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Allotopic RNA expression strategy to rescue an endogenous mitochondrial ATP6[1] mutation in Drosophila

Towheed, Atif (2015) Allotopic RNA expression strategy to rescue an endogenous mitochondrial ATP6[1] mutation in Drosophila. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Mitochondria are essential organelles in the cell. One of their most critical functions is the generation of cellular energy in the form of ATP. The presence of DNA in the mitochondrial matrix makes this organelle semi-autonomous. However, it relies heavily on the nucleus and cytosol to import ~99% of its proteins and some RNA molecules for its normal functioning. Mutations in the mitochondrial DNA (mtDNA) cause several devastating disorders. Due to their complexity and our incomplete understanding of mitochondrial disease pathogenesis, these disorders are difficult to diagnose and currently no pharmacological treatment exists. Further, gene therapy for these devastating disorders is impeded due to lack of mitochondrial genome manipulation techniques. Understanding the mechanism of pathogenesis and developing mtDNA manipulation strategies are key to developing remedial therapies.

In my thesis, I investigated an RNA allotopic strategy of targeting RNA into the mitochondria in vivo in flies. In my first aim, I improved an in vivo mitochondrial-targeting tool (mtTRES vector) to manipulate proteins encoded by the mitochondrial DNA. This vector integrates into the nuclear genome and results in the transcription of a chimeric RNA consisting of a mitochondrial targeting signal sequence and a small non-coding antisense RNA.

Previous studies have attempted allotopic expression via both protein and RNA import with mixed results. Only a few of them, however, have been tested in vivo and none have been examined for rescue in an animal model of mitochondrial disease. Since our lab has a well characterized mtDNA mutation fly model, ATP6[1], I had a unique opportunity to investigate rescue strategies in these models. In my second aim, I improved a unique set of mtTRESPro vectors for both flies and humans to target long coding RNAs into mitochondria. Once imported these long RNAs are designed to be endogenously translated in mitochondria. By targeting a wild type copy of the mutant ATP6 gene, I explored the rescuing potential of allotopic RNA import in vivo. Our data suggest the mtTRES and mtTRESPro mitochondrial manipulation tools have genuine potential to be developed into a mitochondrial disease gene therapy.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorPalladino, Michael Jmjp44@pitt.eduMJP44
Committee ChairRomero, Guillermoggr@pitt.eduGGR
Committee MemberDeFranco, Donalddod1@pitt.eduDOD1
Committee MemberBurton, Edward Aeab25@pitt.eduEAB25
Committee MemberJiang, Yuyuj5@pitt.eduYUJ5
Date: 26 February 2015
Date Type: Publication
Defense Date: 20 January 2015
Approval Date: 26 February 2015
Submission Date: 25 February 2015
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Number of Pages: 152
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Pharmacology and Chemical Biology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Mitochondrial disorders Mitochondria Gene therapy Drosophila RNA Translational Inhibitors
Date Deposited: 26 Feb 2015 19:56
Last Modified: 26 Feb 2020 06:15


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