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William A Beltran, Natalia Dolgova, Valerie L Dufour, Iwabe Simone, Ryan F. Boyd, Joshua T Bartoe, Gustavo D Aguirre, David L schaffer, John Gerard Flannery, Leah Byrne; AAV capsid variants target canine and primate bipolar cells after subretinal and intravitreal delivery. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4489.
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© ARVO (1962-2015); The Authors (2016-present)
To identify, via directed evolution screening conducted in dogs, AAV capsid variants with tropism towards bipolar cells (BP). To validate the top AAV variant candidates' ability to transduce ON BP following subretinal (SR) and/or intravitreal (IVT) delivery in a WT dog, a mutant dog, as well as in a WT NHP.
Four AAV libraries (AAV2 error prone, AAV2-7mer, loop swap, SCHEMA) each containing >106 different AAV capsid variants were packaged so that each AAV particle contained the genome encoding its own capsid, and IVT injected in WT dogs. Following the last (6th) round of selection, high throughput Illumina sequencing was done to track convergence of variants. A bar coded library that included the top 12 candidates and AAV24YF+ TV was developed in which an ON BP specific promoter was used to drive expression of the GFP-barcode constructs. This library was screened in 2 WT dogs after both IVT and SR delivery. Validation of the best IVT and SR variants was done in a WT and a mutant NPHP5-LCA dog at late stage retinal degeneration. Both variants were also tested via both IVT and SR injection in a NHP. In vivo cSLO imaging and IHC were used to detect, respectively, spatial retinal distribution and cellular site expression of reporter genes.
Screening of the bar coded library identified the best variant for IVT (K9#12) and SR (K9#4) delivery which both surpassed in performance AAV24YF+ TV, previously shown to target multiple cell populations following intravitreal injection in dogs. K9#12 injected IVT (380-460 µL at 2E+13 vg/mL) transduced 22-86% of ON BP in central to mid-peripheral retina of a WT dog, while transduction efficiency in NPHP5-LCA dog varied from 0% to 50% (center to periphery). SR injection of K9#4 transduced 57-80% of ON BP in bleb/treated area of WT dog, and 31-48% in NPHP5-LCA dog. Following IVT injection in NHP, both vectors transduced a ring of parafoveal and mid-peripheral ON BP, while SR injection delivered to the macular region targeted efficiently parafoveal and perifoveal ON BP.
We have identified two novel AAV capsid variants that transduce ON BP in both dog and NHP. This paves the way for treatment of complete CSNB due to mutations causing dysfunction in ON BP, as well as vision restoration through delivery of optogenetic tools to ON BP.
This is a 2020 ARVO Annual Meeting abstract.
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