Gene therapy for hemophilia b using pinctm formulated plasmid delivered to muscle with electroporation

Abstract

We have achieved therapeutic circulating levels of human Factor IX (hFIX) in mice and dogs using plasmid-based gene delivery and electroporation. Plasmid encoding CMV-hFIX formulated with 5% polyvinylpyrrolidone (PVP) was injected into the tibialis and the gastrocnemius muscles of C57BL/6 mice, then electroporated using 1 cm2 caliper electrodes. hFIX expression peaked at 144 ng mL−1 on day 7, but was undetectable at day 10. hFIX expression was dose-dependent. In SCID mice, plasma hFIX levels peaked at ∼120 ng mL−1, were maintained for 3 months, then slowly diminished to ∼30 ng mL−1 at day 125. Redosing to the same muscle groups at day 153 injection increased hFIX expression levels to ∼90 ng mL−1. In normal canines, multiple muscles of the rear and front legs were injected followed by electroporation with a six-needle array electrode. Peak mean levels of 36 ng mL−1 were achieved in the plasma of ∼10 kg dogs at 21 days, but was undetectable at day 28 due to antibody formation, confirmed by Western blot. Some animals showed transient increases (3–6x) in creatine kinase levels at day 3, indicating mild muscle trauma. Studies in dogs with hemophilia B are in progress. Plasmid-based gene delivery with a PINCTM polymer augmented with electroporation is scalable for large animals and represents a promising approach for prophylactic treatment of individuals with hemophilia B.