May 2003
Volume 44, Issue 13
ARVO Annual Meeting Abstract  |   May 2003
Ribozymes Targeting Mitochondrial ATP6 to Create a Mouse Cell Culture Model of NARP
Author Affiliations & Notes
  • J.A. Bongorno
    Molecular Genetics/Biology, University Florida, Gainesville, FL, United States
  • J. Guy
    Ophthalmology, University Florida, Gainesville, FL, United States
  • A.S. Lewin
    Ophthalmology, University Florida, Gainesville, FL, United States
  • Footnotes
    Commercial Relationships  J.A. Bongorno, None; J. Guy, None; A.S. Lewin, None.
  • Footnotes
    Support  EY11596
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 4523. doi:
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      J.A. Bongorno, J. Guy, A.S. Lewin; Ribozymes Targeting Mitochondrial ATP6 to Create a Mouse Cell Culture Model of NARP . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4523.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : Purpose: The goal is to create a mouse model for NARP (Neuropathy, Ataxia and Retinitis Pigmentosa), a disease caused by mutations in the mitochondrial ATP6 gene. Methods: Ribozymes were designed to target the mouse ATP6 mRNA. Three ribozymes were tested in vitro on short RNA oligonucleotide targets. The two most efficient ribozymes were linked to the 3’ end of the 5 S ribosomal RNA gene, an RNA that is naturally imported into the mitochondria. Mouse NIH3T3 cells were transfected with the ribozyme-containing plasmids and selected with G418. Clonal isolates were analyzed for ribozyme transcript levels and ATP6 mRNA levels by RT-PCR. For cells whose target mRNA levels were reduced, growth rates were measured in glucose and galactose media. Results: Two ribozymes were found to cleave short oligonucliotide targets efficiently: Kcat = 0.61 min-1 and Km = 1.8 µM. In cell culture, the clonal isolate with the highest levels of ribozyme transcript also contained the lowest levels of ATP6 mRNA. This corresponded to a reduction of about 70 percent relative to control vector and mock transfected cell isolates. In galactose media these ribozyme-containing cells grew significantly slower than control cells. Conclusions: At least one of the ribozymes tested is effective at reducing the mitochondrial ATP6 mRNA to levels sufficient to produce a slow-growth phenotype in cultured mouse cells. This is a promising method for creating an ATP6 deficient mouse, a model for NARP.

Keywords: mitochondria • gene transfer/gene therapy • metabolism 

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