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Novel lentiviral vectors with mutated reverse transcriptase for mRNA delivery of TALE nucleases.
- Source :
-
Scientific reports [Sci Rep] 2014 Sep 18; Vol. 4, pp. 6409. Date of Electronic Publication: 2014 Sep 18. - Publication Year :
- 2014
-
Abstract
- TAL-effector nucleases (TALENs) are attractive tools for sequence-specific genome modifications, but their delivery still remains problematic. It is well known that the presence of multiple sequence repeats in TALEN genes hampers the use of lentiviral vectors. We report that lentiviral vectors readily package full-length vector mRNAs encoding TALENs, but recombination during reverse transcription prevents successful delivery. We reasoned that preventing reverse transcription of lentiviral-vector RNA would allow transfer of TALENs as mRNA. We demonstrate that lentiviral particles containing genetically inactivated reverse transcriptase (RT) mediated efficient transduction of cultured cells and supported transient transgene expression. For proof-of-principle, we transferred CCR5- and TCR-specific TALEN pairs for efficient targeted genome editing and abrogated expression for each of the receptor proteins in different cell lines. Combining the high specificity of TALENs with efficient lentiviral gene delivery should advance genome editing in vitro and potentially in vivo, and RT-deficient lentiviral vectors may be useful for transient expression of various other genes-of-interest.
- Subjects :
- Drug Delivery Systems
Endonucleases genetics
Gene Expression Regulation
Gene Transfer Techniques
Genome, Human
HEK293 Cells
Humans
RNA, Messenger genetics
Receptors, CCR5 genetics
Receptors, CCR5 metabolism
Transduction, Genetic
Endonucleases metabolism
Genetic Engineering
Genetic Vectors
HIV Reverse Transcriptase genetics
Lentivirus genetics
Mutation genetics
RNA, Messenger metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 2045-2322
- Volume :
- 4
- Database :
- MEDLINE
- Journal :
- Scientific reports
- Publication Type :
- Academic Journal
- Accession number :
- 25230987
- Full Text :
- https://doi.org/10.1038/srep06409