Journal
JAIDS-JOURNAL OF ACQUIRED IMMUNE DEFICIENCY SYNDROMES
Volume 74, Issue 2, Pages 229-241Publisher
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1097/QAI.0000000000001190
Keywords
HIV-1; CCR5; genome editing; TALEN; and ZFN
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Funding
- Beijing Municipal Science and Technology Commission [Z121100005212002]
- National Natural Science Foundation Award [81530065]
- Ministry of Science and Technology of China [2014CB5425000-03]
- Janssen Investigator Award
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Transcription activator-like effector nuclease (TALEN) represents a valuable tool for genomic engineering due to its single-nucleotide precision, high nuclease activity, and low cytotoxicity. We report here systematic design and characterization of 28 novel TALENs targeting multiple regions of CCR5 gene (CCR5-TALEN) which encodes the co-receptor critical for entry of human immunodeficiency virus type I (HIV-1). By systemic characterization of these CCR5-TALENs, we have identified one (CCR5-TALEN-515) with higher nuclease activity, specificity, and lower cytotoxicity compared with zinc-finger nuclease (CCR5-ZFN) currently undergoing clinical trials. Sequence analysis of target cell line GHOST-CCR5-CXCR4 and human primary CD4(+) T cells showed that the double-strand breaks at the TALEN targeted sites resulted in truncated or nonfunctional CCR5 proteins thereby conferring protection against HIV-1 infection in vitro. None of the CCR5-TALENs had detectable levels of off-target nuclease activity against the homologous region in CCR2 although substantial level was identified for CCR5-ZFN in the primary CD4(+) T cells. Our results suggest that the CCR5-TALENs identified here are highly functional nucleases that produce protective genetic alterations to human CCR5. Application of these TALENs directly to the primary CD4(+) T cells and CD34(+) hematopoietic stem cells (HSCs) of infected individuals could help to create an immune system resistant to HIV-1 infection, recapitulating the success of Berlin patient and serving as an essential first step towards a functional cure of AIDS.
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