Altered bioavailability of platelet-derived factor VIII during thrombocytosis reverses phenotypic efficacy in haemophilic mice

Abstract

Ectopic delivery of factor VIII (FVIII) to megakaryocytes (Mk) represents a viable approach for localized tenase generation by concentrating the FVIIIa/FIXa enzyme-cofactor complex onto activated platelet membranes. We utilized a core rat platelet factor 4 (PF4) promoter for Mk/platelet-restricted expression of human B-domain-deleted (hBDD) FVIII within the background of a haemophilia A mouse (rPF4/hBDD/FVIII-/-). Platelets from rPF4/hBDD/FVIII(-/-) mice contained approximately 122 mU FVIII:C/1 x 10(9) platelets/ml with no detectable plasmatic FVIII:C, and with no effect on alpha-granule-derived platelet factor V/Va function. Paired tenase assays (+/- thrombin) confirmed that platelet (pt) FVIII (unlike platelet FV) required thrombin cleavage for complete activation. rPF4/hBDD/FVIII(-/-) mice exposed to a thrombocytotic stimulus (thrombopoietin, TPO) demonstrated a statistically-significant 66% reduction in molar ptFVIII activity with a non-significant reduction in total ptFVIII biomass. Decreased molar ptFVIII concentration correlated with loss of phenotypic correction as evaluated using a haemostatic tail-snip assay. Comparative studies using a transgenic mouse expressing human amyloid-beta-precursor protein (hAbetaPP) from the rPF4 promoter confirmed diminished hAbetaPP expression without affecting endogenous alpha-granule PF4, establishing generalizability of these observations. While Mk/platelet-released ptFVIII (unlike pFV) is proteolytically inactive, we also conclude that thrombocytotic stimuli negatively affect ptFVIII bioavailability and phenotypic efficacy, results which correlate best with molar ptFVIII concentration, and not systemically available ptFVIII.

Platelet gene therapy corrects the hemophilic phenotype in immunocompromised hemophilia A mice transplanted with genetically manipulated human cord blood stem cells

“…Previous studies by our group 10,11,16,17 and others [20][21][22][23] using mouse models have demonstrated that platelet-targeted expression of FVIII may be a promising approach for gene therapy of hemophilia A and hemophilia A with inhibitors. In this study, we established a novel immunocompromised hemophilia A mouse model and used humanized mouse models to explore the feasibility and efficacy of targeting FVIII expression to human platelets for hemophilia A gene therapy.…”

Computationally designed adeno-associated virus (AAV) Rep 78 is efficiently maintained within an adenovirus vector

“…Rep 78 immunodetection was established in 293 cells transfected with FLAG-tagged Rep plasmids (pFLAG/wtRep, pFLAG/dRep, or pFLAG/sRep), and at 48 h, protein-solubilized lysates were prepared for 4% to 15% SDS/PAGE and immunoblotting as previously described (14). Immunodetection was performed by enhanced chemiluminescence by using anti-FLAG M2 (1:1,000; Sigma) and anti-GAPDH MAB374 (1:1,000; Millipore) as a loading control.…”

“…Although the AAV integrating elements (i.e., TRs or p5IEE) are readily incorporated into herpesvirus (12,13) or Ad vectors (14)(15)(16), Rep 78/68 is poorly tolerated. Moderate success has been achieved with the use of complex homologous recombination strategies (17) and helper-dependent (18) or tightly regulated (19) expression systems, although the latter two approaches are additionally restricted by the helper-dependent nature of Rep 78-containing Ad.…”

Platelets as delivery systems for disease treatments

“…The other is that they are less likely to activate the host-cell defense machinery than constitutive viral promoters and therefore, the development of immune response to transgene product may be reduced or circumvented [43]. The promoters that have been employed to direct megakaryocyte/platelet lineage-specific transgene expression include those that control the expression of genes encoding GPIIb (αIIb) [44-54], GPIbα [55-57], GPVI [57], platelet factor 4 (PF4) [58-60], and c-mpl [61,62]. All of these promoters are activate in both megakaryocytes and platelets.…”

“…The studies demonstrated that ectopic expression of FVIII in platelets under control of the PF4 promoter had no effect on α-granule-derived platelet factor V/Va function. The amount of FVIII per platelet was decreased, with a parallel decrease in efficacy when transgenic mice were infused with thrombopoietin [60]. …”