Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2020 May 12.
Published in final edited form as: Adv Exp Med Biol. 2019;1185:113–118. doi: 10.1007/978-3-030-27378-1_19

Progress in Gene Therapy for Rhodopsin Autosomal Dominant Retinitis Pigmentosa

Raghavi Sudharsan 1, William A Beltran 1
PMCID: PMC7217593  NIHMSID: NIHMS1575652  PMID: 31884598

Abstract

This brief review summarizes the major proof-of-concept gene therapy studies for autosomal dominant retinitis pigmentosa (RP) caused by mutations in the rhodopsin gene (RHO-adRP) that have been conducted over the past 20 years in various animal models. We have listed in tabular form the various approaches, gene silencing reagents, gene delivery strategies, and salient results from these studies.

Keywords: Autosomal dominant retinitis pigmentosa, Rhodpsin, Gene therapy, Knockdown and replacement

19.1. Introduction

The P23H mutation in rhodopsin (RHO) was the first genetic mutation identified to be causally associated with retinitis pigmentosa (RP) (Dryja et al. 1990). Currently, over 150 unique mutations in RHO are known to cause ~40% of all autosomal dominant forms of RP (adRP), and RHO P23H accounts for ~10% of RHO-adRP among the US Caucasian population (https://sph.uth.edu/Retnet). Mutant RHO proteins cause disease via either a dominant negative or a toxic gain-of-function effect. While gene augmentation with a wild-type copy of RHO may be sufficient to dilute out the effects of a dominant negative mutant protein, a gene knockdown strategy is more likely to be beneficial for the toxic gain-of-function mutations. In the past 20 years, significant progress has been made in the field of gene therapy with very promising results in preclinical animal models. A number of transgenic rodent and pig models of RHO-adRP (listed in Table 19.1) have been used to evaluate gene knockdown, gene augmentation, gene editing, or combined gene knockdown and replacement strategies. The latter approach delivered within a single AAV vector was recently shown to successfully prevent the onset of photoreceptor degeneration in the RHO-T4R dog, the only currently available naturally occurring animal model of this disease (Cideciyan et al. 2018). Gene editing using CRISPR-Cas9 may be an elegant approach to specifically correct common RHO mutants such as P23H; however, due to the wide mutational heterogeneity in RHO, a mutation-independent strategy that combines knockdown with gene replacement could be an economically attractive therapy to target all forms of RHO-adRP. This review presents a tabular summary of all preclinical studies in this field, spanning 20 years, from 1998 to present.

Table 19.1.

Summary of all major gene therapy studies for treatment of RHO-adRP, grouped by therapeutic strategy

Allele specificity (target) Silencing and/or replacement reagents Delivery vector Animal model Salient results References
Treatment strategy: knockdown
Mutation dependent (mouse P23H) Ribozyme Hp11 AAV2-BOPS-Hp11 P23H-3 rat 15% KD of mutant RNA compared to control eye. 12% ONL loss at P60–90 vs. 40% in control eyes. Scotopic ERG b-wave 30% >control eye (Lewin et al. 1998)
Ribozyme Hh13 AAV2-BOPS-Hh13 11% KD of mutant RNA compared to control eye. 20% ONL loss at P60–90 vs. 40% in control eyes. Scotopic ERG b-wave 45% >control eye
Mutation dependent (mouse P23H) Ribozyme Hp11
Ribozyme Hh13 		AAV2-BOPS-Hp11;
AAV2-BOPS-Hh13 		P23H-3 rat Long-term (8 months) ONL and ERG rescue PI at P15 (before RD onset) 0.3-month ONL and ERG rescue PI at P60–P90 (40% PR loss) (LaVail et al. 2000)
Mutation dependent (mouse P23H) siRNA0, shRNA0 (shP23H) AAV2/5-U1-shP23H P23H-3 rat 68% KD (at 3–4 months)
61% KD (at 4–7 months) of mouse P23H RNA; ERG decline and no ONL rescue		 (Tessitore et al. 2006)
Mutation dependent (mouse P23H; mouse RHO) Antisense oligonucleotide: ASO2, ASO3 Intravitreal ASO injection WT RHO+/+mouse 50% (ASO3) −70% (ASO2) KD of mouse RHO (Murray et al. 2015)
P23H-1 rat 30% KD of mouse P23H RHO (ASO3); limited ERG rescue; ONL and OS rescue
Mutation independent (mouse, dog RHO) Ribozyme Rz397 AAV2-mOP-Rz397 RHO+/+ mouse 50% KD of RHO protein (compared to control eye); reduced ERG b-wave amplitude but no ONL or OS loss (Gorbatyuk et al. 2005)
RHO+/− mouse 80% KD of RHO protein (compared to control eye); reduced ERG b-wave amplitude and 30% ONL loss
Mutation independent (mouse, dog, human RHO) Ribozyme: Rz525 AAV2/5-mOP-Rz525 P23H-3 rat 46% KD of mouse P23H RNA; no change in protein levels; ONL rescue; ERG rescue but decline over time (Gorbatyuk et al. 2007a)
Mutation independent (mouse, dog, human RHO) shRNA: shRNA301 AAV2/5-H1-shRNA301 RHO+/+ mouse 49% KD of mouse RHO RNA (Gorbatyuk et al. 2007b)
RHO+/− mouse 30% KD of mouse RHO RNA; 60% KD of RHO protein; reduced ERG amplitudes and ONL loss
Mutation independent (human RHO) shRNA: shBB AAV2/5-H1-shBB NHR+/−
RHO−/− mouse		 90% KD of human RHO RNA in FACS sorted PRs (O’Reilly et al. 2007)
Mutation independent (human RHO) shRNA: shQ1 AAV2/5-H1-shQ1 NHR+/−
Rho−/− mouse		 95% KD of human RHO RNA in FACs sorted PRs; reduced ERG and loss of rod OS and RHO immunostaining (Chadderton et al. 2009)
hP347S+/−
RHO+/− 		Improved ONL thickness and ERG up to 10 weeks PI but not stable: loss of ONL thickness between 5 and 10 weeks PI
Mutation independent (human RHO CRE) Zinc finger artificial transcription factors: ZF-R2; ZF-R6 AAV2/8-RKp-ZF-R6 hP347S+/−
Rho+/+ mouse		 26% KD of hP347S RHO RNA in Tx area; partial ERG and ONL rescue (Mussolino et al. 2011)
Mutation independent (human and pig RHO CRE) Zinc finger DNA-binding domain: ZF6-DB AAV2/8-CMV-ZF6 RHO+/+ pig 45% KD of WT pig RHO at 15 days PI, collapse of OS (Botta et al. 2016)
hP347S+/−
Rho+/+ mouse		 ERG rescue at P30 (injection at P14)
Mutation independent (dog, human RHO) shRNA: shRNA820 scAAV2/5-H1-shRNA820 RHO+/+ dog 8 weeks PI: RHO RNA 0–3%, RHO protein 15% of control at highest safe viral dose. Shortening of OS, loss of immunolabeling (Cideciyan et al. 2018)
Light sensitive RHOT4R/+ dog 6–8 weeks PI: RHO RNA and protein levels, structural changes, similar to seen in treated RHO+/+. ONL preservation in treated area after 8–10 weeks PI, 2 weeks after light exposure
Treatment strategy: replacement
Mutation independent RHO-M (resistant human RHO) Tg RHO-M mouse RHO-M+/−
RHO−/− mouse		 Rescue of rod ONL and ERG loss (O’Reilly et al. 2007)
Single copy of resistant human RHO transgene rescues ONL, OS, and ERG loss; leads to RHO RNA expression (∼ 75% of RHO+/+) and expression of RHO in OS (O’Reilly et al. 2008)
Mutation independent Various RHO-BB (resistant human RHO) AAV-mOP RHO-BB24 Rho−/− mouse ONL rescue + OS formation; rescue of rod ERG but decline from 6 to 12 weeks of age (Palfi et al. 2010)
Treatment strategy: augmentation
Mutation independent RHO301 (mouse RHO resistant to shRNA301) AAV2/5-mOP-RHO301 hP23H+/−
RHO+/+ mouse		 Twofold increase in total RHO RNA and 58% increase in RHO monomer protein; ERG and ONL rescue up to 6-month PI (at Pl5) (Mao et al. 2011)
Mutation independent RHO-BB (human RHO resistant to shBB) AV2/8–1.7 RHOp-RHO-BB; AAV2/rh10–1.7 RHOp-RHO-BB RHO−/− mouse 75% of RHO RNA levels as in NHR+/− Rho−/−; ONL rescue; rod expression in OS, formation of OS, ERG rescue, visual acuity rescue (Palfi et al. 2015)
Treatment strategy: knockdown and replacement
Mutation independent (mouse RHO) shRNA: shMR3
siRNA: siMR3
Resistant RHO: MR7 		shMR3 and resistant RHO MR7 (as plasmids) WT mouse (liver) shMR3 + mouse RHO, 90% KD (in liver); shMR3 + MR7, 0% KD (Kiang et al. 2005)
Mutation independent (human RHO) shRNA: shBB
Resistant RHO: rBB 		AAV2/5-H1-shBB-mOP-rBB hP23H+/−
Rho+/− mouse		 ONL: 33% thicker than control eye at P10 (O’Reilly et al. 2007)
shRNA: shQ1
Resistant RHO: rQ1 		AAV2/5-H1-shQ1-mOP-rQ1 WT mouse (liver) ONL: 33% thicker than control eye at P10
Mutation independent (mouse, dog, human RHO) shRNA: shRNA301
Resistant mouse RHO: RHO301 		AAV2/5-H1-shRNA301-mOP-RHO301 hP23H+/−
RHO+/− mouse		 74% KD of endogenous (human P23H and mouse RHO) RNA; 2X increase in total RHO RNA (compared to control eye); 2X increase in RHO protein (compared to control eye); long-term (9 months) ERG, and ONL and OS rescue (Mao et al. 2012)
Mutation independent ZF6 and hRHO AAV2/8-RHOΔ-ZF6-GNAT1-hRHO-WPRE RHO+/+ pig 38% KD of pig RHO; replacement with hRHO protein; OS structure better preserved than with ZF6 alone (Botta et al. 2016)
Mutation independent (dog, human RHO) shRNA: shRNA820
Resistant human RHO: human RHO820 		scAAV2/5-hOP-RHO820-H1-shRNA820 Light-sensitive RHOT4R/+ dog; complete ONL degeneration in 2 weeks post light exposure. 9 weeks PI: Dog RHO RNA 15% of untreated control eye; human RHO RNA 5–9% of canine RHO in untreated control eyes. Total RHO protein: 18% compared to untreated area
13 weeks PI: Dog RHO RNA 1–2% of untreated control eye; human RHO RNA 118–132% of canine RHO in untreated control eyes. 32% compared to untreated area
Preservation of ONL, OS, and ERG in the treated area even after repeated light exposure (light exposure at 11, 15, 25, and 37 weeks PI; retinal assessment at 13, 17, 27, and 37 weeks)		 (Cideciyan et al. 2018)
Treatment strategy: CRISPR-Cas9 gene editing
Mutation dependent (mouse RHO, S334 locus) spCas9/sgRNA sgRNA-spCAs9 plasmid S334ter-3 rat Cleavage efficiency: 33–36%; ONL rescue (8 rows vs 1 in Ctrl); OS formation, improved optokinetic response, no ERG rescue (Bakondi et al. 2016)
Mutation independent (human RHO) hSpCas9/sgRNA1, sgRNA3, or 2 sgRNAs CRISPR-Cas9-2sgRNA plasmid hP23H+/−
RHO−/− (very fast RD) mouse		 Editing efficiency, 4–33% in transfected rods; KD of RHO protein, 56–77% in transfected rods; no structural or functional rescue shown (Latella et al. 2016)
Mutation dependent (human P23H) saCas9/sgH23 AAV2/5-sgH23–2-saCas9 hP23H Tg pig NHEJ editing in 2 out of 5 pigs but low efficiency (3.4–4.4% alleles showed NHEJ) (Burnight et al. 2017)
Open in a new tab

KD knockdown, PR photoreceptors, Tg transgenic, ONL outer nuclear layer, OS outer segment, PI postinjection, CRE cis-regulatory element, WPRE woodchuck hepatitis virus posttranscriptional regulatory element. Promoters listed: BOPS bovine opsin promoter, mOP mouse proximal opsin promoter, U1 human U1 small nuclear RNA promoter, H1 human H1 RNA polymerase III promoter, GNAT1 human guanine nucleotide-binding protein 1 promoter, CMV cyto-megalovirus promoter, RKp human rhodopsin kinase promoter, 1.7 RHOp 1.7 kb mouse rhodopsin promoter

References

  1. Bakondi B, Lv W, Lu B et al. (2016) In vivo CRISPR/Cas9 gene editing corrects retinal dystrophy in the S334ter-3 rat model of autosomal dominant retinitis pigmentosa. Mol Ther 24:556–563 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Botta S, Marrocco E, de Prisco N et al. (2016) Rhodopsin targeted transcriptional silencing by DNA-binding. elife 5:e12242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burnight ER, Gupta M, Wiley LA et al. (2017) Using CRISPR-Cas9 to generate gene-corrected autologous iPSCs for the treatment of inherited retinal degeneration. Mol Ther 25:1999–2013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chadderton N, Millington-Ward S, Palfi A et al. (2009) Improved retinal function in a mouse model of dominant retinitis pigmentosa following AAV-delivered gene therapy. Mol Ther 17:593–599 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cideciyan AV, Sudharsan R, Dufour VL et al. (2018) Mutation-independent rhodopsin gene therapy by knockdown and replacement with a single AAV vector. Proc Natl Acad Sci U S A 115:E8547–E8556 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dryja TP, McGee TL, Reichel E et al. (1990) A point mutation of the rhodopsin gene in one form of retinitis pigmentosa. Nature 343:364–366 [DOI] [PubMed] [Google Scholar]
  7. Gorbatyuk M, Justilien V, Liu J et al. (2007a) Preservation of photoreceptor morphology and function in P23H rats using an allele independent ribozyme. Exp Eye Res 84:44–52 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gorbatyuk M, Justilien V, Liu J et al. (2007b) Suppression of mouse rhodopsin expression in vivo by AAV mediated siRNA delivery. Vis Res 47:1202–1208 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gorbatyuk MS, Pang JJ, Thomas J Jr et al. (2005) Knockdown of wild-type mouse rhodopsin using an AAV vectored ribozyme as part of an RNA replacement approach. Mol Vis 11:648–656 [PubMed] [Google Scholar]
  10. Kiang AS, Palfi A, Ader M et al. (2005) Toward a gene therapy for dominant disease: validation of an RNA interference-based mutation-independent approach. Mol Ther 12:555–561 [DOI] [PubMed] [Google Scholar]
  11. Latella MC, Di Salvo MT, Cocchiarella F et al. (2016) In vivo editing of the human mutant rhodopsin gene by electroporation of plasmid-based CRISPR/Cas9 in the mouse retina. Mol Ther Nucleic Acids 5:e389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. LaVail MM, Yasumura D, Matthes MT et al. (2000) Ribozyme rescue of photoreceptor cells in P23H transgenic rats: long-term survival and late-stage therapy. Proc Natl Acad Sci U S A 97:11488–11493 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lewin AS, Drenser KA, Hauswirth WW et al. (1998) Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa. Nat Med 4:967–971 [DOI] [PubMed] [Google Scholar]
  14. Mao H, Gorbatyuk MS, Rossmiller B et al. (2012) Long-term rescue of retinal structure and function by rhodopsin RNA replacement with a single adeno-associated viral vector in P23H RHO transgenic mice. Hum Gene Ther 23:356–366 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mao H, James T Jr, Schwein A et al. (2011) AAV delivery of wild-type rhodopsin preserves retinal function in a mouse model of autosomal dominant retinitis pigmentosa. Hum Gene Ther 22:567–575 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Murray SF, Jazayeri A, Matthes MT et al. (2015) Allele-specific inhibition of rhodopsin with an antisense oligonucleotide slows photoreceptor cell degeneration. Invest Ophthalmol Vis Sci 56:6362–6375 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mussolino C, Sanges D, Marrocco E et al. (2011) Zinc-finger-based transcriptional repression of rhodopsin in a model of dominant retinitis pigmentosa. EMBO Mol Med 3:118–128 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. O’Reilly M, Millington-Ward S, Palfi A et al. (2008) A transgenic mouse model for gene therapy of rhodopsin-linked retinitis pigmentosa. Vis Res 48:386–391 [DOI] [PubMed] [Google Scholar]
  19. O’Reilly M, Palfi A, Chadderton N et al. (2007) RNA interference-mediated suppression and replacement of human rhodopsin in vivo. Am J Hum Genet 81:127–135 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Palfi A, Chadderton N, O’Reilly M et al. (2015) Efficient gene delivery to photoreceptors using AAV2/rh10 and rescue of the Rho(−/−) mouse. Mol Ther Methods Clin Dev 2:15016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Palfi A, Millington-Ward S, Chadderton N et al. (2010) Adeno-associated virus-mediated rhodopsin replacement provides therapeutic benefit in mice with a targeted disruption of the rhodopsin gene. Hum Gene Ther 21:311–323 [DOI] [PubMed] [Google Scholar]
  22. Tessitore A, Parisi F, Denti MA et al. (2006) Preferential silencing of a common dominant rhodopsin mutation does not inhibit retinal degeneration in a transgenic model. Mol Ther 14:692–699 [DOI] [PubMed] [Google Scholar]

RESOURCES