Dataset Information

CCR5 editing by Staphylococcus aureus Cas9 in human primary CD4⁺ T cells and hematopoietic stem/progenitor cells promotes HIV-1 resistance and CD4⁺ T cell enrichment in humanized mice.

ABSTRACT:

Background

The chemokine receptor CCR5, which belongs to the superfamily of G protein-coupled receptors, is the major co-receptor for HIV-1 entry. Individuals with a homozygous CCR5?32 mutation have a long lasting and increased resistance to HIV-1 infection. Therefore, CCR5 represents an optimal target for HIV-1/AIDS gene therapy. The CRISPR/Cas9 system has been developed as one of the most efficacious gene editing tools in mammalian cells and the small-sized version from Staphylococcus aureus (SaCas9) has an advantage of easier delivery compared to the most commonly used version from Streptococcus pyogenes Cas9 (SpCas9).

Results

Here, we demonstrated that CCR5 could be specifically and efficiently edited by CRISPR/SaCas9 together with two sgRNAs, which were identified through a screening of 13 sgRNAs. Disruption of CCR5 expression by lentiviral vector-mediated CRISPR/SaCas9 led to increased resistance against HIV-1 infection in human primary CD4⁺ T cells. Moreover, humanized mice engrafted with CCR5-disrupted CD4⁺ T cells showed selective survival and enrichment when challenged with CCR5 (R5)-tropic HIV-1 in comparison to mock-treated CD4⁺ T cells. We also observed CCR5 could be targeted by CRISPR/SaCas9 in human CD34⁺ hematopoietic stem/progenitor cells without obvious differentiation deficiencies.

Conclusions

This work provides an alternative approach to disrupt human CCR5 by CRISPR/SaCas9 for a potential gene therapy strategy against HIV-1/AIDS.

SUBMITTER: Xiao Q

PROVIDER: S-EPMC6560749 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

CCR5 editing by Staphylococcus aureus Cas9 in human primary CD4<sup>+</sup> T cells and hematopoietic stem/progenitor cells promotes HIV-1 resistance and CD4<sup>+</sup> T cell enrichment in humanized mice.

Xiao Qiaoqiao Q Chen Shuliang S Wang Qiankun Q Liu Zhepeng Z Liu Shuai S Deng Huan H Hou Wei W Wu Dongcheng D Xiong Yong Y Li Jiafu J Guo Deyin D Guo Deyin D

Retrovirology 20190611 1

<h4>Background</h4>The chemokine receptor CCR5, which belongs to the superfamily of G protein-coupled receptors, is the major co-receptor for HIV-1 entry. Individuals with a homozygous CCR5Δ32 mutation have a long lasting and increased resistance to HIV-1 infection. Therefore, CCR5 represents an optimal target for HIV-1/AIDS gene therapy. The CRISPR/Cas9 system has been developed as one of the most efficacious gene editing tools in mammalian cells and the small-sized version from Staphylococcus ...[more]

PMID: 31186067

Similar Datasets

Project description:UnlabelledGene-engineered CD34(+) hematopoietic stem and progenitor cells (HSPCs) can be used to generate an HIV-1-resistant immune system. However, a certain threshold of transduced HSPCs might be required for transplantation into mice for creating an HIV-resistant immune system. In this study, we combined CCR5 knockdown by a highly efficient microRNA (miRNA) lentivector with pretransplantation selection of transduced HSPCs to obtain a rather pure population of gene engineered CD34(+) cells. Low-level transduction of HSPCs and subsequent sorting by flow cytometry yielded >70% transduced cells. Mice transplanted with these cells showed functional and persistent resistance to a CCR5-tropic HIV strain: viral load was significantly decreased over months, and human CD4(+) T cells were preserved. In one mouse, viral mutations, resulting presumably in a CXCR4-tropic strain, overcame HIV resistance. Our results suggest that HSPC-based CCR5 knockdown may lead to efficient control of HIV in vivo. We overcame a major limitation of previous HIV gene therapy in humanized mice in which only a proportion of the cells in chimeric mice in vivo are anti-HIV engineered. Our strategy underlines the promising future of gene engineering HIV-resistant CD34(+) cells that produce a constant supply of HIV-resistant progeny.ImportanceMajor issues in experimental long-term in vivo HIV gene therapy have been (i) low efficacy of cell transduction at the time of transplantation and (ii) transduction resulting in multiple copies of heterologous DNA in target cells. In this study, we demonstrated the efficacy of a transplantation approach with a selection step for transduced cells that allows transplantation of an enriched population of HSPCs expressing a single (low) copy of a CCR5 miRNA. Efficient maintenance of CD4(+) T cells and a low viral titer resulted only when at least 70% of the HIV target cells were genetically modified. These findings imply that clinical protocols of HIV gene therapy require a selective enrichment of genetically targeted cells because positive selection of modified cells is likely to be insufficient below this threshold. This selection approach may be beneficial not only for HIV patients but also for other patients requiring transplantation of genetically modified cells.

Dataset Information

CCR5 editing by Staphylococcus aureus Cas9 in human primary CD4⁺ T cells and hematopoietic stem/progenitor cells promotes HIV-1 resistance and CD4⁺ T cell enrichment in humanized mice.

Background

Results

Conclusions

Publications

CCR5 editing by Staphylococcus aureus Cas9 in human primary CD4<sup>+</sup> T cells and hematopoietic stem/progenitor cells promotes HIV-1 resistance and CD4<sup>+</sup> T cell enrichment in humanized mice.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

CCR5 editing by Staphylococcus aureus Cas9 in human primary CD4+ T cells and hematopoietic stem/progenitor cells promotes HIV-1 resistance and CD4+ T cell enrichment in humanized mice.

Background

Results

Conclusions

Publications

CCR5 editing by Staphylococcus aureus Cas9 in human primary CD4<sup>+</sup> T cells and hematopoietic stem/progenitor cells promotes HIV-1 resistance and CD4<sup>+</sup> T cell enrichment in humanized mice.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

CCR5 editing by Staphylococcus aureus Cas9 in human primary CD4⁺ T cells and hematopoietic stem/progenitor cells promotes HIV-1 resistance and CD4⁺ T cell enrichment in humanized mice.