| Summary: | <p>Inherited retinal diseases (IRDs) are a diverse family of disorders caused by genetic mutations that lead to dysfunction and death of cells in the outer retina. Together, they are the most common cause of untreatable blindness in the working age population. While gene supplementation therapies using adeno-associated viral (AAV) vectors have shown restoration of visual function in IRDs, they are limited to the treatment of recessively inherited diseases caused by small genes. The engineering of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) nuclease system has revolutionized the field of molecular biology and generated excitement for the potential of novel therapeutic approaches to treat retinal diseases. Here, I explore the development of CRISPR-Cas9 tools and their therapeutic adaptation towards the treatment of retinal disease.</p>
<p>This thesis firstly investigated the development and optimization of a CRISPR vector system towards in vivo application. I assessed different Cas9 orthologues and regulatory elements that permit minimalization of the construct size for efficient delivery. I demonstrated in vitro that a construct backbone comprised of the Staphylococcus aureus derived SaCas9 driven by elongation factor 1α short promoter mediated robust levels of gene knockdown. Subsequently, I investigated the use of DNA-wrapped gold nanoparticles as an alternative modality for non-viral gene delivery. I demonstrated the ability of gold nanoparticles to efficiently deliver genes in vitro and assessed aspects of their safety including effects on retinal structure and function, although I was not able to detect gene expression in vivo.</p>
<p>This thesis secondly investigated the development of a dCas9-epigenetic editing system for transcriptional repression. I characterized novel transcriptional repressor domains and demonstrated that a novel bipartite repressor comprised of SaCas9 fused to the Krüppel-associated box (KRAB) repressor and the transcriptional repressor domain of Methyl-CpG Binding Protein 2 (MeCP2) facilitated high levels of transcriptional repression in vitro. I used AAV-delivered dCas9-repressors to successfully mediate robust and targeted gene repression in patient iPSC derived 3D retinal organoids. Finally, I evaluated the epigenetic editing system in a pilot in vivo experiment in an Nrl.EGFP+/-mouse model. This represents the first use of an all-in-one bipartite dCas9-repressor system to induce gene knockdown of photoreceptor genes.</p>
<p>The final set of work completed in this thesis focused on exploration of DNA base editing and prime editing technologies, with an eye towards their therapeutic translation for the treatment of IRDs. I demonstrated robust on-target adenine base editing in a novel reporter assay. Moreover, I engineered an enhanced minimal prime editor by truncating the Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) and demonstrated efficient correction of the homologous Ush2a (p.W3947X) mouse mutation in a dual-luciferase assay.</p>
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