected on day 160. This figure is reproduced from Ref [10]

Fig. 2

Comparison of hFVIII levels in plasma and/or platelets after a single IO infusion of E-F8-LVs and G-F8-LVs. a 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. No FVIII activity or anti-FVIII antibody was detected in the PBS treated control mice (data not shown). b-d 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. b 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. 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. * P < 0.05. 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. This figure is reproduced from Ref [10]

Fig. 3

hFVIII expression in platelets of G-F8-LV treated inhibitor HemA mice corrected their hemophilia A phenotype. Inhibitor HemA mice were established by repeated intraperitoneal injection (3×/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. a Platelets were isolated from peripheral blood and marked with CD42d⁺, and their GFP expression levels at 5 months post infusion. b Platelets from 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 post infusion. c The phenotypic correction of G-F8-LV treated HemA inhibitor mice (n = 7) was examined by tail clip assays on day 160 post 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. * P < 0.05, ** P < 0.005. This figure is reproduced from Ref [10]