Optimisation of a TALE nuclease targeting the HIV co-receptor CCR5 for clinical application.

Gene Therapy
Lea Isabell SchwarzeBoris Fehse

Abstract

Disruption of the C-C-Chemokine-receptor-5 (CCR5) gene induces resistance towards CCR5-tropic HIV. Here we optimised our previously described CCR5-Uco-TALEN and its delivery by mRNA electroporation. The novel variant, CCR5-Uco-hetTALEN features an obligatory heterodimeric Fok1-cleavage domain, which resulted in complete abrogation of off-target activity at previously found homodimeric as well as 7/8 in silico predicted, potential heterodimeric off-target sites, the only exception being highly homologous CCR2. Prevailing 18- and 10-bp deletions at the on-target site revealed microhomology-mediated end-joining as a major repair pathway. Notably, the CCR5Δ55-60 protein resulting from the 18-bp deletion was almost completely retained in the cytosol. Simultaneous cutting at CCR5 and CCR2 induced rearrangements, mainly 15-kb deletions between the cut sites, in up to 2% of T cells underlining the necessity to restrict TALEN expression. We optimised in vitro mRNA production and showed that CCR5-on- and CCR2 off-target activities of CCR5-Uco-hetTALEN were limited to the first 72 and 24-48 h post-mRNA electroporation, respectively. Using single-cell HRMCA, we discovered high rates of TALEN-induced biallelic gene editing of CCR5, which tr...Continue Reading

References

Dec 1, 1980·Proceedings of the National Academy of Sciences of the United States of America·B J PoieszR C Gallo
Sep 2, 1998·Proceedings of the National Academy of Sciences of the United States of America·J BitinaiteI Schildkraut
May 27, 2003·Clinical Chemistry·Carl T WittwerRobert J Pryor
Nov 23, 2006·Nature Medicine·Jason M BrenchleyDaniel C Douek
Mar 26, 2008·Molecular Therapy : the Journal of the American Society of Gene Therapy·Kristoffer WeberBoris Fehse
Jun 21, 2008·Blood·Sebastian NewrzelaDorothee von Laer
Aug 9, 2008·The Journal of Clinical Investigation·Salima Hacein-Bey-AbinaMarina Cavazzana-Calvo
Feb 14, 2009·The New England Journal of Medicine·Gero HütterEckhard Thiel
Oct 3, 2012·Proceedings of the National Academy of Sciences of the United States of America·Daniel F CarlsonScott C Fahrenkrug
Dec 18, 2012·Human Gene Therapy Methods·Ulrike MockStefan Horn
Oct 1, 2013·Cell Death and Differentiation·J ZhangYufang Shi
Jan 31, 2014·Nucleic Acids Research·Kerstin CornilsBoris Fehse
Mar 7, 2014·The New England Journal of Medicine·Pablo TebasCarl H June
Mar 19, 2014·Virology·Caroline P Passaes, Asier Sáez-Cirión
May 6, 2014·Nucleic Acids Research·Claudio MussolinoToni Cathomen
Jun 9, 2014·Lancet·Gary MaartensSharon R Lewin
Jun 28, 2014·Nature Methods·Sangsu BaeJin-Soo Kim
Dec 17, 2014·Nature Biotechnology·Richard L FrockFrederick W Alt
Dec 17, 2014·Nature Biotechnology·Shengdar Q TsaiJ Keith Joung

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Citations

Sep 23, 2021·Gene Therapy·Claudio Mussolino, Patrick Harrison

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