Subcutaneous peripheral injection of cationized gelatin/DNA polyplexes as a platform for non-viral gene transfer to sensory neurons.
Selective modulation of sensory neuron gene expression could have numerous applications for the peripheral nervous system. Here, we report that subcutaneous peripheral injection of plasmid DNA complexed with a non-viral cationized gelatin (CG) vector led to transgene expression in rat lumbar dorsal root ganglia (DRGs). CG/DNA polyplexes appeared to undergo rapid retrograde transport through sciatic and spinal nerves, with reporter gene messenger RNA (mRNA) expression detectable in L4 and L5 DRGs within 60 hours. Maximum transgene expression was observed for polyplexes formed at 7.5:1 CG-to-DNA weight ratio under salt-free conditions, which generated 615 +/- 112 nm nanoparticles with zeta-potential of 9.4 +/- 0.19 mV. Six days after injection of the CG/DNA polypex, reporter gene protein immunofluorescence was observed in 1,164 +/- 176 DRG neurons, representing an estimated transfection rate of 47% of targeted neurons. Reporter gene expression was not detected in heart, lung, or liver tissues, suggesting a lack of systemic uptake. Measurements of tactile sensitivity indicate that CG/DNA injection did not cause behavioral toxicity. The injection platform was further used for plasmid-driven short hairpin RNA-mediated suppression of glyceraldehyde-3-phosphate dehydrogenase. This non-invasive gene delivery system could be used for the mechanistic study and targeted molecular evaluation of peripheral nervous system pathologies such as neuropathic pain.
Thakor
Devang
D
Jane and Jerry Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials, and Hospital Dentistry, School of Dentistry, University of California, Los Angeles, California 90095, USA.
Spigelman
Igor
I
Tabata
Yasuhiko
Y
Nishimura
Ichiro
I
D000818:Animals; D001483:Base Sequence; D001522:Behavior, Animal; D002412:Cations; D004247:DNA / Q000008:administration & dosage*; D017931:DNA Primers; D005455:Fluorescent Antibody Technique; D005780:Gelatin / Q000008:administration & dosage*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D008297:Male; D009475:Neurons, Afferent / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D019076:Transgenes
D002412:Cations; D017931:DNA Primers; D005780:Gelatin; D004247:DNA