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

To evaluate the potential of the ex vivo bone marrow stromal cell. (BMSC) s ystem as a gene therapy for hemophilia A, we studied the irt vitro expressi on of human factor VIII (hFVIII) in canine BMSCs following transfection wit h plasmid vectors and transduction with retroviral vectors. Vectors were co mposed of B domain-deleted forms of hFVIII that either retain or delete the proteolytic site at amino acid 1648, On transfection of BMSCs, vectors sup ported expression and secretion of similar levels of up to 386 mU/10(6) cel ls/24 hr, even though only 3-9% of the cells expressed hFVIII while 42-48% of transfected cells harbored plasmid vector, Much higher percentages (simi lar to 70%) of cells expressing hFVIII were achieved when BMSCs were transd uced by retroviral vectors, resulting in expression and secretion as high a s 1000-4000 mU/10(6) cells/24 hr, Western analysis demonstrated that the B domain-deleted forms possessing the proteolytic site were secreted predomin antly as heavy and light chain heterodimers that resemble native forms foun d in plasma. In contrast, the hFVIII lacking the proteolytic site was expre ssed mostly as unprocessed, single heavy-light chains. Both hFVIII forms we re correctly cleaved and activated by thrombin, The proteolyzed hFVIII form possessed greater than or equal to 93% normal biological activity while th e unproteolyzed form possessed consistently less than 55% normal biological activity and was therefore considered less suitable for therapeutic applic ation. These results demonstrate that the BMSC system has potential utility in gene therapy for hemophilia A and stress the importance of selecting th e appropriate hFVIII structure for prospective clinical use.