Skip to main content
SpringerLink
Log in

SubILM Injection of AAV for Gene Delivery to the Retina

  • Protocol
  • First Online:
Adeno-Associated Virus Vectors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1950))

Abstract

Adeno-associated virus (AAV) has emerged as the vector of choice for delivering genes to the retina. Indeed, the first gene therapy to receive FDA approval in the United States is an AAV-based treatment for the inherited retinal disease, Leber congenital amaurosis-2. Voretigene neparvovec (Luxturna™) is delivered to patients via subretinal (SR) injection, an invasive surgical procedure that requires detachment of photoreceptors (PRs) from the retinal pigment epithelium (RPE). It has been reported that subretinal administration of vector under the cone-exclusive fovea leads to a loss of central retinal structure and visual acuity in some patients. Due to its technical difficulty and potential risks, alternatives to SR injection have been explored in primates. Intravitreally (Ivt) delivered AAV transduces inner retina and foveal cones, but with low efficiency. Novel AAV capsid variants identified via rational design or directed evolution have offered only incremental improvements, and have failed to promote pan-inner retinal transduction or significant outer retinal transduction beyond the fovea. Problems with retinal transduction by Ivt-delivered AAV include dilution in the vitreous, potential antibody-mediated neutralization of capsid in this nonimmune privileged space, and the presence of the inner limiting membrane (ILM), a basement membrane separating the vitreous from the neural retina. We have developed an alternative “subILM” injection method that overcomes all three hurdles. Specifically, vector is placed in a surgically induced, hydrodissected space between the ILM and neural retina. We have shown that subILM injection promotes more efficient retinal transduction by AAV than Ivt injection, and results in uniform and extensive transduction of retinal ganglion cells (RGCs) beneath the subILM bleb. We have also demonstrated transduction of Muller glia, ON bipolar cells, and photoreceptors by subILM injection. Our results confirm that the ILM is a major barrier to transduction by AAV in primate retina and that, when it is circumvented, the efficiency and depth to which AAV2 promotes transduction of multiple retinal cell classes is greatly enhanced. Here we describe in detail methods for vector preparation, vector dilution, and subILM injection as performed in macaque (Macaca sp.)

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Boye SE, Alexander JJ, Boye SL, Witherspoon CD, Sandefer KJ, Conlon TJ, Erger K, Sun J, Ryals R, Chiodo VA, Clark ME, Girkin CA, Hauswirth WW, Gamlin PD (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):1101–1115. https://doi.org/10.1089/hum.2012.125

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Vandenberghe LH, Bell P, Maguire AM, Cearley CN, Xiao R, Calcedo R, Wang L, Castle MJ, Maguire AC, Grant R, Wolfe JH, Wilson JM, Bennett J (2011) Dosage thresholds for AAV2 and AAV8 photoreceptor gene therapy in monkey. Sci Transl Med 3(88):88ra54. https://doi.org/10.1126/scitranslmed.3002103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Vandenberghe LH, Bell P, Maguire AM, Xiao R, Hopkins TB, Grant R, Bennett J, Wilson JM (2013) AAV9 targets cone photoreceptors in the nonhuman primate retina. PLoS One 8(1):e53463. https://doi.org/10.1371/journal.pone.0053463

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Jacobson SG, Cideciyan AV, Ratnakaram R, Heon E, Schwartz SB, Roman AJ, Peden MC, Aleman TS, Boye SL, Sumaroka A, Conlon TJ, Calcedo R, Pang JJ, Erger KE, Olivares MB, Mullins CL, Swider M, Kaushal S, Feuer WJ, Iannaccone A, Fishman GA, Stone EM, Byrne BJ, Hauswirth WW (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(1):9–24. https://doi.org/10.1001/archophthalmol.2011.298

    Article  CAS  PubMed  Google Scholar 

  5. Yanoff M, Kertesz Rahn E, Zimmerman LE (1968) Histopathology of juvenile retinoschisis. Arch Ophthalmol 79(1):49–53

    Article  CAS  PubMed  Google Scholar 

  6. Condon GP, Brownstein S, Wang NS, Kearns JA, Ewing CC (1986) Congenital hereditary (juvenile X-linked) retinoschisis. Histopathologic and ultrastructural findings in three eyes. Arch Ophthalmol 104(4):576–583

    Article  CAS  PubMed  Google Scholar 

  7. Yin L, Greenberg K, Hunter JJ, Dalkara D, Kolstad KD, Masella BD, Wolfe R, Visel M, Stone D, Libby RT, Diloreto D Jr, Schaffer D, Flannery J, Williams DR, Merigan WH (2011) Intravitreal injection of AAV2 transduces macaque inner retina. Invest Ophthalmol Vis Sci 52(5):2775–2783. https://doi.org/10.1167/iovs.10-6250

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Dalkara D, Byrne LC, Klimczak RR, Visel M, Yin L, Merigan WH, Flannery JG, Schaffer DV (2013) In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous. Sci Transl Med 5(189):189ra176. https://doi.org/10.1126/scitranslmed.3005708

    Article  CAS  Google Scholar 

  9. Ye GJ, Budzynski E, Sonnentag P, Miller PE, Sharma AK, Ver Hoeve JN, Howard K, Knop DR, Chulay JD (2015) Safety and biodistribution evaluation in cynomolgus macaques of rAAV2tYF-CB-hRS1, a recombinant adeno-associated virus vector expressing retinoschisin. Hum Gene Ther Clin Dev 26(3):165–176. https://doi.org/10.1089/humc.2015.076

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kotterman MA, Yin L, Strazzeri JM, Flannery JG, Merigan WH, Schaffer DV (2015) Antibody neutralization poses a barrier to intravitreal adeno-associated viral vector gene delivery to non-human primates. Gene Ther 22(2):116–126. https://doi.org/10.1038/gt.2014.115

    Article  CAS  PubMed  Google Scholar 

  11. Dalkara D, Kolstad KD, Caporale N, Visel M, Klimczak RR, Schaffer DV, Flannery JG (2009) Inner limiting membrane barriers to AAV-mediated retinal transduction from the vitreous. Mol Ther 17(12):2096–2102. https://doi.org/10.1038/mt.2009.181

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Boye SL, Bennett A, Scalabrino ML, McCullough KT, Van Vliet K, Choudhury S, Ruan Q, Peterson J, Agbandje-McKenna M, Boye SE (2016) Impact of heparan sulfate binding on transduction of retina by recombinant adeno-associated virus vectors. J Virol 90(8):4215–4231. https://doi.org/10.1128/JVI.00200-16

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Matsumoto B, Blanks JC, Ryan SJ (1984) Topographic variations in the rabbit and primate internal limiting membrane. Invest Ophthalmol Vis Sci 25(1):71–82

    CAS  PubMed  Google Scholar 

  14. Cehajic-Kapetanovic J, Le Goff MM, Allen A, Lucas RJ, Bishop PN (2011) Glycosidic enzymes enhance retinal transduction following intravitreal delivery of AAV2. Mol Vis 17:1771–1783

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Boye SE, Alexander JJ, Witherspoon CD, Boye SL, Peterson JJ, Clark ME, Sandefer KJ, Girkin CA, Hauswirth WW, Gamlin PD (2016) Highly efficient delivery of adeno-associated viral vectors to the primate retina. Hum Gene Ther 27(8):580–597. https://doi.org/10.1089/hum.2016.085

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Li J, Samulski RJ, Xiao X (1997) Role for highly regulated rep gene expression in adeno-associated virus vector production. J Virol 71(7):5236–5243

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Xiao W, Chirmule N, Berta SC, McCullough B, Gao G, Wilson JM (1999) Gene therapy vectors based on adeno-associated virus type 1. J Virol 73(5):3994–4003

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Xiao X, Li J, Samulski RJ (1998) Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol 72(3):2224–2232

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Grimm D, Kern A, Rittner K, Kleinschmidt JA (1998) Novel tools for production and purification of recombinant adenoassociated virus vectors. Hum Gene Ther 9(18):2745–2760. https://doi.org/10.1089/hum.1998.9.18-2745

    Article  CAS  PubMed  Google Scholar 

  20. Burger C, Gorbatyuk OS, Velardo MJ, Peden CS, Williams P, Zolotukhin S, Reier PJ, Mandel RJ, Muzyczka N (2004) Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Mol Ther 10(2):302–317

    Article  CAS  PubMed  Google Scholar 

  21. Gray JT, Zolotukhin S (2011) Design and construction of functional AAV vectors. Methods Mol Biol 807:25–46. https://doi.org/10.1007/978-1-61779-370-7_2

    Article  CAS  PubMed  Google Scholar 

  22. Beltran WA, Cideciyan AV, Lewin AS, Iwabe S, Khanna H, Sumaroka A, Chiodo VA, Fajardo DS, Roman AJ, Deng WT, Swider M, Aleman TS, Boye SL, Genini S, Swaroop A, Hauswirth WW, Jacobson SG, Aguirre GD (2012) Gene therapy rescues photoreceptor blindness in dogs and paves the way for treating human X-linked retinitis pigmentosa. Proc Natl Acad Sci U S A 109(6):2132–2137. https://doi.org/10.1073/pnas.1118847109

    Article  PubMed  PubMed Central  Google Scholar 

  23. Bennicelli J, Wright JF, Komaromy A, Jacobs JB, Hauck B, Zelenaia O, Mingozzi F, Hui D, Chung D, Rex TS, Wei Z, Qu G, Zhou S, Zeiss C, Arruda VR, Acland GM, Dell'Osso LF, High KA, Maguire AM, Bennett J (2008) Reversal of blindness in animal models of leber congenital amaurosis using optimized AAV2-mediated gene transfer. Mol Ther 16(3):458–465. https://doi.org/10.1038/sj.mt.6300389

    Article  CAS  PubMed  Google Scholar 

  24. Zolotukhin S, Potter M, Zolotukhin I, Sakai Y, Loiler S, Fraites TJ Jr, Chiodo VA, Phillipsberg T, Muzyczka N, Hauswirth WW, Flotte TR, Byrne BJ, Snyder RO (2002) Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28(2):158–167

    Article  CAS  PubMed  Google Scholar 

  25. Veldwijk MR, Topaly J, Laufs S, Hengge UR, Wenz F, Zeller WJ, Fruehauf S (2002) Development and optimization of a real-time quantitative PCR-based method for the titration of AAV-2 vector stocks. Mol Ther 6(2):272–278

    Article  CAS  PubMed  Google Scholar 

  26. Lock M, Alvira MR, Chen SJ, Wilson JM (2014) Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR. Hum Gene Ther Methods 25(2):115–125. https://doi.org/10.1089/hgtb.2013.131

    Article  CAS  PubMed  Google Scholar 

  27. Day TP, Byrne LC, Flannery JG, Schaffer DV (2018) Screening for neutralizing antibodies against natural and engineered AAV capsids in nonhuman primate retinas. Methods Mol Biol 1715:239–249. https://doi.org/10.1007/978-1-4939-7522-8_17

    Article  CAS  PubMed  Google Scholar 

  28. Desrosiers M, Dalkara D (2018) Neutralizing antibodies against adeno-associated virus (aav): measurement and influence on retinal gene delivery. Methods Mol Biol 1715:225–238. https://doi.org/10.1007/978-1-4939-7522-8_16

    Article  CAS  PubMed  Google Scholar 

  29. AVMA Guidelines for the Euthanasia of Animals: 2013 Edition

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Ophthalmology and Visual Sciences, University of Alabama at Birmingham, Birmingham, AL, USA

    Paul D. Gamlin & C. Douglas Witherspoon

  2. Department of Human Genetics, Emory University, Atlanta, GA, USA

    John J. Alexander

  3. Department of Pediatrics and the Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL, USA

    Sanford L. Boye

  4. Department of Ophthalmology, University of Florida College of Medicine, Gainesville, FL, USA

    Shannon E. Boye

Authors
  1. Paul D. Gamlin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. John J. Alexander
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sanford L. Boye
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. C. Douglas Witherspoon
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shannon E. Boye
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Paul D. Gamlin .

Editor information

Editors and Affiliations

  1. Department of Neurosciences, University of California, San Diego, La Jolla, CA, USA

    Michael J. Castle

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Gamlin, P.D., Alexander, J.J., Boye, S.L., Witherspoon, C.D., Boye, S.E. (2019). SubILM Injection of AAV for Gene Delivery to the Retina. In: Castle, M. (eds) Adeno-Associated Virus Vectors. Methods in Molecular Biology, vol 1950. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9139-6_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9139-6_14

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9138-9

  • Online ISBN: 978-1-4939-9139-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation