# An in vitro/in vivo system for targeted retinal ganglion cell subtype manipulation

> **NIH NIH R21** · BAYLOR COLLEGE OF MEDICINE · 2022 · $240,000

## Abstract

PROJECT SUMMARY
Glaucoma is characterized by the gradual degeneration of retinal ganglion cells (RGCs). RGCs are highly
heterogeneous, and greater than 40 RGC subtypes in the mouse retina have been identified. Currently, our
understanding of RGC subtypes is hindered by the general inaccessibility of RGCs due to their small number.
Therefore, techniques that enrich the population of viable adult RGCs and RGC subtypes for experimentation,
such as immunopanning, are of great value. In this new application, we will develop a novel in vitro/in vivo
system for targeted RGC subtype manipulation based on modifications of the immunopanning technique. Our
overall hypothesis is that Cre-dependent ectopic expression of avian tumor virus receptor A (TVA) in RGCs
and RGC subtypes will allow for the isolation and culture of highly purified RGC populations. This will be
achieved through TVA-mediated immunopanning (TVAMI), a new technique we will develop which integrates a
positive immunopanning step based on protein binding to TVA. Furthermore, through transduction with
lentivirus pseudotyped with EnvA, a selective TVA ligand, we will attempt to induce gene expression in the
same TVA-expressing RGC populations both in vitro and in vivo. There are two Specific Aims: 1) establish and
optimize the TVAMI system for RGC isolation and culture; and 2) manipulate gene expression in targeted RGC
subpopulations. Throughout Aim 1, we will study transgenic mice that express TVA on the cell surface of
nearly all adult RGCs. We will compare several TVAMI variations against standard immunopanning both after
isolation and one week of cell culture with cell staining and immunocytochemistry to develop an optimized
protocol. Throughout Aim 2, we will study transgenic animals that express TVA in αRGCs, a population of
RGCs which includes four RGC subtypes. First, we will isolate αRGCs with TVAMI and confirm the identity,
validate the purity, and measure the relative survival of the four αRGC subtypes after isolation and one week
of cell culture with cell staining and immunocytochemistry. We will also perform single cell RNA sequencing on
this enriched population to define the transcriptional signature of αRGCs. Second, after αRGC isolation, we will
assess the specificity and efficiency of αRGC-specific virus transduction in vitro with EnvA-pseudotyped
lentivirus or AAV2 vector containing an RGC-specific GFP expression cassette. Third, we will introduce the
same viruses to RGCs via intravitreal injection and compare rates of αRGC transduction in vivo with
immunohistochemistry in whole mounted and sectioned retinas and after subsequent TVAMI. If successful, this
system has the potential to unlock multiple new approaches to study RGCs and RGC subtypes in normal and
disease states, and enable a broad range of novel applications with potential therapeutic value.

## Key facts

- **NIH application ID:** 10354977
- **Project number:** 1R21EY033458-01
- **Recipient organization:** BAYLOR COLLEGE OF MEDICINE
- **Principal Investigator:** Benjamin J Frankfort
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $240,000
- **Award type:** 1
- **Project period:** 2022-02-01 → 2024-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10354977

## Citation

> US National Institutes of Health, RePORTER application 10354977, An in vitro/in vivo system for targeted retinal ganglion cell subtype manipulation (1R21EY033458-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10354977. Licensed CC0.

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