Targeting FVIII expression to endothelial cells regenerates a releasable pool of FVIII and restores hemostasis in a mouse model of hemophilia A

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Key Points• Platelet-specific lentiviral gene delivery to human hematopoietic stem cells can efficiently introduce FVIII expression in human platelets.• Human platelet-derived FVIII can ameliorate the hemophilic phenotype in an immunocompromised hemophilia A mouse model.Our previous studies have demonstrated that platelet FVIII (2bF8) gene therapy can improve hemostasis in hemophilia A mice, even in the presence of inhibitory antibodies, but none of our studies has targeted human cells. Here, we evaluated the feasibility for lentivirus (LV)-mediated human platelet gene therapy of hemophilia A. Human platelet FVIII expression was introduced by 2bF8LV-mediated transduction of human cord blood (hCB) CD34 1 cells followed by xenotransplantation into immunocompromised NSG mice or NSG mice in an FVIII null background (NSGF8KO). Platelet FVIII was detected in all recipients that received 2bF8LV-transduced hCB cells as long as human platelet chimerism persisted. All NSGF8KO recipients (n 5 7) that received 2bF8LV-transduced hCB cells survived tail clipping if animals had greater than 2% of platelets derived from 2bF8LV-transduced hCB cells, whereas 5 of 7 survived when human platelets were 0.3% to 2%. Whole blood clotting time analysis confirmed that hemostasis was improved in NSGF8KO mice that received 2bF8LV-transduced hCB cells. We demonstrate, for the first time, the feasibility of 2bF8LV gene delivery to human hematopoietic stem cells to introduce FVIII expression in human platelets and that human platelet-derived FVIII can improve hemostasis in hemophilia A. (Blood. 2014;123(3):395-403)

Section: Fviii Expressed In Human Platelets Has Functional Activitymentioning

confidence: 70%

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

Shi

Kuether

Chen

et al. 2014

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“…[19][20][21][22] Previously, it was demonstrated that overexpression of FVIII in endothelial cells results in FVIII targeting to WPBs. [23][24][25][26] The targeting signal for FVIII sorting to WPBs is currently unclear, although the typical high-affinity interaction with VWF seems not required. 25,26 Interestingly, WPBs containing FVIII do not have their characteristic elongated shape but appear as rounded vesicles.…”

Section: Introductionmentioning

confidence: 99%

Factor VIII alters tubular organization and functional properties of von Willebrand factor stored in Weibel-Palade bodies

Bouwens

Mourik

Biggelaar

et al. 2011

In endothelial cells, von Willebrand factor (VWF) multimers are packaged into tubules that direct biogenesis of elongated Weibel-Palade bodies (WPBs). WPB release results in unfurling of VWF tubules and assembly into strings that serve to recruit platelets. By confocal microscopy, we have previously observed a rounded morphology of WPBs in blood outgrowth endothelial cells transduced to express factor VIII (FVIII). Using correlative lightelectron microscopy and tomography, we now demonstrate that FVIII-containing WPBs have disorganized, short VWF tubules. Whereas normal FVIII and FVIII Y1680F interfered with formation of ultralarge VWF multimers, release of the WPBs resulted in VWF strings of equal length as those from nontransduced blood outgrowth endothelial cells. After release, both WPB-derived FVIII and FVIII Y1680F remained bound to VWF strings, which however had largely lost their ability to recruit platelets. Strings from nontransduced cells, however, were capable of simultaneously recruiting exogenous FVIII and platelets. These findings suggest that the interaction of FVIII with VWF during WPB formation is independent of Y1680, is maintained after WPB release in FVIIIcovered VWF strings, and impairs recruitment of platelets. Apparently, intracellular and extracellular assembly of FVIII-VWF complex involves distinct mechanisms, which differ with regard to their implications for platelet binding to released VWF strings. (Blood. 2011; 118(22):5947-5956) IntroductionWeibel-Palade bodies (WPBs) are secretory organelles specific for vascular endothelial cells with a typical elongated shape of 100 to 200 nm in diameter and up to 5 m in length. 1 These organelles are characterized by striations running along the longitudinal axis consisting of condensed von Willebrand factor (VWF). Stimulation with agonists, such as thrombin and epinephrine, releases VWF to arrests bleeding by recruiting blood platelets to sites of vascular perturbation. 2 VWF is synthesized as a prepropolypeptide of 2813 residues that is cleaved into a propeptide (D1-D2) and mature VWF monomer (DЈ-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK) of approximately 250 kDa. 3 VWF forms multimers through C-and N-terminal disulfide linkage. [4][5][6] Multimers assemble into long, slightly twisted tubules that determine the typical cigar-shaped appearance of WPBs. [7][8][9] On fusion of WPBs with the plasma membrane, VWF tubules are exposed to physiologic pH. This results in rapid disassembly of VWF tubules, which is presumably triggered by dissociation of propeptide from DЈ-D3 regions at neutral pH. 8,10 The released VWF multimers assemble into extended string-like structures on the surface of endothelial cells onto which platelets adhere. [10][11][12] The tubular organization of VWF is thought to be essential for orderly secretion of long VWF strings without tangling. 13 Besides recruiting platelets to sites of vascular damage, VWF functions as a chaperone for factor VIII (FVIII), a cofactor for activated factor IX in the factor Xase complex. It is thus far uncl...

“…31 Finally, 2 recent studies using murine conditional knockout approaches indicated endothelial cells are the major, if not exclusive, source of FVIII, 32,33 and gene transfer of FVIII to endothelial cells in mice normalized plasma FVIII and restored the releasable FVIII pool. 34 Recent studies have also documented the presence of FVIII on the VWF strings that are released from the endothelium. 35 However, the documentation of FVIII expression in lymphatic endothelium and certain types of fenestrated endothelium (in the liver sinusoids and renal glomerulus), with little or no coexpression of VWF, 36 suggests that there is still more to learn about the precise endothelial sources of production of these 2 proteins.…”

Section: Biology Of Vwf and Fviiimentioning

confidence: 99%

Life in the shadow of a dominant partner: the FVIII-VWF association and its clinical implications for hemophilia A

Pipe

Montgomery

Pratt

et al. 2016

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174

186

A normal hemostatic response to vascular injury requires both factor VIII (FVIII) and von Willebrand factor (VWF). In plasma, VWF and FVIII normally circulate as a noncovalent complex, and each has a critical function in the maintenance of hemostasis. Furthermore, the interaction between VWF and FVIII plays a crucial role in FVIII function, immunogenicity, and clearance, with VWF essentially serving as a chaperone for FVIII. Several novel recombinant FVIII (rFVIII) therapies for hemophilia A have been in clinical development, which aim to increase the half-life of FVIII (∼12 hours) and reduce dosing frequency by utilizing bioengineering techniques including PEGylation, Fc fusion, and single-chain design. However, these approaches have achieved only moderate increases in halflife of 1.5-to 2-fold compared with marketed FVIII products. Clearance of PEGylated rFVIII, rFVIIIFc, and rVIII-SingleChain is still regulated to a large extent by interaction with VWF. Therefore, the half-life of VWF (∼15 hours) appears to be the limiting factor that has confounded attempts to extend the half-life of rFVIII. A greater understanding of the interaction between FVIII and VWF is required to drive novel bioengineering strategies for products that either prolong the survival of VWF or limit VWF-mediated clearance of FVIII. (Blood.