CRISPRi-Mediated Treatment of Dominant Rhodopsin-Associated Retinitis Pigmentosa

Overview

Journal CRISPR J

Specialty Molecular Biology

Date 2023 Dec 18

PMID 38108516

Authors

Erin R Burnight

Luke A Wiley

Nathaniel K Mullin

Malavika K Adur

Mallory J Lang

Cathryn M Cranston

Chunhua Jiao

Stephen R Russell

Elliot H Sohn

Ian C Han

Jason W Ross

Edwin M Stone

Robert F Mullins

Budd A Tucker

Affiliations

Soon will be listed here.

Abstract

Rhodopsin () mutations such as Pro23His are the leading cause of dominantly inherited retinitis pigmentosa in North America. As with other dominant retinal dystrophies, these mutations lead to production of a toxic protein product, and treatment will require knockdown of the mutant allele. The purpose of this study was to develop a CRISPR-Cas9-mediated transcriptional repression strategy using catalytically inactive Cas9 (dCas9) fused to the Krüppel-associated box (KRAB) transcriptional repressor domain. Using a reporter construct carrying green fluorescent protein (GFP) cloned downstream of the promoter fragment (nucleotides -1403 to +73), we demonstrate a ∼74-84% reduction in promoter activity in CRISPRi-treated versus plasmid-only controls. After subretinal transduction of human retinal explants and transgenic Pro23His mutant pigs, significant knockdown of rhodopsin protein was achieved. Suppression of mutant transgene was associated with a reduction in endoplasmic reticulum (ER) stress and apoptosis markers and preservation of photoreceptor cell layer thickness.

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References

Maguire A, High K, Auricchio A, Wright J, Pierce E, Testa F . Age-dependent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-escalation trial. Lancet. 2009; 374(9701):1597-605. PMC: 4492302. DOI: 10.1016/S0140-6736(09)61836-5. View

Cideciyan A, Hauswirth W, Aleman T, Kaushal S, Schwartz S, Boye S . Human RPE65 gene therapy for Leber congenital amaurosis: persistence of early visual improvements and safety at 1 year. Hum Gene Ther. 2009; 20(9):999-1004. PMC: 2829287. DOI: 10.1089/hum.2009.086. View

Tan E, Wang Q, Quiambao A, Xu X, Qtaishat N, Peachey N . The relationship between opsin overexpression and photoreceptor degeneration. Invest Ophthalmol Vis Sci. 2001; 42(3):589-600. View

LaVail M, Yasumura D, Matthes M, Drenser K, Flannery J, Lewin A . Ribozyme rescue of photoreceptor cells in P23H transgenic rats: long-term survival and late-stage therapy. Proc Natl Acad Sci U S A. 2000; 97(21):11488-93. PMC: 17227. DOI: 10.1073/pnas.210319397. View

OReilly M, Palfi A, Chadderton N, Millington-Ward S, Ader M, Cronin T . RNA interference-mediated suppression and replacement of human rhodopsin in vivo. Am J Hum Genet. 2007; 81(1):127-35. PMC: 1950918. DOI: 10.1086/519025. View

Hartong D, McGee T, Sandberg M, Berson E, Asselbergs F, van der Harst P . Search for a correlation between telomere length and severity of retinitis pigmentosa due to the dominant rhodopsin Pro23His mutation. Mol Vis. 2009; 15:592-7. PMC: 2661004. View

Cideciyan A, Sudharsan R, Dufour V, Massengill M, Iwabe S, Swider M . Mutation-independent rhodopsin gene therapy by knockdown and replacement with a single AAV vector. Proc Natl Acad Sci U S A. 2018; 115(36):E8547-E8556. PMC: 6130384. DOI: 10.1073/pnas.1805055115. View

Nishimasu H, Cong L, Yan W, Ran F, Zetsche B, Li Y . Crystal Structure of Staphylococcus aureus Cas9. Cell. 2015; 162(5):1113-26. PMC: 4670267. DOI: 10.1016/j.cell.2015.08.007. View

Bainbridge J, Mehat M, Sundaram V, Robbie S, Barker S, Ripamonti C . Long-term effect of gene therapy on Leber's congenital amaurosis. N Engl J Med. 2015; 372(20):1887-97. PMC: 4497809. DOI: 10.1056/NEJMoa1414221. View

10.

Komeima K, Rogers B, Lu L, Campochiaro P . Antioxidants reduce cone cell death in a model of retinitis pigmentosa. Proc Natl Acad Sci U S A. 2006; 103(30):11300-5. PMC: 1544081. DOI: 10.1073/pnas.0604056103. View

11.

Hauswirth W, Aleman T, Kaushal S, Cideciyan A, Schwartz S, Wang L . Treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results of a phase I trial. Hum Gene Ther. 2008; 19(10):979-90. PMC: 2940541. DOI: 10.1089/hum.2008.107. View

12.

Han I, Wiley L, Ochoa D, Lang M, Harman B, Sheehan K . Characterization of a novel Pde6b-deficient rat model of retinal degeneration and treatment with adeno-associated virus (AAV) gene therapy. Gene Ther. 2022; 30(3-4):362-368. DOI: 10.1038/s41434-022-00365-y. View

13.

Gray S, Choi V, Asokan A, Haberman R, McCown T, Samulski R . Production of recombinant adeno-associated viral vectors and use in in vitro and in vivo administration. Curr Protoc Neurosci. 2011; Chapter 4:Unit 4.17. PMC: 3209619. DOI: 10.1002/0471142301.ns0417s57. View

14.

Sohn E, Jiao C, Kaalberg E, Cranston C, Mullins R, Stone E . Allogenic iPSC-derived RPE cell transplants induce immune response in pigs: a pilot study. Sci Rep. 2015; 5:11791. PMC: 4490339. DOI: 10.1038/srep11791. View

15.

Gorbatyuk M, Justilien V, Liu J, Hauswirth W, Lewin A . Suppression of mouse rhodopsin expression in vivo by AAV mediated siRNA delivery. Vision Res. 2007; 47(9):1202-8. PMC: 1892214. DOI: 10.1016/j.visres.2006.11.026. View

16.

Han I, Burnight E, Ulferts M, Worthington K, Russell S, Sohn E . Helper-Dependent Adenovirus Transduces the Human and Rat Retina but Elicits an Inflammatory Reaction When Delivered Subretinally in Rats. Hum Gene Ther. 2019; 30(11):1371-1384. PMC: 6854516. DOI: 10.1089/hum.2019.159. View

17.

Stone E, Andorf J, Whitmore S, DeLuca A, Giacalone J, Streb L . Clinically Focused Molecular Investigation of 1000 Consecutive Families with Inherited Retinal Disease. Ophthalmology. 2017; 124(9):1314-1331. PMC: 5565704. DOI: 10.1016/j.ophtha.2017.04.008. View

18.

Love M, Huber W, Anders S . Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15(12):550. PMC: 4302049. DOI: 10.1186/s13059-014-0550-8. View

19.

Ripps H . Cell death in retinitis pigmentosa: gap junctions and the 'bystander' effect. Exp Eye Res. 2002; 74(3):327-36. DOI: 10.1006/exer.2002.1155. View

20.

Lewin A, Drenser K, Hauswirth W, Nishikawa S, Yasumura D, Flannery J . Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa. Nat Med. 1998; 4(8):967-71. DOI: 10.1038/nm0898-967. View