The impact of GPIbα on platelet-targeted FVIII gene therapy in hemophilia A mice with pre-existing anti-FVIII immunity

Abstract

Essentials Platelet-specific FVIII gene therapy is effective in hemophilia A mice even with inhibitors. The impact of platelet adherence via VWF/GPIbα binding on platelet gene therapy was investigated. GPIbα does not significantly affect platelet gene therapy of hemophilia A with inhibitors. Platelet gene therapy induces immune tolerance in hemophilia A mice with pre-existing immunity. SUMMARY: Background We have previously demonstrated that von Willebrand factor (VWF) is essential in platelet-specific FVIII (2bF8) gene therapy of hemophilia A (HA) with inhibitory antibodies (inhibitors). At the site of injury, platelet adherence is initiated by VWF binding to the platelet GPIb complex. Objective To investigate the impact of GPIbα on platelet gene therapy of HA with inhibitors. Methods Platelet-FVIII expression was introduced by 2bF8 lentivirus (2bF8LV) transduction of hematopoietic stem cells (HSCs) from GPIbα^null (Ib^null ) mice or rhF8-primed FVIII^null (F8^null ) mice followed by transplantation into lethally irradiated rhF8-primed F8^null recipients. Animals were analyzed by flow cytometry, FVIII assays and the tail bleeding test. Results After transplantation, 99% of platelets were derived from donors. The macrothrombocytopenia phenotype was maintained in F8^null mice that received 2bF8LV-transduced Ib^null HSCs (2bF8-Ib^null /F8^null ). The platelet-FVIII expression level in 2bF8-Ib^null /F8^null recipients was similar to that obtained from F8^null mice that received 2bF8LV-transduced F8^null HSCs (2bF8-F8^null /F8^null ). The tail bleeding test showed that the remaining hemoglobin level in the 2bF8-Ib^null /F8^null group was significantly higher than in the F8^null control group, but there was no significant difference between the 2bF8-Ib^null /F8^null and 2bF8-F8^null /F8^null groups. The half-life of inhibitor disappearance time was comparable between the 2bF8-Ib^null /F8^null and 2bF8-F8^null /F8^null groups. The rhF8 re-challenge did not elicit a memory immune response once inhibitor titers dropped to undetectable levels after 2bF8 gene therapy. Conclusion GPIbα does not significantly impact platelet gene therapy of HA with inhibitors. 2bF8 gene therapy restores hemostasis and promotes immune tolerance in HA mice with pre-existing immunity.

Keywords: FVIII; GPIbα; gene therapy; hemophilia A; platelet.

Fig 1.. The diagram depicts the experimental design.

A. The 2bF8-Ib^null inhibitor model in which rhF8-primed F8^null mice received 2bF8LV-transduced Sca-1⁺ cells from GPIbα^null donors. B. The 2bF8-F8^null inhibitor model in which rhF8-primed F8^null mice received 2bF8LV-transduced Sca-1⁺ cells from rhF8-primed F8^null donors. C. The 2bF8-F8^null non-inhibitor model in which F8^null mice received 2bF8LV-transduced Sca-1⁺ cells from F8^null donors. D. The untransduced transplanted Ib^null control model in which F8^null mice received bone marrow (BM) cells from GPIbα^null donors without 2bF8LV transduction.

Fig 2.. Flow cytometry analysis of chimerism in transduced recipients.

Pre-immunized F8^null recipients received whole body irradiation at 1100 cGy followed by transplantation of 2bF8LV-transduced Sca-1⁺ cells from GPIbα^null donors. Blood samples were collected after transplantation and platelets were stained with CD41 and CD42. Leukocytes were stained with CD45.1 and CD45.2. (A) Leukocyte chimerism. (B) Platelet chimerism. Data were expressed as the mean ± SD.

Fig 3.. Cell phenotype analysis for hemophilia A mice with the GPIbα deficiency.

Pre-immunized F8^null recipients received whole body irradiation at 1100 cGy followed by transplantation of 2bF8LV-transduced Sca-1⁺ cells from GPIbα^null or F8^null donors. Blood samples were collected starting at 3 weeks after transplantation and platelet number and platelet volume were measured using the Vet ABC Hematology Analyzer. (A) Platelet number counts. (B) Mean platelet volume. Data for individual mice were analyzed more than once over the study, and the average counts were calculated. Bars represent mean ± SD. Statistical comparisons of experimental groups were evaluated by the one-way ANOVA and the Tukey test was used for pairwise multiple comparison. ****P < 0.0001. “ns” indicates no statistically significant difference between the two groups.

Fig 4.. FVIII expression analysis for hemophilia A mice after 2bF8 lentiviral gene delivery to HSCs.

(A) PCR analysis of the 2bF8 expression cassette. Blood samples were collected from 2bF8LV-transduced recipients. DNA purified from leukocytes was used as the template for PCR analysis of the 2bF8 expression cassette. DNA from 2bF8 transgenic mice was used as a positive control and dH2O was used as a negative control. Mouse Vwf was used as an internal control. Shown is a representative image that has been repeated more than 3 times. (B) Average platelet-FVIII expression in 2bF8LV-transduced recipients. Blood samples were collected from 2bF8LV-transduced recipients. Platelets were isolated and lysed in 0.5% CHAPS. Functional FVIII activity levels in platelet lysates were determined by a modified chromogenic assay. (C) Platelet-derived FVIII activity levels in whole blood. Data were calculated based on platelet counts in whole blood and average platelet-FVIII level. Individual mice were analyzed more than once over the study and the average platelet-FVIII level was calculated. Bars represent mean ± SD. Statistical comparisons of experimental groups were evaluated by the Kruskal-Wallis test and the Dunn’s multiple comparison test was used for pairwise multiple comparison. “ns” indicates no statistically significant difference between the two groups.

Fig 5.. Tail bleeding test assessed the bleeding phenotype in treated animals.

The tail tip was clipped using a 1.6 mm diameter template and monitored for 6 hours. A small amount of blood was collected from eye bleed before and after the test for whole blood count. Hemoglobin level before the tail bleeding test was defined as 100% and remaining hemoglobin level after the test was calculated based on blood count. Bars represent mean ± SD. Statistical comparisons of experimental groups were evaluated by the one-way ANOVA and the Tukey test was used for pairwise multiple comparison. *P < 0.05. **P < 0.01. “ns” indicates no statistically significant difference between the two groups.

Fig 6.. Immune responses in 2bF8LV-transduced recipients.

The inhibitor model was established by rhF8 immunization of F8^null mice before 2bF8 gene therapy. Blood samples were collected from recipients before and various time points after gene therapy and inhibitor titers were determined by a chromogenic-based Bethesda assay. (A) Average inhibitor titers during the study course. N = 5 in each group. The two-way ANOVA test was used to analyze the data. There was no significant difference between the two groups. (B) The half-life of inhibitor disappearance time. The unpaired student t-test was used to compare the two groups. “ns” indicates no statistically significant difference between the two groups. (C) The inhibitor titers after rhF8 rechallenge in rhF8-primed F8^null recipients (n = 3) that received 2bF8LV-transduced F8^null Sca-1⁺ cells (2bF8-F8^null inhibitor model). When inhibitor titers dropped to undetectable levels in recipients after 2bF8 gene therapy, animals were rechalleged with rhF8 (50 U/kg/week IV x 4) and inhibitor titers were determined by Bethesda assay. (D) Platelet-FVIII expression was stable in transduced recipients even after rhF8 rechallenge. Secondary transplant into pre-immunized F8^null recipients was carried out using bone marrow mononuclear cells from an inhibitor model (2bF8-F8^null) primary recipient that had received 2bF8LV-transduced F8^null Sca-1⁺ cells and been reimmunized with rhF8. Platelet-FVIII expression was determined by chromogenic assay. Bars represent mean of platelet-FVIII expression from the primary and secondary recipients. Shown is one representative serial BMT experiment that had been performed in two trials.