Developing novel viral vectors to facilitate gene therapies for neuropathic pain

Abstract

Neuropathic pain (NeuP) is a chronic pain condition arising following injury or disease to the somatosensory system and affects 7-10% of the population worldwide. Despite its prevalence, currently available treatments for NeuP have limited efficacy and are often complicated by issues such as addiction. Spontaneous activity, a hallmark of NeuP, occurs in both injured and intact primary afferents and their relative contribution to pain remains a controversial subject, with the majority of evidence pointing towards an important role for injured afferents. Adeno associated viral (AAV) vector-based gene therapies are an attractive option for treating NeuP, however, their use is limited by immunogenicity and dorsal root ganglion (DRG) toxicity concerns. In this thesis, we designed viral vectors capable of delivering therapeutic transgenes and restricting their expression to injured, but not intact primary afferents. To achieve this, we initially developed a pipeline of in vitro and in vivo model systems to test the targeting of our vectors. Following this, we first investigated the capacity of the endogenous activating transcription factor 3 (ATF3) P1 promoter to target AAV gene expression. While the ATF3P1 AAV9 selectively limited transgene expression to injured sensory neurons in vitro, these results were not replicated in vivo. We next generated an epigenomic atlas of naïve DRG neurons using single nucleus Assay for Transposase Accessible Chromatin sequencing (snATAC-seq) and gained insight into the epigenomic signatures of injured and intact primary afferents. This data provides a resource for exploring the epigenomic mechanisms governing gene expression in the DRG and can be used to identify putative regulatory elements capable of targeting AAV gene expression to injured primary afferents. Finally, we trialled a microRNA (miRNA)-based approach, whereby miR-182 target sites were introduced into an AAV-PHP.S vector and successfully restricted expression to injured primary afferents in vivo