Sleeping Beauty Gene Therapy from Liver to BOECs

University Of Minnesota Twin Cities, Minneapolis MN

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Abstract

DESCRIPTION (provided by applicant): Gene therapy using viral vectors has had limited success in the treatment of the various types of hemophilia. Both the host response and the unique characteristics of the vector systems have made it difficult to achieve persistent long-term transgene expression. An additional complication in the treatment of hemophilia by replacement therapy is the development of inhibitory antibodies to the clotting factor. In fact, this has prompted the use of rFVIIa in an effort to induce hemostasis via tissue factor (TF) independent FX activation at the platelet surface. The main objective of this research project is to evaluate the nonviral Sleeping Beauty transposon (SB-Tn) vector for gene therapy in a mouse model of factor IX deficiency. This objective tests our hypothesis that separate or combined expression of FIX, rFVIIa and soluble TF (sTF) transgenes will improve the disease phenotype. The first specific aim is designed to optimize in cell culture (i) the Tn design for maximal transposition and persistent gene expression of the coagulation factors IX, an engineered rFVIIa and sTF; and (ii) the nonviral delivery systems to hepatocytes via the asialoglycoprotein receptor by several different ligand moieties. The second specific aim evaluates the capacity of the best Tn vectors/delivery systems to promote Tn insertion and persistent transgene expression in vivo. The therapeutic effects in vivo for expression of FIX, rFVIIa, and/or sTF will be monitored and quantitated by the relevant metabolic parameters. The dosing regimen, the delivery vehicle and route of administration will be optimized for efficiency of transposition and safety. The third specific aim is designed to evaluate the potential use of blood outgrowth endothelial cells (BOECs) as a vehicle of transgene expression via SB transposition. The transposition and expression characteristics of the different SB-Tns expressing FIX, TF and/or rFVIIa will be examined in BOECs both individually and in pair wise combinations. The ex vivo engineered BOECs will be infused back into hemophilia B mice, and analyzed for long-term expression and effect on the bleeding diathesis of the FIX deficient animals. The major goals of this research proposal are to (i) optimize Tn designs and nonviral delivery systems that provide therapeutic levels of FIX, rFVIIa and/or sTF with SB-mediated gene transfer; (ii) identify the best in vivo conditions for correction of the clotting disorder in animal models of | hemophilia B; and (iii) characterize the BOECs potential as an autologous vehicle for persistent transgene expression.