July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
RPGR gene editing with CRISPR-Cas 9 system in Retinal degeneration 9 (Rd9) mice to correct a disease mutation and phenotype
Author Affiliations & Notes
  • Amal Alsufyani
    Retinal Cell Biology and Degeneration Section, The National Eye institute , Bethesda , Maryland, United States
    Department of Chemistry and Biochemistry , George Mason University, Fairfax, Virginia, United States
  • Wenhan Yu
    Ocular Gene Therapy Core, The National Eye Institute, Bethesda , Maryland, United States
  • Jessica Gumerson
    Retinal Cell Biology and Degeneration Section, The National Eye institute , Bethesda , Maryland, United States
  • Lijin Dong
    Genetic Engineering Core , The National Eye Institute , Bethesda , Maryland, United States
  • Zhijian Wu
    Ocular Gene Therapy Core, The National Eye Institute, Bethesda , Maryland, United States
  • Tiansen Li
    Retinal Cell Biology and Degeneration Section, The National Eye institute , Bethesda , Maryland, United States
  • Footnotes
    Commercial Relationships   Amal Alsufyani, None; Wenhan Yu, None; Jessica Gumerson, None; Lijin Dong, None; Zhijian Wu, None; Tiansen Li, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 41. doi:
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      Amal Alsufyani, Wenhan Yu, Jessica Gumerson, Lijin Dong, Zhijian Wu, Tiansen Li; RPGR gene editing with CRISPR-Cas 9 system in Retinal degeneration 9 (Rd9) mice to correct a disease mutation and phenotype. Invest. Ophthalmol. Vis. Sci. 2018;59(9):41.

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

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Abstract

Purpose : Retinitis Pigmentosa (RP) is a degenerative disease that is characterized by progressive death of photoreceptors. RPGR gene mutations account for about 70% of all cases of X-linked RP (XLRP). The retinal degeneration 9 (Rd9) mouse is a naturally occurring animal model of XLRP in which a 32 base pair duplication causes a frame shift in the ORF15 region of RPGR that results in a truncated RPGR protein. ORF15 is essential for RPGR function and contains a repetitive region that tolerates substantial variations to its length as long as the reading frame is maintained. Thus a gene correction scheme that can restore the reading frame may be therapeutic.

Methods : We took advantage of the highly repetitive nature of the ORF15 region and introduce a break that when corrected by spontaneous non-homologous end joining (NHEJ), will randomly add or remove a limited number of bases, thus potentially altering the reading frame. we took two complementary approaches, one germline (fertilized egg injection) and the other somatic (subretinal injection of AAV vectors). We predict that up to 1/3 of chromosomes (and cells) that have undergone NHEJ will be converted into an open RPGR reading frame thus restoring the protein coding ability of the mutant allele. This simplified genome editing strategy may be suitable for RPGR ORF15 mutations and provides a useful model for the use of CRISPR for genome editing.

Results : In this study CRISPR-Cas9 mediated excision has restore the normal RPGR reading frame and rescued Rd9 photoreceptors. We tested the efficacy of the CRISPR-Cas9 system to mediate site-specific DNA cleavage in the ORF15 locus of RPGR, and whether Cas9 dsDNA break and NHEJ, introduced by CRISPR immediately upstream of the duplication in Rd9 mice, will restore normal RPGR expression in adult photoreceptors. With both of this study approaches, we were able to detect corrected RPGR expression.

Conclusions : RPGR-ORF15 is a purine rich region, thus it can tolerate in frame deletion and insertion mutations. RPGR exact sequence is not required for functionally folded folding but the correct reading frame is. RPGR specific CRISPR-Cas9 AAV-mediated transduction and activity in Rd9 mouse retina restored RPGR reading frame and protein expression in photoreceptor cilia. CRISPR-Cas9 mediated gene editing of RPGR-ORF15 offers a unique approach for therapeutic intervention in XLRP.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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