Improving Transcriptional Termination of Self-inactivating Gamma-retroviral and Lentiviral Vectors
Open accessAbstract
Adverse events relating to insertional mutagenesis have reinforced the interest in self-inactivating (SIN) gamma-retroviral and lentiviral vectors without enhancer-promoter sequences in the U3 region of the long terminal repeats. However, SIN vectors suffer from leaky transcriptional termination, increasing the probability of read-through into cellular genes. To improve 3' end processing, we incorporated seven upstream polyadenylation enhancer elements (or upstream sequence elements, USEs) derived from viral or cellular genes into the 3' U3 region of gamma-retroviral and lentiviral SIN vectors. A 100-base-pair sequence representing a recombinant direct repeat of the USE derived from simian virus 40 (2xSV USE) gave the best results, improving both titer and gene expression. In both gamma-retroviral and lentiviral SIN vectors, the 2xSV USE partially substituted for effects provided by the much larger post-transcriptional regulatory element derived from woodchuck hepatitis virus (wPRE). By northern blot and reporter assays, we found that the 2xSV USE greatly improved proper messenger RNA (mRNA) processing at the retroviral termination signal. Importantly, the 2xSV USE was superior to the wPRE in suppressing transcriptional read-through, improving not only vector efficiency but potentially also biosafety.
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The upstream element in the late SV40 polyadenylation signal enhances function to a greater extent in a tandem arrangement than when alone.
“A previous study using lentiviral vectors showed that the upstream element in the late SV40 polyadenylation signal enhances the function of the polyadenylation signal to a greater extent in a tandem arrangement than when alone [21].”
From Optimization of AAV expression cassettes to improve packaging capacity and transgene expression in neurons (2014) · cites this paper as [21]
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“Polyadenylation Size Description, Strength References SV40 late 135 +++ [139] 2x SV40 late 100 ++/+++ [169] bGHpolyA 250 ++ [149] 2x sNRP1 34 +/++ [167] Rabbit gbpA 56 Rabbit β-globin [149] spA 49 +/++ (7x lower than bGHpolyA, 3x lower than SV40 late) [139,149] hGHpolyA 624 + [41,170,171] 1x sNRP1 17 + [167]”
Almarza, D., Bussadori, G., Navarro, M., Mavilio, F., Larcher, F., & Murillas, R. (2011) · Gene Therapy · doi:10.1038/gt.2011.12 · Cited by 56 · see citing works
Hager, S., Frame, F. M., Collins, A. T., Burns, J. E., & Maitland, N. J. (2008) · Human Gene Therapy · doi:10.1089/hum.2007.165 · Cited by 30 · see citing works
- Authors
- Schambach, A., Galla, M., Maetzig, T., Loew, R., & Baum, C.
- Affiliations
- Medizinische Hochschule Hannover (DE)
- Cincinnati Children's Hospital Medical Center (US)
- Corresponding author
- Baum, Christopher
- Venue
- Molecular Therapy
- Publisher
- Elsevier BV
- Published
- 2007-06
- Volume
- 15
- Issue
- 6
- Pages
- 1167-1173
- ISSN
- 1525-0016 (ISSN-L)
- OpenAlex
- W2075706751
- Cited by
- 127
- References
- 0
- Citations by year
- 2026: 3 · 2025: 3 · 2024: 3 · 2023: 2 · 2022: 2 · 2021: 5 · 2020: 4 · 2019: 6 · 2018: 3 · 2017: 8 · 2016: 5 · 2015: 5 · 2014: 5 · 2013: 8 · 2012: 8
- FWCI
- 6.63
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Molecular Therapy (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Funders
- European Commission
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Keywords
- gammaretroviral vectors, lentiviral vectors, transcription termination, simian virus 40, woodchuck hepatitis virus, long terminal repeat, insertional mutagenesis, read-through transcription, vector titer, gene expression, biosafety
- MeSH terms
- Animals, Cell Line, Cells, Cultured, Enhancer Elements, Genetic, Flow Cytometry, Genetic Vectors, Humans, Mice, Inbred C57BL, Plasmids, Promoter Regions, Genetic, Retroviridae, RNA, Messenger, Simian virus 40, Transcription, Genetic, Virus Replication, Lentivirus, Polymerase Chain Reaction, Terminal Repeat Sequences, Polyadenylation, Virus Inactivation, Mice
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