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Adeno-Associated Virus Vectors pp 249–262Cite as

SubILM Injection of AAV for Gene Delivery to the Retina

Part of the Methods in Molecular Biology book series (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.)

Key words

  • Inner limiting membrane
  • AAV
  • Gene delivery
  • Gene replacement
  • Retinal ganglion cells
  • Bipolar cells
  • Photoreceptors
  • Novel surgical technique

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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
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  2. John J. Alexander
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  3. Sanford L. Boye
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  4. C. Douglas Witherspoon
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  5. Shannon E. Boye
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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

    Ph.D. Michael J. Castle

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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

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

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