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Outer retinal transduction by AAV2-7m8 following intravitreal injection in a sheep model of CNGA3 achromatopsia

Abstract

Sheep carrying a mutated CNGA3 gene exhibit diminished cone function and provide a naturally occurring large animal model of achromatopsia. Subretinal injection of a vector carrying the CNGA3 transgene resulted in long-term recovery of cone function and photopic vision in these sheep. Research is underway to develop efficacious vectors that would enable safer transgene delivery, while avoiding potential drawbacks of subretinal injections. The current study evaluated two modified vectors, adeno-associated virus 2-7m8 (AAV2-7m8) and AAV9-7m8. Intravitreal injection of AAV2-7m8 carrying enhanced green fluorescent protein under a cone-specific promoter resulted in moderate photoreceptor transduction in wild-type sheep, whereas peripheral subretinal delivery of AAV9-7m8 resulted in the radial spread of the vector beyond the point of deposition. Intravitreal injection of AAV2-7m8 carrying human CNGA3 in mutant sheep resulted in mild photoreceptor transduction, but did not lead to the clinical rescue of photopic vision, while day-blind sheep treated with a subretinal injection exhibited functional recovery of photopic vision. Transgene messenger RNA levels in retinas of intravitreally treated eyes amounted to 4–23% of the endogenous CNGA3 levels, indicating that expression levels >23% are needed to achieve clinical rescue. Overall, our results indicate intravitreal injections of AAV2.7m8 transduce ovine photoreceptors, but not with sufficient efficacy to achieve clinical rescue in CNGA3 mutant sheep.

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Fig. 1: eGFP expression in sheep retinas evaluated in vivo by fluorescent fundus photography and postmortem by immunohistochemistry (IHC).
Fig. 2: Composite fundus photography and fluorescent fundus photography showing the radial spread of AAV9-7m8-PR.1.7-eGFP injected SR in two peripheral locations.
Fig. 3: Electroretinography and behavioral maze test results of CNGA3 mutant sheep injected IVT and SR with AAV2-7m8-PR.1.7-hCNGA3.
Fig. 4: Immunohistochemistry of a representative CNGA3 mutant sheep retina (8730) injected IVT with AAV2-7m8-PR.1.7-hCNGA3.
Fig. 5: Relative quantification of hCNGA3 transgene expression following SR and IVT injections of AAV2-7m8-PR.1.7-hCNGA3.

References

  1. Zobor D, Zobor G, Kohl S. Achromatopsia: on the doorstep of a possible therapy. Ophthalmic Res. 2015;54:103–8.

    PubMed  Google Scholar 

  2. Zelinger L, Cideciyan AV, Kohl S, Schwartz SB, Rosenmann A, Eli D, et al. Genetics and disease expression in the CNGA3 form of achromatopsia: steps on the path to gene therapy. Ophthalmology. 2015;122:997–1007.

    PubMed  Google Scholar 

  3. Sun W, Li S, Xiao X, Wang P, Zhang Q. Genotypes and phenotypes of genes associated with achromatopsia: a reference for clinical genetic testing. Mol Vis. 2020;26:588–602.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Ramlogan-Steel CA, Murali A, Andrzejewski S, Dhungel B, Steel JC, Layton CJ. Gene therapy and the adeno-associated virus in the treatment of genetic and acquired ophthalmic diseases in humans: trials, future directions and safety considerations. Clin Exp Ophthalmol. 2019;47:521–36.

    PubMed  Google Scholar 

  5. Ye GJ, Komáromy AM, Zeiss C, Calcedo R, Harman CD, Koehl KL, et al. Safety and efficacy of AAV5 vectors expressing human or canine CNGB3 in CNGB3-mutant dogs. Hum Gene Ther Clin Dev. 2017;28:197–207.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Komáromy AM, Alexander JJ, Rowlan JS, Garcia MM, Chiodo VA, Kaya A, et al. Gene therapy rescues cone function in congenital achromatopsia. Hum Mol Genet. 2010;19:2581–93.

    PubMed  PubMed Central  Google Scholar 

  7. Carvalho LS, Xu J, Pearson RA, Smith AJ, Bainbridge JW, Morris LM, et al. Long-term and age-dependent restoration of visual function in a mouse model of CNGB3-associated achromatopsia following gene therapy. Hum Mol Genet. 2011;20:3161–75.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Pang JJ, Deng WT, Dai X, Lei B, Everhart D, Umino Y, et al. AAV-mediated cone rescue in a naturally occurring mouse model of CNGA3-achromatopsia. PLoS ONE. 2012;7:e35250.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Gootwine E, Ofri R, Banin E, Obolensky A, Averbukh E, Ezra-Elia R, et al. Safety and efficacy evaluation of rAAV2tYF-PR1.7-hCNGA3 vector delivered by subretinal injection in CNGA3 mutant achromatopsia sheep. Hum Gene Ther Clin Dev. 2017;28:96–107.

  10. Shamir MH, Ofri R, Bor A, Brenner O, Reicher S, Obolensky A, et al. A novel day blindness in sheep: epidemiological, behavioural, electrophysiological and histopathological studies. Vet J. 2010;185:130–7.

    PubMed  Google Scholar 

  11. Reicher S, Seroussi E, Gootwine E. A mutation in gene CNGA3 is associated with day blindness in sheep. Genomics. 2010;95:101–4.

    CAS  PubMed  Google Scholar 

  12. Ezra-Elia R, Banin E, Honig H, Rosov A, Obolensky A, Averbukh E, et al. Flicker cone function in normal and day blind sheep: a large animal model for human achromatopsia caused by CNGA3 mutation. Doc Ophthalmol. 2014;129:141–50.

    PubMed  PubMed Central  Google Scholar 

  13. Ross M, Ofri R, Aizenberg I, Abu-Siam M, Pe’er O, Arad D, et al. Naturally-occurring myopia and loss of cone function in a sheep model of achromatopsia. Sci Rep. 2020;10:19314.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Banin E, Gootwine E, Obolensky A, Ezra-Elia R, Ejzenberg A, Zelinger L, et al. Gene augmentation therapy restores retinal function and visual behavior in a sheep model of CNGA3 achromatopsia. Mol Ther. 2015;23:1423–33.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Gootwine E, Abu-Siam M, Obolensky A, Rosov A, Honig H, Nitzan T, et al. Gene augmentation therapy for a missense substitution in the cGMP-binding domain of ovine CNGA3 gene restores vision in day-blind sheep. Invest Ophthalmol Vis Sci. 2017;58:1577–84.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Ofri R, Averbukh E, Ezra-Elia R, Ross M, Honig H, Obolensky A, et al. Six years and counting: restoration of photopic retinal function and visual behavior following gene augmentation therapy in a sheep model of CNGA3 achromatopsia. Hum Gene Ther. 2018;29:1376–86.

  17. Ochakovski GA, Bartz-Schmidt KU, Fischer MD. Retinal gene therapy: surgical vector delivery in the translation to clinical trials. Front Neurosci. 2017;11:174.

    PubMed  PubMed Central  Google Scholar 

  18. Li Q, Miller R, Han P-Y, Pang J, Dinculescu A, Chiodo V, et al. Intraocular route of AAV2 vector administration defines humoral immune response and therapeutic potential. Mol Vis. 2008;14:1760.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Kotterman MA, Yin L, Strazzeri JM, Flannery JG, Merigan WH, Schaffer DV. Antibody neutralization poses a barrier to intravitreal adeno-associated viral vector gene delivery to non-human primates. Gene Ther. 2015;22:116–26.

    CAS  PubMed  Google Scholar 

  20. Willett KL, Bennett J. Immunology of AAV-mediated gene transfer in the eye. Front Immunol. 2013;4:261.

    PubMed  PubMed Central  Google Scholar 

  21. Bainbridge JW, Smith AJ, Barker SS, Robbie S, Henderson R, Balaggan K, et al. Effect of gene therapy on visual function in Leber’s congenital amaurosis. N Engl J Med. 2008;358:2231–9.

    CAS  PubMed  Google Scholar 

  22. Bainbridge JW, Mehat MS, Sundaram V, Robbie SJ, Barker SE, Ripamonti C, et al. Long-term effect of gene therapy on Leber’s congenital amaurosis. N Engl J Med. 2015;372:1887–97.

    PubMed  PubMed Central  Google Scholar 

  23. Le Meur G, Lebranchu P, Billaud F, Adjali O, Schmitt S, Bézieau S, et al. Safety and long-term efficacy of AAV4 gene therapy in patients with RPE65 leber congenital amaurosis. Mol Ther. 2018;26:256–68.

    PubMed  Google Scholar 

  24. Hauswirth WW, Aleman TS, Kaushal S, Cideciyan AV, Schwartz SB, Wang L, et al. 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;19:979–90.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Dalkara D, Sahel JA. Gene therapy for inherited retinal degenerations. C R Biol. 2014;337:185–92.

    PubMed  Google Scholar 

  26. Ross M, Ofri R. The future of retinal gene therapy: evolving from subretinal to intravitreal vector delivery. Neural Regen Res. 2021;16:1751–9.

    PubMed  PubMed Central  Google Scholar 

  27. Gamlin PD, Alexander JJ, Boye SL, Witherspoon CD, Boye SE. SubILM injection of AAV for gene delivery to the retina. Methods Mol Biol. 2019;1950:249–62.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Zeng Y, Boyd R, Bartoe J, Wiley HE, Marangoni D, Wei LL, et al. “Para-retinal” vector administration into the deep vitreous enhances retinal transgene expression. Mol Ther Methods Clin Dev. 2020;18:422–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Comander J, Carvalho L, Wassmer S, Xiao R, Plovie E, Langsdorf A, et al. 29. Novel surgical method for intravitreal AAV administration overcomes transduction barriers in non-human primates. Mol Ther. 2016;24:S13–S4.

    Google Scholar 

  30. Byrne LC, Day TP, Visel M, Strazzeri JA, Fortuny C, Dalkara D, et al. In vivo-directed evolution of adeno-associated virus in the primate retina. JCI Insight. 2020;5:e135112.

  31. Dalkara D, Byrne LC, Klimczak RR, Visel M, Yin L, Merigan WH, et al. In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous. Sci Transl Med. 2013;5:189ra76.

    PubMed  Google Scholar 

  32. Cronin T, Vandenberghe LH, Hantz P, Juttner J, Reimann A, Kacsó ÁE, et al. Efficient transduction and optogenetic stimulation of retinal bipolar cells by a synthetic adeno‐associated virus capsid and promoter. EMBO Mol Med. 2014;6:1175–90.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Gruntman AM, Flotte TR. The rapidly evolving state of gene therapy. FASEB J. 2018;32:1733–40.

    CAS  PubMed  Google Scholar 

  34. Hickey DG, Edwards TL, Barnard AR, Singh MS, de Silva SR, McClements ME, et al. Tropism of engineered and evolved recombinant AAV serotypes in the rd1 mouse and ex vivo primate retina. Gene Ther. 2017;24:787–800.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Lee EJ, Guenther CM, Suh J. Adeno-associated virus (AAV) vectors: rational design strategies for capsid engineering. Curr Opin Biomed Eng. 2018;7:58–63.

    PubMed  PubMed Central  Google Scholar 

  36. Khabou H, Desrosiers M, Winckler C, Fouquet S, Auregan G, Bemelmans AP, et al. Insight into the mechanisms of enhanced retinal transduction by the engineered AAV2 capsid variant -7m8. Biotechnol Bioeng. 2016;113:2712–24.

    CAS  PubMed  Google Scholar 

  37. Bennett A, Keravala A, Makal V, Kurian J, Belbellaa B, Aeran R, et al. Structure comparison of the chimeric AAV2.7m8 vector with parental AAV2. J Struct Biol. 2020;209:107433.

    CAS  PubMed  Google Scholar 

  38. Kleine Holthaus SM, Ribeiro J, Abelleira-Hervas L, Pearson RA, Duran Y, Georgiadis A, et al. Prevention of photoreceptor cell loss in a Cln6. Mol Ther. 2018;26:1343–53.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Byrne LC, Oztürk BE, Lee T, Fortuny C, Visel M, Dalkara D, et al. Retinoschisin gene therapy in photoreceptors, Müller glia or all retinal cells in the Rs1h−/− mouse. Gene Ther. 2014;21:585–92.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Vandenberghe LH, Bell P, Maguire AM, Xiao R, Hopkins TB, Grant R, et al. AAV9 targets cone photoreceptors in the nonhuman primate retina. PLoS ONE. 2013;8:e53463.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Shen S, Berry GE, Rivera RMC, Cheung RY, Troupes AN, Brown SM, et al. Functional analysis of the putative integrin recognition motif on adeno-associated virus 9. J Biol Chem. 2015;290:1496–504.

    PubMed  Google Scholar 

  42. Khabou H, Garita-Hernandez M, Chaffiol A, Reichman S, Jaillard C, Brazhnikova E, et al. Noninvasive gene delivery to foveal cones for vision restoration. JCI Insight. 2018;3:e96029.

  43. Khabou H, Cordeau C, Pacot L, Fisson S, Dalkara D. Dosage thresholds and influence of transgene cassette in adeno-associated virus-related toxicity. Hum Gene Ther. 2018;29:1235–41.

    CAS  PubMed  Google Scholar 

  44. Ye GJ, Budzynski E, Sonnentag P, Nork TM, Sheibani N, Gurel Z, et al. Cone-specific promoters for gene therapy of achromatopsia and other retinal diseases. Hum Gene Ther. 2016;27:72–82.

    CAS  PubMed  Google Scholar 

  45. Aurnhammer C, Haase M, Muether N, Hausl M, Rauschhuber C, Huber I, et al. Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Hum Gene Ther Part B. 2012;23:18–28.

    CAS  Google Scholar 

  46. Desrosiers M, Dalkara D. Neutralizing antibodies against adeno-associated virus (AAV): measurement and influence on retinal gene delivery. Methods Mol Biol. 2018;1715:225–38.

    CAS  PubMed  Google Scholar 

  47. Ross M, Obolensky A, Averbukh E, Ezra-Elia R, Yamin E, Honig H, et al. Evaluation of photoreceptor transduction efficacy of capsid-modified adeno-associated viral vectors following intravitreal and subretinal delivery in sheep. Hum Gene Ther. 2020;31:719–29.

  48. Ross M, Honig H, Ezra-Elia R, Banin E, Obolensky A, Averbukh E, et al. Consecutive unilateral recording of the two eyes affects dark-adapted ERG responses, when compared to simultaneous bilateral recording. Doc Ophthalmol. 2018;137:183–92.

    PubMed  Google Scholar 

  49. Winkler PA, Occelli LM, Petersen-Jones SM. Large animal models of inherited retinal degenerations: a review. Cells. 2020;9:882.

  50. Macé E, Caplette R, Marre O, Sengupta A, Chaffiol A, Barbe P, et al. Targeting channelrhodopsin-2 to ON-bipolar cells with vitreally administered AAV restores ON and OFF visual responses in blind mice. Mol Ther. 2015;23:7–16.

    PubMed  Google Scholar 

  51. Ramachandran PS, Lee V, Wei Z, Song JY, Casal G, Cronin T, et al. Evaluation of dose and safety of AAV7m8 and AAV8BP2 in the non-human primate retina. Hum Gene Ther. 2017;28:154–67.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Gauvain G, Akolkar H, Chaffiol A, Arcizet F, Khoei MA, Desrosiers M, et al. Optogenetic therapy: high spatiotemporal resolution and pattern discrimination compatible with vision restoration in non-human primates. Commun Biol. 2021;4:125.

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Dose-escalation Study to Evaluate the Safety and Tolerability of GS030 in Subjects With Retinitis Pigmentosa (PIONEER) ClinicalTrials.gov identifier: NCT03326336. 2021. http://clinicaltrials.gov/. Accessed November 25, 2021.

  54. Sahel JA, Boulanger-Scemama E, Pagot C, Arleo A, Galluppi F, Martel JN, et al. Partial recovery of visual function in a blind patient after optogenetic therapy. Nat Med. 2021;27:1223–9.

  55. Peynshaert K, Devoldere J, Minnaert AK, De Smedt SC, Remaut K. Morphology and composition of the inner limiting membrane: species-specific variations and relevance toward drug delivery research. Curr Eye Res. 2019;44:465–75.

    CAS  PubMed  Google Scholar 

  56. Slijkerman RW, Song F, Astuti GD, Huynen MA, van Wijk E, Stieger K, et al. The pros and cons of vertebrate animal models for functional and therapeutic research on inherited retinal dystrophies. Prog Retin Eye Res. 2015;48:137–59.

    PubMed  Google Scholar 

  57. Mowat FM, Gornik KR, Dinculescu A, Boye SL, Hauswirth WW, Petersen-Jones SM, et al. Tyrosine capsid-mutant AAV vectors for gene delivery to the canine retina from a subretinal or intravitreal approach. Gene Ther. 2014;21:96–105.

    CAS  PubMed  Google Scholar 

  58. Petrs-Silva H, Dinculescu A, Li Q, Deng WT, Pang JJ, Min SH, et al. Novel properties of tyrosine-mutant AAV2 vectors in the mouse retina. Mol Ther. 2011;19:293–301.

    CAS  PubMed  Google Scholar 

  59. Reid CA, Ertel KJ, Lipinski DM. Improvement of photoreceptor targeting via intravitreal delivery in mouse and human retina using combinatory rAAV2 capsid mutant vectors. Invest Ophthalmol Vis Sci. 2017;58:6429–39.

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Penaud-Budloo M, Le Guiner C, Nowrouzi A, Toromanoff A, Chérel Y, Chenuaud P, et al. Adeno-associated virus vector genomes persist as episomal chromatin in primate muscle. J Virol. 2008;82:7875–85.

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Zhong G, Wang H, He W, Li Y, Mou H, Tickner ZJ, et al. A reversible RNA on-switch that controls gene expression of AAV-delivered therapeutics in vivo. Nat Biotechnol. 2020;38:169–75.

    CAS  PubMed  Google Scholar 

  62. Le Guiner C, Stieger K, Toromanoff A, Guilbaud M, Mendes-Madeira A, Devaux M, et al. Transgene regulation using the tetracycline-inducible TetR-KRAB system after AAV-mediated gene transfer in rodents and nonhuman primates. PLoS ONE. 2014;9:e102538.

    PubMed  PubMed Central  Google Scholar 

  63. Broeders M, Herrero-Hernandez P, Ernst MPT, van der Ploeg AT, Pijnappel WWMP. Sharpening the molecular scissors: advances in gene-editing technology. iScience. 2020;23:100789.

    CAS  PubMed  Google Scholar 

  64. Baliou S, Adamaki M, Kyriakopoulos AM, Spandidos DA, Panayiotidis M, Christodoulou I, et al. Role of the CRISPR system in controlling gene transcription and monitoring cell fate (Review). Mol Med Rep. 2018;17:1421–7.

    CAS  PubMed  Google Scholar 

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Funding

This study was funded by grants from the Israel Science Foundation (1257/15) and the Chief Scientist Office, Ministry of Health (3-15068), awarded to RO, and ERC Starting Grants (REGENETHER 639888/D.D.), the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), Sorbonne Université, the Agence Nationale pour la Recherche—Recherche Hospitalo-Universitaire en santé (RHU; Light4Deaf), LabEx LIFESENSES (ANR-10-LABX-65), *IHU FOReSIGHT (ANR-18-IAHU-01), awarded to DD, and from Research to Prevent Blindness (USA) awarded to EB.

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MR and RO wrote the manuscript, which was reviewed by all authors; MR and AO conducted the IHC studies; EA, EB, HH, and EY operated on the animals; MD and DD designed and produced the vectors, and developed the serological tests; MR and RE-E conducted the ERG recordings; RM conducted the PCR and serology studies; EG, HD, and AR were responsible for breeding and maintaining the experimental animals and conducting the maze tests; RO, DD, EB, and EG wrote the grants that funded this study.

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Correspondence to R. Ofri.

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DD is co-inventor on patent #9193956 (Adeno-associated virus virions with variant capsid and methods of use thereof), with royalties paid to Adverum Biotechnologies and on pending patent applications on noninvasive methods to target cone photoreceptors (EP17306429.6 and EP17306430.4) licensed to Gamut Tx. DD is a founder and acting CSO of Gamut Tx. The other authors declare no competing interests.

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Ross, M., Obolensky, A., Averbukh, E. et al. Outer retinal transduction by AAV2-7m8 following intravitreal injection in a sheep model of CNGA3 achromatopsia. Gene Ther (2021). https://doi.org/10.1038/s41434-021-00306-1

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