Sustained Gene Expression in the Retina by Improved Episomal Vectors
Open accessAbstract
Gene and cellular therapies are nowadays part of therapeutic strategies for the treatment of diverse pathologies. The drawbacks associated with gene therapy-low levels of transgene expression, vector loss during mitosis, and gene silencing-need to be addressed. The pEPI-1 and pEPito family of vectors was developed to overcome these limitations. It contains a scaffold/matrix attachment region, which anchors its replication to cell division in eukaryotic cells while in an extrachromosomal state and is less prone to silencing, due to a lower number of CpG motifs. Recent success showed that ocular gene therapy is an important tool for the treatment of several diseases, pending the overcome of the aforementioned limitations. To achieve sustained gene delivery in the retina, we evaluated several vectors based on pEPito and pEPI-1 for their ability to sustain transgene expression in retinal cells. These vectors stably transfected and replicated in retinal pigment epithelial (RPE) cells. Expression levels were promoter dependent with constitutive promoters cytomegalovirus immediate early promoter (CMV) and human CMV enhancer/human elongation factor 1 alpha promoter yielding the highest levels of transgene expression compared with the retina-specific RPE65 promoter. When injected in C57Bl6 mice, transgene expression was sustained for at least 32 days. Furthermore, the retina-specific RPE65 promoter showed higher efficiency in vivo compared to in vitro. In this study, we demonstrate that by combining tissue-specific promoters with a mitotic stable system, less susceptible to epigenetic silencing such as pEPito-based plasmids, we can achieve prolonged gene expression and a sustained therapeutic effect.
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Both pEPI-1 and pEPito plasmids were able to transduce RPE cells, with sustained expression observed for up to 3 months in vitro and 32 days in vivo.
“Both plasmids were able to transduce RPE cells, with a sustained expression observed for up to 3 months in vitro and 32 days in vivo (the last observation time-point).”
From Immune responses to gene editing by viral and non-viral delivery vectors used in retinal gene therapy (2022) · cites this paper as [70]
In vivo, GFP expression was lower in the pEPI-1 group compared to the pEPito group, which the authors attributed to less CpG motifs in pEPito.
“In vivo, the GFP expression was lower in the pEPI-1 group compared to the pEPito group, which the authors attributed to less CpG motifs in pEPito.”
From Immune responses to gene editing by viral and non-viral delivery vectors used in retinal gene therapy (2022) · cites this paper as [70]
Other aspects of inflammation were not observed in the pEPI-1/pEPito study, most likely because the study used an immunosuppressive regimen.
“However, other aspects of inflammation were not observed, most likely because this study used an immunosuppressive regimen [70].”
From Immune responses to gene editing by viral and non-viral delivery vectors used in retinal gene therapy (2022) · cites this paper as [70]
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- Authors
- Calado, S. M., Oliveira, A. V., Machado, S., Haase, R., & Silva, G. A.
- Affiliations
- University of Algarve (PT)
- Ludwig-Maximilians-Universität München (DE)
- Corresponding author
- Silva, Gabriela A.
- Venue
- Tissue Engineering Part A
- Publisher
- SAGE Publications
- Published
- 2014-10
- Volume
- 20
- Issue
- 19-20
- Pages
- 2692-2698
- ISSN
- 1937-3341 (ISSN-L)
- 1937-335X
- PMC
- PMC4195477
- OpenAlex
- W2019124146
- Cited by
- 24
- References
- 26
- Citations by year
- 2026: 1 · 2024: 5 · 2023: 1 · 2022: 4 · 2021: 2 · 2020: 2 · 2019: 1 · 2018: 1 · 2017: 2 · 2016: 2 · 2015: 2 · 2014: 1
- FWCI
- 0.96
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Tissue Engineering Part A (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Portuguese National Funding Agency for Science, Research and Technology (RCAAP Project by FCT) (submittedVersion, other-oa)
- Funders
- European Commission (SFRH/BD/70318/2010, 249314)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Retinal Development and Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Keywords
- episomal vectors, ocular gene therapy, retinal gene expression, retinal pigment epithelium, transgene silencing, epigenetic silencing, tissue-specific promoters, CMV promoter, sustained transgene expression, extrachromosomal plasmids
- MeSH terms
- Animals, Genetic Vectors, Humans, Plasmids, Transfection, Genetic Therapy, Gene Expression, Mice, Retinal Pigment Epithelium
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