Zinc finger nuclease gene repair as a treatment for cystinosis

dc.check.date10000-01-01
dc.check.embargoformatE-thesis on CORA onlyen
dc.check.entireThesisEntire Thesis Restricted
dc.check.infoIndefiniteen
dc.check.opt-outYesen
dc.check.reasonThis thesis is due for publication or the author is actively seeking to publish this materialen
dc.contributor.advisorHarrison, Patricken
dc.contributor.advisorScallan, Martinaen
dc.contributor.authorKaschig, Katrin
dc.contributor.funderCystinosis Foundation Irelanden
dc.contributor.funderHealth Research Boarden
dc.date.accessioned2014-01-27T15:46:26Z
dc.date.issued2013
dc.date.submitted2013
dc.description.abstractCystinosis is a multi-system autosomal recessive disorder caused by mutations and/or deletions in both alleles of CTNS, a gene encoding for the low pH dependent lysosomal cystine exporter cystinosin. Cystinosis occurs in approximately 1:200,000 newborns worldwide and is characterised by an accumulation of cystine in the lysosomes. The most severe form of the disorder is nephropathic cystinosis presenting Fanconi syndrome and leads without treatment to an end-stage renal failure before the age of ten. The only treatment available so far is cysteamine therapy, which delays disease progression by five years, but does not provide a cure for cystinosis patients. Current gene and cell based therapeutic approaches have not yet provided a suitable alternative. A potentially approach for a long-term treatment could be to generate autologous gene–modified stem cells by repairing the gene. Zinc Finger Nucleases (ZFNs) serve as a tool to increase HDR up to a 200,000-fold by introducing a double-stranded break (DSB). Thus, simple mutations in the CTNS gene could be corrected by introduction of a double-stranded break using ZFNs to boost the process of HDR with a suitable donor DNA sequence. A permanent repair of the most common lesion CTNS, a 57 kb deletion, could be achieved by ZFN-mediated HDR using a minigene CTNS promoter/cDNA construct. The thesis describes the design and testing of seven zinc finger nuclease pairs for their cleavage activity in vitro and in cellulo.. A highly sensitive assay to detect even low levels of ZFN-mediated HDR was also developed. Finally, to further investigate the role of autophagy in tissue injury in cystinotic cells an assay to monitor autophagy levels in the cells was successfully developed. This assay provides the opportunity to demonstrate functional restoration of CTNS after successful ZFN-HDR in cystinotic cells.en
dc.description.statusNot peer revieweden
dc.description.versionAccepted Version
dc.format.mimetypeapplication/pdfen
dc.identifier.citationKaschig, K. 2013. Zinc finger nuclease gene repair as a treatment for cystinosis. PhD Thesis, University College Cork.en
dc.identifier.urihttps://hdl.handle.net/10468/1337
dc.language.isoenen
dc.publisherUniversity College Corken
dc.rights© 2013, Katrin Kaschig.en
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/3.0/en
dc.subjectZinc finger nucleasesen
dc.subjectHomology directed repairen
dc.subject.lcshCystinosisen
dc.thesis.opt-outtrue
dc.titleZinc finger nuclease gene repair as a treatment for cystinosisen
dc.typeDoctoral thesisen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD (Science)en
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