A Facile Nonviral Method for Delivering Genes and siRNAs to Skeletal Muscle of Mammalian Limbs

Abstract

Delivery is increasingly being recognized as the critical hurdle holding back the tremendous promise of nucleic acid-based therapies that include gene therapy and more recently siRNA-based therapeutics. While numerous candidate genes (and siRNA sequences) with therapeutic potential have been identified, their utility has not yet been realized because of inefficient and/or unsafe delivery technologies. We now describe an intravascular, nonviral methodology that enables efficient and repeatable delivery of nucleic acids to muscle cells (myofibers) throughout the limb muscles of mammals. The procedure involves the injection of naked plasmid DNA or siRNA into a distal vein of a limb that is transiently isolated by a tourniquet or blood pressure cuff. Nucleic acid delivery to myofibers is facilitated by its rapid injection in sufficient volume to enable extravasation of the nucleic acid solution into muscle tissue. High levels of transgene expression in skeletal muscle were achieved in both small and large animals with minimal toxicity. Evidence of siRNA delivery to limb muscle was also obtained. The simplicity, effectiveness, and safety of the procedure make this methodology well suited to limb muscle gene therapy applications.

Localization of exogenous DNA to mitochondria in skeletal muscle following hydrodynamic limb vein injection

“…Our results are consistent with previous reports by Wolff's group where effective nuclear transgene was observed in muscle near the injection site [20].…”

“…This technique was first reported by Liu's group and Wollf's group in 1999, where effective nuclear transgene expression in hepatocytes in mice was achieved by hydrodynamic tail vein injection. In 2004, Wolff's group showed that the rapid injection of large volumes of naked pDNA into the great saphenous vein of the distal hind limb of a rat (HLV injection) resulted in effective nuclear transgene expression in skeletal muscle [20]. Based on previous great reports, we attempted to achieve mitochondrial delivery of pDNA targeted to skeletal muscle using HLV injection.…”

“…Vascular delivery procedures have recently been used to deliver plasmid DNA (pDNA) to the skeletal muscle of rodents and nonhuman primates by hydrodynamic limb vein (HLV) injection [17][18][19][20]. The hydrodynamic injection method originally was reported by Liu et al [21] and Zhang et al [22], and was used to achieve effective nuclear transgene expression in hepatocytes in mice by the rapid injection of large volumes of naked pDNA into the tail vein.…”

“…In the HLV injection procedure, a tourniquet is used to limit the delivery area to one limb per injection and naked pDNA is rapidly injected into the vein in the anterograde direction [20]. A sufficient volume of saline is used to facilitate extravasation of the pDNA from the vasculature and into the muscle tissue through multiple physical barriers.…”

Longevity of rAAV vector and plasmid DNA in blood after intramuscular injection in nonhuman primates: implications for gene doping

“…[10][11][12] Moreover, recombinant adeno-associated viral (rAAV) vectors and naked plasmid are two different gene transfer systems used for IM delivery in animal models [13][14][15][16][17] and in humans. 18,19 Recently, regional vascular infusion of a vector to achieve skeletal muscle transduction has been reported for plasmid DNA (pDNA) 20,21 and for rAAV vectors. 8,22 Among the rAAV serotypes analyzed to date, rAAV1 and rAAV8 are among the most efficient for muscle transduction.…”