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Steven H. DeVries, Yongling Zhu, Vince A. Chiodo, William W. Hauswirth; Selective Transduction Of Ground Squirrel Bipolar Cells With A Capsid Mutated AAV2. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4317.
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The ability to target and genetically manipulate specific neuron types holds great promise for elucidating the functional circuitry of the retina. Such targeting can be achieved through germ line manipulation in the mouse, but viral transduction is generally required for studies on other mammals. Towards this end, we have screened AAV2s with tyrosine mutant capsids for their ability to target specific retinal cell types following intravitreal injection in the ground squirrel.
The AAV2 capsid was modified to contain phe (F) instead of tyr (Y) at 1-6 sites (Petrs-Silva H et al. 2011. Mol Ther 19:293-301). The virus contained single stranded DNA which coded for GFP under the control of a chicken β-actin promoter. Intravitreal injections were made in 13-lined ground squirrels (Ictidomys tridecemlineatus) by approved procedures (Northwestern University IACUC). Animals were anesthetized with ketamine/xylazine, topical anesthetic applied to the eye, and a 27 gauge needle used to puncture the medial epicanthal sclera. 15 µl of virus solution (1012-1013/ml) was injected while advancing a 30 gauge blunt tip syringe needle toward the back of the eye. Animals were sacrificed 6+ weeks after injection. Retinal flat-mounts or slices were fixed and labeled with antibodies to GFP.
Intravitreal injection of a quad capsid mutant (Y272,444,500,730F) led to uniform labeling in the RGC layer. Injection of a sextuple mutant (quad + Y252,704F) produced sporadic but intense labeling in bipolar cells and Muller cells and less labeling in the RGC layer. An individual slice could contain up to 100 labeled bipolar cells. The percentage distribution of labeling among 11 cone bipolar cell types was determined in 4 retinal flat-mounts and favored Off vs On types (Off: cb1a, 25; cb1b, 14; cb2, 8; cb3a, 10; cb3b, 19; On: cb5a, 3; cb5b, 3; cb6a, 10; cb6b, 7; cb7a, 1; cb7b, 7). Rod and S-cone bipolar cells were also labeled.
We identified a sextuple Y-F mutant AAV2 that has a tropism for bipolar cells in the ground squirrel when intravitreally injected. Labeling involved all bipolar cell types, with a numerical bias towards Off cells. Paradoxically Off bipolar cells have their axon terminals furthest from the inner limiting membrane. A virus that selectively targets bipolar cells in the ground squirrel will enable genetic manipulation of cell properties. Examples include the selective suppression of glutamate receptor subunits or the expression of light-sensitive membrane proteins such as channel- or halo-rhodopsin.
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