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Updated: Apr 18, 2026
Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)
Published on: March 16, 2012
Enhancement of chitosan-mediated gene delivery through combination with phiC31 integrase
Ana Vanessa Vieira Oliveira1, Gabriela Araújo Silva2, Daniel C Chung3
1Centre for Molecular and Structural Biomedicine, Institute for Biotechnology and Bioengineering (CBME/IBB), University of Algarve, Faro 8005-139, Portugal; Doctoral Program in Biomedical Sciences, Department of Biomedical Sciences and Medicine, University of Algarve, Faro 8005-139, Portugal; F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, United States.
Abstract:
Gene transfer efficiency and expression stability are key factors to a successful gene therapy approach. In the present work we have developed a combined system for gene transfer that integrates well established non-viral polymeric vectors based on chitosan particles with the properties of phiC31-integrase that promotes a relatively non-immunogenic, site-specific integration, with sustained gene expression. Simultaneously, to overcome one of the major limitations in adeno-associated virus mediated gene transfer--the delivery of large genes--we have tested the capacity of our non-viral vectors to incorporate a large (8 Kb) transgene. Polyplexes were extensively characterized for their size, surface charge, morphology, pDNA complexation, transfection efficiency and transgene expression in vitro using HEK293 cells. Co-transfection with integrase was done by complexation in a single polyplex preparation or the use of two separate polyplex preparations. Transgene expression, GFP and CEP290 (1Kb and 8 Kb, respectively), was evaluated by fluorescence microscopy, flow cytometry and Western blot analysis. DNA complexation efficiency, particle size and morphology were consistent with gene delivery for all formulations. In contrast, transfection efficiency and transgene expression varied with polymer and polyplex size. Following delivery by chitosan polyplexes, high levels of GFP expression were still visible 16 weeks post-transfection and over-expression of the large transgene was detected at least 6 weeks post-transfection. Polyplexes incorporating phiC1 integrase demonstrate prolonged gene expression of both small (GFP, 1 Kb) and large genes (CEP290, 8Kb). This approach, using a combined strategy of polymers and integrase may overcome the size limitation found in commonly used adeno-associated virus mediated gene transfer techniques, while maintaining a high safety profile and prolonged, sustained gene expression, thus constituting an alternative for gene delivery.
