| 1426 Transgene expression in sensory neurons after peripheral vector injection | ||
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D.K. THAKOR1, I. SPIGELMAN1, Y. TABATA2, and I. NISHIMURA1, 1University of California - Los Angeles, USA, 2Kyoto University, Japan Objectives: Current methods for gene delivery in the peripheral nervous system are limited by suboptimal vectors and invasive delivery techniques. We have used subcutaneous injection of a nonviral biodegradable gelatin vector to achieve a noninvasive gene transfer to sensory neurons. Methods: Red fluorescent protein (RFP)-expressing DNA plasmids complexed to polyethylenimine (PEI) or cationized gelatin (CG) vectors were subcutaneously injected into the glabrous rat hindpaw. Post-injection RFP expression in sensory neurons was confirmed using RT-PCR, Western blots, and fluorescence imaging of dissociated L4/L5 dorsal root ganglion (DRG) neurons. Relative fluorescence intensities (RFPfi) were quantified for imaging data. Paw withdrawal thresholds to thermal and mechanical stimuli, paw edema, and paw temperature were measured for 3 weeks after injection. Transgene expression was compared for vector/DNA complexes formed at several PEI/DNA nitrogen/phosphate ratios and CG/DNA mass ratios, and for CG/DNA formation under saline and salt-free conditions. Results: Salt-free CG/DNA formation produced greater RFPfi than saline formation (saline 2.04x107, salt-free 6.06x107, p<0.001). In addition, CG/DNA at a mass ratio of 7.5:1 showed stronger RFPfi compared to all other mass ratios (5:1 CG/DNA 6.06x107, 7.5:1 CG/DNA 9.71x107, p<0.001) and compared to PEI at all nitrogen/phosphate ratios (15:1 PEI/DNA 5.32x107, 7.5:1 CG/DNA 9.71x107, p<0.001) Behavioral measurements did not show any lasting differences between injected and noninjected paws. Conclusions: Our studies establish peripheral subcutaneous CG/DNA injection as a noninvasive technique for sensory neuron gene delivery that does not cause lasting pain or inflammation. We also introduce a salt-free condition for CG/DNA complex formation that enhances transfection, and for this application, we demonstrate superior transfection of the recently developed CG vector over polyethylenimine, a standard nonviral vector. This novel gene transfer paradigm could potentially be used to study basic sensory mechanisms or for molecular therapy of sensory neuron disorders such as trigeminal neuralgia. Supported by NSF IGERT DGE9972802 | ||
| Seq #152 - Sensory-Motor Systems and Mechanisms 12:30 PM-2:30 PM, Thursday, 11 March 2004 Hawaii Convention Center 328 | ||
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