Bone Marrow Stromal Cell-Mediated Gene Therapy for Hemophilia A: In Vitro Expression of Human Factor VIII with High Biological Activity Requires the Inclusion of the Proteolytic Site at Amino Acid 1648

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Abstract
To evaluate the potential of the ex vivo bone marrow stromal cell (BMSC) system as a gene therapy for he- mophilia A, we studied the in vitro expression of human factor VIII (hFVIII) in canine BMSCs following tran- fection with plasmid vectors and transduction with retroviral vectors. Vectors were composed of B domain- deleted forms of hFVIII that either retain or delete the proteolytic site at amino acid 1648. On transfection of BMSCs, vectors supported expression and secretion of similar levels of up to 386 mU/10 6 cells/24 hr, even though only 3-9% of the cells expressed hFVIII while 42-48% of transfected cells harbored plasmid vector. Much higher percentages (~70%) of cells expressing hFVIII were achieved when BMSCs were transduced by retroviral vectors, resulting in expression and secretion as high as 1000-4000 mU/10 6 cells/24 hr. Western analysis demonstrated that the B domain-deleted forms possessing the proteolytic site were secreted pre- dominantly as heavy and light chain heterodimers that resemble native forms found in plasma. In contrast, the hFVIII lacking the proteolytic site was expressed mostly as unprocessed, single heavy-light chains. Both hFVIII forms were correctly cleaved and activated by thrombin. The proteolyzed hFVIII form possessed 93% normal biological activity while the unproteolyzed form possessed consistently less than 55% normal biological activity and was therefore considered less suitable for therapeutic application. These results demon- strate that the BMSC system has potential utility in gene therapy for hemophilia A and stress the importance of selecting the appropriate hFVIII structure for prospective clinical use.