Generation of SIV-resistant T cells and macrophages from nonhuman primate induced pluripotent stem cells with edited CCR5 locus

D'Souza, Saritha S.; Kumar, Akhilesh; Weinfurter, Jason T.; Park, Mi Ae; Maufort, John P.; Tao, Lihong; Kang, Ho Jung; Dettle, Samuel T; Golos, Thaddeus G.; Thomson, James A.; Reynolds, Matthew R.; Slukvin, Igor I.

doi:10.1016/j.stemcr.2022.03.003

Cited by 9 publications

(9 citation statements)

References 40 publications

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“…To functionally delete CCR5 in macaque embryos, we designed gRNAs that would encompass a 24-bp deletion that has previously been shown to be essential for expressing CCR5 in non-human primates ( Chen et al, 1998 ). Our previous cell-based editing experiments confirmed successful on-target editing with functional deletion of the CCR5 gene in both human ( Kang et al, 2015 ) and macaque ( D'Souza et al, 2022 ) iPSCs. A schematic diagram of the targeting region is shown in Figure 1A .…”

Section: Resultssupporting

confidence: 59%

Whole genome sequencing of CCR5 CRISPR-Cas9-edited Mauritian cynomolgus macaque blastomeres reveals large-scale deletions and off-target edits

et al. 2023

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Introduction: Genome editing by CRISPR-Cas9 approaches offers promise for introducing or correcting disease-associated mutations for research and clinical applications. Nonhuman primates are physiologically closer to humans than other laboratory animal models, providing ideal candidates for introducing human disease-associated mutations to develop models of human disease. The incidence of large chromosomal anomalies in CRISPR-Cas9-edited human embryos and cells warrants comprehensive genotypic investigation of editing outcomes in primate embryos. Our objective was to evaluate on- and off-target editing outcomes in CCR5 CRISPR-Cas9-targeted Mauritian cynomolgus macaque embryos.Methods: DNA isolated from individual blastomeres of two embryos, along with paternal and maternal DNA, was subjected to whole genome sequencing (WGS) analysis.Results: Large deletions were identified in macaque blastomeres at the on-target site that were not previously detected using PCR-based methods. De novo mutations were also identified at predicted CRISPR-Cas9 off-target sites.Discussion: This is the first report of WGS analysis of CRISPR-Cas9-targeted nonhuman primate embryonic cells, in which a high editing efficiency was coupled with the incidence of editing errors in cells from two embryos. These data demonstrate that comprehensive sequencing-based methods are warranted for evaluating editing outcomes in primate embryos, as well as any resultant offspring to ensure that the observed phenotype is due to the targeted edit and not due to unidentified off-target mutations.

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Section: Resultssupporting

confidence: 59%

Whole genome sequencing of CCR5 CRISPR-Cas9-edited Mauritian cynomolgus macaque blastomeres reveals large-scale deletions and off-target edits

et al. 2023

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“…As a co-receptor, CCR5 facilitates cellular entry for CCR5-tropic HIV-1 viruses [ 9 , 70 ], making it an attractive target for designing therapeutic approaches for HIV. Unsurprisingly, CCR5 has been targeted through a myriad of CRISPR-based approaches in different animal models [ 71 , 72 ], but also in human cell lines and primary human cells (summarized in Table 2 ). Early reports indicated low genome editing frequencies [ 22 ] and high off-target activity [ 73 ] in plasmid reporter studies.…”

Section: Targeting Ccr5 Via Crispr/cas9-mediated Genome Editingmentioning

confidence: 99%

Closing the Door with CRISPR: Genome Editing of CCR5 and CXCR4 as a Potential Curative Solution for HIV

Heeren¹

2022

BioTech

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Human immunodeficiency virus (HIV) infection can be controlled by anti-retroviral therapy. Suppressing viral replication relies on life-long medication, but anti-retroviral therapy is not without risks to the patient. Therefore, it is important that permanent cures for HIV infection are developed. Three patients have been described to be completely cured from HIV infection in recent years. In all cases, patients received a hematopoietic stem cell (HSC) transplantation due to a hematological malignancy. The HSCs were sourced from autologous donors that expressed a homozygous mutation in the CCR5 gene. This mutation results in a non-functional receptor, and confers resistance to CCR5-tropic HIV strains that rely on CCR5 to enter host cells. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is one of the methods of choice for gene editing, and the CRISPR/Cas system has been employed to target loci of interest in the context of HIV. Here, the current literature regarding CRISPR-mediated genome editing to render cells resistant to HIV (re)-infection by knocking out the co-receptors CCR5 and CXCR4 is summarized, and an outlook is provided regarding future (research) directions.

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“…Non-human primates (NHPs), such as rhesus macaques, currently play an important role in pre-clinical PSC studies ( 106 , 107 ). NHPs are useful for evaluation of human PSC-based therapies and associated immunosuppression requirements ( 108 ), as well as useful for gene editing studies ( 109 ). We recently developed a BLT-type “primatized” mouse model ( 110 ) for evaluation of the NHP immune response prior to conducting full-scale large animal studies.…”

Section: Modeling the In Vivo Immune Response To P...mentioning

confidence: 99%

Genetic Engineering of Immune Evasive Stem Cell-Derived Islets

et al. 2022

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Genome editing has the potential to revolutionize many investigative and therapeutic strategies in biology and medicine. In the field of regenerative medicine, one of the leading applications of genome engineering technology is the generation of immune evasive pluripotent stem cell-derived somatic cells for transplantation. In particular, as more functional and therapeutically relevant human pluripotent stem cell-derived islets (SCDI) are produced in many labs and studied in clinical trials, there is keen interest in studying the immunogenicity of these cells and modulating allogeneic and autoimmune immune responses for therapeutic benefit. Significant experimental work has already suggested that elimination of Human Leukocytes Antigen (HLA) expression and overexpression of immunomodulatory genes can impact survival of a variety of pluripotent stem cell-derived somatic cell types. Limited work published to date focuses on stem cell-derived islets and work in a number of labs is ongoing. Rapid progress is occurring in the genome editing of human pluripotent stem cells and their progeny focused on evading destruction by the immune system in transplantation models, and while much research is still needed, there is no doubt the combined technologies of genome editing and stem cell therapy will profoundly impact transplantation medicine in the future.

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Generation of SIV-resistant T cells and macrophages from nonhuman primate induced pluripotent stem cells with edited CCR5 locus

Cited by 9 publications

References 40 publications

Whole genome sequencing of CCR5 CRISPR-Cas9-edited Mauritian cynomolgus macaque blastomeres reveals large-scale deletions and off-target edits

Whole genome sequencing of CCR5 CRISPR-Cas9-edited Mauritian cynomolgus macaque blastomeres reveals large-scale deletions and off-target edits

Closing the Door with CRISPR: Genome Editing of CCR5 and CXCR4 as a Potential Curative Solution for HIV

Genetic Engineering of Immune Evasive Stem Cell-Derived Islets

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