June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Development of a CRISPR dCas9-KRAB based strategy for treatment for dominant retinal degenerative blindness
Author Affiliations & Notes
  • Erin R Burnight
    University of Iowa Institute for Vision Research, Iowa, United States
    Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa, United States
  • Luke A Wiley
    University of Iowa Institute for Vision Research, Iowa, United States
    Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa, United States
  • Malavika K. Adur
    Department of Animal Science, Iowa State University, Iowa, United States
  • Dalyz Ochoa
    University of Iowa Institute for Vision Research, Iowa, United States
    Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa, United States
  • Emily E. Kaalberg
    University of Iowa Institute for Vision Research, Iowa, United States
    Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa, United States
  • Mallory Lang
    University of Iowa Institute for Vision Research, Iowa, United States
    Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa, United States
  • Jeremy M. Hoffman
    University of Iowa Institute for Vision Research, Iowa, United States
    Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa, United States
  • Chunhua Jiao
    University of Iowa Institute for Vision Research, Iowa, United States
    Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa, United States
  • Stephen R Russell
    University of Iowa Institute for Vision Research, Iowa, United States
    Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa, United States
  • Ian Han
    University of Iowa Institute for Vision Research, Iowa, United States
    Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa, United States
  • Elliott H Sohn
    University of Iowa Institute for Vision Research, Iowa, United States
    Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa, United States
  • Jason W. Ross
    Department of Animal Science, Iowa State University, Iowa, United States
  • Robert F Mullins
    University of Iowa Institute for Vision Research, Iowa, United States
    Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa, United States
  • Edwin M Stone
    University of Iowa Institute for Vision Research, Iowa, United States
    Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa, United States
  • Budd A. Tucker
    University of Iowa Institute for Vision Research, Iowa, United States
    Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa, United States
  • Footnotes
    Commercial Relationships   Erin Burnight None; Luke Wiley None; Malavika Adur None; Dalyz Ochoa None; Emily Kaalberg None; Mallory Lang None; Jeremy Hoffman None; Chunhua Jiao None; Stephen Russell None; Ian Han None; Elliott Sohn None; Jason Ross None; Robert Mullins None; Edwin Stone None; Budd Tucker None
  • Footnotes
    Support  NIH Grant EY024588; NIH Grant EY026008, Institute for Vision Research
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 1775 – F0324. doi:
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    • Get Citation

      Erin R Burnight, Luke A Wiley, Malavika K. Adur, Dalyz Ochoa, Emily E. Kaalberg, Mallory Lang, Jeremy M. Hoffman, Chunhua Jiao, Stephen R Russell, Ian Han, Elliott H Sohn, Jason W. Ross, Robert F Mullins, Edwin M Stone, Budd A. Tucker; Development of a CRISPR dCas9-KRAB based strategy for treatment for dominant retinal degenerative blindness. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1775 – F0324.

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

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Abstract

Purpose : Inherited retinal disease is a major cause of blindness worldwide. The most common disease-causing mutation in patients with RHO-associated retinitis pigmentosa (RP) is a dominant gain-of-function missense variant at amino acid residue 23 (Pro23His). Treatment for this disease and other dominantly inherited retinal dystrophies will require knockdown or ablation of mutant allele expression. To that end the purpose of this study was to develop a CRISPR-Cas9-mediated transcriptional repression strategy capable of suppressing RHO expression.

Methods : We created CRISPR-Cas9 transcriptional knockdown reagents using catalytically inactive S. aureus Cas9 (dCas9) fused to the Krüppel associated box (KRAB) transcriptional repressor domain and evaluated four guides targeted to transcriptional regulatory elements in the promoter region of RHO. Using a reporter construct carrying GFP cloned downstream of the RHO promoter fragment (nucleotides -1403 to +73), we assayed GFP knockdown in cells treated with our RHO promoter-targeted CRISPR-dCas9 reagents. Following functional confirmation, reagents were packaged into AAV5 vectors and delivered to human retinal explants (N=3) and Pro23His mutant retinal degenerative swine (N=4). RHO knockdown was demonstrated via Western blot analysis and immunocytochemistry.

Results : Quantitative RT-PCR analysis of cells treated with CRISPR-dCas9 and reporter plasmids demonstrated ~74%-84% reduction in GFP expression when compared to control cells treated with reporter plasmid only. The guide with the greatest knockdown in this assay (sag94) was cloned into AAV5 cassette plasmids along with dCas9-KRAB and used in downstream ex vivo and in vivo experiments. Western blot analysis demonstrated significant RHO knockdown (50%) in human retinal explants treated with AAV5-RHOpCRISPRi vector compared to untreated controls. When we extended these studies into the Pro23His rhodopsin mutant swine model, we observed a 20% knockdown of rhodopsin in CRISPRi-treated retinae compared with untreated contralateral control retinae at two-weeks post injection.

Conclusions : We have generated a dCas9-KRAB gene repression system suitable for suppressing RHO expression in vivo. This work may provide a paradigm from which to develop CRISPR-dCas9 therapies to treat dominantly inherited retinal dystrophies.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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