Gene Therapy for Efficient Suppression of T-Type Channels in Treating Diabetic Neuropathy

Abstract

Painful diabetic neuropathy (PDN), a chronic and often incurable syndrome, is one of the most common and unpleasant complications of diabetes. Effective clinical interventions for PDN are very limited and already developed approaches are characterized by lack of molecular or cellular target specificity and a short duration of therapeutic effects. Numerous investigations causally link upregulation of the Cav3.2 T-type Ca2+ channels in peripheral nociceptive neurons to painful symptoms of PDN. Here we suggest an approach to alleviate these symptoms based on implementation of virus-mediated cell-specific delivery of vectors expressing small hairpin RNAs (shRNAs). Processed by Dicer into specific small interfering RNA (siRNA), they would suppress the expression of T-type Ca2+ channels. In order to experimentally validate this approach, we have initially confirmed the ability of designed Dicer-substrate small interfering RNAs (DsiRNAs) to suppress expression of T-type channels in neurons of primary hippocampal and dorsal root ganglia (DRG) cultures. Target sequences of the effectively interfering DsiRNA were then used to design shRNAs and the coding sequences of shRNAs were cloned into the vector pAAV under U6 promotor. This plasmid was also proved to be effective in interference with expression of the T-type channels in the rat cultured DRG neurons. The expression cassette of this plasmid will be packed into AAV6 particles with tropism to unmyelinated fibers to suppress T-type channel expression in nociceptive DRG neurons and to alleviate painful symptoms of PDN.