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Development of non-viral vectors for gene therapy for pathologies of the retina

Publication . Oliveira, Ana V.; Silva, Gabriela

The success of gene therapy relies on efficient gene transfer and stable transgene expression. Our goal was to develop non-viral vectors optimized for retinal gene therapy with continued gene expression. Polymers, chitosan and hyaluronic acid or the modified polymers (thiolated chitosan and aminated hyaluronic acid) were chosen considering their biocompatibility and biodegradability to prepare several formulations. Vectors were formulated and characterized regarding their physical properties, biocompatibility and gene transfer efficiency in vitro on both retinal pigment epithelial and HEK293 cells and gene expression in the mouse retina. Our results show that our vectors exhibit size and surface charge consistent with gene delivery. They also present long-term stability in both storage and physiological conditions, remain stable after several freeze-thaw cycles and are capable of efficiently protecting DNA from nuclease degradation. Transfection studies show that transfection efficiency and transgene expression is affected by cell type, polymer molecular weight and mode of integrase delivery with the polyplexes. The incorporation of hyaluronic acid affected formulation stability, as expected, but it did not affect DNA loading and protection. The combination of chitosan and hyaluronic acid in polyplexes showed a significant improvement of transfection efficiency compared to chitosan-based vectors. In order to achieve sustained gene transfer vectors were combined with phiC31-integrase to promote transgene integration. The combined strategy of chitosan-based delivery and integrase demonstrate prolonged gene expression of both small (GFP, 1 Kb) and large genes (CEP290, 8Kb) several weeks post-transfection. In vivo sub-retinal administration of our vectors showed efficient transfection and sustained transgene expression in RPE cells at least 6 months post- injection. Our results indicate this chitosan-based approach may overcome size limitations found in commonly used adeno-associated viruses mediated gene transfer, while maintaining a high safety profile and prolonged, sustained gene expression, thus constituting an alternative for retinal gene delivery.

2015-12-13Doctoral thesis

Open Access