Show simple item record

dc.contributor.advisorWen Xue
dc.contributor.advisorRobert H Brown Jr.
dc.contributor.authorKennedy, Zachary C.
dc.date2022-08-11T08:08:38.000
dc.date.accessioned2022-08-23T16:02:08Z
dc.date.available2022-08-23T16:02:08Z
dc.date.issued2019-08-09
dc.date.submitted2019-08-30
dc.identifier.doi10.13028/9qjz-qq07
dc.identifier.urihttp://hdl.handle.net/20.500.14038/31268
dc.description.abstractMutations in the SOD1 gene are the best characterized genetic cause of amyotrophic lateral sclerosis (ALS) and account for ~20% of inherited cases and 1-3% of sporadic cases. The gene-editing tool Cas9 can silence mutant genes that cause disease, but effective delivery of CRISPR-Cas9 to the central nervous system (CNS) remains challenging. Here, I developed strategies using canonical Streptococcus pyogenes Cas9 to silence SOD1. In the first strategy, I demonstrate effectiveness of systemic delivery of guide RNA targeting SOD1 to the CNS in a transgenic mouse model expressing human mutant SOD1 and Cas9. Silencing was observed in both the brain and the spinal cord. In the second strategy, I demonstrate the effectiveness of delivering both guide RNA and Cas9 via two AAVs into the ventricles of the brain of SOD1G93A mice. Silencing was observed in the brain and in motor neurons within the spinal cord. For both strategies, treated mice had prolonged survival when compared to controls. Treated mice also had improvements in grip strength and rotarod function. For ICV treated mice, we detected a benefit of SOD1 silencing using net axonal transport assays, a novel method to detect motor neuron function in mice before onset of motor symptoms. These studies demonstrate that Cas9-mediated genome editing can mediate disease gene silencing in motor neurons and warrants further development for use as a therapeutic intervention for SOD1-linked ALS patients.
dc.language.isoen_US
dc.rightsCopyright is held by the author, with all rights reserved.
dc.subjectCRISPR/Cas9
dc.subjectCas9
dc.subjectAmyotrophic lateral sclerosis
dc.subjectALS
dc.subjectMotor Neuron Disease
dc.subjectSOD1
dc.subjectsuperoxide dismutase
dc.subjectgene therapy
dc.subjectgene editing
dc.subjectAAV
dc.subjectadeno associated vector
dc.subjectadeno-associated vector
dc.subjectAAV9
dc.subjectBiology
dc.subjectBiotechnology
dc.subjectMolecular and Cellular Neuroscience
dc.subjectMusculoskeletal Diseases
dc.subjectNervous System Diseases
dc.subjectNucleic Acids, Nucleotides, and Nucleosides
dc.subjectOther Neuroscience and Neurobiology
dc.subjectTherapeutics
dc.titleOptimizing CRISPR/Cas9 for Gene Silencing of SOD1 in Mouse Models of ALS
dc.typeDoctoral Dissertation
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=2056&context=gsbs_diss&unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_diss/1047
dc.legacy.embargo2020-02-28T00:00:00-08:00
dc.identifier.contextkey15236305
refterms.dateFOA2022-08-26T04:19:10Z
html.description.abstract<p>Mutations in the <em>SOD1</em> gene are the best characterized genetic cause of amyotrophic lateral sclerosis (ALS) and account for ~20% of inherited cases and 1-3% of sporadic cases. The gene-editing tool Cas9 can silence mutant genes that cause disease, but effective delivery of CRISPR-Cas9 to the central nervous system (CNS) remains challenging. Here, I developed strategies using canonical <em>Streptococcus pyogenes</em> Cas9 to silence <em>SOD1</em>. In the first strategy, I demonstrate effectiveness of systemic delivery of guide RNA targeting <em>SOD1</em> to the CNS in a transgenic mouse model expressing human mutant SOD1 and Cas9. Silencing was observed in both the brain and the spinal<em> </em>cord. In the second strategy, I demonstrate the effectiveness of delivering both guide RNA and Cas9 via two AAVs into the ventricles of the brain of <em>SOD1<sup>G93A</sup></em> mice. Silencing was observed in the brain and in motor neurons within the spinal cord. For both strategies, treated mice had prolonged survival when compared to controls. Treated mice also had improvements in grip strength and rotarod function. For ICV treated mice, we detected a benefit of SOD1 silencing using net axonal transport assays, a novel method to detect motor neuron function in mice before onset of motor symptoms.<em> </em> These studies demonstrate that Cas9-mediated genome editing can mediate disease gene silencing in motor neurons and warrants further development for use as a therapeutic intervention for <em>SOD1</em>-linked ALS patients.</p>
dc.identifier.submissionpathgsbs_diss/1047
dc.contributor.departmentRNA Therapeutics Institute
dc.description.thesisprogramNeuroscience
dc.identifier.orcid0000-0003-2476-1624


Files in this item

Thumbnail
Name:
Thesis_Dissertation__190830.pdf
Size:
1.968Mb
Format:
PDF

This item appears in the following Collection(s)

Show simple item record