Christian Mueller PhDRobert H. Brown DPhil, MDToro, Gabriela2022-08-232022-08-232019-03-282019-05-0810.13028/kw7m-9a26https://hdl.handle.net/20.500.14038/31239Amyotrophic lateral sclerosis (ALS) is a terminal neurodegenerative disease that affects motor neurons causing progressive muscle weakness and respiratory failure. In 2011, the presence of a hexanucleotide repeat expansion within chromosome 9 open reading frame 72(C9ORF72) was identified in ALS patient samples, becoming the major known genetic cause for ALS and frontotemporal dementia (FTD). Carriers of this mutation present reduced levels of C9ORF72 mRNA, RNA foci produced by the aggregating expansion and toxic dipeptides generated through repeat-associated non-ATG translation. These findings have led to multiple hypotheses on the pathogenesis of C9ORF72: 1) Haploinsufficiency, 2) RNA gain-of-function, 3) RAN Translation, and 4) Disrupted nucleocytoplasmic trafficking. Due to lack of treatments for this disease, we have pursued an AAV-RNAi dependent gene therapy approach, using an artificial microRNA (amiR) packaged in a recombinant adeno-associated virus (rAAV). After validating our in vitro results, we advanced to in vivo experiments using transgenic mice that recapitulate the major histopathological features seen in human ALS/FTD patients. Adult and neonate mice were injected through clinically relevant routes and our results indicate that AAV9-mediated amiR silencing not only reduced mRNA and protein levels of C9ORF72 but also the expansion derived toxic GP dipeptides. Although our amiR is not targeting the expansion itself but exon 3, we illustrate here that the evident dipeptide decrease is achievable due to the presence of aberrant transcripts in the cytoplasm containing miss-spliced Intron-HRE-C9ORF72 species. These encouraging results have led to the continued testing of this treatment as a therapeutic option for C9ORF72 - ALS patients.en-USCopyright is held by the author, with all rights reserved.ALSC9ORF72Gene therapy for ALSmicroRNAsEpigenetics in ALSC9ORF72 mouse modelSilencing of C9ORF72Molecular and Cellular NeuroscienceNervous System DiseasesVirologyDoctoral Dissertationhttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=2029&context=gsbs_diss&unstamped=1https://escholarship.umassmed.edu/gsbs_diss/102014456538gsbs_diss/10200000-0002-8905-1589