Extensive Methylation of Promoter Sequences Silences Lentiviral Transgene Expression During Stem Cell Differentiation In Vivo
Open accessAbstract
Lentiviral vectors (LV) are widely used to stably transfer genes into target cells investigating or treating gene functions. In addition, gene transfer into early murine embryos may be improved to efficiently generate transgenic mice. We applied lentiviral gene transfer to generate a mouse model transgenic for SET binding protein-1 (Setbp1) and enhanced green fluorescent protein (eGFP). Neither transgenic founders nor their vector-positive offspring transcribed or expressed the transgenes. Bisulfite sequencing of the internal spleen focus-forming virus (SFFV) promoter demonstrated extensive methylation of all analyzed CpGs in the transgenic mice. To analyze the impact of Setbp1 on epigenetic silencing, embryonic stem cells (ESC) were differentiated into cardiomyocytes (CM) in vitro. In contrast to human promoters in LV, virally derived promoter sequences were strongly methylated during differentiation, independent of the transgene. Moreover, the commonly used SFFV promoter (SFFVp) was highly methylated with remarkable strength and frequency during hematopoietic differentiation in vivo in LV but less in γ-retroviral (γ-RV) backbones. In summary, we conclude that LV using an internal SFFVp are not suitable to generate transgenic mice or perform constitutive expression studies in differentiating cells. Choosing the appropriate promoter is also crucial to allow stable transgene expression in clinical gene therapy.
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Several methods have been described to limit continuous expression of lentiviral transgenes, such as natural promoter silencing and self-limiting CRISPR-Cas genetic circuits.
“Several methods have been described to limit continuous expression of lentiviral transgenes as natural promoter silencing and self-limiting CRISPR-Cas genetic circuits41,59,60.”
From Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing (2019) · cites this paper as [60]
The Discussion (page not provided) reports that methylation of the lentiviral SFFV promoter silenced transgene expression, supporting natural promoter silencing as a way expression can cease. It does not describe that silencing as a deliberate method to limit expression or discuss self-limiting CRISPR-Cas circuits. (LLM, conf=0.86)
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- Authors
- Herbst, F., Ball, C. R., Tuorto, F., Nowrouzi, A., Wang, W., Zavidij, O., Dieter, S. M., Fessler, S., van der Hoeven, F., Kloz, U., Lyko, F., Schmidt, M., von Kalle, C., & Glimm, H.
- Affiliations
- German Cancer Research Center (DE)
- Corresponding author
- Glimm, Hanno
- ORCID
- Venue
- Molecular Therapy
- Publisher
- Elsevier BV
- Published
- 2012-05
- Volume
- 20
- Issue
- 5
- Pages
- 1014-1021
- ISSN
- 1525-0016 (ISSN-L)
- PMC
- PMC3345972
- OpenAlex
- W2031887473
- Cited by
- 104
- References
- 46
- Citations by year
- 2026: 6 · 2025: 4 · 2024: 12 · 2023: 8 · 2022: 9 · 2021: 13 · 2020: 9 · 2019: 5 · 2018: 4 · 2017: 7 · 2016: 8 · 2015: 6 · 2014: 4 · 2013: 6 · 2012: 2
- FWCI
- 3.47
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Molecular Therapy (publishedVersion, cc-by-nc-nd)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Wellcome Trust
- Bundesministerium für Bildung und Forschung
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Animal Genetics and Reproduction
- Keywords
- lentiviral vectors, DNA methylation, promoter methylation, epigenetic silencing, transgene expression, SETBP1, embryonic stem cell differentiation, cardiomyocyte differentiation, hematopoietic differentiation, transgenic mice, gammaretroviral vectors, CpG methylation, gene therapy
- MeSH terms
- Animals, Carrier Proteins, Cell Differentiation, Genetic Vectors, Humans, Mice, Transgenic, Promoter Regions, Genetic, Spleen Focus-Forming Viruses, Stem Cells, Lentivirus, Sequence Analysis, DNA, Founder Effect, CpG Islands, Transgenes, DNA Methylation, Genes, Essential, Gene Silencing, Myocytes, Cardiac, Epigenesis, Genetic, Green Fluorescent Proteins, Mice
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