PhD Dissertation: Mehri Ahmadiangollajeh

Development and Evaluation of dCas9-Based Artificial Transcription Factors for Neuroprotection: In Vitro and Ex Vivo Studies and In Vivo Evaluation in the RhoP23H Retinitis Pigmentosa Model

Mehri Ahmadiangollajeh
Molecular Biology, Genetics, and Bioengineering,

PhD Dissertation, 2025

Thesis Jury

Asst. Prof. Dr. Cavit Ağca (Thesis Advisor)

Asst. Prof. Dr. Stuart J. Lucas

Asst. Prof. Dr. Alex Lyakhovich

Assoc. Prof. Dr. Med. Markus Tschopp

Asst. Prof. Dr. Ahsen Morva Yılmaz

Date & Time: 23rd, July 2025 – 5:00 PM

Place: FENS G032

Zoom Link: https://sabanciuniv.zoom.us/j/9853862933?omn=93334618339

Keywords : Retinitis Pigmentosa, Artificial Transcription Factor,  AAV Mediated Gene Therapy, Neuroprotection, CRISPR-Cas9

Abstract

Inherited retinal degenerations, such as Retinitis Pigmentosa (RP), cause progressive loss of photoreceptors and vision impairment, with limited treatment options. This study presents a novel gene therapy approach based on transcriptional activation of endogenous neuroprotective genes using CRISPR-dCas9 artificial transcription factors (ATFs) to promote retinal cell survival. We designed and evaluated multiple guide RNAs (gRNAs) to achieve controlled, multiplexed overexpression of key neuroprotective factors, including Lif, Cntf, Manf, Fgf2, Bdnf, and Clcf1, using in vitro, in vivo, and ex vivo models.

Initial gRNA screening was performed in Muller glia-derived dCas9-VP64 and dCas9-SPH immortalized cell lines. Flow cytometry was used to sort double-positive cells, confirming the co-expression of gRNA and dCas9. ddPCR analysis demonstrated dose-dependent upregulation of target genes, with dCas9-SPH achieving higher expression than VP64, showing the importance of activator strength in achieving therapeutic levels of gene expression. Based on these results, the most effective gRNAs were cloned into AAV vectors under U6 promoters, and viral titers were determined for in vivo delivery. To evaluate therapeutic potential, we utilized retinosphere cultures and an advanced transgenic mouse model (Rho-P23H^ki/wt; dCas9-SPH^ki/wt), which allowed targeted delivery and assessment of gene activation in a slow-degeneration animal model. AAV vectors using both ShH10 and AAV8 capsids were evaluated in retinospheres via fluorescence imaging and ddPCR analysis for target gene upregulation. Retinospheres showed significant overexpression of target genes, supporting the feasibility of ATF-dependent neuroprotection as a therapeutic strategy. To evaluate the in vivo efficiency of ATFs, in the Rho-P23H^ki/wt; dCas9-SPH^ki/wt model, in vivo fluorescent and Optical Coherence Tomography (OCT) imaging techniques were performed before and after AAV injections to follow up the degeneration and the neuroprotective effects of the key neurotrophic factors. AAV transduction was confirmed by GFP expression, and OCT imaging showed reduced retinal thinning. However, this did not translate into measurable functional recovery, highlighting the need for further optimization of disease model, dosing strategies, and evaluation metrics in future studies.

Overall, our findings establish a flexible and controllable platform for CRISPR-based gene activation therapies in retinal disease. The ability to regulate endogenous neuroprotective gene expression in a dose-dependent manner via CRISPR-dCas9 and AAV vectors offers a promising alternative to traditional gene supplementation approaches, facilitating the way for precise gene regulation therapies in inherited retinal disorders.