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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

T28 - CRISPR-based model of a retinal dystrophy to investigate drivers of epithelial-to-mesenchymal transition in progressive blindness.

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Ally O'Shea

Undergraduate Researcher
University of Virginia
Glen Allen, Virginia, United States

Co-Author(s)

RB

Rishi Bhogaraju

Undergraduate Researcher
University of Virginia, United States
Grace Burns

Undergraduate Researcher
University of Virginia
Aiken, South Carolina, United States
EB

Emily Baum

PhD student
University of Virginia, United States
JS

Jagathpala Shetty

Research Scientist
University of Virginia, United States

Primary Investigator(s)

TB

Thomas H. Barker, PhD

Biomedical Engineering Professor
University of Virginia
Charlottesville, Virginia, United States

Introduction: : Choroideremia (CHM), a rare X-linked monogenic retinal disease affecting 1 in 50,000 to 100,000 individuals, leads to progressive loss of vision and blindness for all patients by adulthood. The disease is caused by mutations in the CHM gene causing loss of function of Rab Escort Protein 1 (REP1). Although REP1 shares 75% sequence identity with the ubiquitously expressed REP2, REP2 cannot compensate for the loss of REP1 in retinal cells of patients with CHM. REP1 facilitates the interaction between Rab GTPases (Rabs), with Rab geranylgeranyltransferase (RGGTase), which catalyzes prenylation, a post translational modification of Rab proteins essential for their membrane localization and vesicular trafficking [1,2].

In CHM, Rab underprenylation, particularly of Rab12, results in mTORC1 overactivation and reduced autophagy [3] diminishing the cells capacity to neutralize reactive oxygen species (ROS) [4]. Oxidative stress from ROS can enhance epithelial-to-mesenchymal transition (EMT) [4]. I hypothesize that defective Rab prenylation in CHM promotes EMT in retinal pigment epithelial (RPE) cells via ROS accumulation, representing a previously uncharacterized mechanism of disease progression. Elucidating this pathway may uncover novel aspects of CHM pathogenesis and inform therapeutic strategies beyond gene replacement.

No current animal model fully replicates the retinal degeneration seen in affected males with CHM, and RPE cells differentiated from stem cells have been shown to lose polarized organization after only a few passages [5,6]. This current study aims to develop a CRISPR-based cellular model of CHM in the more stable ARPE-19 cell line that recapitulates in vivo RPE phenotype to better understand disease pathophysiology.

Materials and

Methods: : ARPE-19 cells (ATCC) were cultured in MEM-Nic (MEM alpha with GlutaMAX, 10% FBS, 10% Penicillin/Streptomycin (P/S), 0.1 mM NEAA, 1% N1 supplement, taurine (0.25 mg/ml), hydrocortisone (20 ng/ml), triiodo-thyronine (0.013 ng/ml), and 10 mM nicotinamide) at 37 °C, 5% CO2 on standard tissue culture-treated plastic. To induce differentiation toward a mature RPE phenotype, cells were transferred to Transwell inserts coated with mouse laminin or fibronectin in 6-well plates. Cells were seeded at 1.5 x 10^5 cells/Transwell in complete MEM-Nic media with 10% FBS. After 24 hours, apical and basal compartments were media switched to complete MEM-Nic media with 1% FBS to induce differentiation, followed by media changes every third day [7].

Generation of a CRISPR KO cell line.
Two distinct guide RNAs (gRNAs) targeting different exons in CHM (REP1) were designed using Integrated DNA Technologies’ CRISPR design tool. Alt-R CRISPR-Cas9 ribonucleoprotein (RNP) complexes were prepared. Each gRNA was a duplex between a crRNA and a fluorescently labeled tracrRNA. ARPE-19 cells (4 x 10^4/well; 96-well plate) were washed with PBS and switched to complete MEM-Nic media without P/S followed by transfection of RNP complexes using Lipofectamine™ CRISPRMAX reagent [8]. Cell sorting was performed at t=24h on a Sony MA900 cell sorter. ATTO-550-positive fluorescent cells were sorted at a density of one cell per well into two 96-well plates containing 100uL equal parts conditioned and fresh media supplemented with 20% FBS. Clones were left to grow, and were switched to fresh media every 5 days after an initial 7 day incubation period.

Results, Conclusions, and Discussions:: To model defective Rab prenylation in CHM, we used the CRISPR-Cas9 system to knock out REP1 in ARPE-19 cells. Two distinct gRNAs were tested individually to optimize editing and transfection efficiency. Fluorescently–labeled tracrRNAs enabled enrichment of edited cells by flow sorting of single cell clones into 96 well plates. Although initial viability was high, post-sort clones exhibited slow growth. A representative FACS plot demonstrating gating strategy and enrichment of ATTO-550–positive cells is shown in Figure 1. Early-stage knockout populations are currently expanding for downstream validation (Figure 2a). Once clonal populations are mature DNA will be isolated for T7E1 mismatch assays and Sanger sequencing with ICE analysis to confirm knockout efficiency [9]. In parallel, array serial dilution plating is being tested as an alternative method for single-cell isolation to potentially increase clonal growth rates [10].

Control ARPE–19 cells grown on TC plastic display cobblestone morphology after 16 days, consistent with a differentiated RPE (Figure 2b). To further establish epithelial identity prior to EMT analysis, ARPE-19 cells were cultured on Transwell inserts, which separate the culture into apical (retinal-facing) and basolateral (choroidal-facing) compartments, effectively mimicking the native polarity of the retinal pigment epithelium monolayer [11]. After 12 days, cells displayed early cobblestone morphology, and maintained strong adhesion, consistent with a partial polarization of the RPE (Figure 2c, 2d). In order to verify epithelial identity, immunostaining for tight junction protein ZO-1 and Western blotting for visual cycle protein RPE65 is ongoing and will ensure correctly differentiated cells for downstream analysis [12].

This study lays the groundwork for a comprehensive investigation of RPE cell EMT in CHM through CRISPR-based ARPE-19 knockouts targeting the CHM gene. The CRISPR-engineered ARPE-19 model will provide a practical and reliable platform for studying disease mechanisms and overcoming the limitations of existing CHM models [5,6]. Ultimately, this study seeks to determine whether impaired Rab prenylation in CHM promotes oxidative stress-driven EMT, potentially revealing a novel mechanism of retinal degeneration and remodeling. Given the limited scope of current therapies, which primarily focus on gene replacement, uncovering additional disease pathways is essential for the development of more comprehensive and durable treatment strategies.

Acknowledgements and/or References (Optional): : [1] Sarkar, H., & Moosajee, M., Choroideremia: molecular mechanisms and therapies. Trends in molecular medicine. 2022;28(5), 378–387. doi: /10.1016/j.molmed.2022.02.011
[2] Pylypenko, O., Hammich, H., Yu, I. M., & Houdusse, A., Rab GTPases and their interacting protein partners: Structural insights into Rab functional diversity. Small GTPases. 2018;9(1-2), 22–48. doi: 10.1080/21541248.2017.1336191
[3] Raeker, M. Ö., Perera, N. D., Karoukis, A. J., Chen, L., Feathers, K. L., Ali, R. R., Thompson, D. A., & Fahim, A. T., Reduced Retinal Pigment Epithelial Autophagy Due to Loss of Rab12 Prenylation in a Human iPSC-RPE Model of Choroideremia. Cells. 2024;13(12), 1068. doi: 10.3390/cells13121068
[4] Yang, Y. C., Chien, Y., Yarmishyn, A. A., Lim, L. Y., Tsai, H. Y., Kuo, W. C., Tsai, P. H., Yang, S. H., Hong, S. I., Chen, S. J., Hwang, D. K., Yang, Y. P., & Chiou, S. H., Inhibition of oxidative stress-induced epithelial-mesenchymal transition in retinal pigment epithelial cells of age-related macular degeneration model by suppressing ERK activation. Journal of advanced research. 2024;60, 141–157. doi: /10.1016/j.jare.2023.06.004
[5] Jazouli, Z., Torriano, S., & Kalatzis, V., Chapter 5—iPSCs for modeling choroideremia. In A. Birbrair (Ed.), Novel Concepts in iPSC Disease Modeling (Vol. 15, pp. 123–151). Academic Press. 2022.doi: 10.1016/B978-0-12-823882-0.00005-9
[6] Singh, R., Phillips, M. J., Kuai, D., Meyer, J., Martin, J. M., Smith, M. A., Perez, E. T., Shen, W., Wallace, K. A., Capowski, E. E., Wright, L. S., & Gamm, D. M., Functional analysis of serially expanded human iPS cell-derived RPE cultures. Investigative ophthalmology & visual science. 2013;54(10), 6767–6778. doi: 10.1167/iovs.13-11943
[7] Hazim, R. A., Volland, S., Yen, A., Burgess, B. L., & Williams, D. S., Rapid differentiation of the human RPE cell line, ARPE-19, induced by nicotinamide. Experimental eye research. 2019;179, 18–24. doi: 10.1016/j.exer.2018.10.009
[8] Kim, S., Kim, D., Cho, S. W., Kim, J., & Kim, J. S., Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome research. 2014; 24(6), 1012–1019. doi:10.1101/gr.171322.113
[9]Yao, B., Yang, Q., Gonçalves, M. A. F. V., Schiffelers, R., Sluijter, J. P. G., & Lei, Z., Comparative Analysis of Methods for Assessing
On-Target Gene Editing Efficiencies. Methods and protocols. 2025; 8(2), 23. doi: 10.3390/mps8020023
[10] Giuliano, C. J., Lin, A., Girish, V., & Sheltzer, J. M., Generating Single Cell-Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9. Current protocols in molecular biology. 2019;128(1), e100. doi: 10.1002/cpmb.100
[11] Sonoda, S., Spee, C., Barron, E., Ryan, S. J., Kannan, R., & Hinton, D. R., A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells. Nature protocols. 2009; 4(5), 662–673. doi: 10.1038/nprot.2009.33
[12]Samuel, W., Jaworski, C., Postnikova, O. A., Kutty, R. K., Duncan, T., Tan, L. X., Poliakov, E., Lakkaraju, A., & Redmond, T. M., Appropriately differentiated ARPE-19 cells regain phenotype and gene expression profiles similar to those of native RPE cells. Molecular vision. 2017; 23, 60–89.