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. 2014 Aug;12(8):1283-93.
doi: 10.1111/jth.12633. Epub 2014 Jul 17.

In vivo enrichment of genetically manipulated platelets corrects the murine hemophilic phenotype and induces immune tolerance even using a low multiplicity of infection

J A Schroeder  1 , Y Chen, J Fang, D A Wilcox, Q Shi
Affiliations

In vivo enrichment of genetically manipulated platelets corrects the murine hemophilic phenotype and induces immune tolerance even using a low multiplicity of infection

J A Schroeder et al. J Thromb Haemost. 2014 Aug.

Abstract

Background: Our previous studies have demonstrated that platelet-specific gene delivery to hematopoietic stem cells can induce sustained therapeutic levels of platelet factor VIII (FVIII) expression in mice with hemophilia A.

Objective: In this study, we aimed to enhance platelet FVIII expression while minimizing potential toxicities.

Methods: A novel lentiviral vector (LV), which harbors dual genes, the FVIII gene driven by the αIIb promoter (2bF8) and a drug-resistance gene, the MGMT(P140K) cassette, was constructed. Platelet FVIII expression in mice with hemophilia A was introduced by transduction of hematopoietic stem cells and transplantation. The recipients were treated with O(6)-benzylguanine followed by 1,3-bis-2 chloroethyl-1-nitrosourea monthly three or four times. Animals were analyzed by using polymerase chain reaction (PCR), quantitative PCR, FVIII:C assays, and inhibitor assays. Phenotypic correction was assessed by tail clipping tests and rotational thromboelastometry analysis.

Results: Even using a low multiplicity of infection of 1 and a non-myeloablative conditioning regimen, after in vivo selection, the levels of platelet FVIII expression in recipients increased to 4.33 ± 5.48 mU per 10(8) platelets (n = 16), which were 19.7-fold higher than the levels obtained from the recipients before treatment. Quantitative PCR results confirmed that 2bF8/MGMT-LV-transduced cells were effectively enriched after drug-selective treatment. Fifteen of 16 treated animals survived tail clipping. Blood loss and whole blood clotting time were normalized in the treated recipients. Notably, no anti-FVIII antibodies were detected in the treated animals even after recombinant human B-domain deleted FVIII challenge.

Conclusion: we have established an effective in vivo selective system that allows us to enrich 2bF8LV-transduced cells, enhancing platelet FVIII expression while reducing the potential toxicities associated with platelet gene therapy.

Keywords: blood platelet; factor VIII; genetic therapy; hemophilia A; immune tolerance.

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Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1. 2bF8 transgene analysis
(A) Diagram of the pWPT-2bF8/MGMT construct. FVIII is driven by the platelet-specific αIIb promoter. MGMTp140k was driven by the MSCV promoter. (B) PCR detection of the 2bF8 transgene. DNA was purified from peripheral white blood cells. A 0.65 kb fragment from the 2bF8 expression cassette was amplified. Wild type (WT) mFVIII and mFVIIInull PCRs were used as controls to confirm that 2bF8/MGMT-transduced recipients were on the FVIIInull background. (C) qPCR determination of the average copy number of the 2bF8/MGMT transgene per cell in transduced recipients. Peripheral blood cell-derived DNA was analyzed for the 2bF8 transgene and normalized to the ApoB gene. These results demonstrate that 2bF8/MGMT genetically modified hematopoietic cells were viable and selectable by BG/BCNU treatments.
Fig. 2
Fig. 2. Platelet-FVIII expression in 2bF8/MGMT-transduced recipients (MOI of 1)
Platelets were isolated from peripheral blood from 2bF8/MGMT-transduced recipients before and after BG/BCNU drug selection treatments. The levels of platelet-FVIII were determined by a chromogenic assay on platelet lysates. The levels of platelet-FVIII expression pre-BCNU treatment were averaged from the time points before BG/BCNU treatments and the levels post-BCNU were averaged from three or more time points after the final BG/BCNU treatment. (A) Platelet-FVIII expression in 2bF8/MGMT-transduced recipients pre-conditioned with 660 cGy TBI. (B) Platelet-FVIII expression in 2bF8/MGMT-transduced recipients pre-conditioned with 440 cGy TBI. These data demonstrate that MGMT-mediated in vivo drug-selection treatments can efficiently enhance platelet-FVIII expression in 2bF8/MGMT-transduced recipients.
Fig. 3
Fig. 3. Phenotypic correction assessment of 2bF8/MGMT-transduced recipients (MOI of 1)
(A) Tail clip survival test. Tail clipping was performed at least 6 weeks after the final BG/BCNU treatment. Fifteen of 16 transduced recipients pre-conditioned with 660 cGy TBI survived tail clipping. (B) Tail bleeding test. Hemoglobin (Hb) levels were measured before and 6 hours after tail clipping. The level of Hb in each animal before the test was defined as 100%. Five of 12 FVIIInull mice survived beyond 6 hours with only 35% Hb remaining, while all BG/BCNU-treated transduced recipients survived with 69% Hb remaining. (C) ROTEM analysis of whole blood clotting time. The clotting time in transduced BG/BCNU-treated animals was significantly shorter than in FVIIInull controls. These results demonstrate that hemostasis was restored in hemophilia A mice after 2bF8/MGMT gene therapy followed by in vivo selection.
Fig. 4
Fig. 4. Sequential transplantation
To ascertain whether platelet-FVIII expression was sustained, secondary (2°) transplant was carried out using BM cells from primary (1°) recipients that had been received 2bF8/MGMT-transduced HSCs. (A) Average copy number of 2bF8 proviral DNA per cell in 1° and 2° recipients. (B) Average FVIII:C levels in 1° and 2° recipients. 1° BMT*: data presented in this group were summarized from the primary recipients post-selection from which bone marrow mononuclear cells were collected and transplanted into the secondary recipients. These results demonstrate that the levels of platelet-FVIII expression in the 2° recipients were similar to those obtained from the 1° recipients.
Fig. 5
Fig. 5. The immune response in 2bF8/MGMT-transduced recipients
To investigate the immune response in 2bF8/MGMT-transduced recipients, plasma samples were collected from 2bF8/MGMT-transduced recipients before and after rhF8 challenge. (A) The titers of anti-FVIII inhibitory antibodies (inhibitors) determined by Bethesda assay. (B) The titers of total anti-FVIII antibodies determined by ELISA assay. (C) The anti-FVIII antibody secreting cells examined by ELISPOT assay. Shown are representative results from 4×106 cells seeded per well. Taken together, figures A, B, and C show that none of the 2bF8/MGMT-transduced BG/BCNU treated recipients pre-conditioned with 660 cGy TBI developed anti-FVIII antibodies even after rhF8 immunization (50 U/kg/week IV × 4). (D) The titers of anti-ovalbumin (OVA) antibodies. To ensure that the immune system was not defective in the 2bF8/MGMT-transduced recipients, we further challenged the animals with OVA and the titers were determined by ELISA. All animals developed high titers of anti-OVA antibodies after OVA challenge. These results demonstrate that immune tolerance developed in the 2bF8/MGMT-transduced recipients and that the immune tolerance is FVIII-specific.
Fig. 6
Fig. 6. The efficacy of high MOI (MOI of 10) in 2bF8/MGMT gene therapy
To compare the efficacy of our novel vector 2bF8/MGMT and our non-selectable 2bF8 construct in platelet gene therapy of hemophilia A, we used an MOI of approximately 10, the MOI typically used in our previous 2bF8LV studies. (A) Platelet-FVIII expression in recipients. Platelet-FVIII expression levels in recipients were monitored before and after each BG/BCNU treatment. The levels of platelet-FVIII expression before BG/BCNU treatment were averaged from 2 time points and the levels after the 3rd treatment were averaged from three time points after the final treatment. (B) Tail bleeding test. Hemoglobin (Hb) levels were measured before and 6 hours after tail clipping. The level of Hb in each animal before the test was defined as 100%.
Fig. 7
Fig. 7. Blood count during study course of 2bF8/MGMT-transduction and BG/BCNU treatment
Blood samples were collected from 2F8/MGMT-transduced recipients using sodium citrate as an anticoagulant. Blood cell number and hemoglobin levels were measured using the Vet blood counter. (A) Leukocyte number; (B) Hemoglobin levels; and (C) Platelet number.
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