Engineering toxins as neuropeptides: targeted pain modulation via recombinant adeno-associated virus (rAAV) vectors delivery to nociceptors

Chronic pain is defined as persistent or recurrent pain lasting longer than three months. It currently affects 30% of the worldwide population and significantly impacts the quality of life. Treatments mostly rely on oral administration of small-molecule analgesics or opioids, which often display poor efficacy and promote severe side effects. Thus, there is an unmet therapeutical need to design new strategies to treat chronic pain. Here, a mechanism for self-modulation of nociceptors, the peripheral sensory neurons responsible for damage detection and signalling, was developed. Two venom peptides, the 𝜔-conotoxin MVIIA and spider toxin GpTx-1-71, were chosen for their selective and efficient antagonism against two voltage gated ion channels involved in pain signalling: Cav2.2 and Nav1.7, respectively. To minimize side effects due to active peptide handling, MVIIA and GpTx-1-71 DNA sequences were manipulated. The insertion of the pre- and pro- sequences derived from brain-derived neurotrophic factor (BDNF) via molecular cloning upstream the sequences for mature peptides induced the two toxins to act as neuropeptides. In vitro experiments demonstrated that PreProBDNF sequence managed to drive the peptides localization into dense-core vesicles (DCVs) as well as being released upon sustained stimulation and inhibit their own release, using a combination of stimulation-dependent exocytosis recording, genetic code expansion through unnatural aminoacids and electrophysiological characterization of vesicle release. Cell-specific gene delivery was then achieved through recombinant adeno-associated viral vectors (rAAV) to transduce nociceptors, were then able to produce and release the peptides. These would then act retroactively on their target channel on nociceptors, thus creating a self-modulation strategy upon the sensory neurons responsible for signalling pain to the dorsal horn of the spinal cord. Upon nociceptor transduction in dorsal root ganglia (DRG) by optimization of administration routes and rAAVs modification, the stimulus for vesicle release from nociceptors was refined through optogenetic; also, the MVIIA toxin, engineered to mimic neuropeptides, was successful in modulating nociception in injected animals. The developed strategy proposes an innovative therapeutical approach based on gene therapy for chronic pain with an important safety switch due to toxins release self-inhibition. Also, it offers a DNA manipulation strategy that allow peptides of interest to be act as neuropeptides, thus providing intriguing insights for protein manipulation.