?
Therapeutic Angiogenesis by a “Dynamic Duo”: Simultaneous Expression of HGF and VEGF165 by Novel Bicistronic Plasmid Restores Blood Flow in Ischemic Skeletal Muscle
Therapeutic angiogenesis is a promising strategy for relief of ischemic conditions, and gene
delivery was used to stimulate blood vessels’ formation and growth. We have previously shown
that intramuscular injection of a mixture containing plasmids encoding vascular endothelial growth
factor (VEGF)165 and hepatocyte growth factor (HGF) leads to restoration of blood flow in mouse
ischemic limb, and ecacy of combined delivery was superior to each plasmid administered alone.
In this work, we evaluated di erent approaches for co-expression of HGF and VEGF165 genes in a
panel of candidate plasmid DNAs (pDNAs) with internal ribosome entry sites (IRESs), a bidirectional
promoter or two independent promoters for each gene of interest. Studies in HEK293T culture
showed that all plasmids provided synthesis of HGF and VEGF165 proteins and stimulated capillary
formation by human umbilical vein endothelial cells (HUVEC), indicating the biological potency of
expressed factors. Tests in skeletal muscle explants showed a dramatic di erence and most plasmids
failed to express HGF and VEGF165 in a significant quantity. However, a bicistronic plasmid with two
independent promoters (cytomegalovirus (CMV) for HGF and chicken b-actin (CAG) for VEGF165)
provided expression of both grow factors in skeletal muscle at an equimolar ratio. Ecacy tests
of bicistronic plasmid were performed in a mouse model of hind limb ischemia. Intramuscular
administration of plasmid induced significant restoration of perfusion compared to an empty vector
and saline. These findings were supported by increased CD31+ capillary density in animals that
received pHGF/VEGF. Overall, our study reports a first-in-class candidate gene therapy drug to
deliver two pivotal angiogenic growth factors (HGF and VEGF165) with properties that provide
basis for future development of treatment for an unmet medical need—peripheral artery disease and
associated limb ischemia.