May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
siRNA–mediated suppression of IMPDH1 transcripts: relevance to therapy for RP10 form of retinitis pigmentosa
Author Affiliations & Notes
  • L.C. S. Tam
    The Ocular Genetics Unit, Department of Genetics, Trinity College Dublin, Dublin, Ireland
  • A.S. Kiang
    The Ocular Genetics Unit, Department of Genetics, Trinity College Dublin, Dublin, Ireland
  • A. Kennan
    The Ocular Genetics Unit, Department of Genetics, Trinity College Dublin, Dublin, Ireland
  • A. Aherne
    The Ocular Genetics Unit, Department of Genetics, Trinity College Dublin, Dublin, Ireland
  • P. Humphries
    The Ocular Genetics Unit, Department of Genetics, Trinity College Dublin, Dublin, Ireland
  • Footnotes
    Commercial Relationships  L.C.S. Tam, None; A.S. Kiang, None; A. Kennan, None; A. Aherne, None; P. Humphries, None.
  • Footnotes
    Support  The Embark Initiative, The Irish Research Council for Science, Engineering and Technology
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 4778. doi:
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      L.C. S. Tam, A.S. Kiang, A. Kennan, A. Aherne, P. Humphries; siRNA–mediated suppression of IMPDH1 transcripts: relevance to therapy for RP10 form of retinitis pigmentosa . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4778.

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

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Abstract

Abstract: : Purpose: The RP10 form of autosomal dominant retinitis pigmentosa (RP) has been mapped to chromosome 7q31.1, and accounts for 5–7% of all dominant cases of RP. Recent mutational studies have identified the gene; IMPDH1 as a candidate gene for the RP10 form of adRP. The aim of the study is to use mutation–independent suppression and replacement approach to suppress autosomal dominant RP caused by mutation in IMPDH1. Methods: Commercial web–based program provided by www.dharmacon.com was used for the design of siRNAs. Five siRNA molecules targeting human IMPDH1 transgenes were cloned into human H1 promoter–driven mammalian expression vectors to give short–hairpin RNAs (shRNAs) and these were transiently transfected into human embryonic kidney cells (HEK293) using Lipofectamine 2000 (Qiagen). Total RNA was extracted from the cells using Trizol as outlined by the manufacturer (Gibco–BRL). The mRNA levels of IMPDH1, IMPDH2 and beta–actin were quantified by RT–PCR on a Light Cycler (Roche). A replacement gene construct containing changes in wobble degenerate position was generated by PCR mutagenesis. Results: A high level of endogenous IMPDH1 transcript has been detected in HEK293. The silencing efficiencies of the five shRNAs have been assessed following transfection into HEK293 cells. A replacement IMPDH1 gene construct has been evaluated for resistance against the shRNAs. Conclusions: The suppression and replacement of IMPDH1 transcripts provides a platform on which to develop a gene therapy for the RP10 form of retinitis pigmentosa.

Keywords: gene transfer/gene therapy • retinal degenerations: hereditary • gene/expression 
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