Adeno-Associated Viral Gene Therapy for Retinal Disorders

  • Samantha R. de Silva
  • Michelle E. McClements
  • Mark W. Hankins
  • Robert E. MacLarenEmail author
Part of the Neuromethods book series (NM, volume 98)


Gene therapy is ideally suited to the treatment of inherited retinal degenerations in order to prevent blindness. The target area of the outer retina is small and relatively immune privileged, which facilitates the delivery of small volumes of vector without generating significant immune reactions. Moreover, most of the currently untreatable forms of blindness have a genetic component, either monogenic such as in retinitis pigmentosa or as a result of several genes interacting along a common pathway, such as age-related macular degeneration, the commonest cause of legal blindness in the developed world. The self-contained nature of the eye also facilitates the application of gene therapy for sustained expression of intraocular proteins, such as inhibitors of angiogenesis or growth factors that might confer neuroprotection to dying cells. In this chapter, we review the indications and applications of gene therapy, concentrating specifically on adeno-associated viral (AAV) vectors, describing the protocols for production and administration of AAV vectors to the eye in the laboratory setting.

Key words

Gene therapy Adeno-associated viral vector Retinal disorders 


  1. 1.
    Tomita T (1970) Electrical activity of vertebrate photoreceptors. Q Rev Biophys 3:179–222PubMedCrossRefGoogle Scholar
  2. 2.
    Korenbrot JI (2012) Speed, sensitivity, and stability of the light response in rod and cone photoreceptors: facts and models. Prog Retin Eye Res 31:442–466PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Rodieck RW (1998) The first steps in seeing. Sinauer Associates, Sunderland, MAGoogle Scholar
  4. 4.
    Bessant DA, Ali RR, Bhattacharya SS (2001) Molecular genetics and prospects for therapy of the inherited retinal dystrophies. Curr Opin Genet Dev 11:307–316PubMedCrossRefGoogle Scholar
  5. 5.
    Ivanova E, Hwang GS, Pan ZH et al (2010) Evaluation of AAV-mediated expression of Chop2-GFP in the marmoset retina. Invest Ophthalmol Vis Sci 51:5288–5296PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Bok D, Yasumura D, Matthes MT et al (2002) Effects of adeno-associated virus-vectored ciliary neurotrophic factor on retinal structure and function in mice with a P216L rds/peripherin mutation. Exp Eye Res 74:719–735PubMedCrossRefGoogle Scholar
  7. 7.
    Redmond TM, Yu S, Lee E et al (1998) Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nat Genet 20:344–351PubMedCrossRefGoogle Scholar
  8. 8.
    Pang JJ, Chang B, Hawes NL et al (2005) Retinal degeneration 12 (rd12): a new, spontaneously arising mouse model for human Leber congenital amaurosis (LCA). Mol Vis 11:152–162PubMedGoogle Scholar
  9. 9.
    Samardzija M, von Lintig J, Tanimoto N et al (2008) R91W mutation in Rpe65 leads to milder early-onset retinal dystrophy due to the generation of low levels of 11-cis-retinal. Hum Mol Genet 17:281–292PubMedCrossRefGoogle Scholar
  10. 10.
    Bennicelli J, Wright JF, Komaromy A et al (2008) Reversal of blindness in animal models of Leber congenital amaurosis using optimized AAV2-mediated gene transfer. Mol Ther 16:458–465PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Pang JJ, Chang B, Kumar A et al (2006) Gene therapy restores vision-dependent behavior as well as retinal structure and function in a mouse model of RPE65 Leber congenital amaurosis. Mol Ther 13:565–572PubMedCrossRefGoogle Scholar
  12. 12.
    Roman AJ, Boye SL, Aleman TS et al (2007) Electroretinographic analyses of Rpe65-mutant rd12 mice: developing an in vivo bioassay for human gene therapy trials of Leber congenital amaurosis. Mol Vis 13:1701–1710PubMedGoogle Scholar
  13. 13.
    Acland GM, Aguirre GD, Ray J et al (2001) Gene therapy restores vision in a canine model of childhood blindness. Nat Genet 28:92–95PubMedGoogle Scholar
  14. 14.
    Bainbridge JW, Smith AJ, Barker SS et al (2008) Effect of gene therapy on visual function in Leber’s congenital amaurosis. N Engl J Med 358:2231–2239PubMedCrossRefGoogle Scholar
  15. 15.
    Maguire AM, Simonelli F, Pierce EA et al (2008) Safety and efficacy of gene transfer for Leber’s congenital amaurosis. N Engl J Med 358:2240–2248PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Hauswirth WW, Aleman TS, Kaushal S et al (2008) 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 19:979–990PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Banin E, Bandah-Rozenfeld D, Obolensky A et al (2010) Molecular anthropology meets genetic medicine to treat blindness in the North African Jewish population: human gene therapy initiated in Israel. Hum Gene Ther 21:1749–1757PubMedCrossRefGoogle Scholar
  18. 18.
    Jacobson SG, Cideciyan AV, Ratnakaram R et al (2012) Gene therapy for Leber congenital amaurosis caused by RPE65 mutations: safety and efficacy in 15 children and adults followed up to 3 years. Arch Ophthalmol 130:9–24PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Bennett J, Ashtari M, Wellman J et al (2012) AAV2 gene therapy readministration in three adults with congenital blindness. Sci Transl Med 4:120ra115Google Scholar
  20. 20.
    Maclaren RE, Groppe M, Barnard AR et al (2014) Retinal gene therapy in patients with choroideremia: initial findings from a phase 1/2 clinical trial. Lancet 383(9923):1129–1137PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Rolling F (2004) Recombinant AAV-mediated gene transfer to the retina: gene therapy perspectives. Gene Ther 11(Suppl 1):S26–S32PubMedCrossRefGoogle Scholar
  22. 22.
    Rabinowitz JE, Samulski J (1998) Adeno-associated virus expression systems for gene transfer. Curr Opin Biotechnol 9:470–475PubMedCrossRefGoogle Scholar
  23. 23.
    Kotin RM, Siniscalco M, Samulski RJ et al (1990) Site-specific integration by adeno-associated virus. Proc Natl Acad Sci U S A 87:2211–2215PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Schnepp BC, Jensen RL, Chen CL et al (2005) Characterization of adeno-associated virus genomes isolated from human tissues. J Virol 79:14793–14803PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Lebherz C, Maguire A, Tang W et al (2008) Novel AAV serotypes for improved ocular gene transfer. J Gene Med 10:375–382PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Gao GP, Alvira MR, Wang L et al (2002) Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci U S A 99:11854–11859PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Samulski RJ, Srivastava A, Berns KI et al (1983) Rescue of adeno-associated virus from recombinant plasmids: gene correction within the terminal repeats of AAV. Cell 33:135–143PubMedCrossRefGoogle Scholar
  28. 28.
    Rabinowitz JE, Rolling F, Li C et al (2002) Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity. J Virol 76:791–801PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Ali RR, Reichel MB, Thrasher AJ et al (1996) Gene transfer into the mouse retina mediated by an adeno-associated viral vector. Hum Mol Genet 5:591–594PubMedCrossRefGoogle Scholar
  30. 30.
    Xu L, Daly T, Gao C et al (2001) CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice. Hum Gene Ther 12:563–573PubMedCrossRefGoogle Scholar
  31. 31.
    Paterna JC, Moccetti T, Mura A et al (2000) Influence of promoter and WHV post-transcriptional regulatory element on AAV-mediated transgene expression in the rat brain. Gene Ther 7:1304–1311PubMedCrossRefGoogle Scholar
  32. 32.
    Brooks AR, Harkins RN, Wang P et al (2004) Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle. J Gene Med 6:395–404PubMedCrossRefGoogle Scholar
  33. 33.
    Le Meur G, Stieger K, Smith AJ et al (2007) Restoration of vision in RPE65-deficient Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium. Gene Ther 14:292–303PubMedCrossRefGoogle Scholar
  34. 34.
    Boye SE, Alexander JJ, Boye SL et al (2012) The human rhodopsin kinase promoter in an AAV5 vector confers rod and cone specific expression in the primate retina. Hum Gene Ther 23(10):1101–1115PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Komaromy AM, Alexander JJ, Cooper AE et al (2008) Targeting gene expression to cones with human cone opsin promoters in recombinant AAV. Gene Ther 15:1049–1055PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Carvalho LS, Xu J, Pearson RA et al (2011) Long-term and age-dependent restoration of visual function in a mouse model of CNGB3-associated achromatopsia following gene therapy. Hum Mol Genet 20:3161–3175PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Doroudchi MM, Greenberg KP, Liu J et al (2011) Virally delivered channelrhodopsin-2 safely and effectively restores visual function in multiple mouse models of blindness. Mol Ther 19:1220–1229PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Yin L, Greenberg K, Hunter JJ et al (2011) Intravitreal injection of AAV2 transduces macaque inner retina. Invest Ophthalmol Vis Sci 52:2775–2783PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Loeb JE, Cordier WS, Harris ME et al (1999) Enhanced expression of transgenes from adeno-associated virus vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: implications for gene therapy. Hum Gene Ther 10:2295–2305PubMedCrossRefGoogle Scholar
  40. 40.
    Kaplitt MG, Feigin A, Tang C et al (2007) Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson’s disease: an open label, phase I trial. Lancet 369:2097–2105PubMedCrossRefGoogle Scholar
  41. 41.
    Kingsman SM, Mitrophanous K, Olsen JC (2005) Potential oncogene activity of the woodchuck hepatitis post-transcriptional regulatory element (WPRE). Gene Ther 12:3–4PubMedCrossRefGoogle Scholar
  42. 42.
    McCarty DM, Fu H, Monahan PE et al (2003) Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo. Gene Ther 10:2112–2118PubMedCrossRefGoogle Scholar
  43. 43.
    Yokoi K, Kachi S, Zhang HS et al (2007) Ocular gene transfer with self-complementary AAV vectors. Invest Ophthalmol Vis Sci 48:3324–3328PubMedCrossRefGoogle Scholar
  44. 44.
    Natkunarajah M, Trittibach P, McIntosh J et al (2008) Assessment of ocular transduction using single-stranded and self-complementary recombinant adeno-associated virus serotype 2/8. Gene Ther 15:463–467PubMedCrossRefGoogle Scholar
  45. 45.
    Petersen-Jones SM, Bartoe JT, Fischer AJ et al (2009) AAV retinal transduction in a large animal model species: comparison of a self-complementary AAV2/5 with a single-stranded AAV2/5 vector. Mol Vis 15:1835–1842PubMedCentralPubMedGoogle Scholar
  46. 46.
    Bartlett JS, Wilcher R, Samulski RJ (2000) Infectious entry pathway of adeno-associated virus and adeno-associated virus vectors. J Virol 74:2777–2785PubMedCentralPubMedCrossRefGoogle Scholar
  47. 47.
    Zhong L, Li B, Mah CS et al (2008) Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses. Proc Natl Acad Sci U S A 105:7827–7832PubMedCentralPubMedCrossRefGoogle Scholar
  48. 48.
    Petrs-Silva H, Dinculescu A, Li Q et al (2009) High-efficiency transduction of the mouse retina by tyrosine-mutant AAV serotype vectors. Mol Ther 17:463–471PubMedCentralPubMedCrossRefGoogle Scholar
  49. 49.
    Petrs-Silva H, Dinculescu A, Li Q et al (2011) Novel properties of tyrosine-mutant AAV2 vectors in the mouse retina. Mol Ther 19:293–301PubMedCentralPubMedCrossRefGoogle Scholar
  50. 50.
    Kong J, Kim SR, Binley K et al (2008) Correction of the disease phenotype in the mouse model of Stargardt disease by lentiviral gene therapy. Gene Ther 15:1311–1320PubMedCentralPubMedCrossRefGoogle Scholar
  51. 51.
    Auricchio A, Kobinger G, Anand V et al (2001) Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: the retina as a model. Hum Mol Genet 10:3075–3081PubMedCrossRefGoogle Scholar
  52. 52.
    Dong JY, Fan PD, Frizzell RA (1996) Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum Gene Ther 7:2101–2112PubMedCrossRefGoogle Scholar
  53. 53.
    Grieger JC, Samulski RJ (2005) Packaging capacity of adeno-associated virus serotypes: impact of larger genomes on infectivity and postentry steps. J Virol 79:9933–9944PubMedCentralPubMedCrossRefGoogle Scholar
  54. 54.
    Monahan PE, Lothrop CD, Sun J et al (2010) Proteasome inhibitors enhance gene delivery by AAV virus vectors expressing large genomes in hemophilia mouse and dog models: a strategy for broad clinical application. Mol Ther 18:1907–1916PubMedCentralPubMedCrossRefGoogle Scholar
  55. 55.
    Allocca M, Doria M, Petrillo M et al (2008) Serotype-dependent packaging of large genes in adeno-associated viral vectors results in effective gene delivery in mice. J Clin Invest 118:1955–1964PubMedCentralPubMedCrossRefGoogle Scholar
  56. 56.
    Dong B, Nakai H, Xiao W (2010) Characterization of genome integrity for oversized recombinant AAV vector. Mol Ther 18:87–92PubMedCentralPubMedCrossRefGoogle Scholar
  57. 57.
    Wu Z, Yang H, Colosi P (2010) Effect of genome size on AAV vector packaging. Mol Ther 18:80–86PubMedCentralPubMedCrossRefGoogle Scholar
  58. 58.
    Lai Y, Yue Y, Duan D (2010) Evidence for the failure of adeno-associated virus serotype 5 to package a viral genome > or =8.2 kb. Mol Ther 18:75–79PubMedCentralPubMedCrossRefGoogle Scholar
  59. 59.
    Duan D, Yue Y, Engelhardt JF (2001) Expanding AAV packaging capacity with trans-splicing or overlapping vectors: a quantitative comparison. Mol Ther 4:383–391PubMedCrossRefGoogle Scholar
  60. 60.
    Halbert CL, Allen JM, Miller AD (2002) Efficient mouse airway transduction following recombination between AAV vectors carrying parts of a larger gene. Nat Biotechnol 20:697–701PubMedCrossRefGoogle Scholar
  61. 61.
    Grose WE, Clark KR, Griffin D et al (2012) Homologous recombination mediates functional recovery of dysferlin deficiency following AAV5 gene transfer. PLoS One 7:e39233PubMedCentralPubMedCrossRefGoogle Scholar
  62. 62.
    Odom GL, Gregorevic P, Allen JM et al (2011) Gene therapy of mdx mice with large truncated dystrophins generated by recombination using rAAV6. Mol Ther 19:36–45PubMedCentralPubMedCrossRefGoogle Scholar
  63. 63.
    Yan Z, Zhang Y, Duan D et al (2000) Trans-splicing vectors expand the utility of adeno-associated virus for gene therapy. Proc Natl Acad Sci U S A 97:6716–6721PubMedCentralPubMedCrossRefGoogle Scholar
  64. 64.
    Lai Y, Yue Y, Liu M et al (2005) Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors. Nat Biotechnol 23:1435–1439PubMedCentralPubMedCrossRefGoogle Scholar
  65. 65.
    Li J, Sun W, Wang B et al (2008) Protein trans-splicing as a means for viral vector-mediated in vivo gene therapy. Hum Gene Ther 19:958–964PubMedCentralPubMedCrossRefGoogle Scholar
  66. 66.
    Ghosh A, Yue Y, Shin JH et al (2009) Systemic trans-splicing adeno-associated viral delivery efficiently transduces the heart of adult mdx mouse, a model for Duchenne muscular dystrophy. Hum Gene Ther 20:1319–1328PubMedCentralPubMedCrossRefGoogle Scholar
  67. 67.
    Reich SJ, Auricchio A, Hildinger M et al (2003) Efficient trans-splicing in the retina expands the utility of adeno-associated virus as a vector for gene therapy. Hum Gene Ther 14:37–44PubMedCrossRefGoogle Scholar
  68. 68.
    Ghosh A, Yue Y, Lai Y et al (2008) A hybrid vector system expands adeno-associated viral vector packaging capacity in a transgene-independent manner. Mol Ther 16:124–130PubMedCrossRefGoogle Scholar
  69. 69.
    Trapani I, Colella P, Sommella A et al (2014) Effective delivery of large genes to the retina by dual AAV vectors. EMBO Mol Med 6:194–211PubMedCentralPubMedGoogle Scholar
  70. 70.
    Palfi A, Chadderton N, McKee AG et al (2012) Efficacy of codelivery of dual AAV2/5 vectors in the murine retina and hippocampus. Hum Gene Ther 23:847–858PubMedCrossRefGoogle Scholar
  71. 71.
    Ghosh A, Yue Y, Duan D (2006) Viral serotype and the transgene sequence influence overlapping adeno-associated viral (AAV) vector-mediated gene transfer in skeletal muscle. J Gene Med 8:298–305PubMedCentralPubMedCrossRefGoogle Scholar
  72. 72.
    Ghosh A, Yue Y, Duan D (2011) Efficient transgene reconstitution with hybrid dual AAV vectors carrying the minimized bridging sequences. Hum Gene Ther 22:77–83PubMedCentralPubMedCrossRefGoogle Scholar
  73. 73.
    Dunn KC, Aotaki-Keen AE, Putkey FR et al (1996) ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp Eye Res 62:155–169PubMedCrossRefGoogle Scholar
  74. 74.
    Ryals RC, Boye SL, Dinculescu A et al (2011) Quantifying transduction efficiencies of unmodified and tyrosine capsid mutant AAV vectors in vitro using two ocular cell lines. Mol Vis 17:1090–1102PubMedCentralPubMedGoogle Scholar
  75. 75.
    Seigel GM (1999) The golden age of retinal cell culture. Mol Vis 5:4PubMedGoogle Scholar
  76. 76.
    Krishnamoorthy RR, Agarwal P, Prasanna G et al (2001) Characterization of a transformed rat retinal ganglion cell line. Brain Res Mol Brain Res 86:1–12PubMedCrossRefGoogle Scholar
  77. 77.
    Biedler JL, Roffler-Tarlov S, Schachner M et al (1978) Multiple neurotransmitter synthesis by human neuroblastoma cell lines and clones. Cancer Res 38:3751–3757PubMedGoogle Scholar
  78. 78.
    You Q, Brown L, McClements M, Hankins MW, Maclaren RE (2012) Tetradecanoylphorbol-13-acetate (TPA) significantly increases AAV2/5 transduction of human neuronal cells in vitro. Exp Eye Res 97(1):148–153PubMedCrossRefGoogle Scholar
  79. 79.
    Garrity-Moses ME, Teng Q, Liu J et al (2005) Neuroprotective adeno-associated virus Bcl-xL gene transfer in models of motor neuron disease. Muscle Nerve 32:734–744PubMedCrossRefGoogle Scholar
  80. 80.
    Charbel Issa P, Singh MS, Lipinski DM et al (2012) Optimization of in vivo confocal autofluorescence imaging of the ocular fundus in mice and its application to models of human retinal degeneration. Invest Ophthalmol Vis Sci 53:1066–1075PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Samantha R. de Silva
    • 1
    • 2
  • Michelle E. McClements
    • 1
    • 2
  • Mark W. Hankins
    • 1
    • 2
  • Robert E. MacLaren
    • 2
    • 3
    Email author
  1. 1.Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical NeurosciencesUniversity of OxfordOxfordUK
  2. 2.NIHR Biomedical Research CentreOxfordUK
  3. 3.Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, John Radcliffe HospitalUniversity of OxfordOxfordUK

Personalised recommendations