Development and evaluation of new strategies for retinal gene therapy using CRISPR-mediated transcriptional activation

Hinrichsmeyer, Klara Sonnie (2022): Development and evaluation of new strategies for retinal gene therapy using CRISPR-mediated transcriptional activation. Dissertation, LMU München: Fakultät für Biologie

Abstract

Inherited retinal dystrophies (IRDs) comprise a large and heterogeneous group of inherited blinding disorders characterised by loss of retinal structure and function. For the vast majority of these disorders including those caused by mutations in large genes or by gain-of-function mutations, no therapy exists. The gene replacement approach with recombinant adeno-associated viral (rAAV) vectors is currently the gold standard for (retinal) gene therapy. However, the rAAV genome packaging capacity (4.7 kb) impedes the delivery of large genes. Moreover, the gene replacement strategy is insufficient for the treatment of gain-of-function mutations which require simultaneous repression of the diseased allele. There is thus a high unmet medical need for the development of new strategies which are suited to overcome the limitations of current gene replacement approaches designed to treat IRDs. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated transcriptional activation (CRISPRa) is independent of the target gene size and can also be modified to allow for treatment of more complex diseases or even to develop gene-independent gene therapy approaches. Nevertheless, CRISPRa modules exceed the genome packaging capacity of rAAVs and require dual rAAV vectors for in vivo expression. In this study, I developed innovative CRISPRa-based gene therapy strategies in combination with dual rAAV vectors designed to reconstitute split genes at the transcript level. This dual rAAV vector strategy termed REVeRT (reconstitution via mRNA trans-splicing) was used to activate the MYO7B gene in vivo. MYO7B is a functional equivalent of MYO7A whose defects are associated with the Usher syndrome, the most frequent type of inherited deafblindness. The MYO7B gene could be efficiently activated in the murine retina and various other organs upon different routes of administration. Furthermore, using a modified multiplexed CRISPRa system that allows concurrent gene activation and knockdown I tested a new approach for the treatment of gain-of-function mutations in the most common mouse model for autosomal dominant retinitis pigmentosa caused by the Pro23His mutation in the rod photoreceptor-specific rhodopsin gene (Rho) (RhoP23H+/-). Although a proof-of-principle for simultaneous activation of the M-Opsin gene (Opn1mw), a cone-specific photopigment that can functionally compensate for the missing rhodopsin function, and repression of Rho were successful in the RhoP23H+/- retina, the retinal phenotype could not be improved in this mouse model. Finally, the strategy of concurrent gene activation and knockdown was also evaluated to develop a new gene-independent gene therapy strategy in the RhoP23H/+ mouse model. For this purpose, the Nrl gene was repressed to reprogramme diseased rod photoreceptors into less degeneration prone cone-like cells combined with transcriptional activation of the neuroprotective Nxnl1 gene. This approach could ameliorate retinal degeneration four weeks after treatment. In conclusion, the new strategies and techniques developed in this study show great potential for the treatment of IRDs and can be modified to treat a variety of other inherited and acquired diseases in different tissues and organs.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 27. Oktober 2022
• 1. Bericht­erstatter:in: Biel, Martin