Efficient clinical scale gene modification via zinc finger nuclease-targeted disruption of the HIV co-receptor CCR5

Hum Gene Ther. 2013 Mar;24(3):245-58. doi: 10.1089/hum.2012.172. Epub 2013 Mar 6.


Since HIV requires CD4 and a co-receptor, most commonly C-C chemokine receptor 5 (CCR5), for cellular entry, targeting CCR5 expression is an attractive approach for therapy of HIV infection. Treatment of CD4(+) T cells with zinc-finger protein nucleases (ZFNs) specifically disrupting chemokine receptor CCR5 coding sequences induces resistance to HIV infection in vitro and in vivo. A chimeric Ad5/F35 adenoviral vector encoding CCR5-ZFNs permitted efficient delivery and transient expression following anti-CD3/anti-CD28 costimulation of T lymphocytes. We present data showing CD3/CD28 costimulation substantially improved transduction efficiency over reported methods for Ad5/F35 transduction of T lymphocytes. Modifications to the laboratory scale process, incorporating clinically compatible reagents and methods, resulted in a robust ex vivo manufacturing process capable of generating >10(10) CCR5 gene-edited CD4+ T cells from healthy and HIV+ donors. CD4+ T-cell phenotype, cytokine production, and repertoire were comparable between ZFN-modified and control cells. Following consultation with regulatory authorities, we conducted in vivo toxicity studies that showed no detectable ZFN-specific toxicity or T-cell transformation. Based on these findings, we initiated a clinical trial testing the safety and feasibility of CCR5 gene-edited CD4+ T-cell transfer in study subjects with HIV-1 infection.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenoviruses, Human / genetics
  • Adoptive Transfer
  • Animals
  • CD28 Antigens / immunology
  • CD3 Complex / immunology
  • DNA Restriction Enzymes / genetics*
  • DNA Restriction Enzymes / metabolism
  • Female
  • Genetic Vectors / administration & dosage
  • Genetic Vectors / adverse effects
  • Genetic Vectors / genetics
  • Genetic Vectors / standards*
  • HIV Infections / genetics*
  • HIV Infections / immunology*
  • HIV Infections / therapy
  • Humans
  • Lymphocyte Activation / immunology
  • Male
  • Mice
  • Phenotype
  • Receptors, CCR5 / genetics*
  • Receptors, CCR5 / immunology
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism
  • Transduction, Genetic / methods
  • Transduction, Genetic / standards
  • Transplantation, Heterologous
  • Zinc Fingers / genetics*


  • CD28 Antigens
  • CD3 Complex
  • Receptors, CCR5
  • DNA Restriction Enzymes