919. Gene Therapy for Platelet Disorders: Targeting Transgene Expression in Canine Platelets

Abstract

Platelets mediate the primary response to vascular injury; as such molecular defects in some platelet proteins can disrupt normal hemostasis and lead to uncontrolled hemorrhage. To develop a large animal model to examine lineage-specific gene therapy for disorders affecting platelets, canine CD34+ peripheral blood cells (PBC) were transduced with a self-inactivating, human immunodeficiency type-1 lentivirus-derived vector controlled by the megakaryocyte-specific human integrin αIIb gene promoter. This construct, αIIb-GFP-WPT, utilized the woodchuck hepatitis virus postregulatory element (W), and the central polypurine tract (PT) to enhance the efficiency of transgene expression. cDNA encoding the green fluorescent protein (GFP) was subcloned into the construct to analyze transgene expression in the progeny of transduced cells. A second vector (EF1-GFP-WPT) under control of the promoter from the housekeeping gene, Elongation Factor-1, which drives constitutively active transgene expression was transduced into CD34+ PBC as a control for lineage non-specific transgene expression. Cells were immuno-magnetically selected for the CD34+ antigen from granulocyte-colony stimulating factor mobilized PBC, which were collected by apheresis of a normal dog. CD34+ PBC were prestimulated for 2 days in media containing cytokines (flk2/flt3-ligand, SCF, IL-3, IL-6 and c-mpl ligand), transduced for 2 days and induced for 5 days with culture media containing the cocktail of cytokines used for prestimulation and IL-11 to induce the cells to differentiate into a population comprised of several lineages including megakaryocytes. Flow cytometric analysis using an antibody specific for the canine β3-subunit revealed that the vectors (EF1- and αIIb-) transduced megakaryocytes with equal efficiency with 60% of megakaryocytes expressing GFP. Only 14% of the cell population expressing GFP was megakaryocytes when using the EF1-promoter, while 60% of the cells expressing GFP under control of the αIIb-promoter were megakaryocytes. When αIIb-GFP transduced PBC were xenotransplanted into immune-compromised (NOD/SCID) mice, flow cytometric analysis demonstrated that GFP-positive platelets were circulating within the vasculature and could be isolated at the site of a tail vein injury at two weeks post-transplant of canine PBC. These studies demonstrate that a lentivirus construct under control of the human αIIb promoter can target expression of GFP in canine megakaryocytes with high specificity compared to the EF1-promoter's ability to drive lineage-specific transgene expression. Furthermore, transduced PBC are capable of forming megakaryocytes and platelet progeny expressing transgene products in vivo. This work suggests that there is potential feasibility for using a lineage-specific αIIb gene promoter to develop gene therapy for disorders affecting platelets.