Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by the progressive loss of upper and lower motor neurons, eventually culminating in patient paralysis and death via asphyxiation. At the time of diagnosis, approximately 50% of patient motor neurons are predicted to have degenerated, resulting in a prognosis of 3-5 years. While most incidences of ALS are sporadic (~90%) with no obvious genetic constituent, approximately 10% of cases are inherited in a dominant manner and are referred to as familial ALS. At the present time there is no cure for ALS and current pharmacological treatments, such as Riluzole, operate with limited efficacy. As such, gaining further insight into pathomechanisms driving ALS is paramount for the development of novel and effective therapeutics. The C9ORF72 hexanucleotide repeat expansion is attributed to 40-50% of familial and approximately 10% of sporadic ALS cases. Investigating pathomechanisms driving familial cases of C9-ALS could elucidate neurodegenerative mechanisms central to both familial and sporadic cases. The aim of this thesis was to investigate transcriptional perturbations in C9-ALS to elucidate de novo candidates rendering motor neurons particularly susceptible to pathology. Evidence from previous studies suggested that nociceptors of ALS patients remain functionally viable due to the presence of non-neuropathic pain in approximately 80% of patients. We subsequently developed a novel protocol to efficiently generate functional sensory neuron cultures predominantly consisting of spinal nociceptors (~75%). Utilizing our protocol, we compared the transcriptomes of C9-ALS nociceptors with C9-ALS motor neurons which elucidated dysregulation of synaptic genes in motor neurons, potentially suggesting increased susceptibility of motor neurons to excitotoxic mechanisms. Finally, we utilised CRISPR systems employing dCas9 fused to epigenetic effectors to selectively demethylate the C9ORF72 repeat expansion and proximally associated regions to investigate epigenetic contributions to pathology. We observed an increase in transcript variant 3 expression and retention of intron 1 was observed upon demethylation of the repeat expansion. While increased expression of C9ORF72 could exacerbate pathomechanisms driven by RNA foci and accumulation of DPR proteins, deficits in synaptic function could be attenuated by reducing C9ORF72 haploinsufficiency. In summary, the work presented in this thesis outlines novel approaches to investigating the mechanisms driving pathology in C9-ALS with the hope of elucidating de novo candidates for therapeutic targeting. Furthermore, the ability to generate functional nociceptors broadens the scope of pain research permitting in vitro experiments to understand the mechanics of pain in human cells.Wellcome Trust