Purchase this article with an account.
Shreyasi Choudhury, Christianne E Strang, Sanford L. Boye, Julie Lynch Hill, Douglas C Witherspoon, Daniel T Kasuga, Paul D Gamlin, Shannon Elizabeth Boye; Novel Methodology for Creating Primate Retina with Sortable Cell Populations. Invest. Ophthalmol. Vis. Sci. 2016;57(12):766.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
With the ability to transduce retinal pigment epithelium for corrective gene therapy, focus has shifted to development and evaluation of viral vectors that effectively target other retinal cells. Given their involvement in most inherited retinal diseases, photoreceptor (PR) targeting is one priority. Targeting retinal ganglion cells (RGCs) is also important due to their role in glaucoma pathology and as an emerging target for optogenetic mediated vision restoration. Transgenic mouse models with constitutively fluorescent retinal cells have been used to quantify cell specific transduction by Adeno Associated Virus (AAV). However, clinical translatability is limited by the significant differences between rodent and primate eyes. In this regard, M. fascicularis, a widely used non-human primate (NHP) model, faithfully recapitulates the anatomy of the human eye. The purpose of our study was to develop a method for creating NHP retinas with sortable PRs and RGCs, isolate these populations by FACS and validate the results.
NHP were subretinally injected with AAV5 containing human rhodopsin kinase promoter (hGRK1)–driving GFP. GFP expression was validated four weeks post-injection using SLO. The lateral geniculate nuclei were then injected bilaterally with TRITC/Biotin-labeled Dextran (micro-ruby) to retrogradely label RGCs. A week later, animals were deeply anesthetized and enucleated. Samples corresponding to macular, superior and inferior retina were dissociated using conditions optimized for NHP retina and FACS sorted. Relative expression of retinal cell specific genes in the GFP+ and micro-ruby+ populations from the respective geographic locations were compared to unlabeled cells by qRT-PCR.
GFP+ and micro-ruby+ labeling of putative PRs and RGCs were verified by retinal whole mount. Gene expression profiles in GFP+ and micro-ruby+ cells indicated isolation of pure PR and RGC populations, respectively. Furthermore, variation in M/L cone, S cone, and rod specific gene expression from sorted PRs correlated with the relative abundance of these cell types in respective retinal regions.
We have established a method for creating and isolating pure populations of PRs and RGCs from NHP retina. This approach can be used to interrogate transduction profiles of novel viral vectors and/or promoters as well as identify those best suited for addressing various forms of inherited retinal disease.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
This PDF is available to Subscribers Only