185. Systemic Protein Delivery by Muscle-Directed Gene Transfer Is Limited by a Local Immune Response
Authors: Morvarid Moayeri, Ali Ramezani, Richard A. Morgan, Teresa S. Hawley, Robert G. Hawley
Journal: Molecular Therapy (2004)

Hemophilia A is an inherited bleeding disorder caused by the lack of functional coagulation factor VIII (fVIII). Although fVIII protein replacement therapy is effective, it is inconvenient, very expensive, and has disadvantages such as unpredictable product shortage and possible transmission of viral blood-borne diseases in the case of plasma-derived product. Considering these limitations, a gene-based treatment for hemophilia A with the potential to cure the disease is very attractive. Hemophilia A is an excellent candidate for gene therapy because it is a monogenic disorder, modest elevation of fVIII levels to 1.5–2% of normal is sufficient to significantly improve the clinical symptoms, and tissue-specific expression is not required. Although much progress has been made in hemophilia A gene therapy, major hurdles that remain are low frequencies of transgene delivery, suboptimal expression of fVIII mRNA and inefficient secretion of fVIII protein, plus the development of inhibitory antibodies to fVIII neoantigen. We have taken several approaches to overcome these difficulties: use of an oncoretroviral vector backbone containing strong transcriptional control elements such as the MSCV LTR; inclusion of introns for more efficient processing and accumulation of fVIII mRNA; construction of a secretion-enhanced variant of fVIII (sfVIII) and use of hematopoietic stem cells (HSCs) as a target population. HSCs are a promising target cell population because they allow long-term expression of an integrating transgene from circulating blood cells. More importantly, their use as the target population may prevent inhibitor formation to recombinant fVIII by inducing immune tolerance during lymphoid reconstitution. To evaluate the potential of HSCs for hemophilia A gene therapy, primary bone marrow cells from immunocompetent (E16; fVIII exon 16 disrupted) and immunocompromised (E12; fVIII exon 16 and B7-2 disrupted) hemophilic mice were transduced with an optimized MSCV-based splice-gag oncoretroviral vector containing an sfVIII-IRES-EGFP cassette. Transplantation of 2–3.5 106 EGFP-expressing bone marrow cells into sublethally irradiated syngeneic hemophilic recipients resulted in persistant expression of EGFP in peripheral blood cells for at least 21 weeks. Therapeutic levels of sfVIII (2 to 101 ng/ml) corresponding to the percentage of peripheral blood EGFP-expressing cells were detected in the plasma of all E12 recipients and led to correction of their hemophilic phenotype as observed by survival of tail-clipping. Transplantation of bone marrow cells obtained from sfVIII-expressing E12 mice (wk 15 post-transplant) into sublethally irradiated secondary E12 recipients has indicated that hematopoietic stem cells are the target population responsible for maintenance of sfVIII production in vivo. Ongoing studies with immunocompetent E16 mice are encouraging, with 30 12 ng/ml of sfVIII detected in the best example for up to 20 wks. Further investigations are focused on strategies to reproducibly induce immune tolerance to curative levels of fVIII neoantigen.