Dose Response in Rodents and Nonhuman Primates After Hydrodynamic Limb Vein Delivery of Naked Plasmid DNA

Abstract

The efficacy of gene therapy mediated by plasmid DNA (pDNA) depends on the selection of suitable vectors and doses. Using hydrodynamic limb vein (HLV) injection to deliver naked pDNA to skeletal muscles of the limbs, we evaluated key parameters that affect expression in muscle from genes encoded in pDNA. Short-term and long-term promoter comparisons demonstrated that kinetics of expression differed between cytomegalovirus (CMV), muscle creatine kinase, and desmin promoters, but all gave stable expression from 2 to 49 weeks after delivery to mouse muscle. Expression from the CMV promoter was highest. For mice, rats, and rhesus monkeys, the linear range for pDNA dose response could be defined by the mass of pDNA relative to the mass of target muscle. Correlation between pDNA dose and expression was linear between a threshold dose of 75 mg/g and maximal expression at approximately 400 mg/g. One HLV injection into rats of a dose of CMV-LacZ yielding maximal expression resulted in an average transfection of 28% of all hind leg muscle and 40% of the gastrocnemius and soleus. Despite an immune reaction to the reporter gene in monkeys, a single injection transfected an average of 10% of all myofibers in the targeted muscle of the arms and legs and an average of 15% of myofibers in the gastrocnemius and soleus.

Localization of exogenous DNA to mitochondria in skeletal muscle following hydrodynamic limb vein 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.…”

Measuring microRNA reporter activity in skeletal muscle using hydrodynamic limb vein injection of plasmid DNA combined with in vivo imaging

“…[41] although the percentage of transfected fibers we report is lower. This may be due to decreased sensitivity of detecting a fluorescent reporter (GFP) versus a colorimetric stain (β-galactosidase).…”

“…Similar to this report, we find the highest percentage (15.9%) of transfected myofibers in the posterior lower leg (gastrocnemius) muscles and the lowest percentage (6.1%) in the anterior lower leg (TA) muscles (Figure 3). Additionally, others have observed reporter gene expression up to 49 weeks after HLV injection [41], suggesting that this method could be used for longer-term regulatory studies, provided that the luciferase and mKATE signals follow similar expression profiles.…”

Nonviral Gene Therapy—The Challenge of Mobilizing DNA

“…Hydrodynamic injection into the limb vein was established also for forced delivery of naked plasmid to the myofibers of nonhuman primates (Hegge et al 2010). By a single hydrodynamic limb vein injection, Wooddell and coworkers were able to transfect an average of 10 % of all myofibers in the targeted muscle of the arms and legs (Wooddell et al 2011), providing hope that this method may have clinical relevance. Despite such promising findings, it is still the overall impression that hydrodynamic injection may need further optimization for effective and safe use in humans.…”