It was recently found that subretinal injection of Adeno associated virus (AAV) in patients with RPE65 Leber congenital amaurosis-2 (LCA2) led to a loss of central retinal thickness and central visual acuity. Because the vast majority of inherited retinal diseases are caused by mutations in photoreceptor (PR)-specific genes, there is an obvious need to develop gene replacement strategies that can more safely target these cells. We propose to develop novel AAV vectors capable of transducing PRs following a safer, surgically less invasive intravitreal injection. In so doing, we will overcome a major hurdle in th field of retinal gene therapy - how to safely deliver genes to rods and cones in fragile, diseased retinas that are prone to further damage upon surgically-induced retinal detachment. The ability to safely target genes to central cones is especially significant because this is the area of the retina responsible for acute, daylight vision. Transduction of inner retina via the vitreous with unmodified AAV serotypes depends on their ability to bind heparin sulfate proteoglycan (HSPG), a glycan present in the inner limiting membrane (ILM), a basement membrane that forms the vitreoretinal interface. Our hypothesis is that the AAV capsid can be simultaneously optimized to confer adhesion to the inner limiting membrane and traffic to the outer retina while maintaining or gaining photoreceptor tropism. To achieve this goal we propose the following three aims.
In Aim 1, we will test novel variants developed via rational design and directed evolution (novel, highly complex AAV capsid libraries) for their ability to transduce mouse PRs via the vitreous. Relative transduction efficiency will be quantified using a mouse model with sortable (GFP-positive) PRs- the Rho-GFP knock in mouse. Because their ocular characteristics are most similar to human, in Aim 2, we will screen and test vectors in non-human primate and determine whether capsid motifs that confer PR transduction to AAV mutants in mouse retina are also important in primate. Sortable NHP PRs will be created via subretinal injection of a vector proven to have exclusive activity in rods and cones of NHP, AAV5-hGRK1-GFP.
In Aim 3, the most efficient variants will be tested for their ability to restore vision to mouse models of CNGB3 achromatopsia and GUCY2D Leber congenital amaurosis (LCA1). Results may directly impact clinical trial designs for both diseases and be applied more broadly to other photoreceptor-mediated retinal disease. In summary, results of this study would transform the way gene therapy agents are administered to inherited retinal disease patients, converting a full blown vitreoretinal surgery into an outpatient procedure akin to that required for wet age related macular degeneration drugs, Lucentis and Avastin. Such vectors could be administered in clinic rather than a surgical suite, thereby increasing accessibility of gene therapies to much larger patient populations. Access to gene therapies would improve as the number of clinical trial study sites qualified to administer study agent would greatly increase.

Public Health Relevance

The overall goal of this proposal is to develop novel Adeno associated viral (AAV) vectors that are capable of transducing photoreceptors following a non-invasive intravitreal injection. In so doing, we will overcome a major hurdle in the field of retinl gene therapy - how to safely deliver genes to rod and cone photoreceptors in fragile, diseased retinas that are prone to further damage upon surgically-induced retinal detachment. Screening will be performed in mouse and non-human primate and most efficient vectors will be tested for their ability to restore vision to mouse models of CNGB3 achromatopsia and GUCY2D Leber congenital amaurosis- 1(LCA1).

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Special Emphasis Panel (DPVS)
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Shen, Grace L
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University of Florida
Schools of Medicine
United States
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Beltran, William A; Cideciyan, Artur V; Boye, Shannon E et al. (2017) Optimization of Retinal Gene Therapy for X-Linked Retinitis Pigmentosa Due to RPGR Mutations. Mol Ther 25:1866-1880
Kanaan, Nicholas M; Sellnow, Rhyomi C; Boye, Sanford L et al. (2017) Rationally Engineered AAV Capsids Improve Transduction and Volumetric Spread in the CNS. Mol Ther Nucleic Acids 8:184-197
Choudhury, Shreyasi; Strang, Christianne E; Alexander, John J et al. (2016) Novel Methodology for Creating Macaque Retinas with Sortable Photoreceptors and Ganglion Cells. Front Neurosci 10:551
Boyd, R F; Sledge, D G; Boye, S L et al. (2016) Photoreceptor-targeted gene delivery using intravitreally administered AAV vectors in dogs. Gene Ther 23:223-30
Boye, Shannon E; Alexander, John J; Witherspoon, C Douglas et al. (2016) Highly Efficient Delivery of Adeno-Associated Viral Vectors to the Primate Retina. Hum Gene Ther 27:580-97
Boye, Sanford L; Bennett, Antonette; Scalabrino, Miranda L et al. (2016) Impact of Heparan Sulfate Binding on Transduction of Retina by Recombinant Adeno-Associated Virus Vectors. J Virol 90:4215-31
Xie, Keqiang; Colgan, Lesley A; Dao, Maria T et al. (2016) NF1 Is a Direct G Protein Effector Essential for Opioid Signaling to Ras in the Striatum. Curr Biol 26:2992-3003
Boyd, R F; Boye, S L; Conlon, T J et al. (2016) Reduced retinal transduction and enhanced transgene-directed immunogenicity with intravitreal delivery of rAAV following posterior vitrectomy in dogs. Gene Ther 23:548-56
Ildefonso, Cristhian J; Jaime, Henrique; Brown, Emily E et al. (2016) Targeting the Nrf2 Signaling Pathway in the Retina With a Gene-Delivered Secretable and Cell-Penetrating Peptide. Invest Ophthalmol Vis Sci 57:372-86
Boye, Sanford L; Peterson, James J; Choudhury, Shreyasi et al. (2015) Gene Therapy Fully Restores Vision to the All-Cone Nrl(-/-) Gucy2e(-/-) Mouse Model of Leber Congenital Amaurosis-1. Hum Gene Ther 26:575-92

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