Skip to main content

Volume 9 Supplement 2

AIDS Vaccine 2012

Engineering SIV-resistant macaque hematopoietic stem cells and CD4+ T cells with CCR5-specific zinc-finger nucleases

Background

CCR5 is the major HIV co-receptor, and individuals homozygous for a 32-bp deletion in CCR5 are resistant to infection by CCR5-tropic HIV-1. Zinc finger nuclease (ZFN) technology is a class of engineered DNA-binding proteins facilitating targeted genome editing by binding to a user-specific locus and causing a double-strand break in the region of interest. As a result, the gene of interest targeted by ZFN cleavage is disrupted. We investigated the ability of a Ccr5 gene-specific ZFN to establish SIV-resistant CD4+ T cells and hematopoietic progenitor cells isolated from macaques.

Methods

Bone marrow and blood samples were obtained from ten naïve uninfected rhesus macaques. Immunomagnetic enrichments were performed to isolate hematopoietic stem/progenitor cells and CD4+ T cells using magnetic-beads. Purified cells were nucleofected with RNAs encoding for a Ccr5-specific ZFNs.

Results

We successfully engineered CCR5-modified macaque CD4+ T lymphocytes that were resistant to in vitro infection with SIVmac239, SIVmac251, and CCR5-tropism SIVagm. CD4+ T lymphocytes that incorporated RNAs encoding for the Ccr5-specific set of ZFNs marked resistance to SIV infection as showed by lack of p27 expression in culture supernatants. Infection of non-modified CD4+ T lymphocytes led to significant p27 production from day 5 post-infection with a pick at day 7 post-infection. We developed and optimized the conditions for proper isolation, expansion and in vitro differentiation of macaque hematopoietic stem cells to be used for the generation of SIV-resistant macaque hematopoietic stem cells using our ZFNs. We successfully engineered CD34+ hematopoietic stem cells using nucleofection of RNAs encoding for a Ccr5-specific ZFNs.

Conclusion

We demonstrated the feasibility of using ZFN technology to establish CD4+ and hematopoietic stem cells resistant to SIV infection in macaque model. The generation of a nonhuman primate model using this modern molecular-based strategy might significantly help in the design of new therapies to prevent viral infection in human.

Author information

Affiliations

Authors

Corresponding author

Correspondence to M Gauduin.

Rights and permissions

Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and Permissions

About this article

Cite this article

Bonello, G., Lentz, P., Salas, M. et al. Engineering SIV-resistant macaque hematopoietic stem cells and CD4+ T cells with CCR5-specific zinc-finger nucleases. Retrovirology 9, P226 (2012). https://doi.org/10.1186/1742-4690-9-S2-P226

Download citation

Keywords

  • Hematopoietic Stem Cell
  • Rhesus Macaque
  • Genome Editing
  • Hematopoietic Progenitor Cell
  • Zinc Finger Nuclease