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Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium
Published on: August 7, 2015
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Sustained gene expression in the retina by improved episomal vectors
Sofia M Calado1, Ana V Oliveira, Susana Machado
11 Doctoral Program in Biomedical Sciences, Department of Biomedical Sciences and Medicine, University of Algarve , Faro, Portugal .
Tissue Engineering. Part A
|April 2, 2014
Summary
New gene therapy vectors, pEPito and pEPI-1, offer sustained transgene expression in retinal cells. These vectors overcome limitations like gene silencing, enabling prolonged therapeutic effects for eye diseases.
Area of Science:
- Ophthalmology
- Molecular Biology
- Gene Therapy
Background:
- Gene and cellular therapies face challenges including low transgene expression, vector loss, and gene silencing.
- The pEPI-1 and pEPito vector families were developed to address these limitations by utilizing a scaffold/matrix attachment region for mitotic stability and reduced CpG motifs to prevent silencing.
- Ocular gene therapy shows promise for treating various eye diseases, but requires overcoming existing delivery and expression challenges.
Purpose of the Study:
- To evaluate pEPito and pEPI-1 based vectors for sustained transgene expression in retinal cells.
- To determine the efficacy of different promoters in achieving long-term gene expression in the retina.
- To assess the in vitro and in vivo performance of these vectors for potential therapeutic applications in ocular diseases.
Main Methods:
- Evaluation of pEPito and pEPI-1 derived vectors for stable transfection and replication in retinal pigment epithelial (RPE) cells.
- Comparison of transgene expression levels driven by constitutive promoters (CMV, hCMV enhancer/hEF1a) versus a retina-specific promoter (RPE65).
- In vivo assessment of transgene expression duration in C57Bl6 mice following vector injection.
Main Results:
- pEPito and pEPI-1 vectors demonstrated stable transfection and replication in RPE cells.
- Constitutive promoters (CMV, hCMV enhancer/hEF1a) yielded higher transgene expression in vitro compared to the RPE65 promoter.
- In vivo, transgene expression was sustained for at least 32 days, with the RPE65 promoter showing enhanced efficiency compared to in vitro studies.
- The combination of tissue-specific promoters with the pEPito mitotic stable system achieved prolonged gene expression.
Conclusions:
- pEPito-based plasmids, combined with tissue-specific promoters, offer a promising strategy for achieving prolonged gene expression in retinal cells.
- This approach can lead to sustained therapeutic effects for ocular diseases by overcoming limitations of current gene therapy vectors.
- The study highlights the potential of mitotic stable, extrachromosomal vectors for effective ocular gene therapy.
