Toualbi, Lyes; (2024) Developing a non-viral gene therapy for CHM and USH2A retinopathy using Scaffold Matrix Attachment Region DNA plasmids. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Gene therapy for inherited retinal diseases (IRDs) has had to overcome many pitfalls to reach the clinic, and still has many challenges to address, such as expanding the carrying capacity of vectors, ensuring long-term transgene expression, and limiting inflammatory reactions. The development of alternative non-viral vectors is therefore crucial to broaden the gene therapy possibilities. In this thesis, non-viral scaffold matrix attachement region (S/MAR) plasmid DNA vectors were investigated in two examples of IRDs, that would benefit from gene augmentation therapy: Choroideremia and Usher syndrome type IIA. S/MAR vectors containing the human CHM (1.9kb) or USH2A (15.6kb) coding sequences, which produce the proteins REP-1 and usherin, respectively, were successfully generated. These vectors were assessed in different patientcell disease models. CHM-S/MAR vectors restored REP-1 expression with a 75% rescue of prenylation function in CHM patient fibroblasts. CHM human induced pluripotent stem cells (hiPSC) were generated and differentiated into RPE, for further experiments. USH2A-S/MAR vectors drove transgene expression in patient fibroblasts, with Usherin presence confirmed by qPCR, western blots, and immunostainings. This work also gave new insights on USH2A patient-derived cell cilia defects and usherin function in focal adhesion and cell migration. S/MAR vectors have shown promise as a novel non-viral retinal gene therapy, warranting further development for both choroideremia and USH2A-related diseases.

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