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1
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Shao L, Zheng Y, Somerville RP, Stroncek DF, Jin P. New insights on potency assays from recent advances and discoveries in CAR T-cell therapy. Front Immunol 2025; 16:1597888. [PMID: 40406092 PMCID: PMC12095010 DOI: 10.3389/fimmu.2025.1597888] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2025] [Accepted: 04/15/2025] [Indexed: 05/26/2025] Open
Abstract
This review explores recent advances in the characteristics and manufacturing of CAR T-cell products. Traditional potency assays have been designed based on well-established CAR T-cell functionalities. However, the advent of innovative tools and methodologies has revealed a broader spectrum of important CAR T-cell characteristics that correlate with function. Furthermore, as manufacturing strategies continue to evolve, conventional potency assays may no longer fully capture the complexity of these products. Therefore, it is essential to examine these emerging characteristics and manufacturing approaches and consider the development of tailored potency assays to ensure products are fully characterized.
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Affiliation(s)
| | | | | | - David F. Stroncek
- Center for Cellular Engineering, Clinical Center, National Institutes of Health, Bethesda, MD, United States
| | - Ping Jin
- Center for Cellular Engineering, Clinical Center, National Institutes of Health, Bethesda, MD, United States
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2
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Xu X, Niu M, Lamberty BG, Emanuel K, Apostol MJF, Fox HS. Transformation of brain myeloid cell populations by SIV in rhesus macaques revealed by multiomics. Commun Biol 2025; 8:100. [PMID: 39838075 PMCID: PMC11751027 DOI: 10.1038/s42003-024-07443-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2024] [Accepted: 12/26/2024] [Indexed: 01/23/2025] Open
Abstract
The primary immune constituents in the brain, microglia and macrophages, are the target for HIV in people and simian immunodeficiency virus (SIV) in nonhuman primates. This infection can lead to neurological dysfunction, known as HIV-associated neurocognitive disorder (HAND). Given the gaps in our knowledge on how these cells respond in vivo to CNS infection, we perform single-cell multiomic sequencing, including gene expression and ATAC-seq, on myeloid cells from the brains of rhesus macaques with SIV-induced encephalitis (SIVE) as well as uninfected controls. We find that SIVE significantly changes the myeloid cell populations. In SIVE, microglia-like cells express high levels of chemoattractants capable of recruiting highly activated CAM-like cells to the site of infection/inflammation. A unique population of microglia-like cells is found in which the chromatin accessibility of genes diverges from their RNA expression. Additionally, we observe a dramatic shift of upstream gene regulators and their targets in brain myeloid cells during SIVE. This study further uncovers the transcriptome, gene regulatory events, and potential roles of different brain myeloid phenotypes in SIVE. This might deepen the understanding of SIVE/HIVE and enlighten the therapeutic development.
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Affiliation(s)
- Xiaoke Xu
- Department of Neurological Sciences, University of Nebraska Medical Center, Omaha, NE, USA
| | - Meng Niu
- Department of Neurological Sciences, University of Nebraska Medical Center, Omaha, NE, USA
| | - Benjamin G Lamberty
- Department of Neurological Sciences, University of Nebraska Medical Center, Omaha, NE, USA
| | - Katy Emanuel
- Department of Neurological Sciences, University of Nebraska Medical Center, Omaha, NE, USA
| | | | - Howard S Fox
- Department of Neurological Sciences, University of Nebraska Medical Center, Omaha, NE, USA.
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3
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Lemmens M, Dorsheimer L, Zeller A, Dietz-Baum Y. Non-clinical safety assessment of novel drug modalities: Genome safety perspectives on viral-, nuclease- and nucleotide-based gene therapies. MUTATION RESEARCH. GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2024; 896:503767. [PMID: 38821669 DOI: 10.1016/j.mrgentox.2024.503767] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/18/2023] [Revised: 04/08/2024] [Accepted: 05/13/2024] [Indexed: 06/02/2024]
Abstract
Gene therapies have emerged as promising treatments for various conditions including inherited diseases as well as cancer. Ensuring their safe clinical application requires the development of appropriate safety testing strategies. Several guidelines have been provided by health authorities to address these concerns. These guidelines state that non-clinical testing should be carried out on a case-by-case basis depending on the modality. This review focuses on the genome safety assessment of frequently used gene therapy modalities, namely Adeno Associated Viruses (AAVs), Lentiviruses, designer nucleases and mRNAs. Important safety considerations for these modalities, amongst others, are vector integrations into the patient genome (insertional mutagenesis) and off-target editing. Taking into account the constraints of in vivo studies, health authorities endorse the development of novel approach methodologies (NAMs), which are innovative in vitro strategies for genotoxicity testing. This review provides an overview of NAMs applied to viral and CRISPR/Cas9 safety, including next generation sequencing-based methods for integration site analysis and off-target editing. Additionally, NAMs to evaluate the oncogenicity risk arising from unwanted genomic modifications are discussed. Thus, a range of promising techniques are available to support the safe development of gene therapies. Thorough validation, comparisons and correlations with clinical outcomes are essential to identify the most reliable safety testing strategies. By providing a comprehensive overview of these NAMs, this review aims to contribute to a better understanding of the genome safety perspectives of gene therapies.
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Affiliation(s)
| | - Lena Dorsheimer
- Research and Development, Preclinical Safety, Sanofi, Industriepark Hoechst, Frankfurt am Main 65926, Germany.
| | - Andreas Zeller
- Pharmaceutical Sciences, pRED Innovation Center Basel, Hoffmann-La Roche Ltd, Basel 4070, Switzerland
| | - Yasmin Dietz-Baum
- Research and Development, Preclinical Safety, Sanofi, Industriepark Hoechst, Frankfurt am Main 65926, Germany
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4
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Zhu M, Han Y, Gu T, Wang R, Si X, Kong D, Zhao P, Wang X, Li J, Zhai X, Yu Z, Lu H, Li J, Huang H, Qian P. Class I HDAC inhibitors enhance antitumor efficacy and persistence of CAR-T cells by activation of the Wnt pathway. Cell Rep 2024; 43:114065. [PMID: 38578828 DOI: 10.1016/j.celrep.2024.114065] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2023] [Revised: 01/18/2024] [Accepted: 03/21/2024] [Indexed: 04/07/2024] Open
Abstract
Epigenetic modification shapes differentiation trajectory and regulates the exhaustion state of chimeric antigen receptor T (CAR-T) cells. Limited efficacy induced by terminal exhaustion closely ties with intrinsic transcriptional regulation. However, the comprehensive regulatory mechanisms remain largely elusive. Here, we identify class I histone deacetylase inhibitors (HDACi) as boosters of CAR-T cell function by high-throughput screening of chromatin-modifying drugs, in which M344 and chidamide enhance memory maintenance and resistance to exhaustion of CAR-T cells that induce sustained antitumor efficacy both in vitro and in vivo. Mechanistically, HDACi decrease HDAC1 expression and enhance H3K27ac activity. Multi-omics analyses from RNA-seq, ATAC-seq, and H3K27ac CUT&Tag-seq show that HDACi upregulate expression of TCF4, LEF1, and CTNNB1, which subsequently activate the canonical Wnt/β-catenin pathway. Collectively, our findings elucidate the functional roles of class I HDACi in enhancing CAR-T cell function, which provides the basis and therapeutic targets for synergic combination of CAR-T cell therapy and HDACi treatment.
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Affiliation(s)
- Meng Zhu
- Center for Stem Cell and Regenerative Medicine and Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; Liangzhu Laboratory, Zhejiang University, 1369 West Wenyi Road, Hangzhou 311121, China; Institute of Hematology, Zhejiang University & Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou 310058, China
| | - Yingli Han
- Center for Stem Cell and Regenerative Medicine and Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; Liangzhu Laboratory, Zhejiang University, 1369 West Wenyi Road, Hangzhou 311121, China; Institute of Hematology, Zhejiang University & Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou 310058, China
| | - Tianning Gu
- Center for Stem Cell and Regenerative Medicine and Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; Liangzhu Laboratory, Zhejiang University, 1369 West Wenyi Road, Hangzhou 311121, China; Institute of Hematology, Zhejiang University & Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou 310058, China; Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Rui Wang
- Center for Stem Cell and Regenerative Medicine and Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; Liangzhu Laboratory, Zhejiang University, 1369 West Wenyi Road, Hangzhou 311121, China; Institute of Hematology, Zhejiang University & Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou 310058, China
| | - Xiaohui Si
- Center for Stem Cell and Regenerative Medicine and Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; Liangzhu Laboratory, Zhejiang University, 1369 West Wenyi Road, Hangzhou 311121, China; Institute of Hematology, Zhejiang University & Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou 310058, China; Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Delin Kong
- Center for Stem Cell and Regenerative Medicine and Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; Liangzhu Laboratory, Zhejiang University, 1369 West Wenyi Road, Hangzhou 311121, China; Institute of Hematology, Zhejiang University & Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou 310058, China; Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Peng Zhao
- Center for Stem Cell and Regenerative Medicine and Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; Liangzhu Laboratory, Zhejiang University, 1369 West Wenyi Road, Hangzhou 311121, China; Institute of Hematology, Zhejiang University & Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou 310058, China
| | - Xiujian Wang
- Center for Stem Cell and Regenerative Medicine and Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; Liangzhu Laboratory, Zhejiang University, 1369 West Wenyi Road, Hangzhou 311121, China; Institute of Hematology, Zhejiang University & Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou 310058, China; Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Jinxin Li
- Center for Stem Cell and Regenerative Medicine and Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; Liangzhu Laboratory, Zhejiang University, 1369 West Wenyi Road, Hangzhou 311121, China; Institute of Hematology, Zhejiang University & Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou 310058, China
| | - Xingyuan Zhai
- Center for Stem Cell and Regenerative Medicine and Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; Liangzhu Laboratory, Zhejiang University, 1369 West Wenyi Road, Hangzhou 311121, China; Institute of Hematology, Zhejiang University & Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou 310058, China; Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Zebin Yu
- Center for Stem Cell and Regenerative Medicine and Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; Liangzhu Laboratory, Zhejiang University, 1369 West Wenyi Road, Hangzhou 311121, China; Institute of Hematology, Zhejiang University & Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou 310058, China
| | - Huan Lu
- Center for Stem Cell and Regenerative Medicine and Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; Liangzhu Laboratory, Zhejiang University, 1369 West Wenyi Road, Hangzhou 311121, China; Institute of Hematology, Zhejiang University & Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou 310058, China
| | - Jingyi Li
- Center for Stem Cell and Regenerative Medicine and Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; Liangzhu Laboratory, Zhejiang University, 1369 West Wenyi Road, Hangzhou 311121, China; Institute of Hematology, Zhejiang University & Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou 310058, China
| | - He Huang
- Center for Stem Cell and Regenerative Medicine and Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; Liangzhu Laboratory, Zhejiang University, 1369 West Wenyi Road, Hangzhou 311121, China; Institute of Hematology, Zhejiang University & Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou 310058, China; Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
| | - Pengxu Qian
- Center for Stem Cell and Regenerative Medicine and Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; Liangzhu Laboratory, Zhejiang University, 1369 West Wenyi Road, Hangzhou 311121, China; Institute of Hematology, Zhejiang University & Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou 310058, China.
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5
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Levstek L, Janžič L, Ihan A, Kopitar AN. Biomarkers for prediction of CAR T therapy outcomes: current and future perspectives. Front Immunol 2024; 15:1378944. [PMID: 38558801 PMCID: PMC10979304 DOI: 10.3389/fimmu.2024.1378944] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2024] [Accepted: 03/04/2024] [Indexed: 04/04/2024] Open
Abstract
Chimeric antigen receptor (CAR) T cell therapy holds enormous potential for the treatment of hematologic malignancies. Despite its benefits, it is still used as a second line of therapy, mainly because of its severe side effects and patient unresponsiveness. Numerous researchers worldwide have attempted to identify effective predictive biomarkers for early prediction of treatment outcomes and adverse effects in CAR T cell therapy, albeit so far only with limited success. This review provides a comprehensive overview of the current state of predictive biomarkers. Although existing predictive metrics correlate to some extent with treatment outcomes, they fail to encapsulate the complexity of the immune system dynamics. The aim of this review is to identify six major groups of predictive biomarkers and propose their use in developing improved and efficient prediction models. These groups include changes in mitochondrial dynamics, endothelial activation, central nervous system impairment, immune system markers, extracellular vesicles, and the inhibitory tumor microenvironment. A comprehensive understanding of the multiple factors that influence therapeutic efficacy has the potential to significantly improve the course of CAR T cell therapy and patient care, thereby making this advanced immunotherapy more appealing and the course of therapy more convenient and favorable for patients.
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Affiliation(s)
| | | | | | - Andreja Nataša Kopitar
- Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
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6
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Ghaffari S, Saleh M, Akbari B, Ramezani F, Mirzaei HR. Applications of single-cell omics for chimeric antigen receptor T cell therapy. Immunology 2024; 171:339-364. [PMID: 38009707 DOI: 10.1111/imm.13720] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2023] [Accepted: 11/13/2023] [Indexed: 11/29/2023] Open
Abstract
Chimeric antigen receptor (CAR) T cell therapy is a promising cancer treatment modality. The breakthroughs in CAR T cell therapy were, in part, possible with the help of cell analysis methods, such as single-cell analysis. Bulk analyses have provided invaluable information regarding the complex molecular dynamics of CAR T cells, but their results are an average of thousands of signals in CAR T or tumour cells. Since cancer is a heterogeneous disease where each minute detail of a subclone could change the outcome of the treatment, single-cell analysis could prove to be a powerful instrument in deciphering the secrets of tumour microenvironment for cancer immunotherapy. With the recent studies in all aspects of adoptive cell therapy making use of single-cell analysis, a comprehensive review of the recent preclinical and clinical findings in CAR T cell therapy was needed. Here, we categorized and summarized the key points of the studies in which single-cell analysis provided insights into the genomics, epigenomics, transcriptomics and proteomics as well as their respective multi-omics of CAR T cell therapy.
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Affiliation(s)
- Sasan Ghaffari
- Department of Immunology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida, USA
| | - Mahshid Saleh
- Wisconsin National Primate Research Center, University of Wisconsin Graduate School, Madison, Wisconsin, USA
| | - Behnia Akbari
- Department of Medical Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Faezeh Ramezani
- Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
- Department of Medical Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Hamid Reza Mirzaei
- Department of Medical Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
- Molecular Imaging and Therapy Service, Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, New York, USA
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7
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Liao YM, Hsu SH, Chiou SS. Harnessing the Transcriptional Signatures of CAR-T-Cells and Leukemia/Lymphoma Using Single-Cell Sequencing Technologies. Int J Mol Sci 2024; 25:2416. [PMID: 38397092 PMCID: PMC10889174 DOI: 10.3390/ijms25042416] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2023] [Revised: 02/02/2024] [Accepted: 02/10/2024] [Indexed: 02/25/2024] Open
Abstract
Chimeric antigen receptor (CAR)-T-cell therapy has greatly improved outcomes for patients with relapsed or refractory hematological malignancies. However, challenges such as treatment resistance, relapse, and severe toxicity still hinder its widespread clinical application. Traditional transcriptome analysis has provided limited insights into the complex transcriptional landscape of both leukemia cells and engineered CAR-T-cells, as well as their interactions within the tumor microenvironment. However, with the advent of single-cell sequencing techniques, a paradigm shift has occurred, providing robust tools to unravel the complexities of these factors. These techniques enable an unbiased analysis of cellular heterogeneity and molecular patterns. These insights are invaluable for precise receptor design, guiding gene-based T-cell modification, and optimizing manufacturing conditions. Consequently, this review utilizes modern single-cell sequencing techniques to clarify the transcriptional intricacies of leukemia cells and CAR-Ts. The aim of this manuscript is to discuss the potential mechanisms that contribute to the clinical failures of CAR-T immunotherapy. We examine the biological characteristics of CAR-Ts, the mechanisms that govern clinical responses, and the intricacies of adverse events. By exploring these aspects, we hope to gain a deeper understanding of CAR-T therapy, which will ultimately lead to improved clinical outcomes and broader therapeutic applications.
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Affiliation(s)
- Yu-Mei Liao
- Division of Hematology-Oncology, Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 807, Taiwan;
| | - Shih-Hsien Hsu
- Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
- Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 807, Taiwan
- Research Center for Environmental Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
- Center of Applied Genomics, Kaohsiung Medical University, Kaohsiung 807, Taiwan
| | - Shyh-Shin Chiou
- Division of Hematology-Oncology, Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 807, Taiwan;
- Research Center for Environmental Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
- Center of Applied Genomics, Kaohsiung Medical University, Kaohsiung 807, Taiwan
- Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
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8
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Tang L, Huang ZP, Mei H, Hu Y. Insights gained from single-cell analysis of chimeric antigen receptor T-cell immunotherapy in cancer. Mil Med Res 2023; 10:52. [PMID: 37941075 PMCID: PMC10631149 DOI: 10.1186/s40779-023-00486-4] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/05/2023] [Accepted: 10/10/2023] [Indexed: 11/10/2023] Open
Abstract
Advances in chimeric antigen receptor (CAR)-T cell therapy have significantly improved clinical outcomes of patients with relapsed or refractory hematologic malignancies. However, progress is still hindered as clinical benefit is only available for a fraction of patients. A lack of understanding of CAR-T cell behaviors in vivo at the single-cell level impedes their more extensive application in clinical practice. Mounting evidence suggests that single-cell sequencing techniques can help perfect the receptor design, guide gene-based T cell modification, and optimize the CAR-T manufacturing conditions, and all of them are essential for long-term immunosurveillance and more favorable clinical outcomes. The information generated by employing these methods also potentially informs our understanding of the numerous complex factors that dictate therapeutic efficacy and toxicities. In this review, we discuss the reasons why CAR-T immunotherapy fails in clinical practice and what this field has learned since the milestone of single-cell sequencing technologies. We further outline recent advances in the application of single-cell analyses in CAR-T immunotherapy. Specifically, we provide an overview of single-cell studies focusing on target antigens, CAR-transgene integration, and preclinical research and clinical applications, and then discuss how it will affect the future of CAR-T cell therapy.
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Affiliation(s)
- Lu Tang
- Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China
- Hubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, 430022, China
- Key Laboratory of Biological Targeted Therapy, The Ministry of Education, Wuhan, 430022, China
| | - Zhong-Pei Huang
- Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China
- Hubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, 430022, China
- Key Laboratory of Biological Targeted Therapy, The Ministry of Education, Wuhan, 430022, China
| | - Heng Mei
- Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
- Hubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, 430022, China.
- Key Laboratory of Biological Targeted Therapy, The Ministry of Education, Wuhan, 430022, China.
- Hubei Key Laboratory of Biological Targeted Therapy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
| | - Yu Hu
- Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
- Hubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, 430022, China.
- Key Laboratory of Biological Targeted Therapy, The Ministry of Education, Wuhan, 430022, China.
- Hubei Key Laboratory of Biological Targeted Therapy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
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9
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Yan KK, Condori J, Ma Z, Metais JY, Ju B, Ding L, Dhungana Y, Palmer LE, Langfitt DM, Ferrara F, Throm R, Shi H, Risch I, Bhatara S, Shaner B, Lockey TD, Talleur AC, Easton J, Meagher MM, Puck JM, Cowan MJ, Zhou S, Mamcarz E, Gottschalk S, Yu J. Integrome signatures of lentiviral gene therapy for SCID-X1 patients. SCIENCE ADVANCES 2023; 9:eadg9959. [PMID: 37801507 PMCID: PMC10558130 DOI: 10.1126/sciadv.adg9959] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/04/2023] [Accepted: 09/06/2023] [Indexed: 10/08/2023]
Abstract
Lentiviral vector (LV)-based gene therapy holds promise for a broad range of diseases. Analyzing more than 280,000 vector integration sites (VISs) in 273 samples from 10 patients with X-linked severe combined immunodeficiency (SCID-X1), we discovered shared LV integrome signatures in 9 of 10 patients in relation to the genomics, epigenomics, and 3D structure of the human genome. VISs were enriched in the nuclear subcompartment A1 and integrated into super-enhancers close to nuclear pore complexes. These signatures were validated in T cells transduced with an LV encoding a CD19-specific chimeric antigen receptor. Intriguingly, the one patient whose VISs deviated from the identified integrome signatures had a distinct clinical course. Comparison of LV and gamma retrovirus integromes regarding their 3D genome signatures identified differences that might explain the lower risk of insertional mutagenesis in LV-based gene therapy. Our findings suggest that LV integrome signatures, shaped by common features such as genome organization, may affect the efficacy of LV-based cellular therapies.
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Affiliation(s)
- Koon-Kiu Yan
- Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Jose Condori
- Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Zhijun Ma
- Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Jean-Yves Metais
- Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Bensheng Ju
- Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Liang Ding
- Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Yogesh Dhungana
- Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
- Graduate School of Biomedical Sciences, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Lance E. Palmer
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Deanna M. Langfitt
- Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Francesca Ferrara
- Vector Development and Production Core, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Robert Throm
- Vector Development and Production Core, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Hao Shi
- Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Isabel Risch
- Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
- Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Sheetal Bhatara
- Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Bridget Shaner
- Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Timothy D. Lockey
- Department of Therapeutics Production and Quality, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Aimee C. Talleur
- Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - John Easton
- Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Michael M. Meagher
- Department of Therapeutics Production and Quality, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Jennifer M. Puck
- Department of Pediatrics, Division of Pediatric Allergy, Immunology and Bone Marrow Transplantation, University of California San Francisco Benioff Children’s Hospital, San Francisco, CA 94158, USA
| | - Morton J. Cowan
- Department of Pediatrics, Division of Pediatric Allergy, Immunology and Bone Marrow Transplantation, University of California San Francisco Benioff Children’s Hospital, San Francisco, CA 94158, USA
| | - Sheng Zhou
- Experimental Cellular Therapeutics Laboratory, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Ewelina Mamcarz
- Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Stephen Gottschalk
- Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Jiyang Yu
- Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
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10
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Wong M, Wei Y, Ho YC. Single-cell multiomic understanding of HIV-1 reservoir at epigenetic, transcriptional, and protein levels. Curr Opin HIV AIDS 2023; 18:246-256. [PMID: 37535039 PMCID: PMC10442869 DOI: 10.1097/coh.0000000000000809] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/04/2023]
Abstract
PURPOSE OF REVIEW The success of HIV-1 eradication strategies relies on in-depth understanding of HIV-1-infected cells. However, HIV-1-infected cells are extremely heterogeneous and rare. Single-cell multiomic approaches resolve the heterogeneity and rarity of HIV-1-infected cells. RECENT FINDINGS Advancement in single-cell multiomic approaches enabled HIV-1 reservoir profiling across the epigenetic (ATAC-seq), transcriptional (RNA-seq), and protein levels (CITE-seq). Using HIV-1 RNA as a surrogate, ECCITE-seq identified enrichment of HIV-1-infected cells in clonally expanded cytotoxic CD4+ T cells. Using HIV-1 DNA PCR-activated microfluidic sorting, FIND-seq captured the bulk transcriptome of HIV-1 DNA+ cells. Using targeted HIV-1 DNA amplification, PheP-seq identified surface protein expression of intact versus defective HIV-1-infected cells. Using ATAC-seq to identify HIV-1 DNA, ASAP-seq captured transcription factor activity and surface protein expression of HIV-1 DNA+ cells. Combining HIV-1 mapping by ATAC-seq and HIV-1 RNA mapping by RNA-seq, DOGMA-seq captured the epigenetic, transcriptional, and surface protein expression of latent and transcriptionally active HIV-1-infected cells. To identify reproducible biological insights and authentic HIV-1-infected cells and avoid false-positive discovery of artifacts, we reviewed current practices of single-cell multiomic experimental design and bioinformatic analysis. SUMMARY Single-cell multiomic approaches may identify innovative mechanisms of HIV-1 persistence, nominate therapeutic strategies, and accelerate discoveries.
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Affiliation(s)
- Michelle Wong
- Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, USA
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11
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Van Emmenis L. Golnaz Vahedi: My environment enables me to achieve impossible goals. J Exp Med 2023; 220:e20231182. [PMID: 37477639 PMCID: PMC10360022 DOI: 10.1084/jem.20231182] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/22/2023] Open
Abstract
Golnaz Vahedi is an associate professor of genetics at the Perelman School of Medicine, University of Pennsylvania. Golnaz runs a multidisciplinary lab that uses cutting-edge computational and experimental approaches to understand the molecular mechanisms by which genomic information in immune cells is interpreted in normal development and during immune-mediated diseases. We talked about her diverse scientific background, the benefits of integrating molecular biology and immunology, and the importance of staying positive in academia.
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12
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Battistello E, Hixon KA, Comstock DE, Collings CK, Chen X, Rodriguez Hernaez J, Lee S, Cervantes KS, Hinkley MM, Ntatsoulis K, Cesarano A, Hockemeyer K, Haining WN, Witkowski MT, Qi J, Tsirigos A, Perna F, Aifantis I, Kadoch C. Stepwise activities of mSWI/SNF family chromatin remodeling complexes direct T cell activation and exhaustion. Mol Cell 2023; 83:1216-1236.e12. [PMID: 36944333 PMCID: PMC10121856 DOI: 10.1016/j.molcel.2023.02.026] [Citation(s) in RCA: 33] [Impact Index Per Article: 16.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2022] [Revised: 01/11/2023] [Accepted: 02/23/2023] [Indexed: 03/23/2023]
Abstract
Highly coordinated changes in gene expression underlie T cell activation and exhaustion. However, the mechanisms by which such programs are regulated and how these may be targeted for therapeutic benefit remain poorly understood. Here, we comprehensively profile the genomic occupancy of mSWI/SNF chromatin remodeling complexes throughout acute and chronic T cell stimulation, finding that stepwise changes in localization over transcription factor binding sites direct site-specific chromatin accessibility and gene activation leading to distinct phenotypes. Notably, perturbation of mSWI/SNF complexes using genetic and clinically relevant chemical strategies enhances the persistence of T cells with attenuated exhaustion hallmarks and increased memory features in vitro and in vivo. Finally, pharmacologic mSWI/SNF inhibition improves CAR-T expansion and results in improved anti-tumor control in vivo. These findings reveal the central role of mSWI/SNF complexes in the coordination of T cell activation and exhaustion and nominate small-molecule-based strategies for the improvement of current immunotherapy protocols.
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Affiliation(s)
- Elena Battistello
- Department of Pathology and Laura and Isaac Perlmutter Cancer Center, NYU Grossman School of Medicine, New York, NY 10016, USA
| | - Kimberlee A Hixon
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02215, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Biological and Biomedical Sciences Program, Harvard Medical School, Boston, MA 02115, USA
| | - Dawn E Comstock
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02215, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Program in Immunology, Harvard Medical School, Boston, MA 02115, USA
| | - Clayton K Collings
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02215, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Xufeng Chen
- Department of Pathology and Laura and Isaac Perlmutter Cancer Center, NYU Grossman School of Medicine, New York, NY 10016, USA
| | - Javier Rodriguez Hernaez
- Department of Pathology and Laura and Isaac Perlmutter Cancer Center, NYU Grossman School of Medicine, New York, NY 10016, USA
| | - Soobeom Lee
- Department of Pathology and Laura and Isaac Perlmutter Cancer Center, NYU Grossman School of Medicine, New York, NY 10016, USA
| | - Kasey S Cervantes
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02215, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Madeline M Hinkley
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02215, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Konstantinos Ntatsoulis
- Department of Pathology and Laura and Isaac Perlmutter Cancer Center, NYU Grossman School of Medicine, New York, NY 10016, USA
| | - Annamaria Cesarano
- Department of Medicine, Division of Hematology/Oncology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Kathryn Hockemeyer
- Department of Pathology and Laura and Isaac Perlmutter Cancer Center, NYU Grossman School of Medicine, New York, NY 10016, USA
| | - W Nicholas Haining
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02215, USA
| | - Matthew T Witkowski
- Department of Pediatrics-HemeOnc and Bone Marrow Transplantation, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Jun Qi
- Department of Cancer Biology, Dana-Farber Cancer Institute and Harvard Medical School, Cambridge, MA, USA
| | - Aristotelis Tsirigos
- Department of Pathology and Laura and Isaac Perlmutter Cancer Center, NYU Grossman School of Medicine, New York, NY 10016, USA; Applied Bioinformatics Laboratories, Office of Science & Research, NYU Grossman School of Medicine, New York, NY, USA
| | - Fabiana Perna
- Department of Medicine, Division of Hematology/Oncology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Iannis Aifantis
- Department of Pathology and Laura and Isaac Perlmutter Cancer Center, NYU Grossman School of Medicine, New York, NY 10016, USA.
| | - Cigall Kadoch
- Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02215, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA.
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13
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Wu VH, Nordin JML, Nguyen S, Joy J, Mampe F, Del Rio Estrada PM, Torres-Ruiz F, González-Navarro M, Luna-Villalobos YA, Ávila-Ríos S, Reyes-Terán G, Tebas P, Montaner LJ, Bar KJ, Vella LA, Betts MR. Profound phenotypic and epigenetic heterogeneity of the HIV-1-infected CD4 + T cell reservoir. Nat Immunol 2023; 24:359-370. [PMID: 36536105 PMCID: PMC9892009 DOI: 10.1038/s41590-022-01371-3] [Citation(s) in RCA: 41] [Impact Index Per Article: 20.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2022] [Accepted: 10/24/2022] [Indexed: 12/24/2022]
Abstract
Understanding the complexity of the long-lived HIV reservoir during antiretroviral therapy (ART) remains a considerable impediment in research towards a cure for HIV. To address this, we developed a single-cell strategy to precisely define the unperturbed peripheral blood HIV-infected memory CD4+ T cell reservoir from ART-treated people living with HIV (ART-PLWH) via the presence of integrated accessible proviral DNA in concert with epigenetic and cell surface protein profiling. We identified profound reservoir heterogeneity within and between ART-PLWH, characterized by new and known surface markers within total and individual memory CD4+ T cell subsets. We further uncovered new epigenetic profiles and transcription factor motifs enriched in HIV-infected cells that suggest infected cells with accessible provirus, irrespective of reservoir distribution, are poised for reactivation during ART treatment. Together, our findings reveal the extensive inter- and intrapersonal cellular heterogeneity of the HIV reservoir, and establish an initial multiomic atlas to develop targeted reservoir elimination strategies.
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Grants
- K08 AI136660 NIAID NIH HHS
- T32 AI007632 NIAID NIH HHS
- R21 AI172629 NIAID NIH HHS
- UM1 AI164570 NIAID NIH HHS
- P30 AI045008 NIAID NIH HHS
- R01 AI031338 NIAID NIH HHS
- U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)
- Support for this study was provided by the following NIH grants: U19-A1-149680-02 (MRB), P01-AI31338 (MRB, KJB), K08-AI136660 (LAV), T32-AI007632 (VW), P30-AI045008 (Penn Center for AIDS Research) (MRB, LAV, KJB, PT, LJM), UM-1AI164570 (BEAT-HIV Collaboratory) which is co-supported by the National Institute of Allergies and Infectious Diseases (NIAID), the National Institute of Mental Health (NIMH), the National Institute of Neurological Disorders and Stroke (NINDS), the National Institute on Drug Abuse (NIDA), and the Robert I. Jacobs Fund of The Philadelphia Foundation (MRB, KJB, PT, LJM). LJM is also supported by the Herbert Kean, M.D., Family Professorship. CIENI-INER is supported by the Mexican Government (Programa Presupuestal P016; Anexo 13 del Decreto del Presupuesto de Egresos de la Federación).
- CIENI-INER is supported by the Mexican Government (Programa Presupuestal P016; Anexo 13 del Decreto del Presupuesto de Egresos de la Federación).
- LJM is also supported by the Herbert Kean, M.D., Family Professorship.
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Affiliation(s)
- Vincent H Wu
- Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Center for AIDS Research, University of Pennsylvania, Philadelphia, PA, USA
| | - Jayme M L Nordin
- Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Center for AIDS Research, University of Pennsylvania, Philadelphia, PA, USA
| | - Son Nguyen
- Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Institute for Medical Engineering and Science, Department of Chemistry, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Jaimy Joy
- Center for AIDS Research, University of Pennsylvania, Philadelphia, PA, USA
- Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Felicity Mampe
- Center for AIDS Research, University of Pennsylvania, Philadelphia, PA, USA
- Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Perla M Del Rio Estrada
- Centro de Investigación en Enfermedades Infecciosas, Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico
| | - Fernanda Torres-Ruiz
- Centro de Investigación en Enfermedades Infecciosas, Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico
| | - Mauricio González-Navarro
- Centro de Investigación en Enfermedades Infecciosas, Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico
| | - Yara Andrea Luna-Villalobos
- Centro de Investigación en Enfermedades Infecciosas, Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico
| | - Santiago Ávila-Ríos
- Centro de Investigación en Enfermedades Infecciosas, Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico
| | - Gustavo Reyes-Terán
- Institutos Nacionales de Salud y Hospitales de Alta Especialidad, Secretaría de Salud de México, Mexico City, Mexico
| | - Pablo Tebas
- Center for AIDS Research, University of Pennsylvania, Philadelphia, PA, USA
- Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Luis J Montaner
- Center for AIDS Research, University of Pennsylvania, Philadelphia, PA, USA
- The Wistar Institute, Philadelphia, PA, USA
| | - Katharine J Bar
- Center for AIDS Research, University of Pennsylvania, Philadelphia, PA, USA
- Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Laura A Vella
- Center for AIDS Research, University of Pennsylvania, Philadelphia, PA, USA.
- Division of Infectious Diseases, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
| | - Michael R Betts
- Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
- Center for AIDS Research, University of Pennsylvania, Philadelphia, PA, USA.
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14
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Shao L, Shi R, Zhao Y, Liu H, Lu A, Ma J, Cai Y, Fuksenko T, Pelayo A, Shah NN, Kochenderfer JN, Norberg SM, Hinrichs C, Highfill SL, Somerville RP, Panch SR, Jin P, Stroncek DF. Genome-wide profiling of retroviral DNA integration and its effect on clinical pre-infusion CAR T-cell products. J Transl Med 2022; 20:514. [DOI: 10.1186/s12967-022-03729-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2022] [Accepted: 10/23/2022] [Indexed: 11/09/2022] Open
Abstract
Abstract
Background
Clinical CAR T-cell therapy using integrating vector systems represents a promising approach for the treatment of hematological malignancies. Lentiviral and γ-retroviral vectors are the most commonly used vectors in the manufacturing process. However, the integration pattern of these viral vectors and subsequent effect on CAR T-cell products is still unclear.
Methods
We used a modified viral integration sites analysis (VISA) pipeline to evaluate viral integration events around the whole genome in pre-infusion CAR T-cell products. We compared the differences of integration pattern between lentiviral and γ-retroviral products. We also explored whether the integration sites correlated with clinical outcomes.
Results
We found that γ-retroviral vectors were more likely to insert than lentiviral vectors into promoter, untranslated, and exon regions, while lentiviral vector integration sites were more likely to occur in intron and intergenic regions. Some integration events affected gene expression at the transcriptional and post-transcriptional level. Moreover, γ-retroviral vectors showed a stronger impact on the host transcriptome. Analysis of individuals with different clinical outcomes revealed genes with differential enrichment of integration events. These genes may affect biological functions by interrupting amino acid sequences and generating abnormal proteins, instead of by affecting mRNA expression. These results suggest that vector integration is associated with CAR T-cell efficacy and clinical responses.
Conclusion
We found differences in integration patterns, insertion hotspots and effects on gene expression vary between lentiviral and γ-retroviral vectors used in CAR T-cell products and established a foundation upon which we can conduct further analyses.
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15
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Jiang P, Zhang Z, Hu Y, Liang Z, Han Y, Li X, Zeng X, Zhang H, Zhu M, Dong J, Huang H, Qian P. Single-cell ATAC-seq maps the comprehensive and dynamic chromatin accessibility landscape of CAR-T cell dysfunction. Leukemia 2022; 36:2656-2668. [PMID: 35962059 DOI: 10.1038/s41375-022-01676-0] [Citation(s) in RCA: 22] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2022] [Revised: 07/28/2022] [Accepted: 08/01/2022] [Indexed: 11/09/2022]
Abstract
Chimeric antigen receptor T cells (CAR-T) therapy has achieved remarkable therapeutic success in treating a variety of hematopoietic malignancies. However, the high relapse rate and poor in vivo persistence, partially caused by CAR-T cell exhaustion, are still important barriers against CAR-T therapy. It remains largely elusive on the mechanisms of CAR-T exhaustion and how to attenuate exhaustion to achieve better therapeutic efficacy. In this study, we initially observed that CAR-T cells showed rapid differentiation and increased exhaustion after co-culture with tumor cells in vitro, and then performed single-cell ATAC-seq to depict the comprehensive and dynamic landscape of chromatin accessibility of CAR-T cells during tumor cell stimulation. Analyses of differential chromatin accessible regions and motif accessibility revealed that TFs were distinct in each cell type and reconstituted a coordinated regulatory network to drive CAR-T exhaustion. Furthermore, we performed scATAC-seq in patient-derived CAR-T cells and identified BATF and IRF4 as pivotal regulators in CAR-T cell exhaustion. Finally, knockdown of BATF or IRF4 enhanced the killing ability, inhibited exhaustion, and prolonged the persistence of CAR-T cells in vivo. Together, our study unraveled the epigenetic regulatory mechanisms of CAR-T exhaustion and provided new insights into CAR-T engineering to achieve better clinical treatment benefits.
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Affiliation(s)
- Penglei Jiang
- Center of Stem Cell and Regenerative Medicine, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China.,Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China.,Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China
| | - Zhaoru Zhang
- Center of Stem Cell and Regenerative Medicine, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China.,Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China.,Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China
| | - Yongxian Hu
- Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China.,Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China.,Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, Zhejiang, China
| | - Zuyu Liang
- Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China.,Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China.,Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, Zhejiang, China
| | - Yingli Han
- Center of Stem Cell and Regenerative Medicine, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China.,Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China.,Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China
| | - Xia Li
- Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China.,Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China.,Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, Zhejiang, China
| | - Xin Zeng
- Center of Stem Cell and Regenerative Medicine, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China.,Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China.,Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China
| | - Hao Zhang
- Department of Hematology, The Third Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325200, Zhejiang, China
| | - Meng Zhu
- Center of Stem Cell and Regenerative Medicine, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China.,Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China.,Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China
| | - Jian Dong
- Center of Stem Cell and Regenerative Medicine, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China.,Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China.,Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China
| | - He Huang
- Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China. .,Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China. .,Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, Zhejiang, China.
| | - Pengxu Qian
- Center of Stem Cell and Regenerative Medicine, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China. .,Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121, China. .,Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China.
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16
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GU T, ZHU M, HUANG H, HU Y. Relapse after CAR-T cell therapy in B-cell malignancies: challenges and future approaches. J Zhejiang Univ Sci B 2022; 23:793-811. [PMID: 36226535 PMCID: PMC9561408 DOI: 10.1631/jzus.b2200256] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
Chimeric antigen receptor-T (CAR-T) cell therapy, as a novel cellular immunotherapy, has dramatically reshaped the landscape of cancer treatment, especially in hematological malignancies. However, relapse is still one of the most troublesome obstacles to achieving broad clinical application. The intrinsic factors and superior adaptability of tumor cells mark a fundamental aspect of relapse. The unique biological function of CAR-T cells governed by their special CAR construction also affects treatment efficacy. Moreover, complex cross-interactions among CAR-T cells, tumor cells, and the tumor microenvironment (TME) profoundly influence clinical outcomes concerning CAR-T cell function and persistence. Therefore, in this review, based on the most recent discoveries, we focus on the challenges of relapse after CAR-T cell therapy in B-cell malignancies from the perspective of tumor cells, CAR-T cells, and the TME. We also discuss the corresponding basic and clinical approaches that may overcome the problem in the future. We aim to provide a comprehensive understanding for scientists and physicians that will help improve research and clinical practice.
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Affiliation(s)
- Tianning GU
- Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou310003, China,Liangzhu Laboratory, Zhejiang University Medical Center, Hangzhou311121, China,Institute of Hematology, Zhejiang University, Hangzhou310058, China,Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou310058, China
| | - Meng ZHU
- Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou310003, China,Liangzhu Laboratory, Zhejiang University Medical Center, Hangzhou311121, China,Institute of Hematology, Zhejiang University, Hangzhou310058, China,Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou310058, China
| | - He HUANG
- Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou310003, China,Liangzhu Laboratory, Zhejiang University Medical Center, Hangzhou311121, China,Institute of Hematology, Zhejiang University, Hangzhou310058, China,Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou310058, China,He HUANG,
| | - Yongxian HU
- Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou310003, China,Liangzhu Laboratory, Zhejiang University Medical Center, Hangzhou311121, China,Institute of Hematology, Zhejiang University, Hangzhou310058, China,Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou310058, China,Yongxian HU,
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17
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Bucktrout SL, Banovich NE, Butterfield LH, Cimen-Bozkus C, Giles JR, Good Z, Goodman D, Jonsson VD, Lareau C, Marson A, Maurer DM, Munson PV, Stubbington M, Taylor S, Cutchin A. Advancing T cell-based cancer therapy with single-cell technologies. Nat Med 2022; 28:1761-1764. [PMID: 36127419 DOI: 10.1038/s41591-022-01986-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Affiliation(s)
| | - Nicholas E Banovich
- Integrated Cancer Genomics Division, Translational Genomics Research Institute, Phoenix, AZ, USA
| | | | - Cansu Cimen-Bozkus
- Parker Institute of Cancer Immunotherapy, San Francisco, CA, USA
- Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Josephine R Giles
- Parker Institute of Cancer Immunotherapy, San Francisco, CA, USA
- Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA, USA
- Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Zinaida Good
- Parker Institute of Cancer Immunotherapy, San Francisco, CA, USA
- Stanford Cancer Institute and Department of Biomedical Data Science, Stanford University School of Medicine, Stanford, CA, USA
| | - Daniel Goodman
- Parker Institute of Cancer Immunotherapy, San Francisco, CA, USA
- Microbiology and Immunology, School of Medicine, University of California San Francisco, San Francisco, CA, USA
| | - Vanessa D Jonsson
- Department of Applied Mathematics, University of California, Santa Cruz, Santa Cruz, CA, USA
- Department of Biomolecular Engineering, University of California, Santa Cruz, Santa Cruz, CA, USA
- Genomics Institute, University of California, Santa Cruz, Santa Cruz, CA, USA
| | - Caleb Lareau
- Parker Institute of Cancer Immunotherapy, San Francisco, CA, USA
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
| | - Alexander Marson
- Parker Institute of Cancer Immunotherapy, San Francisco, CA, USA
- Gladstone-UCSF Institute for Genomic Immunology, San Francisco, CA, USA
| | - Deena M Maurer
- Parker Institute of Cancer Immunotherapy, San Francisco, CA, USA
| | - Paul V Munson
- Parker Institute of Cancer Immunotherapy, San Francisco, CA, USA
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18
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Palamenghi M, De Luca M, De Rosa L. The steep uphill path leading to ex vivo gene therapy for genodermatoses. Am J Physiol Cell Physiol 2022; 323:C896-C906. [PMID: 35912986 DOI: 10.1152/ajpcell.00117.2022] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Cell therapy, gene therapy and tissue engineering have the potential to revolutionize the field of regenerative medicine. In particular, gene therapy is understood as the therapeutical correction of mutated genes by addition of a correct copy of the gene or site-specific gene modifications. Gene correction of somatic stem cells sustaining renewing tissues is critical to ensure long-term clinical success of ex vivo gene therapy. To date, remarkable clinical outcomes arose from combined ex vivo cell and gene therapy of different genetic diseases, such as immunodeficiencies and genodermatoses. Despite the efforts of researchers around the world, only few of these advanced approaches has yet made it to routine therapy. In fact, gene therapy poses one of the greatest technical challenges in modern medicine, spanning safety and efficacy issues, regulatory constraints, registration and market access, all of which need to be addressed to make the therapy available to rare disease patients. In this review, we survey at some of the main challenges in the development of combined cell and gene therapy of genetic skin diseases.
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Affiliation(s)
- Michele Palamenghi
- Centre for Regenerative Medicine "Stefano Ferrari", University of Modena and Reggio Emilia, Modena, Italy
| | - Michele De Luca
- Centre for Regenerative Medicine "Stefano Ferrari", University of Modena and Reggio Emilia, Modena, Italy
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The third-generation anti-CD30 CAR T-cells specifically homing to the tumor and mediating powerful antitumor activity. Sci Rep 2022; 12:10488. [PMID: 35729339 PMCID: PMC9213494 DOI: 10.1038/s41598-022-14523-0] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2021] [Accepted: 06/08/2022] [Indexed: 12/23/2022] Open
Abstract
CAR T-cell therapy is well tolerated and effective in patients with Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL). However, even second- generation anti-CD30 CAR T-cells with CD28 (28z) costimulatory domains failed to achieve the desired rate of complete responses. In the present study, we developed second-generation (CD28z) and third-generation (CD28BBz) CAR T-cells targeting CD30 and investigated their efficacy in vitro and in vivo. Both of CD28z and CD28BBz anti-CD30 CAR T cells were similar regarding amplification, T cell subsets distribution, T cell activity, effector/memory and exhaustion. However, we found that the 28BBz anti-CD30 CAR T-cells persist long-term, specifically homing to the tumor and mediating powerful antitumor activity in tumor xenograft models. Subsequently, we also demonstrated that the third generation anti-CD30 CAR T-cells have miner side effects or potential risks of tumorigenesis. Thus, anti-CD30 CAR T-cells represent a safe and effective treatment for Hodgkin lymphoma.
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20
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Bai Z, Woodhouse S, Zhao Z, Arya R, Govek K, Kim D, Lundh S, Baysoy A, Sun H, Deng Y, Xiao Y, Barrett DM, Myers RM, Grupp SA, June CH, Fan R, Camara PG, Melenhorst JJ. Single-cell antigen-specific landscape of CAR T infusion product identifies determinants of CD19-positive relapse in patients with ALL. SCIENCE ADVANCES 2022; 8:eabj2820. [PMID: 35675405 PMCID: PMC9177075 DOI: 10.1126/sciadv.abj2820] [Citation(s) in RCA: 97] [Impact Index Per Article: 32.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 05/02/2023]
Abstract
A notable number of acute lymphoblastic leukemia (ALL) patients develop CD19-positive relapse within 1 year after receiving chimeric antigen receptor (CAR) T cell therapy. It remains unclear if the long-term response is associated with the characteristics of CAR T cells in infusion products, hindering the identification of biomarkers to predict therapeutic outcomes. Here, we present 101,326 single-cell transcriptomes and surface protein landscape from the infusion products of 12 ALL patients. We observed substantial heterogeneity in the antigen-specific activation states, among which a deficiency of T helper 2 function was associated with CD19-positive relapse compared with durable responders (remission, >54 months). Proteomic data revealed that the frequency of early memory T cells, rather than activation or coinhibitory signatures, could distinguish the relapse. These findings were corroborated by independent functional profiling of 49 patients, and an integrative model was developed to predict the response. Our data unveil the molecular mechanisms that may inform strategies to boost specific T cell function to maintain long-term remission.
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Affiliation(s)
- Zhiliang Bai
- Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA
| | - Steven Woodhouse
- Department of Genetics and Institute for Biomedical Informatics, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Ziran Zhao
- Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
- Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
- Parker Institute for Cancer Immunotherapy at University of Pennsylvania, Philadelphia, PA 19104, USA
- Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44106, USA
| | - Rahul Arya
- Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
- Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - Kiya Govek
- Department of Genetics and Institute for Biomedical Informatics, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Dongjoo Kim
- Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA
| | - Stefan Lundh
- Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Alev Baysoy
- Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA
| | - Hongxing Sun
- Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Yanxiang Deng
- Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA
| | - Yang Xiao
- Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA
| | - David M. Barrett
- Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
- Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - Regina M. Myers
- Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - Stephan A. Grupp
- Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
- Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - Carl H. June
- Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
- Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
- Parker Institute for Cancer Immunotherapy at University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Rong Fan
- Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA
- Yale Stem Cell Center and Yale Cancer Center, Yale School of Medicine, New Haven, CT 06520, USA
- Human and Translational Immunology, Yale School of Medicine, New Haven, CT 06520, USA
- Corresponding author. (R.F.); (P.G.C.); (J.J.M.)
| | - Pablo G. Camara
- Department of Genetics and Institute for Biomedical Informatics, University of Pennsylvania, Philadelphia, PA 19104, USA
- Corresponding author. (R.F.); (P.G.C.); (J.J.M.)
| | - J. Joseph Melenhorst
- Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
- Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
- Parker Institute for Cancer Immunotherapy at University of Pennsylvania, Philadelphia, PA 19104, USA
- Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44106, USA
- Corresponding author. (R.F.); (P.G.C.); (J.J.M.)
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21
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Garcia-Prieto CA, Villanueva L, Bueno-Costa A, Davalos V, González-Navarro EA, Juan M, Urbano-Ispizua Á, Delgado J, Ortiz-Maldonado V, del Bufalo F, Locatelli F, Quintarelli C, Sinibaldi M, Soler M, Castro de Moura M, Ferrer G, Urdinguio RG, Fernandez AF, Fraga MF, Bar D, Meir A, Itzhaki O, Besser MJ, Avigdor A, Jacoby E, Esteller M. Epigenetic Profiling and Response to CD19 Chimeric Antigen Receptor T-Cell Therapy in B-Cell Malignancies. J Natl Cancer Inst 2022; 114:436-445. [PMID: 34581788 PMCID: PMC8902331 DOI: 10.1093/jnci/djab194] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2021] [Revised: 07/11/2021] [Accepted: 09/22/2021] [Indexed: 11/12/2022] Open
Abstract
BACKGROUND Chimeric antigen receptor (CAR) T cells directed against CD19 (CART19) are effective in B-cell malignancies, but little is known about the molecular factors predicting clinical outcome of CART19 therapy. The increasingly recognized relevance of epigenetic changes in cancer immunology prompted us to determine the impact of the DNA methylation profiles of CART19 cells on the clinical course. METHODS We recruited 114 patients with B-cell malignancies, comprising 77 patients with acute lymphoblastic leukemia and 37 patients with non-Hodgkin lymphoma who were treated with CART19 cells. Using a comprehensive DNA methylation microarray, we determined the epigenomic changes that occur in the patient T cells upon transduction of the CAR vector. The effects of the identified DNA methylation sites on clinical response, cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, event-free survival, and overall survival were assessed. All statistical tests were 2-sided. RESULTS We identified 984 genomic sites with differential DNA methylation between CAR-untransduced and CAR-transduced T cells before infusion into the patient. Eighteen of these distinct epigenetic loci were associated with complete response (CR), adjusting by multiple testing. Using the sites linked to CR, an epigenetic signature, referred to hereafter as the EPICART signature, was established in the initial discovery cohort (n = 79), which was associated with CR (Fisher exact test, P < .001) and enhanced event-free survival (hazard ratio [HR] = 0.36; 95% confidence interval [CI] = 0.19 to 0.70; P = .002; log-rank P = .003) and overall survival (HR = 0.45; 95% CI = 0.20 to 0.99; P = .047; log-rank P = .04;). Most important, the EPICART profile maintained its clinical course predictive value in the validation cohort (n = 35), where it was associated with CR (Fisher exact test, P < .001) and enhanced overall survival (HR = 0.31; 95% CI = 0.11 to 0.84; P = .02; log-rank P = .02). CONCLUSIONS We show that the DNA methylation landscape of patient CART19 cells influences the efficacy of the cellular immunotherapy treatment in patients with B-cell malignancy.
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Affiliation(s)
- Carlos A Garcia-Prieto
- Cancer and Leukemia Epigenetics and Biology Program (PEBCL), Josep Carreras Leukaemia Research Institute (IJC), Badalona, Spain
- Life Sciences Department, Barcelona Supercomputing Center (BSC), Barcelona, Spain
| | - Lorea Villanueva
- Cancer and Leukemia Epigenetics and Biology Program (PEBCL), Josep Carreras Leukaemia Research Institute (IJC), Badalona, Spain
| | - Alberto Bueno-Costa
- Cancer and Leukemia Epigenetics and Biology Program (PEBCL), Josep Carreras Leukaemia Research Institute (IJC), Badalona, Spain
| | - Veronica Davalos
- Cancer and Leukemia Epigenetics and Biology Program (PEBCL), Josep Carreras Leukaemia Research Institute (IJC), Badalona, Spain
| | | | - Manel Juan
- Department of Immunology, Hospital Clinic, Barcelona, Spain
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
| | - Álvaro Urbano-Ispizua
- Cancer and Leukemia Epigenetics and Biology Program (PEBCL), Josep Carreras Leukaemia Research Institute (IJC), Badalona, Spain
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
- Department of Hematology, University of Barcelona (UB), Barcelona, Spain
| | - Julio Delgado
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
- Centro de Investigación Biomédica en Red de Cancer (CIBERONC), Madrid, Spain
| | | | - Francesca del Bufalo
- Department of Paediatric Haematology and Oncology, Cell and Gene Therapy, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
| | - Franco Locatelli
- Department of Paediatric Haematology and Oncology, Cell and Gene Therapy, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
- Department of Pediatrics, Sapienza University of Rome, Rome, Italy
| | - Concetta Quintarelli
- Department of Paediatric Haematology and Oncology, Cell and Gene Therapy, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
- Department of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy
| | - Matilde Sinibaldi
- Department of Paediatric Haematology and Oncology, Cell and Gene Therapy, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
| | - Marta Soler
- Cancer and Leukemia Epigenetics and Biology Program (PEBCL), Josep Carreras Leukaemia Research Institute (IJC), Badalona, Spain
| | - Manuel Castro de Moura
- Cancer and Leukemia Epigenetics and Biology Program (PEBCL), Josep Carreras Leukaemia Research Institute (IJC), Badalona, Spain
| | - Gerardo Ferrer
- Cancer and Leukemia Epigenetics and Biology Program (PEBCL), Josep Carreras Leukaemia Research Institute (IJC), Badalona, Spain
| | - Rocio G Urdinguio
- Nanomaterials and Nanotechnology Research Center (CINNCSIC), Health Research Institute of Asturias (ISPA), Institute of Oncology of Asturias (IUOPA), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Department of Organisms and Systems Biology (BOS), University of Oviedo, Oviedo, Spain
| | - Agustin F Fernandez
- Nanomaterials and Nanotechnology Research Center (CINNCSIC), Health Research Institute of Asturias (ISPA), Institute of Oncology of Asturias (IUOPA), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Department of Organisms and Systems Biology (BOS), University of Oviedo, Oviedo, Spain
| | - Mario F Fraga
- Nanomaterials and Nanotechnology Research Center (CINNCSIC), Health Research Institute of Asturias (ISPA), Institute of Oncology of Asturias (IUOPA), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Department of Organisms and Systems Biology (BOS), University of Oviedo, Oviedo, Spain
| | - Diana Bar
- Division of Pediatric Hematology and Oncology, The Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel
| | - Amilia Meir
- Division of Pediatric Hematology and Oncology, The Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel
| | - Orit Itzhaki
- Ella Lemelbaum Institute for Immuno Oncology, Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel
| | - Michal J Besser
- Ella Lemelbaum Institute for Immuno Oncology, Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel
- Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
| | - Abraham Avigdor
- Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
- Institute of Hematology, Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel
| | - Elad Jacoby
- Division of Pediatric Hematology and Oncology, The Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel
- Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
| | - Manel Esteller
- Cancer and Leukemia Epigenetics and Biology Program (PEBCL), Josep Carreras Leukaemia Research Institute (IJC), Badalona, Spain
- Centro de Investigación Biomédica en Red de Cancer (CIBERONC), Madrid, Spain
- Institucio Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain
- Physiological Sciences Department, School of Medicine and Health Sciences, University of Barcelona (UB), Spain
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22
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Einkauf KB, Osborn MR, Gao C, Sun W, Sun X, Lian X, Parsons EM, Gladkov GT, Seiger KW, Blackmer JE, Jiang C, Yukl SA, Rosenberg ES, Yu XG, Lichterfeld M. Parallel analysis of transcription, integration, and sequence of single HIV-1 proviruses. Cell 2022; 185:266-282.e15. [PMID: 35026153 PMCID: PMC8809251 DOI: 10.1016/j.cell.2021.12.011] [Citation(s) in RCA: 147] [Impact Index Per Article: 49.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2021] [Revised: 10/17/2021] [Accepted: 12/10/2021] [Indexed: 01/09/2023]
Abstract
HIV-1-infected cells that persist despite antiretroviral therapy (ART) are frequently considered "transcriptionally silent," but active viral gene expression may occur in some cells, challenging the concept of viral latency. Applying an assay for profiling the transcriptional activity and the chromosomal locations of individual proviruses, we describe a global genomic and epigenetic map of transcriptionally active and silent proviral species and evaluate their longitudinal evolution in persons receiving suppressive ART. Using genome-wide epigenetic reference data, we show that proviral transcriptional activity is associated with activating epigenetic chromatin features in linear proximity of integration sites and in their inter- and intrachromosomal contact regions. Transcriptionally active proviruses were actively selected against during prolonged ART; however, this pattern was violated by large clones of virally infected cells that may outcompete negative selection forces through elevated intrinsic proliferative activity. Our results suggest that transcriptionally active proviruses are dynamically evolving under selection pressure by host factors.
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Affiliation(s)
- Kevin B Einkauf
- Infectious Disease Division, Brigham and Women's Hospital, Boston, MA 02115, USA; Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA
| | - Matthew R Osborn
- Infectious Disease Division, Brigham and Women's Hospital, Boston, MA 02115, USA; Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA
| | - Ce Gao
- Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA
| | - Weiwei Sun
- Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA
| | - Xiaoming Sun
- Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA; Department of Immunology and Microbiology, Hangzhou Normal University, Zhejiang, P.R. China
| | - Xiaodong Lian
- Infectious Disease Division, Brigham and Women's Hospital, Boston, MA 02115, USA; Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA
| | - Elizabeth M Parsons
- Infectious Disease Division, Brigham and Women's Hospital, Boston, MA 02115, USA; Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA
| | | | - Kyra W Seiger
- Infectious Disease Division, Brigham and Women's Hospital, Boston, MA 02115, USA; Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA
| | - Jane E Blackmer
- Infectious Disease Division, Brigham and Women's Hospital, Boston, MA 02115, USA; Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA
| | - Chenyang Jiang
- Infectious Disease Division, Brigham and Women's Hospital, Boston, MA 02115, USA; Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA
| | - Steven A Yukl
- San Francisco VA Medical Center, University of California at San Francisco, San Francisco, CA 94121, USA
| | - Eric S Rosenberg
- Infectious Disease Division, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Xu G Yu
- Infectious Disease Division, Brigham and Women's Hospital, Boston, MA 02115, USA; Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA
| | - Mathias Lichterfeld
- Infectious Disease Division, Brigham and Women's Hospital, Boston, MA 02115, USA; Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA.
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23
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The immune niche of the liver. Clin Sci (Lond) 2021; 135:2445-2466. [PMID: 34709406 DOI: 10.1042/cs20190654] [Citation(s) in RCA: 59] [Impact Index Per Article: 14.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2021] [Revised: 09/17/2021] [Accepted: 10/08/2021] [Indexed: 12/19/2022]
Abstract
The liver is an essential organ that is critical for the removal of toxins, the production of proteins, and the maintenance of metabolic homeostasis. Behind each liver functional unit, termed lobules, hides a heterogeneous, complex, and well-orchestrated system. Despite parenchymal cells being most commonly associated with the liver's primary functionality, it has become clear that it is the immune niche of the liver that plays a central role in maintaining both local and systemic homeostasis by propagating hepatic inflammation and orchestrating its resolution. As such, the immunological processes that are at play in healthy and diseased livers are being investigated thoroughly in order to understand the underpinnings of inflammation and the potential avenues for restoring homeostasis. This review highlights recent advances in our understanding of the immune niche of the liver and provides perspectives for how the implementation of new transcriptomic, multimodal, and spatial technologies can uncover the heterogeneity, plasticity, and location of hepatic immune populations. Findings from these technologies will further our understanding of liver biology and create a new framework for the identification of therapeutic targets.
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24
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Lin H, Cheng J, Mu W, Zhou J, Zhu L. Advances in Universal CAR-T Cell Therapy. Front Immunol 2021; 12:744823. [PMID: 34691052 PMCID: PMC8526896 DOI: 10.3389/fimmu.2021.744823] [Citation(s) in RCA: 105] [Impact Index Per Article: 26.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2021] [Accepted: 09/13/2021] [Indexed: 12/27/2022] Open
Abstract
Chimeric antigen receptor T (CAR-T) cell therapy achieved extraordinary achievements results in antitumor treatments, especially against hematological malignancies, where it leads to remarkable, long-term antineoplastic effects with higher target specificity. Nevertheless, some limitations persist in autologous CAR-T cell therapy, such as high costs, long manufacturing periods, and restricted cell sources. The development of a universal CAR-T (UCAR-T) cell therapy is an attractive breakthrough point that may overcome most of these drawbacks. Here, we review the progress and challenges in CAR-T cell therapy, especially focusing on comprehensive comparison in UCAR-T cell therapy to original CAR-T cell therapy. Furthermore, we summarize the developments and concerns about the safety and efficiency of UCAR-T cell therapy. Finally, we address other immune cells, which might be promising candidates as a complement for UCAR-T cells. Through a detailed overview, we describe the current landscape and explore the prospect of UCAR-T cell therapy.
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Affiliation(s)
- Haolong Lin
- Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Jiali Cheng
- Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Wei Mu
- Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Jianfeng Zhou
- Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Li Zhu
- Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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25
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Li X, Shao M, Zeng X, Qian P, Huang H. Signaling pathways in the regulation of cytokine release syndrome in human diseases and intervention therapy. Signal Transduct Target Ther 2021; 6:367. [PMID: 34667157 PMCID: PMC8526712 DOI: 10.1038/s41392-021-00764-4] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2021] [Revised: 08/09/2021] [Accepted: 09/05/2021] [Indexed: 01/08/2023] Open
Abstract
Cytokine release syndrome (CRS) embodies a mixture of clinical manifestations, including elevated circulating cytokine levels, acute systemic inflammatory symptoms and secondary organ dysfunction, which was first described in the context of acute graft-versus-host disease after allogeneic hematopoietic stem-cell transplantation and was later observed in pandemics of influenza, SARS-CoV and COVID-19, immunotherapy of tumor, after chimeric antigen receptor T (CAR-T) therapy, and in monogenic disorders and autoimmune diseases. Particularly, severe CRS is a very significant and life-threatening complication, which is clinically characterized by persistent high fever, hyperinflammation, and severe organ dysfunction. However, CRS is a double-edged sword, which may be both helpful in controlling tumors/viruses/infections and harmful to the host. Although a high incidence and high levels of cytokines are features of CRS, the detailed kinetics and specific mechanisms of CRS in human diseases and intervention therapy remain unclear. In the present review, we have summarized the most recent advances related to the clinical features and management of CRS as well as cutting-edge technologies to elucidate the mechanisms of CRS. Considering that CRS is the major adverse event in human diseases and intervention therapy, our review delineates the characteristics, kinetics, signaling pathways, and potential mechanisms of CRS, which shows its clinical relevance for achieving both favorable efficacy and low toxicity.
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Affiliation(s)
- Xia Li
- grid.13402.340000 0004 1759 700XBone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China ,grid.13402.340000 0004 1759 700XLiangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121 People’s Republic of China ,grid.13402.340000 0004 1759 700XInstitute of Hematology, Zhejiang University, Hangzhou, Zhejiang People’s Republic of China ,grid.13402.340000 0004 1759 700XZhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou, Zhejiang People’s Republic of China
| | - Mi Shao
- grid.13402.340000 0004 1759 700XBone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China ,grid.13402.340000 0004 1759 700XLiangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121 People’s Republic of China ,grid.13402.340000 0004 1759 700XInstitute of Hematology, Zhejiang University, Hangzhou, Zhejiang People’s Republic of China ,grid.13402.340000 0004 1759 700XZhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou, Zhejiang People’s Republic of China
| | - Xiangjun Zeng
- grid.13402.340000 0004 1759 700XBone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China ,grid.13402.340000 0004 1759 700XLiangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121 People’s Republic of China ,grid.13402.340000 0004 1759 700XInstitute of Hematology, Zhejiang University, Hangzhou, Zhejiang People’s Republic of China ,grid.13402.340000 0004 1759 700XZhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou, Zhejiang People’s Republic of China
| | - Pengxu Qian
- grid.13402.340000 0004 1759 700XBone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China ,grid.13402.340000 0004 1759 700XLiangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121 People’s Republic of China ,grid.13402.340000 0004 1759 700XInstitute of Hematology, Zhejiang University, Hangzhou, Zhejiang People’s Republic of China ,grid.13402.340000 0004 1759 700XZhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou, Zhejiang People’s Republic of China ,grid.13402.340000 0004 1759 700XCenter of Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China
| | - He Huang
- grid.13402.340000 0004 1759 700XBone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China ,grid.13402.340000 0004 1759 700XLiangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, 311121 People’s Republic of China ,grid.13402.340000 0004 1759 700XInstitute of Hematology, Zhejiang University, Hangzhou, Zhejiang People’s Republic of China ,grid.13402.340000 0004 1759 700XZhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou, Zhejiang People’s Republic of China
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26
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Irving M, Lanitis E, Migliorini D, Ivics Z, Guedan S. Choosing the Right Tool for Genetic Engineering: Clinical Lessons from Chimeric Antigen Receptor-T Cells. Hum Gene Ther 2021; 32:1044-1058. [PMID: 34662233 PMCID: PMC8697565 DOI: 10.1089/hum.2021.173] [Citation(s) in RCA: 38] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
T cell modification with genes that encode chimeric antigen receptors (CAR-T cells) has shown tremendous promise for the treatment of B cell malignancies. The successful translation of CAR-T cell therapy to other tumor types, including solid tumors, is the next big challenge. As the field advances from second- to next-generation CAR-T cells comprising multiple genetic modifications, more sophisticated methods and tools to engineer T cells are being developed. Viral vectors, especially γ-retroviruses and lentiviruses, are traditionally used for CAR-T cell engineering due to their high transduction efficiency. However, limited genetic cargo, high costs of production under good manufacturing practice (GMP) conditions, and the high regulatory demands are obstacles for widespread clinical translation. To overcome these limitations, different nonviral approaches are being explored at a preclinical or clinical level, including transposon/transposase systems and mRNA electroporation and nonintegrating DNA nanovectors. Genome editing tools that allow efficient knockout of particular genes and/or site-directed integration of the CAR and/or other transgenes into the genome are also being evaluated for CAR-T cell engineering. In this review, we discuss the development of viral and nonviral vectors used to generate CAR-T cells, focusing on their advantages and limitations. We also discuss the lessons learned from clinical trials using the different genetic engineering tools, with special focus on safety and efficacy.
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Affiliation(s)
- Melita Irving
- Department of Oncology, Ludwig Institute for Cancer Research Lausanne, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland
| | - Evripidis Lanitis
- Department of Oncology, Ludwig Institute for Cancer Research Lausanne, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland
| | - Denis Migliorini
- Department of Oncology, Geneva University Hospitals, Geneva, Switzerland.,Center for Translational Research in Onco-Hematology, University of Geneva, Geneva, Switzerland.,Swiss Cancer Center Léman, Geneva and Lausanne, Switzerland
| | - Zoltán Ivics
- Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany
| | - Sonia Guedan
- Department of Hematology and Oncology, Hospital Clinic, August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Barcelona, Spain
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27
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Abstract
Dysfunction in T cells limits the efficacy of cancer immunotherapy. We profiled the epigenome, transcriptome, and enhancer connectome of exhaustion-prone GD2-targeting HA-28z chimeric antigen receptor (CAR) T cells and control CD19-targeting CAR T cells, which present less exhaustion-inducing tonic signaling, at multiple points during their ex vivo expansion. We found widespread, dynamic changes in chromatin accessibility and three-dimensional (3D) chromosome conformation preceding changes in gene expression, notably at loci proximal to exhaustion-associated genes such as PDCD1, CTLA4, and HAVCR2, and increased DNA motif access for AP-1 family transcription factors, which are known to promote exhaustion. Although T cell exhaustion has been studied in detail in mice, we find that the regulatory networks of T cell exhaustion differ between species and involve distinct loci of accessible chromatin and cis-regulated target genes in human CAR T cell exhaustion. Deletion of exhaustion-specific candidate enhancers of PDCD1 suppress the expression of PD-1 in an in vitro model of T cell dysfunction and in HA-28z CAR T cells, suggesting enhancer editing as a path forward in improving cancer immunotherapy.
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28
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Bode D, Cull AH, Rubio-Lara JA, Kent DG. Exploiting Single-Cell Tools in Gene and Cell Therapy. Front Immunol 2021; 12:702636. [PMID: 34322133 PMCID: PMC8312222 DOI: 10.3389/fimmu.2021.702636] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2021] [Accepted: 06/28/2021] [Indexed: 12/12/2022] Open
Abstract
Single-cell molecular tools have been developed at an incredible pace over the last five years as sequencing costs continue to drop and numerous molecular assays have been coupled to sequencing readouts. This rapid period of technological development has facilitated the delineation of individual molecular characteristics including the genome, transcriptome, epigenome, and proteome of individual cells, leading to an unprecedented resolution of the molecular networks governing complex biological systems. The immense power of single-cell molecular screens has been particularly highlighted through work in systems where cellular heterogeneity is a key feature, such as stem cell biology, immunology, and tumor cell biology. Single-cell-omics technologies have already contributed to the identification of novel disease biomarkers, cellular subsets, therapeutic targets and diagnostics, many of which would have been undetectable by bulk sequencing approaches. More recently, efforts to integrate single-cell multi-omics with single cell functional output and/or physical location have been challenging but have led to substantial advances. Perhaps most excitingly, there are emerging opportunities to reach beyond the description of static cellular states with recent advances in modulation of cells through CRISPR technology, in particular with the development of base editors which greatly raises the prospect of cell and gene therapies. In this review, we provide a brief overview of emerging single-cell technologies and discuss current developments in integrating single-cell molecular screens and performing single-cell multi-omics for clinical applications. We also discuss how single-cell molecular assays can be usefully combined with functional data to unpick the mechanism of cellular decision-making. Finally, we reflect upon the introduction of spatial transcriptomics and proteomics, its complementary role with single-cell RNA sequencing (scRNA-seq) and potential application in cellular and gene therapy.
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Affiliation(s)
- Daniel Bode
- Wellcome Medical Research Council (MRC) Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom
- Department of Haematology, University of Cambridge, Cambridge, United Kingdom
| | - Alyssa H. Cull
- York Biomedical Research Institute, Department of Biology, University of York, York, United Kingdom
| | - Juan A. Rubio-Lara
- York Biomedical Research Institute, Department of Biology, University of York, York, United Kingdom
| | - David G. Kent
- York Biomedical Research Institute, Department of Biology, University of York, York, United Kingdom
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29
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Swanson E, Lord C, Reading J, Heubeck AT, Genge PC, Thomson Z, Weiss MDA, Li XJ, Savage AK, Green RR, Torgerson TR, Bumol TF, Graybuck LT, Skene PJ. Simultaneous trimodal single-cell measurement of transcripts, epitopes, and chromatin accessibility using TEA-seq. eLife 2021; 10:e63632. [PMID: 33835024 PMCID: PMC8034981 DOI: 10.7554/elife.63632] [Citation(s) in RCA: 138] [Impact Index Per Article: 34.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2020] [Accepted: 03/11/2021] [Indexed: 01/04/2023] Open
Abstract
Single-cell measurements of cellular characteristics have been instrumental in understanding the heterogeneous pathways that drive differentiation, cellular responses to signals, and human disease. Recent advances have allowed paired capture of protein abundance and transcriptomic state, but a lack of epigenetic information in these assays has left a missing link to gene regulation. Using the heterogeneous mixture of cells in human peripheral blood as a test case, we developed a novel scATAC-seq workflow that increases signal-to-noise and allows paired measurement of cell surface markers and chromatin accessibility: integrated cellular indexing of chromatin landscape and epitopes, called ICICLE-seq. We extended this approach using a droplet-based multiomics platform to develop a trimodal assay that simultaneously measures transcriptomics (scRNA-seq), epitopes, and chromatin accessibility (scATAC-seq) from thousands of single cells, which we term TEA-seq. Together, these multimodal single-cell assays provide a novel toolkit to identify type-specific gene regulation and expression grounded in phenotypically defined cell types.
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Affiliation(s)
| | - Cara Lord
- Allen Institute for ImmunologySeattleUnited States
| | | | | | | | | | | | - Xiao-jun Li
- Allen Institute for ImmunologySeattleUnited States
| | | | - Richard R Green
- Allen Institute for ImmunologySeattleUnited States
- Department of Biomedical Informatics and Medical Education (BIME), University of WashingtonSeattleUnited States
| | - Troy R Torgerson
- Allen Institute for ImmunologySeattleUnited States
- Department of Pediatrics, University of WashingtonSeattleUnited States
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30
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Jefferys SR, Burgos SD, Peterson JJ, Selitsky SR, Turner AMW, James LI, Tsai YH, Coffey AR, Margolis DM, Parker J, Browne EP. Epigenomic characterization of latent HIV infection identifies latency regulating transcription factors. PLoS Pathog 2021; 17:e1009346. [PMID: 33635929 PMCID: PMC7946360 DOI: 10.1371/journal.ppat.1009346] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2020] [Revised: 03/10/2021] [Accepted: 01/29/2021] [Indexed: 12/12/2022] Open
Abstract
Transcriptional silencing of HIV in CD4 T cells generates a reservoir of latently infected cells that can reseed infection after interruption of therapy. As such, these cells represent the principal barrier to curing HIV infection, but little is known about their characteristics. To further our understanding of the molecular mechanisms of latency, we characterized a primary cell model of HIV latency in which infected cells adopt heterogeneous transcriptional fates. In this model, we observed that latency is a stable, heritable state that is transmitted through cell division. Using Assay of Transposon-Accessible Chromatin sequencing (ATACseq) we found that latently infected cells exhibit greatly reduced proviral accessibility, indicating the presence of chromatin-based structural barriers to viral gene expression. By quantifying the activity of host cell transcription factors, we observe elevated activity of Forkhead and Kruppel-like factor transcription factors (TFs), and reduced activity of AP-1, RUNX and GATA TFs in latently infected cells. Interestingly, latency reversing agents with different mechanisms of action caused distinct patterns of chromatin reopening across the provirus. We observe that binding sites for the chromatin insulator CTCF are highly enriched in the differentially open chromatin of infected CD4 T cells. Furthermore, depletion of CTCF inhibited HIV latency, identifying this factor as playing a key role in the initiation or enforcement of latency. These data indicate that HIV latency develops preferentially in cells with a distinct pattern of TF activity that promotes a closed proviral structure and inhibits viral gene expression. Furthermore, these findings identify CTCF as a novel regulator of HIV latency. HIV is able to persist during antiviral therapy by entering a state of viral latency, in which viral gene expression is greatly reduced. These latently infected cells can re-seed infection if therapy is interrupted, and thus represent a major obstacle to an HIV cure. Identifying the mechanisms that lead to this state will help to identify strategies to block or eliminate HIV latency, leading to a cure for infection. By observing HIV gene expression in infected CD4 T cells, we isolated cells in which HIV has entered latency and identified characteristics that distinguish them from cells with active viral replication. We found that latently infected cells have elevated activity of specific transcription factors including Forkhead TFs and Kruppel-like factors. We also identify CTCF, a protein responsible for mediating insulation of genome domains from each other, as being required for the establishment of HIV latency. Developing agents to target these factors may lead to new strategies to eliminate the HIV reservoir.
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Affiliation(s)
- Stuart R. Jefferys
- Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Samuel D. Burgos
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Jackson J. Peterson
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Sara R. Selitsky
- Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Anne-Marie W. Turner
- Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Lindsey I. James
- Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Yi-Hsuan Tsai
- Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Alisha R. Coffey
- Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - David M. Margolis
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Joel Parker
- Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Edward P. Browne
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- * E-mail:
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31
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Abstract
Chimeric antigen receptor-T (CAR-T) cell therapy is a promising frontier of immunoengineering and cancer immunotherapy. Methods that detect, quantify, track, and visualize the CAR, have catalyzed the rapid advancement of CAR-T cell therapy from preclinical models to clinical adoption. For instance, CAR-staining/labeling agents have enabled flow cytometry analysis, imaging applications, cell sorting, and high-dimensional clinical profiling. Molecular assays, such as quantitative polymerase chain reaction, integration site analysis, and RNA-sequencing, have characterized CAR transduction, expression, and in vivo CAR-T cell expansion kinetics. In vitro visualization methods, including confocal and total internal reflection fluorescence microscopy, have captured the molecular details underlying CAR immunological synapse formation, signaling, and cytotoxicity. In vivo tracking methods, including two-photon microscopy, bioluminescence imaging, and positron emission tomography scanning, have monitored CAR-T cell biodistribution across blood, tissue, and tumor. Here, we review the plethora of CAR detection methods, which can operate at the genomic, transcriptomic, proteomic, and organismal levels. For each method, we discuss: (1) what it measures; (2) how it works; (3) its scientific and clinical importance; (4) relevant examples of its use; (5) specific protocols for CAR detection; and (6) its strengths and weaknesses. Finally, we consider current scientific and clinical needs in order to provide future perspectives for improved CAR detection.
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Affiliation(s)
- Yifei Hu
- Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL, United States
- Pritzker School of Medicine, University of Chicago, Chicago, IL, United States
| | - Jun Huang
- Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL, United States
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32
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Zhou M, Hu Z, Zhang C, Wu L, Li Z, Liang D. Gene Therapy for Hemophilia A: Where We Stand. Curr Gene Ther 2020; 20:142-151. [PMID: 32767930 DOI: 10.2174/1566523220666200806110849] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2020] [Revised: 06/25/2020] [Accepted: 07/13/2020] [Indexed: 01/19/2023]
Abstract
Hemophilia A (HA) is a hereditary hemorrhagic disease caused by a deficiency of coagulation factor VIII (FVIII) in blood plasma. Patients with HA usually suffer from spontaneous and recurrent bleeding in joints and muscles, or even intracerebral hemorrhage, which might lead to disability or death. Although the disease is currently manageable via delivery of plasma-derived or recombinant FVIII, this approach is costly, and neutralizing antibodies may be generated in a large portion of patients, which render the regimens ineffective and inaccessible. Given the monogenic nature of HA and that a slight increase in FVIII can remarkably alleviate the phenotypes, HA has been considered to be a suitable target disease for gene therapy. Consequently, the introduction of a functional F8 gene copy into the appropriate target cells via viral or nonviral delivery vectors, including gene correction through genome editing approaches, could ultimately provide an effective therapeutic method for HA patients. In this review, we discuss the recent progress of gene therapy for HA with viral and nonviral delivery vectors, including piggyBac, lentiviral and adeno-associated viral vectors, as well as new raising issues involving liver toxicity, pre-existing neutralizing antibodies of viral approach, and the selection of the target cell type for nonviral delivery.
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Affiliation(s)
- Miaojin Zhou
- Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan 410078, China
| | - Zhiqing Hu
- Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan 410078, China
| | - Chunhua Zhang
- Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan 410078, China
| | - Lingqian Wu
- Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan 410078, China
| | - Zhuo Li
- Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan 410078, China
| | - Desheng Liang
- Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan 410078, China
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