Bone marrow stromal cells as a genetic platformfor systemic delivery of therapeutic proteins in vivo: human factor IX model

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Abstract
Background: Hemophilia B is an X‐linked bleeding disorder that results from a deficiency in functional coagulation factor IX (hFIX). In patients lacking FIX, the intrinsic coagulation pathway is disrupted leading to a lifelong, debilitating and sometimes fatal disease.Methods: We have developed an ex vivo gene therapy system using genetically modified bone marrow stromal cells (BMSCs) as a platform for sustained delivery of therapeutic proteins into the general circulation. This model exploits the ability of BMSCs to form localized ectopic ossicles when transplanted in vivo. BMSCs were transduced with MFG‐hFIX, a retroviral construct directing the expression of hFIX. The biological activity of hFIX expressed by these cells was assessed in vitro and in vivo.Results: Transduced cells produced biologically active hFIX in vitro with a specific activity of 90% and expressed hFIX at levels of ∼497 ng/106 cells/24 h and 322 ng/106 cells/24 h for human and porcine cells, respectively. The secretion of hFIX was confirmed by Western blot analysis of the conditioned medium using a hFIX‐specific antibody. Transduced BMSCs (8 × 106 cells per animal) were transplanted within scaffolds into subcutaneous sites in immunocompromised mice. At 1 week post‐implantation, serum samples contained hFIX at levels greater than 25 ng/ml. Circulating levels of hFIX gradually decreased to 11.5 ng/ml at 1 month post‐implantation and declined to a stable level at 6.1 ng/ml at 4 months.Conclusions: These findings demonstrate that genetically modified BMSCs can continuously secrete biologically active hFIX from self‐contained ectopic ossicles in vivo, and thus represent a novel delivery system for releasing therapeutic proteins into the circulation. Copyright © 2002 John Wiley & Sons, Ltd.