Selective block of CaV3.2 in primary sensory neurons for treatment of chronic neuropathic pain Summary/Abstract: Chronic pain is a devastating problem, while opioid treatment of chronic pain has numerous risks, including misuse, overdose, and addiction, highlighting the need for new analgesic targets. The peripheral sensory nervous system (PSNS) is a particularly accessible site for devising new treatments, where the sensory neurons of the dorsal root ganglia (DRG) initiate nociception and have a central role in the development and maintenance of painful neuropathy. Sensory neuronal CaV3.2 T-type calcium channels regulate neuronal excitability and are a promising target for treatment of pain, but the development of selective CaV3.2 inhibitors has proved elusive. Small peptides, especially those derived from the natural proteins as functionally inhibitory peptide aptamers (iPAs), are recognized as being highly effective and selective, allowing blockade of specific pain molecular interactomes to reduce pain with minimal off-target effects. The analgesic iPA-sustained expression in the PSNS delivered by adeno-associated viral vectors (AAV) in the DRG is a safe and feasible path to chronic pain treatment with minimal side effects and no abuse/addiction liability. Molecular signaling interactions are often mediated by regions of proteins lacking a defined tertiary structure, known as protein intrinsically disordered regions (IDRs). Using established IDR prediction algorithms, we localized highly disordered regions in CaV3.2, and identified a novel 17mer peptide that produces sustained CaV3.2 T-type current inhibition and pain attenuation in a neuropathic pain rat model, demonstrating its therapeutic potential for pain treatment. The proposed Two-Phase study is to develop evidence that candidate CaV3.2iPAs delivered by AAV into DRG neurons have sufficient biological activity to justify further development as a novel analgesic approach. In Phase I (R66), a combined computational and experimental strategy will be used to design candidate CaV3.2iPAs and test their inhibition of T-type current by in vitro of cell-based studies. The milestone for advancement from R66 Phase to Phase II (R33) is identification of the top CaV3.2iPA leads that meet our selection criteria (higher potency and selectivity in block of CaV3.2) and of production of therapeutic AAV- CaV3.2iPAs. Phase II aims to apply AAV-CaV3.2iPAs in vivo by intraganglionic injection to evaluate critical features of AAV-CaV3.2iPAs in nerve injury-induced rat pain model, compared to controls The milestone for the end of Phase II is generation of sufficient data of in vivo efficacy and safety which will help us to make a go/no- go informed decision for further translational development. Our ultimate goal is to develop a novel therapeutics combined AAV-targeted gene delivery with potent and selective iPA block of CaV3.2 in anatomically segmental DRG to treat chronic neuropathic pain.