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Research Article

Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection

Lin Ye, Jiaming Wang, Ashley I. Beyer, Fernando Teque, Thomas J. Cradick, Zhongxia Qi, Judy C. Chang, Gang Bao, Marcus O. Muench, Jingwei Yu, Jay A. Levy, and Yuet Wai Kan
  1. aDepartment of Medicine and Institute for Human Genetics,
  2. fThe Liver Center,
  3. bDepartment of Laboratory Medicine, and
  4. dDivision of Hematology/Oncology, University of California, San Francisco, CA 94143;
  5. cBlood Systems Research Institute, San Francisco, CA 94118; and
  6. eDepartment of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322

See allHide authors and affiliations

PNAS first published June 9, 2014; https://doi.org/10.1073/pnas.1407473111
Lin Ye
aDepartment of Medicine and Institute for Human Genetics,
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  • For correspondence: lin.ye@ucsf.edu yw.kan@ucsf.edu
Jiaming Wang
aDepartment of Medicine and Institute for Human Genetics,
bDepartment of Laboratory Medicine, and
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Ashley I. Beyer
cBlood Systems Research Institute, San Francisco, CA 94118; and
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Fernando Teque
aDepartment of Medicine and Institute for Human Genetics,
dDivision of Hematology/Oncology, University of California, San Francisco, CA 94143;
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Thomas J. Cradick
eDepartment of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322
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Zhongxia Qi
bDepartment of Laboratory Medicine, and
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Judy C. Chang
aDepartment of Medicine and Institute for Human Genetics,
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Gang Bao
eDepartment of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322
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Marcus O. Muench
bDepartment of Laboratory Medicine, and
cBlood Systems Research Institute, San Francisco, CA 94118; and
fThe Liver Center,
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Jingwei Yu
bDepartment of Laboratory Medicine, and
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Jay A. Levy
dDivision of Hematology/Oncology, University of California, San Francisco, CA 94143;
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Yuet Wai Kan
aDepartment of Medicine and Institute for Human Genetics,
bDepartment of Laboratory Medicine, and
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  • For correspondence: lin.ye@ucsf.edu yw.kan@ucsf.edu
  1. Contributed by Yuet Wai Kan, April 29, 2014 (sent for review March 26, 2014)

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Significance

Patients homozygous for the C-C chemokine receptor type 5 (CCR5) gene with 32-bp deletions (Δ32) are resistant to HIV infection. Using the piggyBac technology plus transcription activator-like effector nucleases or clustered regularly interspaced short palindromic repeats-Cas9, the authors report, to our knowledge, for the first time in induced pluripotent stem cells (iPSCs) the efficient and seamless derivation of a homozygous CCR5Δ32 mutation, exactly mimicking the natural mutation. Monocytes and macrophages differentiated from these mutated iPSCs in vitro are resistant to HIV infection. This approach can be applied in the future toward the functional cure of HIV infection. The findings are also of great interest to researchers in many fields who wish to correct or introduce mutations in specific genes.

Abstract

Individuals homozygous for the C-C chemokine receptor type 5 gene with 32-bp deletions (CCR5Δ32) are resistant to HIV-1 infection. In this study, we generated induced pluripotent stem cells (iPSCs) homozygous for the naturally occurring CCR5Δ32 mutation through genome editing of wild-type iPSCs using a combination of transcription activator-like effector nucleases (TALENs) or RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 together with the piggyBac technology. Remarkably, TALENs or CRISPR-Cas9–mediated double-strand DNA breaks resulted in up to 100% targeting of the colonies on one allele of which biallelic targeting occurred at an average of 14% with TALENs and 33% with CRISPR. Excision of the piggyBac using transposase seamlessly reproduced exactly the naturally occurring CCR5Δ32 mutation without detectable exogenous sequences. We differentiated these modified iPSCs into monocytes/macrophages and demonstrated their resistance to HIV-1 challenge. We propose that this strategy may provide an approach toward a functional cure of HIV-1 infection.

  • homologous recombination
  • TTAA site
  • off-site target
  • cellular therapy

Footnotes

  • ↵1To whom correspondence may be addressed. E-mail: lin.ye{at}ucsf.edu or yw.kan{at}ucsf.edu.
  • Author contributions: L.Y., J.A.L., and Y.W.K. designed research; L.Y., J.W., A.I.B., F.T., T.J.C., Z.Q., and J.C.C. performed research; G.B., M.O.M., J.Y., and J.A.L. contributed new reagents/analytic tools; L.Y., T.J.C., Z.Q., J.A.L., and Y.W.K. analyzed data; and L.Y. and Y.W.K. wrote the paper.

  • The authors declare no conflict of interest.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1407473111/-/DCSupplemental.

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Seamless CCR5∆32 mutation resistant to HIV
Lin Ye, Jiaming Wang, Ashley I. Beyer, Fernando Teque, Thomas J. Cradick, Zhongxia Qi, Judy C. Chang, Gang Bao, Marcus O. Muench, Jingwei Yu, Jay A. Levy, Yuet Wai Kan
Proceedings of the National Academy of Sciences Jun 2014, 201407473; DOI: 10.1073/pnas.1407473111

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Seamless CCR5∆32 mutation resistant to HIV
Lin Ye, Jiaming Wang, Ashley I. Beyer, Fernando Teque, Thomas J. Cradick, Zhongxia Qi, Judy C. Chang, Gang Bao, Marcus O. Muench, Jingwei Yu, Jay A. Levy, Yuet Wai Kan
Proceedings of the National Academy of Sciences Jun 2014, 201407473; DOI: 10.1073/pnas.1407473111
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