Novel lentiviral vectors with mutated reverse transcriptase for mRNA delivery of TALE nucleases

• Published in: Scientific reports Vol. 4; no. 1; p. 6409
• Main Authors: Mock, Ulrike, Riecken, Kristoffer, Berdien, Belinda, Qasim, Waseem, Chan, Emma, Cathomen, Toni, Fehse, Boris
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 18.09.2014; Nature Publishing Group
• Subjects: 42; 42/41; 42/44; 631/61/201/2110; 631/61/2300/1514; CCR5 protein; Cell lines; Drug Delivery Systems; Endonucleases - genetics; Endonucleases - metabolism; Expression vectors; Gene Expression Regulation; Gene transfer; Gene Transfer Techniques; Genetic Engineering; Genetic Vectors; Genome editing; Genome, Human; Genomes; HEK293 Cells; HIV Reverse Transcriptase - genetics; Humanities and Social Sciences; Humans; Lentivirus - genetics; multidisciplinary; Mutation - genetics; Nuclease; Nucleotide sequence; Receptors, CCR5 - genetics; Receptors, CCR5 - metabolism; Recombination; Reverse transcription; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA-directed DNA polymerase; Science; Transcription activator-like effector nucleases; Transduction, Genetic
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep06409

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.