Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Jul 9.
Published in final edited form as: Adv Exp Med Biol. 2012;723:199–205. doi: 10.1007/978-1-4614-0631-0_27

Gene Delivery of Wild-Type Rhodopsin Rescues Retinal Function in an Autosomal Dominant Retinitis Pigmentosa Mouse Model

Haoyu Mao 1, Marina S Gorbatyuk 3, William W Hauswirth 1,2, Alfred S Lewin 1
PMCID: PMC3392039  NIHMSID: NIHMS377527  PMID: 22183334

Abstract

Autosomal dominant retinitis pigmentosa (ADRP) is frequently caused by mutations within the gene for the opsin of rod photoreceptor cells. Studies on transgenic mice, carrying mutated rhodopsin (RHO) transgene on different genetic backgrounds suggested that that an increased amount of wild-type RHO in ADRP photoreceptors attenuated the impact of the mutant transgene. Therefore, we employed a gene therapy approach with a help of Adeno-associated virus (AAV) to treat mice expressing a P23H mutant human RHO transgene. Knowing that AAV5 primarily transduces photoreceptor cells, we designed “hardened” form of the rhodopsin gene (RHO301) that expressed normal rhodopsin and was specifically resistant to degradation by the previously tested siRNA301. AAV5 RHO301 was subretinaly injected into the right eyes of P23H RHO mice at post-natal day 15. Animals were analyzed monthly by electroretinography (ERG) for 6 months. Analysis of the full field scotopic electroretinogram (ERG) demonstrated that increased expression of opsin slowed the rate of retinal degeneration in P23H mice with increased amplitudes in both a-wave and b-wave amplitudes compared to control eyes. An increase in the ERG amplitudes was correlated with improvement of retinal structure. The thickness of the outer nuclear layer in AAV-RHO301 injected eyes was increased by 80% compared to control eyes. This finding indicates that wild -type RHO could rescue the retinal degeneration in transgenic mice carrying a dominant RHO mutation and that increased production of normal rhodopsin could suppress the effect of the mutant protein. These findings suggest that wild-type RHO can used as a therapeutic agent to retard retinal degeneration in ADRP caused by different mutations of RHO via increased production of normal rhodopsin protein.

XX.1 Introduction

Retinitis pigmentosa (RP) is a neurodegenerative disease with the prevalence of 1/4000 and almost 1.5 million patients globally (Hartong et al. 2006; Daiger et al. 2007), with autosomal dominant retinitis pigmentosa (ADRP) accounting for 40% of clinical cases. The primary symptoms of ADRP are gradual loss of night vision followed by loss of peripheral vision, but central vision is diminished late in the disease (van Soest et al. 1999; Farrar et al. 2002). Currently there is no cure for the disease. Mutations in rhodopsin (RHO) are associated with over 25% of ADRP cases (Daiger et al. 2007). The first identified RHO mutation, P23H (proline 23 substituted by histidine), is associated with 12% ADRP patients in the U.S. (Dryja et al. 1990; van Soest et al. 1999; Daiger et al. 2007). Several P23H transgenic animal models, including mouse, rat, fly and frog models, have been developed and used to test gene and pharmacological therapies (Olsson et al. 1992; Roof et al. 1994; Bush et al. 2000; Organisciak et al. 2003; Ranchon et al. 2003; Galy et al. 2005; Gorbatyuk et al. 2005a; Tam and Moritz 2006; Gorbatyuk et al. 2007a; Gorbatyuk et al. 2008). Despite the genetic heterogeneity of ADRP, our group and others have explored a “resect and replace” gene therapy employing RNA interference (RNAi) or catalytic RNA enzymes (ribozymes) (Georgiadis et al. 2010; Lewin et al. 1998; Sullivan et al. 2002; Gorbatyuk et al. 2005b; Kiang et al. 2005; Gorbatyuk et al. . 2007b; Gorbatyuk et al. 2008). For thisallele -independent method, expression of both the mutant and wild-type gene is suppressed and a resistant allele is introduced (Sullivan et al. 2002; Cashman et al. 2005; Kiang et al. 2005; Tessitore et al. 2006; Gorbatyuk et al. 2007a, 2007b, Gorbatyuk et al. 2008). This approach should be applicable to ADRP genes, like RHO which are affected by many different mutations. In present studies, however, we introduced a single gene, RHO301, to express normal rhodopsin protein. The increased production of normal rhodopsin rescued photoreceptor function in P23H mice, suggesting that gene therapy with normal gene is possible for treatment of this class of ADRP mutation.

XX.2 Materials and Methods

XX.2.1 RHO301 Gene Cloning

The mouse RHO (Genbank, BC013125) cDNA we employed contained 109 bp 5’ UTR and 159 bp 3’ UTR. It also contained 5 silent mutations to eliminate siRNA301 recognition site. Expression was driven by a mouse opsin proximal promoter, and the insert was packaged in AAV serotype 5 capsids at a titer of 2×10 12vg/ml.

XX.2.2 Experiment with Animal Models

All animal procedures were approved by the University of Florida Institutional Animal Care and Use Committee in accordance with the ARVO statement for the “Use Animals in Ophthalmic and Vision Research”. P23H mice (line 37) containing a human P23H RHO transgene (Dryja et al. 1990) were bred with C57BL/6J wild-type mice (Jackson Laboratories) to obtain the transgenic P23H RHO, mouse RHO +/+ mice. Subretinal AAV injections with 1μl AAV5 RHO301 (2 X 109 viral particles) were performed as described by Timmers et al. (Dryja et al. 1990; Olsson et al. 1992; Timmers et al. 2001). Only right eyes were injected with virus.

XX.2.3 Electroretinography (ERG)

After subretinal injection, retinal function was measured at 1, 2, 3 and 6 months time points post injection. AAV5-RHO301 injected P23H mice were analyzed by simultaneous full-field ERG of a UTAS-E 2000 Visual Electrodiagnostic System (LKC). The a-wave amplitudes were measured from baseline to the peak in the cornea-negative direction, and b-wave amplitudes were measured from cornea-negative peak to major cornea-positive peak. The results from each group of mice were averaged and the means were compared statistically by using Student’s t-test for paired data and ANOVA for multiple groups.

XX.2.4 Histological Analysis for t he Outer Nuclear Layer

At 6 months post injection, the mice were euthanized, and the retinas were fixed and prepared for plastic sectioning after perfusion. Dissected tissues were post-fixed in 1% osmium tetroxide and maintained in 0.1M cacodylate buffer overnight. Tissues were embedded in an epoxy resin and stained with toluidine blue. The thickness of the outer nuclear layer was measured at 10 equally spaced superior and inferior loci using the MBF Stereo investigator on a Zeiss microscope. Mean readings were averaged from 10 measurements. Differences between the ONL thickness of left control eyes and that of right treated eyes were analyzed by using Student’s t-test for paired samples.

XX.3 Results

XX.3.1 Expression of AAV-delivered RHO301

The RHO301 was constructed by introducing 5 silent mismatches into a normal mouse RHO cDNA to eliminate the binding site of siRNA301 (Gorbatyuk et al. 2007b; Gorbatyuk et al. 2008). At postnatal day 15, RHO301 was delivered by subretinal injection. We analyzed mRNA and protein levels of RHO301 one month post subretinal injection. Compared to RHO mRNA levels in untreated left eyes, we obtained an almost 2-fold increase of total RHO mRNA in treated right eyes indicating successful RHO301 delivery (data not shown). Opsin protein was increased by 50% in right injected eyes as detected by immunoblot using mouse anti-rhodopsin monoclonal antibodies( 1D4, data not shown).

XX.3.2 Retinal Structure Integrity in AAV Gene Delivered P23H Mice

To analyze the structural integrity of retina, the outer nuclear layer (ONL) thickness was measured to determine the photoreceptors survival. In eyes expressing RHO301, ONL thickness was remarkably elevated compared to untreated eyes (Fig. XX.1A, 1B). 6- month survival of photoreceptors was approximately double in treated eyes when averaged over the entire retina. (Fig.XX.1C).

Fig. XX.1.

Fig. XX.1

Open in a new tab

The structural integrity of P23H retinas via RHO301 gene transfer. Six months after injection, the thickness of ONL was measured in (A) AAV-RHO301 injected P23H right retinas and compared with (B) untreated left retinas. The white caliper bar is 10μm. (C ) Significant increase of the ONL thickness was detected in the RHO301 injected (right) eyes of P23H mice (labeled as R RHO301)compared with the ONL of untreated (left) eyes ( labeled as L control) in the whole retina (p<0.05).

XX.3.3 Rescued retinal function observed in AAV-RHO301 injected P23H eyes

But were surviving rod cells functional? The full-field electroretinogram (ERG) was measured to assess retinal function in P23H transgenic mice. RHO301 gene transfer consistently improved both the a-wave and b-wave responses of P23H mice compared to untreated control eyes in a 6 month time-course with different intensities readings (Fig. XX.2A, 2B). At 6 months post injection, there was an almost 3-fold increase of a-wave amplitude in injected eyes compared with untreated eyes at the highest light intensity with 2.68 cd-s/m2 luminance (Fig. XX.2A). At same time point, 80% of the b-wave amplitude was maintained in AAV-RHO301 treated P23H eyes (Fig. XX.2B). Therefore, the retinal degeneration in P23H mice was dramatically slowed by delivery of functional RHO. These results are consistent with the preserved retinal structure in AAV-RHO301 injected P23H eyes.

Fig. XX.2.

Fig. XX.2

Open in a new tab

RHO301 gene delivery protects retinas of P23H mice. The scotopic ERG a- and b- wave amplitudes measured at 0.18 and 2.68 cd-s/m2 luminance, from 1 to 6 months following injection of RHO301 (gray circles) in right eyes. Untreated left eyes of transgenic mice (black diamonds) showed retinal degeneration with decreased amplitudes. (A) In ERG a-wave response, the amplitudes of right treated eyes were significantly increased compared with that of left untreated eyes at different time points (*p<0.05, shown as statistical significance). (B) At both 0.18 and 2.68 cd-s/m2 luminance, the b-wave amplitudes of right injected eyes significantly higher than those of left untreated eyes at all 4 different time points (*p <0.05, shown as statistical significance at 1, 2, 3 and 6 months time points).

XX.4 Discussion

In this study we used viral mediated gene transfer in P23H RHO transgenic mice model to test the hypothesis that supplementation of degenerating retina with functional RHO would reduce the rate of retinal degeneration in photoreceptors affected by a dominant rhodopsin mutation. Since we have already tested its cognate siRNA301 in vitro and in vivo , these experiments also validated the resistant cDNA, necessary for the replacement step of the “resect and replace” strategy. Our results not only confirmed those of Fredrick et al. that transgenic over expression of RHO could benefit mice carrying the P23H RHO gene (Fredrick et al. 2001), but also demonstrated that viral delivery of normal RHO can be beneficial to reduce the rate of retinal degeneration in ADRP photoreceptors.

Human clinical trials of gene therapy for a recessive retinal degeneration have shown promise in restoring vision (Bainbridge et al. 2008; Cideciyan et al. 2009; Simonelli et al. 2010)and suggest that gene transfer to the retina is safe. It is possible that gene transfer of RHO may be applicable to other Class II rhodopsin mutations that lead to retinitis pigmentosa by mechanisms similar to the P23H mutation (Mendes et al. 2005). Therefore, the increase of rhodopsin expression by wild-type gene delivery using AAV is a potential treatment for a large number of people with ADRP.

References

  1. Bainbridge JWB, Smith AJ, Barker SS, et al. Effect of gene therapy on visual function in Leber's congenital amaurosis. New England Journal of Medicine. 2008;358:2231–2239. doi: 10.1056/NEJMoa0802268. [DOI] [PubMed] [Google Scholar]
  2. Bush RA, Kononen L, Machida S, et al. The effect of calcium channel blocker diltiazem on photoreceptor degeneration in the rhodopsin Pro23His rat. Investigative Ophthalmology & Visual Science. 2000;41:2697–2701. [PubMed] [Google Scholar]
  3. Cashman SM, Binkley EA, Kumar-Singh R. Towards mutation-independent silencing of genes involved in retinal degeneration by RNA interference. Gene Therapy. 2005;12:1223–1228. doi: 10.1038/sj.gt.3302512. [DOI] [PubMed] [Google Scholar]
  4. Cideciyan AV, Hauswirth WW, Aleman TS, et al. Human RPE65 gene therapy for Leber congenital amaurosis: persistence of early visual improvements and safety at 1 year. Hum Gene Ther. 2009;20:999–1004. doi: 10.1089/hum.2009.086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Daiger SP, Bowne SJ, Sullivan LS. Perspective on genes and mutations causing retinitis pigmentosa. Arch Ophthalmol. 2007;125:151–158. doi: 10.1001/archopht.125.2.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dryja TP, McGee TL, Reichel E, et al. A point mutation of the rhodopsin gene in one form of retinitis pigmentosa. Nature. 1990;343:364–366. doi: 10.1038/343364a0. [DOI] [PubMed] [Google Scholar]
  7. Farrar GJ, Kenna PF, Humphries P. On the genetics of retinitis pigmentosa and on mutation-independent approaches to therapeutic intervention. EMBO J. 2002;21:857–864. doi: 10.1093/emboj/21.5.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Frederick JM, Krasnoperova NV, Hoffmann K, Church-Kopish J, Ruther K, Howes K, Lem J, Baehr W. Mutant rhodopsin transgene expression on a null background. Invest Ophthalmol Vis Sci. 2001;42:826–833. [PubMed] [Google Scholar]
  9. Galy A, Roux MJ, Sahel JA, et al. Rhodopsin maturation defects induce photoreceptor death by apoptosis: a fly model for Rhodopsin Pro23 His human retinitis pigmentosa. Hum Mol Genet. 2005;14:2547–2557. doi: 10.1093/hmg/ddi258. [DOI] [PubMed] [Google Scholar]
  10. Georgiadis A, Tschernutter M, Bainbridge JW, et al. AAV-mediated knockdown of peripherin-2 in vivo using miRNA-based hairpins. Gene Ther. 2010;17:486–493. doi: 10.1038/gt.2009.162. [DOI] [PubMed] [Google Scholar]
  11. Gorbatyuk M, Justilien V, Liu J, et al. Preservation of photoreceptor morphology P23H rats using an allele independent and function in ribozyme. Experimental Eye Research. 2007a;84:44–52. doi: 10.1016/j.exer.2006.08.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gorbatyuk M, Justilien V, Liu J, et al. Suppression of mouse rhodopsin expression in vivo by AAV mediated siRNA delivery. Vision Research. 2007b;47:1202–1208. doi: 10.1016/j.visres.2006.11.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gorbatyuk MS, Hauswirth WW, Lewin AS. Gene therapy for mouse models of ADRP. Adv Exp Med Biol. 2008;613:107–112. doi: 10.1007/978-0-387-74904-4_11. [DOI] [PubMed] [Google Scholar]
  14. Gorbatyuk MS, Timmers AMM, Pang JJ, et al. Rescue of vision in P23H rats with an rAAV delivered ribozyme targeting mouse opsin. Investigative Ophthalmology & Visual Science. 2005a;46 [Google Scholar]
  15. Gorbatyuk MS, Pang JJ, Thomas J, et al. Knockdown of wild-type mouse rhodopsin using an AAV vectored ribozyme as part of an RNA replacement approach. Molecular Vision. 2005b;11:648–656. [PubMed] [Google Scholar]
  16. Hartong DT, Berson EL, Dryja TP. Retinitis pigmentosa. Lancet. 2006;368:1795–1809. doi: 10.1016/S0140-6736(06)69740-7. [DOI] [PubMed] [Google Scholar]
  17. Kiang AS, Palfi A, Ader M, et al. Toward a gene therapy for dominant disease: validation of an RNA interference-based mutation-independent approach. Mol Ther. 2005;12:555–561. doi: 10.1016/j.ymthe.2005.03.028. [DOI] [PubMed] [Google Scholar]
  18. Lewin AS, Drenser KA, Hauswirth WW, et al. Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa. Nat Med. 1998;4:967–971. doi: 10.1038/nm0898-967. [DOI] [PubMed] [Google Scholar]
  19. Mendes HF, van der Spuy J, Chapple JP, et al. Mechanisms of cell death in rhodopsin retinitis pigmentosa: implications for therapy. Trends in Molecular Medicine. 2005;11:177–185. doi: 10.1016/j.molmed.2005.02.007. [DOI] [PubMed] [Google Scholar]
  20. Olsson JE, Gordon JW, Pawlyk BS, et al. Transgenic mice with a rhodopsin mutation (Pro23His): a mouse model of autosomal dominant retinitis pigmentosa. Neuron. 1992;9:815–830. doi: 10.1016/0896-6273(92)90236-7. [DOI] [PubMed] [Google Scholar]
  21. Organisciak DT, Darrow RM, Barsalou L, et al. Susceptibility to retinal light damage in transgenic rats with rhodopsin mutations. Invest Ophthalmol Vis Sci. 2003;44:486–492. doi: 10.1167/iovs.02-0708. [DOI] [PubMed] [Google Scholar]
  22. Ranchon I, LaVail MM, Kotake Y, et al. Free radical trap phenyl-N-tert-butylnitrone protects against light damage but does not rescue P23H and S334ter rhodopsin transgenic rats from inherited retinal degeneration. Journal of Neuroscience. 2003;23:6050–6057. doi: 10.1523/JNEUROSCI.23-14-06050.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Roof DJ, Adamian M, Hayes A. Rhodopsin accumulation at abnormal sites in retinas of mice with a human P23H rhodopsin transgene. Invest Ophthalmol Vis Sci. 1994;35:4049–4062. [PubMed] [Google Scholar]
  24. Simonelli F, Maguire AM, Testa F, et al. Gene Therapy for Leber's Congenital Amaurosis is Safe and Effective Through 1.5 Years After Vector Administration. Molecular Therapy. 2010;18:643–650. doi: 10.1038/mt.2009.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sullivan JM, Pietras KM, Shin BJ, et al. Hammerhead ribozymes designed to cleave all human rod opsin mRNAs which cause autosomal dominant retinitis pigmentosa. Mol Vis. 2002;8:102–113. [PubMed] [Google Scholar]
  26. Tam BM, Moritz OL. Characterization of rhodopsin P23H-induced retinal degeneration in a Xenopus laevis model of retinitis pigmentosa. Invest Ophthalmol Vis Sci. 2006;47:3234–3241. doi: 10.1167/iovs.06-0213. [DOI] [PubMed] [Google Scholar]
  27. Tessitore A, Parisi F, Denti MA, et al. Preferential silencing of a common dominant rhodopsin mutation does not inhibit retinal degeneration in a transgenic model. Molecular Therapy. 2006;14:692–699. doi: 10.1016/j.ymthe.2006.07.008. [DOI] [PubMed] [Google Scholar]
  28. Timmers A, Zhang H, Squitieri A, et al. Subretinal injections in rodent eyes: effects on electrophysiology and histology of rat retina. Molecular Vision. 2001;7:131–137. [PubMed] [Google Scholar]
  29. van Soest S, Westerveld A, de Jong PT, et al. Retinitis pigmentosa: defined from a molecular point of view. Surv Ophthalmol. 1999;43:321–334. doi: 10.1016/s0039-6257(98)00046-0. [DOI] [PubMed] [Google Scholar]

RESOURCES