title: Novel lentiviral vectors with mutated reverse transcriptase for mRNA delivery of TALE nucleases.
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.
authors.affiliation: Research Dept. Cell and Gene Therapy, Clinic for Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Mock
authors.affiliation: Research Dept. Cell and Gene Therapy, Clinic for Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg, Germany.
authors.forename: Kristoffer
authors.initials: K
authors.lastname: Riecken
authors.affiliation: Research Dept. Cell and Gene Therapy, Clinic for Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg, Germany.
authors.forename: Belinda
authors.initials: B
authors.lastname: Berdien
authors.affiliation: Molecular Immunology Unit, UCL Institute of Child Health, London, WC1N 1EH, United Kingdom.
authors.forename: Waseem
authors.initials: W
authors.lastname: Qasim
authors.affiliation: Molecular Immunology Unit, UCL Institute of Child Health, London, WC1N 1EH, United Kingdom.
authors.forename: Emma
authors.initials: E
authors.lastname: Chan
authors.affiliation: 1] Institute for Cell and Gene Therapy, University Medical Centre Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany [2] Centre for Chronic Immunodeficiency, University Medical Centre Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany.
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
authors.affiliation: Research Dept. Cell and Gene Therapy, Clinic for Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg, Germany.
authors.forename: Boris
authors.initials: B
authors.lastname: Fehse
mesh_terms: D016503:Drug Delivery Systems; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D005822:Genetic Vectors; D015894:Genome, Human; D057809:HEK293 Cells; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D019713:Receptors, CCR5 / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: D012333:RNA, Messenger; D019713:Receptors, CCR5; D054303:HIV Reverse Transcriptase; D004720:Endonucleases