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.; Ma, Park; Maufort, John P.; Tao, Ling; Kang, Ho Jung; Golos, Thaddeus G.; Ja, Thomson; Reynolds, Matthew R.; Slukvin,

doi:10.1101/2021.05.03.442446

Cited by 3 publications

(3 citation statements)

References 35 publications

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“…To functionally delete CCR5 in macaque embryos, we designed gRNAs that would encompass a 24-bp deletion that Frontiers in Genome Editing frontiersin.org 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: Wgs Of Individual Blastomeres Produces Variable Sequence Cov...mentioning

confidence: 77%

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: Wgs Of Individual Blastomeres Produces Variable Sequence Cov...mentioning

confidence: 77%

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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“…To confirm that CRISPR/Cas9-targeted CCR5 edits also protects macaque cells from SIV infection, we generated iPSCs from MCM fibroblasts, edited their CCR5 locus, and derived T cells and macrophages. We found that T cells and macrophages produced from CCR5-edited fibroblast-derived iPSCs did not support replication of the CCR5 T cell-tropic SIVmac239 and macrophagetropic SIVmac316 simian immunodeficiency viruses, thus validating our CCR5 editing strategy [76].…”

Section: Editing Ccr5 In Mauritian Cynomolgus Macaque Embryosmentioning

confidence: 80%

CRISPR/Cas9 genome editing to create nonhuman primate models for studying stem cell therapies for HIV infection

et al. 2022

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Nonhuman primates (NHPs) are well-established basic and translational research models for human immunodeficiency virus (HIV) infections and pathophysiology, hematopoietic stem cell (HSC) transplantation, and assisted reproductive technologies. Recent advances in CRISPR/Cas9 gene editing technologies present opportunities to refine NHP HIV models for investigating genetic factors that affect HIV replication and designing cellular therapies that exploit genetic barriers to HIV infections, including engineering mutations into CCR5 and conferring resistance to HIV/simian immunodeficiency virus (SIV) infections. In this report, we provide an overview of recent advances and challenges in gene editing NHP embryos and discuss the value of genetically engineered animal models for developing novel stem cell-based therapies for curing HIV.

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“…High expression of CCR5 causes tumor cell migration and vascular invasion, which can result in distant metastasis of several types of tumor such as in breast cancers [7][8][9] , hepatocellular carcinoma [10] and prostate cancers [11][12][13] . Given its prominence in the regulation of immunity, several clinical trials targeting CCR5 are under way, involving cancers [7,13] , COVID-19 [14] ,immune de ciency diseases like HIV [15][16][17][18] , etc.…”

Section: Introductionmentioning

confidence: 99%

CT-based machine learning radiomics predict CCR5 expression level and survival in ovarian cancer

Wan

Zhou

Che

et al. 2022

Preprint

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Objective We aimed to evaluate the prognostic value of C-C motif chemokine receptor type 5 (CCR5) expression level for patients with ovarian cancer and to establish a radiomic model that can predict CCR5 expression level using The Cancer Imaging Archive (TCIA) and The Cancer Genome Atlas (TCGA) database. Methods A total of 343 cases of ovarian cancer from the TCGA were used for the gene-based prognostic analysis. 57 cases had preoperative computed tomography (CT) images stored in TCIA with genomic data in TCGA were used for radiomic feature extraction and model construction. 89 cases with both TCGA and TCIA clinical data were used for radiomic model evaluation. Results CCR5 was identified as a differentially expressed prognosis-related gene in tumor and normal sample, which were involved in the regulation of immune response and tumor invasion and metastasis. Four optimal radiomic features were selected to predict overall survival. The performance of the radiomic model for predicting the CCR5 expression level with 10-fold cross- validation achieved Area Under Curve (AUCs) of 0.770 and of 0.726, respectively, in the training and validation sets. A predictive nomogram was generated based on the total risk score of each patient, the AUCs of the time-dependent receiver operating characteristic (ROC) curve of the model was 0.8, 0.673 and 0.792 for 1-year, 3-year and 5-year, respectively. Along with clinical features, important imaging biomarkers could improve the overall survival accuracy of the prediction model. Conclusion The expression levels of CCR5 can affect the prognosis of patients with ovarian cancer. CT-based radiomics could serve as a new tool for prognosis prediction.

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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 3 publications

References 35 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

CRISPR/Cas9 genome editing to create nonhuman primate models for studying stem cell therapies for HIV infection

CT-based machine learning radiomics predict CCR5 expression level and survival in ovarian cancer

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