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Overcoming promoter competition in packaging cells improves production of self-inactivating retroviral vectors
- Source :
- Gene Therapy. 13:1524-1533
- Publication Year :
- 2006
- Publisher :
- Springer Science and Business Media LLC, 2006.
-
Abstract
- Retroviral vectors with self-inactivating (SIN) long-terminal repeats not only increase the autonomy of the internal promoter but may also reduce the risk of insertional upregulation of neighboring alleles. However, gammaretroviral as opposed to lentiviral packaging systems produce suboptimal SIN vector titers, a major limitation for their clinical use. Northern blot data revealed that low SIN titers were associated with abundant transcription of internal rather than full-length transcripts in transfected packaging cells. When using the promoter of Rous sarcoma virus or a tetracycline-inducible promoter to generate full-length transcripts, we obtained a strong enhancement in titer (up to 4 x 10(7) transducing units per ml of unconcentrated supernatant). Dual fluorescence vectors and Northern blots revealed that promoter competition is a rate-limiting step of SIN vector production. SIN vector stocks pseudotyped with RD114 envelope protein had high transduction efficiency in human and non-human primate cells. This study introduces a new generation of efficient gammaretroviral SIN vectors as a platform for further optimizations of retroviral vector performance.
- Subjects :
- Male
T-Lymphocytes
viruses
Genetic enhancement
Genetic Vectors
Antigens, CD34
Transfection
Cell Line
Viral vector
Transduction (genetics)
Transduction, Genetic
Transcription (biology)
Genetics
Animals
Northern blot
Promoter Regions, Genetic
Molecular Biology
Rous sarcoma virus
biology
Terminal Repeat Sequences
Genetic Therapy
Flow Cytometry
biology.organism_classification
Macaca mulatta
Molecular biology
Titer
Gene Expression Regulation
Virus Inactivation
Molecular Medicine
Gammaretrovirus
Genetic Engineering
Plasmids
Subjects
Details
- ISSN :
- 14765462 and 09697128
- Volume :
- 13
- Database :
- OpenAIRE
- Journal :
- Gene Therapy
- Accession number :
- edsair.doi.dedup.....66cf2e98d81f15e2ac73c13bb478df62
- Full Text :
- https://doi.org/10.1038/sj.gt.3302807