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Oliveira BC, Bari S, Melenhorst JJ. Leveraging Vector-Based Gene Disruptions to Enhance CAR T-Cell Effectiveness. Cancers (Basel) 2025; 17:383. [PMID: 39941752 PMCID: PMC11815729 DOI: 10.3390/cancers17030383] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2024] [Revised: 01/21/2025] [Accepted: 01/21/2025] [Indexed: 02/16/2025] Open
Abstract
Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy represents a breakthrough in the treatment of relapsed and refractory B-cell malignancies, such as chronic lymphocytic leukemia (CLL), inducing long-term, sometimes curative, responses. However, fewer than 30% of CLL patients achieve such outcomes. It has been shown that a smaller subset of T cells capable of expansion and persistence is crucial for treatment effectiveness. Notably, a pre-existing mutation in the epigenetic regulator TET2, combined with CAR vector-induced disruption of the other intact allele, significantly enhanced the potency of the CAR-engineered T-cell clone in one CLL patient. This finding aligns with independent research, suggesting that the CAR gene's genomic insertion site influences tumor-targeting capability. Thus, it is plausible that vector-induced gene disruptions affect CAR T-cell function. This review synthesizes existing knowledge on vector integration into the host genome and its impact on clinical outcomes in CAR T-cell therapy patients. Our aim is to inform the development of improved therapies and enhance their overall efficacy.
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Affiliation(s)
| | | | - J. Joseph Melenhorst
- Cell Therapy & Immuno-Engineering Program, Center for Immunotherapy and Precision Immuno-Oncology, Lerner College of Medicine, Cleveland Clinic, Cleveland, OH 44016, USA; (B.C.O.); (S.B.)
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2
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Stevens CS, Carmichael JC, Watkinson R, Kowdle S, Reis RA, Hamane K, Jang J, Park A, Pernet O, Khamaikawin W, Hong P, Thibault P, Gowlikar A, An DS, Lee B. A temperature-sensitive and less immunogenic Sendai virus for efficient gene editing. J Virol 2024; 98:e0083224. [PMID: 39494910 PMCID: PMC11650993 DOI: 10.1128/jvi.00832-24] [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: 05/09/2024] [Accepted: 10/10/2024] [Indexed: 11/05/2024] Open
Abstract
The therapeutic potential of gene editing technologies hinges on the development of safe and effective delivery methods. In this study, we developed a temperature-sensitive and less immunogenic Sendai virus (ts SeV) as a novel delivery vector for CRISPR-Cas9 and for efficient gene editing in sensitive human cell types with limited induction of an innate immune response. ts SeV demonstrates high transduction efficiency in human CD34+ hematopoietic stem and progenitor cells (HSPCs) including transduction of the CD34+/CD38-/CD45RA-/CD90+(Thy1+)/CD49fhigh stem cell enriched subpopulation. The frequency of CCR5 editing exceeded 90% and bi-allelic CCR5 editing exceeded 70% resulting in significant inhibition of HIV-1 infection in primary human CD14+ monocytes. These results demonstrate the potential of the ts SeV platform as a safe, efficient, and flexible addition to the current gene-editing tool delivery methods, which may help further expand the possibilities in personalized medicine and the treatment of genetic disorders. IMPORTANCE Gene editing has the potential to be a powerful tool for the treatment of human diseases including HIV, β-thalassemias, and sickle cell disease. Recent advances have begun to overcome one of the major limiting factors of this technology, namely delivery of the CRISPR-Cas9 gene editing machinery, by utilizing viral vectors. However, gene editing therapies have yet to be implemented due to inherent risks associated with the DNA viral vectors typically used for delivery. As an alternative strategy, we have developed an RNA-based Sendai virus CRISPR-Cas9 delivery vector that does not integrate into the genome, is temperature sensitive, and does not induce a significant host interferon response. This recombinant SeV successfully delivered CRISPR-Cas9 in primary human CD14+ monocytes ex vivo resulting in a high level of CCR5 editing and inhibition of HIV infection.
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Affiliation(s)
- Christian S. Stevens
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Jillian C. Carmichael
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Ruth Watkinson
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Shreyas Kowdle
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Rebecca A. Reis
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Kory Hamane
- UCLA School of Nursing, Los Angeles, California, USA
- UCLA AIDS Institute, Los Angeles, California, USA
| | - Jason Jang
- UCLA School of Nursing, Los Angeles, California, USA
- UCLA AIDS Institute, Los Angeles, California, USA
| | - Arnold Park
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Olivier Pernet
- UCLA School of Nursing, Los Angeles, California, USA
- UCLA AIDS Institute, Los Angeles, California, USA
| | - Wannisa Khamaikawin
- UCLA School of Nursing, Los Angeles, California, USA
- UCLA AIDS Institute, Los Angeles, California, USA
| | - Patrick Hong
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Patricia Thibault
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Aditya Gowlikar
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Dong Sung An
- UCLA School of Nursing, Los Angeles, California, USA
- UCLA AIDS Institute, Los Angeles, California, USA
| | - Benhur Lee
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
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3
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Karimi-Sani I, Molavi Z, Naderi S, Mirmajidi SH, Zare I, Naeimzadeh Y, Mansouri A, Tajbakhsh A, Savardashtaki A, Sahebkar A. Personalized mRNA vaccines in glioblastoma therapy: from rational design to clinical trials. J Nanobiotechnology 2024; 22:601. [PMID: 39367418 PMCID: PMC11453023 DOI: 10.1186/s12951-024-02882-x] [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: 06/03/2024] [Accepted: 09/26/2024] [Indexed: 10/06/2024] Open
Abstract
Glioblastomas (GBMs) are the most common and aggressive malignant brain tumors, presenting significant challenges for treatment due to their invasive nature and localization in critical brain regions. Standard treatment includes surgical resection followed by radiation and adjuvant chemotherapy with temozolomide (TMZ). Recent advances in immunotherapy, including the use of mRNA vaccines, offer promising alternatives. This review focuses on the emerging use of mRNA vaccines for GBM treatment. We summarize recent advancements, evaluate current obstacles, and discuss notable successes in this field. Our analysis highlights that while mRNA vaccines have shown potential, their use in GBM treatment is still experimental. Ongoing research and clinical trials are essential to fully understand their therapeutic potential. Future developments in mRNA vaccine technology and insights into GBM-specific immune responses may lead to more targeted and effective treatments. Despite the promise, further research is crucial to validate and optimize the effectiveness of mRNA vaccines in combating GBM.
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Affiliation(s)
- Iman Karimi-Sani
- Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Zahra Molavi
- Proteomics Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Samaneh Naderi
- Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Seyedeh-Habibeh Mirmajidi
- Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Iman Zare
- Research and Development Department, Sina Medical Biochemistry Technologies Co. Ltd., Shiraz, 7178795844, Iran
| | - Yasaman Naeimzadeh
- Department of Molecular Medicine, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Atena Mansouri
- Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran
| | - Amir Tajbakhsh
- Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Amir Savardashtaki
- Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran.
- Infertility Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
| | - Amirhossein Sahebkar
- Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
- Applied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
- Department of Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.
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4
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Wang F, Huang Y, Li J, Zhou W, Wang W. Targeted gene delivery systems for T-cell engineering. Cell Oncol (Dordr) 2024; 47:1537-1560. [PMID: 38753155 DOI: 10.1007/s13402-024-00954-6] [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] [Accepted: 04/28/2024] [Indexed: 06/27/2024] Open
Abstract
T lymphocytes are indispensable for the host systems of defense against pathogens, tumors, and environmental threats. The therapeutic potential of harnessing the cytotoxic properties of T lymphocytes for antigen-specific cell elimination is both evident and efficacious. Genetically engineered T-cells, such as those employed in CAR-T and TCR-T cell therapies, have demonstrated significant clinical benefits in treating cancer and autoimmune disorders. However, the current landscape of T-cell genetic engineering is dominated by strategies that necessitate in vitro T-cell isolation and modification, which introduce complexity and prolong the development timeline of T-cell based immunotherapies. This review explores the complexities of gene delivery systems designed for T cells, covering both viral and nonviral vectors. Viral vectors are known for their high transduction efficiency, yet they face significant limitations, such as potential immunogenicity and the complexities involved in large-scale production. Nonviral vectors, conversely, offer a safer profile and the potential for scalable manufacturing, yet they often struggle with lower transduction efficiency. The pursuit of gene delivery systems that can achieve targeted gene transfer to T cell without the need for isolation represents a significant advancement in the field. This review assesses the design principles and current research progress of such systems, highlighting the potential for in vivo gene modification therapies that could revolutionize T-cell based treatments. By providing a comprehensive analysis of these systems, we aim to contribute valuable insights into the future development of T-cell immunotherapy.
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Affiliation(s)
- Fengling Wang
- Department of Biotherapy, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, 610041, People's Republic of China
| | - Yong Huang
- Department of Biotherapy, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, 610041, People's Republic of China
| | - JiaQian Li
- Department of Biotherapy, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, 610041, People's Republic of China
| | - Weilin Zhou
- Department of Biotherapy, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, 610041, People's Republic of China
| | - Wei Wang
- Department of Biotherapy, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, 610041, People's Republic of China.
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5
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Graham JP, Castro JG, Werba LC, Fardone LC, Francis KP, Ramamurthi A, Layden M, McCarthy HO, Gonzalez-Fernandez T. Versatile Cell Penetrating Peptide for Multimodal CRISPR Gene Editing in Primary Stem Cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.09.23.614499. [PMID: 39386541 PMCID: PMC11463527 DOI: 10.1101/2024.09.23.614499] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 10/12/2024]
Abstract
CRISPR gene editing offers unprecedented genomic and transcriptomic control for precise regulation of cell function and phenotype. However, delivering the necessary CRISPR components to therapeutically relevant cell types without cytotoxicity or unexpected side effects remains challenging. Viral vectors risk genomic integration and immunogenicity while non-viral delivery systems are challenging to adapt to different CRISPR cargos, and many are highly cytotoxic. The arginine-alanine-leucine-alanine (RALA) cell penetrating peptide is an amphiphilic peptide that self-assembles into nanoparticles through electrostatic interactions with negatively charged molecules before delivering them across the cell membrane. This system has been used to deliver DNAs, RNAs, and small anionic molecules to primary cells with lower cytotoxicity compared to alternative non-viral approaches. Given the low cytotoxicity, versatility, and competitive transfection rates of RALA, we aimed to establish this peptide as a new CRISPR delivery system in a wide range of molecular formats across different editing modalities. We report that RALA was able to effectively encapsulate and deliver CRISPR in DNA, RNA, and ribonucleic protein (RNP) formats to primary mesenchymal stem cells (MSCs). Comparisons between RALA and commercially available reagents revealed superior cell viability leading to higher numbers of transfected cells and the maintenance of cell proliferative capacity. We then used the RALA peptide for the knock-in and knock-out of reporter genes into the MSC genome as well as for the transcriptional activation of therapeutically relevant genes. In summary, we establish RALA as a powerful tool for safer and effective delivery of CRISPR machinery in multiple cargo formats for a wide range of gene editing strategies.
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Affiliation(s)
- Josh P. Graham
- Department of Bioengineering, Lehigh University, Bethlehem, PA, USA
| | | | - Lisette C. Werba
- Department of Bioengineering, Lehigh University, Bethlehem, PA, USA
| | - Luke C. Fardone
- Department of Bioengineering, Lehigh University, Bethlehem, PA, USA
| | | | - Anand Ramamurthi
- Department of Bioengineering, Lehigh University, Bethlehem, PA, USA
| | - Michael Layden
- Department of Biological Sciences, Lehigh University, Bethlehem, PA, USA
| | - Helen O. McCarthy
- School of Pharmacy, Queen’s University Belfast, Northern Ireland, United Kingdom
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6
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Afzal SY, MacDougall MS, McGhee SA. Immunodeficiency: Gene therapy for primary immune deficiency. Allergy Asthma Proc 2024; 45:384-388. [PMID: 39294901 DOI: 10.2500/aap.2024.45.240054] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/21/2024]
Abstract
Current gene therapy for inborn errors of immunity have involved the use of gene addition approaches with viral delivery. This main strategy has had demonstrated success mainly in severe combined immune deficiency, Wiskott-Aldrich syndrome, and chronic granulomatous disease. Despite the increasing success of gene therapy, there are limitations of gene therapy, and, therefore, hematopoietic stem cell transplantation continues to be the preferred option. With improvements in viral delivery through next-generation lentiviral vectors and the advent of gene editing with CRISPR-Cas9, the efficacy and safety of gene therapy may soon surpass hematopoietic stem cell transplantation. Furthermore, these advances improve the viability of gene therapy for inborn errors of immunity primarily through decreased risk of transplantation-related complications. Therefore, despite current limitations, gene therapy for inborn errors of immunity is poised to continue to expand to more patients and indications.
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7
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Rozenbaum M, Fluss R, Marcu-Malina V, Sarouk I, Meir A, Elitzur S, Zinger T, Jacob-Hirsch J, Saar EG, Rechavi G, Jacoby E. Genotoxicity Associated with Retroviral CAR Transduction of ATM-Deficient T Cells. Blood Cancer Discov 2024; 5:267-275. [PMID: 38747501 PMCID: PMC11215369 DOI: 10.1158/2643-3230.bcd-23-0268] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2023] [Revised: 02/26/2024] [Accepted: 05/08/2024] [Indexed: 07/02/2024] Open
Abstract
Somatic variants in DNA damage response genes such as ATM are widespread in hematologic malignancies. ATM protein is essential for double-strand DNA break repair. Germline ATM deficiencies underlie ataxia-telangiectasia (A-T), a disease manifested by radiosensitivity, immunodeficiency, and predisposition to lymphoid malignancies. Patients with A-T diagnosed with malignancies have poor tolerance to chemotherapy or radiation. In this study, we investigated chimeric antigen receptor (CAR) T cells using primary T cells from patients with A-T (ATM-/-), heterozygote donors (ATM+/-), and healthy donors. ATM-/- T cells proliferate and can be successfully transduced with CARs, though functional impairment of ATM-/- CAR T-cells was observed. Retroviral transduction of the CAR in ATM-/- T cells resulted in high rates of chromosomal lesions at CAR insertion sites, as confirmed by next-generation long-read sequencing. This work suggests that ATM is essential to preserve genome integrity of CAR T-cells during retroviral manufacturing, and its lack poses a risk of chromosomal translocations and potential leukemogenicity. Significance: CAR T-cells are clinically approved genetically modified cells, but the control of genome integrity remains largely uncharacterized. This study demonstrates that ATM deficiency marginally impairs CAR T-cell function and results in high rates of chromosomal aberrations after retroviral transduction, which may be of concern in patients with DNA repair deficiencies.
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Affiliation(s)
- Meir Rozenbaum
- Cell Therapy Lab, Sheba Medical Center, Tel Hashomer, Israel.
| | - Reut Fluss
- Cancer Research Center, Sheba Medical Center, Tel Hashomer, Israel.
- Wohl Centre for Translational Medicine, Sheba Medical Center, Tel Hashomer, Israel.
| | | | - Ifat Sarouk
- National A-T Center, Pediatric Pulmonology Unit, The Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Tel Hashomer, Israel.
| | - Amilia Meir
- Cell Therapy Lab, Sheba Medical Center, Tel Hashomer, Israel.
| | - Sarah Elitzur
- Department of Pediatric Hematology-Oncology, Schneider Children’s Medical Center, Petah Tikva, Israel.
- Faculty of Medicinal & Health Sciences, Tel Aviv University, Tel Aviv, Israel.
| | - Tal Zinger
- Cancer Research Center, Sheba Medical Center, Tel Hashomer, Israel.
| | - Jasmine Jacob-Hirsch
- Cancer Research Center, Sheba Medical Center, Tel Hashomer, Israel.
- Wohl Centre for Translational Medicine, Sheba Medical Center, Tel Hashomer, Israel.
| | - Efrat G. Saar
- Cancer Research Center, Sheba Medical Center, Tel Hashomer, Israel.
- Wohl Centre for Translational Medicine, Sheba Medical Center, Tel Hashomer, Israel.
| | - Gideon Rechavi
- Cancer Research Center, Sheba Medical Center, Tel Hashomer, Israel.
- Wohl Centre for Translational Medicine, Sheba Medical Center, Tel Hashomer, Israel.
- Faculty of Medicinal & Health Sciences, Tel Aviv University, Tel Aviv, Israel.
| | - Elad Jacoby
- Cell Therapy Lab, Sheba Medical Center, Tel Hashomer, Israel.
- Faculty of Medicinal & Health Sciences, Tel Aviv University, Tel Aviv, Israel.
- Division of Pediatric Hematology and Oncology, The Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Tel Hashomer, Israel.
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8
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McIvor RS, Eaton EJ, Webber BR, Moriarity BS. Advancing gene targeting for primary immune deficiencies: Adenine base editing of the human IL2RG locus for correction of SCID-X1. Mol Ther 2024; 32:1606-1608. [PMID: 38781958 PMCID: PMC11184398 DOI: 10.1016/j.ymthe.2024.05.026] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2024] [Revised: 05/10/2024] [Accepted: 05/10/2024] [Indexed: 05/25/2024] Open
Affiliation(s)
- R Scott McIvor
- Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA.
| | - Ella J Eaton
- Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
| | - Beau R Webber
- Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA; Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Institute for Engineering in Medicine, University of Minnesota, Minneapolis, MN 55455, USA
| | - Branden S Moriarity
- Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA; Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Institute for Engineering in Medicine, University of Minnesota, Minneapolis, MN 55455, USA
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9
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Stevens CS, Carmichael J, Watkinson R, Kowdle S, Reis RA, Hamane K, Jang J, Park A, Pernet O, Khamaikawin W, Hong P, Thibault P, Gowlikar A, An DS, Lee B. A temperature-sensitive and interferon-silent Sendai virus vector for CRISPR-Cas9 delivery and gene editing in primary human cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.05.03.592383. [PMID: 38746439 PMCID: PMC11092779 DOI: 10.1101/2024.05.03.592383] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/16/2024]
Abstract
The transformative potential of gene editing technologies hinges on the development of safe and effective delivery methods. In this study, we developed a temperature-sensitive and interferon-silent Sendai virus (ts SeV) as a novel delivery vector for CRISPR-Cas9 and for efficient gene editing in sensitive human cell types without inducing IFN responses. ts SeV demonstrates unprecedented transduction efficiency in human CD34+ hematopoietic stem and progenitor cells (HSPCs) including transduction of the CD34+/CD38-/CD45RA-/CD90+(Thy1+)/CD49fhigh stem cell enriched subpopulation. The frequency of CCR5 editing exceeded 90% and bi-allelic CCR5 editing exceeded 70% resulting in significant inhibition of HIV-1 infection in primary human CD14+ monocytes. These results demonstrate the potential of the ts SeV platform as a safe, efficient, and flexible addition to the current gene-editing tool delivery methods, which may help to further expand the possibilities in personalized medicine and the treatment of genetic disorders.
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Affiliation(s)
- Christian S Stevens
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Jillian Carmichael
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Ruth Watkinson
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Shreyas Kowdle
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Rebecca A Reis
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Kory Hamane
- UCLA School of Nursing, Los Angeles, California, 90095
- UCLA AIDS Institute, Los Angeles, California, 90095
| | - Jason Jang
- UCLA School of Nursing, Los Angeles, California, 90095
- UCLA AIDS Institute, Los Angeles, California, 90095
| | - Arnold Park
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Olivier Pernet
- UCLA School of Nursing, Los Angeles, California, 90095
- UCLA AIDS Institute, Los Angeles, California, 90095
| | - Wannisa Khamaikawin
- UCLA School of Nursing, Los Angeles, California, 90095
- UCLA AIDS Institute, Los Angeles, California, 90095
| | - Patrick Hong
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Patricia Thibault
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Aditya Gowlikar
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Dong Sung An
- UCLA School of Nursing, Los Angeles, California, 90095
- UCLA AIDS Institute, Los Angeles, California, 90095
| | - Benhur Lee
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
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10
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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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11
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Cesana D, Cicalese MP, Calabria A, Merli P, Caruso R, Volpin M, Rudilosso L, Migliavacca M, Barzaghi F, Fossati C, Gazzo F, Pizzi S, Ciolfi A, Bruselles A, Tucci F, Spinozzi G, Pais G, Benedicenti F, Barcella M, Merelli I, Gallina P, Giannelli S, Dionisio F, Scala S, Casiraghi M, Strocchio L, Vinti L, Pacillo L, Draghi E, Cesana M, Riccardo S, Colantuono C, Six E, Cavazzana M, Carlucci F, Schmidt M, Cancrini C, Ciceri F, Vago L, Cacchiarelli D, Gentner B, Naldini L, Tartaglia M, Montini E, Locatelli F, Aiuti A. A case of T-cell acute lymphoblastic leukemia in retroviral gene therapy for ADA-SCID. Nat Commun 2024; 15:3662. [PMID: 38688902 PMCID: PMC11061298 DOI: 10.1038/s41467-024-47866-5] [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: 10/13/2023] [Accepted: 04/10/2024] [Indexed: 05/02/2024] Open
Abstract
Hematopoietic stem cell gene therapy (GT) using a γ-retroviral vector (γ-RV) is an effective treatment for Severe Combined Immunodeficiency due to Adenosine Deaminase deficiency. Here, we describe a case of GT-related T-cell acute lymphoblastic leukemia (T-ALL) that developed 4.7 years after treatment. The patient underwent chemotherapy and haploidentical transplantation and is currently in remission. Blast cells contain a single vector insertion activating the LIM-only protein 2 (LMO2) proto-oncogene, confirmed by physical interaction, and low Adenosine Deaminase (ADA) activity resulting from methylation of viral promoter. The insertion is detected years before T-ALL in multiple lineages, suggesting that further hits occurred in a thymic progenitor. Blast cells contain known and novel somatic mutations as well as germline mutations which may have contributed to transformation. Before T-ALL onset, the insertion profile is similar to those of other ADA-deficient patients. The limited incidence of vector-related adverse events in ADA-deficiency compared to other γ-RV GT trials could be explained by differences in transgenes, background disease and patient's specific factors.
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Affiliation(s)
- Daniela Cesana
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Maria Pia Cicalese
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
- Paediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy
- Università Vita-Salute San Raffaele, Milan, Italy
| | - Andrea Calabria
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Pietro Merli
- IRCCS Bambino Gesù Children's Hospital, Rome, Italy
| | | | - Monica Volpin
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Laura Rudilosso
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Maddalena Migliavacca
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
- Paediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Federica Barzaghi
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
- Paediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Claudia Fossati
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Francesco Gazzo
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Simone Pizzi
- Molecular Genetics and Functional Genomics, IRCCS Bambino Gesù Children's Hospital, Rome, Italy
| | - Andrea Ciolfi
- Molecular Genetics and Functional Genomics, IRCCS Bambino Gesù Children's Hospital, Rome, Italy
| | - Alessandro Bruselles
- Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy
| | - Francesca Tucci
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
- Paediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Giulio Spinozzi
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Giulia Pais
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Fabrizio Benedicenti
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Matteo Barcella
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
- National Research Council, Institute for Biomedical Technologies, Segrate, Italy
| | - Ivan Merelli
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
- National Research Council, Institute for Biomedical Technologies, Segrate, Italy
| | - Pierangela Gallina
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Stefania Giannelli
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Francesca Dionisio
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Serena Scala
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Miriam Casiraghi
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
| | | | | | - Lucia Pacillo
- Immune and Infectious Diseases Division, Research Unit of Primary Immunodeficiencies, Academic Department of Pediatrics, IRCCS Bambino Gesù Children's Hospital, Rome, Italy
| | - Eleonora Draghi
- Immunogenetics, Leukemia Genomics and Immunobiology Unit, Division of Immunology, Transplantation and Infectious Diseases, Ospedale San Raffaele Scientific Institute, 20132, Milan, Italy
| | - Marcella Cesana
- Telethon Institute of Genetics and Medicine (TIGEM), Armenise/Harvard Laboratory of Integrative Genomics, Pozzuoli, Italy
- Department of Advanced Biomedical Sciences, University of Naples "Federico II", Naples, Italy
| | - Sara Riccardo
- Telethon Institute of Genetics and Medicine (TIGEM), Armenise/Harvard Laboratory of Integrative Genomics, Pozzuoli, Italy
- NEGEDIA S.r.l., Pozzuoli, Italy
| | - Chiara Colantuono
- Telethon Institute of Genetics and Medicine (TIGEM), Armenise/Harvard Laboratory of Integrative Genomics, Pozzuoli, Italy
- NEGEDIA S.r.l., Pozzuoli, Italy
| | - Emmanuelle Six
- Laboratory of Human Lympho-hematopoiesis, INSERM, Paris, France
| | | | - Filippo Carlucci
- Department of Medical Biotechnologies, University of Siena, Siena, Italy
| | | | - Caterina Cancrini
- Immune and Infectious Diseases Division, Research Unit of Primary Immunodeficiencies, Academic Department of Pediatrics, IRCCS Bambino Gesù Children's Hospital, Rome, Italy
- Department of Systems Medicine University of Rome Tor Vergata, Rome, Italy
| | - Fabio Ciceri
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
- Università Vita-Salute San Raffaele, Milan, Italy
- Haematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milan, Italy
| | - Luca Vago
- Università Vita-Salute San Raffaele, Milan, Italy
- Immunogenetics, Leukemia Genomics and Immunobiology Unit, Division of Immunology, Transplantation and Infectious Diseases, Ospedale San Raffaele Scientific Institute, 20132, Milan, Italy
- Haematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milan, Italy
| | - Davide Cacchiarelli
- Telethon Institute of Genetics and Medicine (TIGEM), Armenise/Harvard Laboratory of Integrative Genomics, Pozzuoli, Italy
- Department of Translational Medicine, University of Naples "Federico II", Naples, Italy
- School for Advanced Studies, Genomics and Experimental Medicine Program, University of Naples "Federico II", Naples, Italy
| | - Bernhard Gentner
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
- Haematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milan, Italy
| | - Luigi Naldini
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
- Università Vita-Salute San Raffaele, Milan, Italy
| | - Marco Tartaglia
- Molecular Genetics and Functional Genomics, IRCCS Bambino Gesù Children's Hospital, Rome, Italy
| | - Eugenio Montini
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Franco Locatelli
- Department of Pediatric Hematology/Oncology and Cell and Gene Therapy, IRCCS Ospedale Pediatrico Bambino Gesù, Rome, Italy
- Università Cattolica del Sacro Cuore, Rome, Italy
| | - Alessandro Aiuti
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy.
- Paediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy.
- Università Vita-Salute San Raffaele, Milan, Italy.
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12
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Rossi A, Brunetti-Pierri N. Gene therapies for mucopolysaccharidoses. J Inherit Metab Dis 2024; 47:135-144. [PMID: 37204267 DOI: 10.1002/jimd.12626] [Citation(s) in RCA: 9] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/12/2023] [Revised: 04/27/2023] [Accepted: 05/15/2023] [Indexed: 05/20/2023]
Abstract
Current specific treatments for mucopolysaccharidoses (MPSs) include enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT). Both treatments are hampered by several limitations, including lack of efficacy on brain and skeletal manifestations, need for lifelong injections, and high costs. Therefore, more effective treatments are needed. Gene therapy in MPSs is aimed at obtaining high levels of the therapeutic enzyme in multiple tissues either by engrafted gene-modified hematopoietic stem progenitor cells (ex vivo) or by direct infusion of a viral vector expressing the therapeutic gene (in vivo). This review focuses on the most recent clinical progress in gene therapies for MPSs. The various gene therapy approaches with their strengths and limitations are discussed.
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Affiliation(s)
- Alessandro Rossi
- Department of Translational Medicine, Federico II University of Naples, Naples, Italy
| | - Nicola Brunetti-Pierri
- Department of Translational Medicine, Federico II University of Naples, Naples, Italy
- Telethon Institute of Genetics and Medicine, Pozzuoli, Italy
- Scuola Superiore Meridionale (SSM, School of Advanced Studies), Genomics and Experimental Medicine Program, University of Naples Federico II, Naples, Italy
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13
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Liu Y, Karlsson S. Perspectives of current understanding and therapeutics of Diamond-Blackfan anemia. Leukemia 2024; 38:1-9. [PMID: 37973818 PMCID: PMC10776401 DOI: 10.1038/s41375-023-02082-w] [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: 08/18/2023] [Revised: 10/20/2023] [Accepted: 11/06/2023] [Indexed: 11/19/2023]
Abstract
ABSTACT Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure disorder characterized by erythroid hypoplasia. It primarily affects infants and is often caused by heterozygous allelic variations in ribosomal protein (RP) genes. Recent studies also indicated that non-RP genes like GATA1, TSR2, are associated with DBA. P53 activation, translational dysfunction, inflammation, imbalanced globin/heme synthesis, and autophagy dysregulation were shown to contribute to disrupted erythropoiesis and impaired red blood cell production. The main therapeutic option for DBA patients is corticosteroids. However, half of these patients become non-responsive to corticosteroid therapy over prolonged treatment and have to be given blood transfusions. Hematopoietic stem cell transplantation is currently the sole curative option, however, the treatment is limited by the availability of suitable donors and the potential for serious immunological complications. Recent advances in gene therapy using lentiviral vectors have shown promise in treating RPS19-deficient DBA by promoting normal hematopoiesis. With deepening insights into the molecular framework of DBA, emerging therapies like gene therapy hold promise for providing curative solutions and advancing comprehension of the underlying disease mechanisms.
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Affiliation(s)
- Yang Liu
- Center for Hematology and Regenerative Medicine, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.
- Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden.
| | - Stefan Karlsson
- Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden.
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14
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Karpov DS, Sosnovtseva AO, Pylina SV, Bastrich AN, Petrova DA, Kovalev MA, Shuvalova AI, Eremkina AK, Mokrysheva NG. Challenges of CRISPR/Cas-Based Cell Therapy for Type 1 Diabetes: How Not to Engineer a "Trojan Horse". Int J Mol Sci 2023; 24:17320. [PMID: 38139149 PMCID: PMC10743607 DOI: 10.3390/ijms242417320] [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: 11/03/2023] [Revised: 12/04/2023] [Accepted: 12/07/2023] [Indexed: 12/24/2023] Open
Abstract
Type 1 diabetes mellitus (T1D) is an autoimmune disease caused by the destruction of insulin-producing β-cells in the pancreas by cytotoxic T-cells. To date, there are no drugs that can prevent the development of T1D. Insulin replacement therapy is the standard care for patients with T1D. This treatment is life-saving, but is expensive, can lead to acute and long-term complications, and results in reduced overall life expectancy. This has stimulated the research and development of alternative treatments for T1D. In this review, we consider potential therapies for T1D using cellular regenerative medicine approaches with a focus on CRISPR/Cas-engineered cellular products. However, CRISPR/Cas as a genome editing tool has several drawbacks that should be considered for safe and efficient cell engineering. In addition, cellular engineering approaches themselves pose a hidden threat. The purpose of this review is to critically discuss novel strategies for the treatment of T1D using genome editing technology. A well-designed approach to β-cell derivation using CRISPR/Cas-based genome editing technology will significantly reduce the risk of incorrectly engineered cell products that could behave as a "Trojan horse".
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Affiliation(s)
- Dmitry S. Karpov
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia; (D.S.K.); (A.O.S.); (M.A.K.); (A.I.S.)
| | - Anastasiia O. Sosnovtseva
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia; (D.S.K.); (A.O.S.); (M.A.K.); (A.I.S.)
| | - Svetlana V. Pylina
- Endocrinology Research Centre, 115478 Moscow, Russia; (S.V.P.); (A.N.B.); (D.A.P.); (A.K.E.)
| | - Asya N. Bastrich
- Endocrinology Research Centre, 115478 Moscow, Russia; (S.V.P.); (A.N.B.); (D.A.P.); (A.K.E.)
| | - Darya A. Petrova
- Endocrinology Research Centre, 115478 Moscow, Russia; (S.V.P.); (A.N.B.); (D.A.P.); (A.K.E.)
| | - Maxim A. Kovalev
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia; (D.S.K.); (A.O.S.); (M.A.K.); (A.I.S.)
| | - Anastasija I. Shuvalova
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia; (D.S.K.); (A.O.S.); (M.A.K.); (A.I.S.)
| | - Anna K. Eremkina
- Endocrinology Research Centre, 115478 Moscow, Russia; (S.V.P.); (A.N.B.); (D.A.P.); (A.K.E.)
| | - Natalia G. Mokrysheva
- Endocrinology Research Centre, 115478 Moscow, Russia; (S.V.P.); (A.N.B.); (D.A.P.); (A.K.E.)
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15
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Uchiyama T, Kawai T, Nakabayashi K, Nakazawa Y, Goto F, Okamura K, Nishimura T, Kato K, Watanabe N, Miura A, Yasuda T, Ando Y, Minegishi T, Edasawa K, Shimura M, Akiba Y, Sato-Otsubo A, Mizukami T, Kato M, Akashi K, Nunoi H, Onodera M. Myelodysplasia after clonal hematopoiesis with APOBEC3-mediated CYBB inactivation in retroviral gene therapy for X-CGD. Mol Ther 2023; 31:3424-3440. [PMID: 37705244 PMCID: PMC10727956 DOI: 10.1016/j.ymthe.2023.09.004] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2022] [Revised: 09/02/2023] [Accepted: 09/07/2023] [Indexed: 09/15/2023] Open
Abstract
Stem cell gene therapy using the MFGS-gp91phox retroviral vector was performed on a 27-year-old patient with X-linked chronic granulomatous disease (X-CGD) in 2014. The patient's refractory infections were resolved, whereas the oxidase-positive neutrophils disappeared within 6 months. Thirty-two months after gene therapy, the patient developed myelodysplastic syndrome (MDS), and vector integration into the MECOM locus was identified in blast cells. The vector integration into MECOM was detectable in most myeloid cells at 12 months after gene therapy. However, the patient exhibited normal hematopoiesis until the onset of MDS, suggesting that MECOM transactivation contributed to clonal hematopoiesis, and the blast transformation likely arose after the acquisition of additional genetic lesions. In whole-genome sequencing, the biallelic loss of the WT1 tumor suppressor gene, which occurred immediately before tumorigenesis, was identified as a potential candidate genetic alteration. The provirus CYBB cDNA in the blasts contained 108 G-to-A mutations exclusively in the coding strand, suggesting the occurrence of APOBEC3-mediated hypermutations during the transduction of CD34-positive cells. A hypermutation-mediated loss of oxidase activity may have facilitated the survival and proliferation of the clone with MECOM transactivation. Our data provide valuable insights into the complex mechanisms underlying the development of leukemia in X-CGD gene therapy.
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Affiliation(s)
- Toru Uchiyama
- Department of Human Genetics, National Center for Child Health and Development, Tokyo, Japan.
| | - Toshinao Kawai
- Division of Immunology, National Center for Child Health and Development, Tokyo, Japan
| | - Kazuhiko Nakabayashi
- Department of Maternal-Fetal Biology, National Center for Child Health and Development, Tokyo, Japan
| | - Yumiko Nakazawa
- Division of Immunology, National Center for Child Health and Development, Tokyo, Japan
| | - Fumihiro Goto
- Division of Immunology, National Center for Child Health and Development, Tokyo, Japan
| | - Kohji Okamura
- Department of Systems BioMedicine, National Center for Child Health and Development, Tokyo, Japan
| | - Toyoki Nishimura
- Division of Pediatrics, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
| | - Koji Kato
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Science, Fukuoka, Japan
| | - Nobuyuki Watanabe
- Department of Human Genetics, National Center for Child Health and Development, Tokyo, Japan
| | - Akane Miura
- Department of Human Genetics, National Center for Child Health and Development, Tokyo, Japan
| | - Toru Yasuda
- Department of Human Genetics, National Center for Child Health and Development, Tokyo, Japan
| | - Yukiko Ando
- Department of Human Genetics, National Center for Child Health and Development, Tokyo, Japan
| | - Tomoko Minegishi
- Department of Human Genetics, National Center for Child Health and Development, Tokyo, Japan
| | - Kaori Edasawa
- Department of Human Genetics, National Center for Child Health and Development, Tokyo, Japan
| | - Marika Shimura
- Department of Human Genetics, National Center for Child Health and Development, Tokyo, Japan
| | - Yumi Akiba
- Department of Human Genetics, National Center for Child Health and Development, Tokyo, Japan
| | - Aiko Sato-Otsubo
- Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Pediatric Hematology and Oncology, National Center for Child Health and Development, Tokyo, Japan
| | - Tomoyuki Mizukami
- Department of Pediatrics, NHO Kumamoto Medical Center, Kumamoto, Japan
| | - Motohiro Kato
- Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Pediatric Hematology and Oncology, National Center for Child Health and Development, Tokyo, Japan
| | - Koichi Akashi
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Science, Fukuoka, Japan
| | - Hiroyuki Nunoi
- Division of Pediatrics, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
| | - Masafumi Onodera
- Department of Human Genetics, National Center for Child Health and Development, Tokyo, Japan
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16
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Voit RA, Corey SJ. Gene therapy for congenital marrow failure syndromes - no longer grasping at straws? Haematologica 2023; 108:2880-2882. [PMID: 37317900 PMCID: PMC10620564 DOI: 10.3324/haematol.2023.283462] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2023] [Accepted: 06/06/2023] [Indexed: 06/16/2023] Open
Affiliation(s)
- Richard A Voit
- Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115
| | - Seth J Corey
- Departments of Pediatrics and Cancer Biology, Cleveland Clinic, Cleveland, OH 44195, USA; Case Comprehensive Cancer Center, Cleveland, OH 44106.
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17
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Riller Q, Fourgeaud J, Bruneau J, De Ravin SS, Smith G, Fusaro M, Meriem S, Magerus A, Luka M, Abdessalem G, Lhermitte L, Jamet A, Six E, Magnani A, Castelle M, Lévy R, Lecuit MM, Fournier B, Winter S, Semeraro M, Pinto G, Abid H, Mahlaoui N, Cheikh N, Florkin B, Frange P, Jeziorski E, Suarez F, Sarrot-Reynauld F, Nouar D, Debray D, Lacaille F, Picard C, Pérot P, Regnault B, Da Rocha N, de Cevins C, Delage L, Pérot BP, Vinit A, Carbone F, Brunaud C, Marchais M, Stolzenberg MC, Asnafi V, Molina T, Rieux-Laucat F, Notarangelo LD, Pittaluga S, Jais JP, Moshous D, Blanche S, Malech H, Eloit M, Cavazzana M, Fischer A, Ménager MM, Neven B. Late-onset enteric virus infection associated with hepatitis (EVAH) in transplanted SCID patients. J Allergy Clin Immunol 2023; 151:1634-1645. [PMID: 36638922 PMCID: PMC10336473 DOI: 10.1016/j.jaci.2022.12.822] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2022] [Revised: 12/14/2022] [Accepted: 12/21/2022] [Indexed: 01/12/2023]
Abstract
BACKGROUND Allogenic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT) are potentially curative treatments for severe combined immunodeficiency (SCID). Late-onset posttreatment manifestations (such as persistent hepatitis) are not uncommon. OBJECTIVE We sought to characterize the prevalence and pathophysiology of persistent hepatitis in transplanted SCID patients (SCIDH+) and to evaluate risk factors and treatments. METHODS We used various techniques (including pathology assessments, metagenomics, single-cell transcriptomics, and cytometry by time of flight) to perform an in-depth study of different tissues from patients in the SCIDH+ group and corresponding asymptomatic similarly transplanted SCID patients without hepatitis (SCIDH-). RESULTS Eleven patients developed persistent hepatitis (median of 6 years after HSCT or GT). This condition was associated with the chronic detection of enteric viruses (human Aichi virus, norovirus, and sapovirus) in liver and/or stools, which were not found in stools from the SCIDH- group (n = 12). Multiomics analysis identified an expansion of effector memory CD8+ T cells with high type I and II interferon signatures. Hepatitis was associated with absence of myeloablation during conditioning, split chimerism, and defective B-cell function, representing 25% of the 44 patients with SCID having these characteristics. Partially myeloablative retransplantation or GT of patients with this condition (which we have named as "enteric virus infection associated with hepatitis") led to the reconstitution of T- and B-cell immunity and remission of hepatitis in 5 patients, concomitantly with viral clearance. CONCLUSIONS Enteric virus infection associated with hepatitis is related to chronic enteric viral infection and immune dysregulation and is an important risk for transplanted SCID patients with defective B-cell function.
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Affiliation(s)
- Quentin Riller
- University of Paris Cité, Paris, France; Laboratory of Immunogenetics of Pediatric Autoimmune Diseases, Imagine Institute, INSERM UMR 1163, Paris, France
| | - Jacques Fourgeaud
- University of Paris Cité, Paris, France; Microbiology Department, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France; Pathogen Discovery Laboratory, Institut Pasteur, Université de Paris, Paris, France; Prise en Charge des Anomalies Congénitales et leur Traitement, Unit 7328, Imagine Institute, University of Paris Cité, Paris, France
| | - Julie Bruneau
- University of Paris Cité, Paris, France; Pathology Department, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France; Imagine Institute, INSERM UMR 1163, Laboratory of Molecular Mechanisms of Hematologic Disorders and Therapeutic Implications, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Suk See De Ravin
- Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md
| | - Grace Smith
- Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Md
| | - Mathieu Fusaro
- Study Center for Primary Immunodeficiencies, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Samy Meriem
- Laboratory of Biostatistics, University of Paris Cité, Paris, France
| | - Aude Magerus
- University of Paris Cité, Paris, France; Laboratory of Immunogenetics of Pediatric Autoimmune Diseases, Imagine Institute, INSERM UMR 1163, Paris, France
| | - Marine Luka
- Labtech Single-Cell@Imagine, Imagine Institute, INSERM UMR 1163, Paris, France
| | - Ghaith Abdessalem
- Labtech Single-Cell@Imagine, Imagine Institute, INSERM UMR 1163, Paris, France
| | - Ludovic Lhermitte
- University of Paris Cité, Paris, France; Laboratory of Onco-Haematology, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France; the Institut Necker-Enfants Malades (INEM), INSERM UMR 1151, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Anne Jamet
- University of Paris Cité, Paris, France; Microbiology Department, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France; the Institut Necker-Enfants Malades (INEM), INSERM UMR 1151, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Emmanuelle Six
- University of Paris Cité, Paris, France; Laboratory of Human Lympho-Hematopoiesis, Imagine Institute, INSERM UMR 1163, Paris, France
| | - Alessandra Magnani
- Department of Biotherapy, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Martin Castelle
- Pediatric Hematology-Immunology and Rheumatology Unit, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Romain Lévy
- University of Paris Cité, Paris, France; Pediatric Hematology-Immunology and Rheumatology Unit, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France; Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Mathilde M Lecuit
- Pediatric Hematology-Immunology and Rheumatology Unit, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Benjamin Fournier
- University of Paris Cité, Paris, France; Pediatric Hematology-Immunology and Rheumatology Unit, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Sarah Winter
- University of Paris Cité, Paris, France; Pediatric Hematology-Immunology and Rheumatology Unit, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Michaela Semeraro
- University of Paris Cité, Paris, France; Clinical Investigation Center, Clinical Research Unit, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Graziella Pinto
- Pediatric Endocrinology, Gynecology, Diabetology, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Hanène Abid
- University of Paris Cité, Paris, France; Microbiology Department, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Nizar Mahlaoui
- Pediatric Hematology-Immunology and Rheumatology Unit, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Nathalie Cheikh
- Pediatric Hematology Oncology Unit, University Hospital of Besançon, Besançon, France
| | - Benoit Florkin
- Immuno-Hémato-Rhumatologie Pédiatrique, Service de Pédiatrie, CHR Citadelle, Liege, Belgium
| | - Pierre Frange
- University of Paris Cité, Paris, France; Microbiology Department, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France; Pediatric Hematology-Immunology and Rheumatology Unit, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Eric Jeziorski
- Department of Pediatrics, Infectious Diseases, and Immunology, University of Montpellier, CHU Montpellier, Montpellier, France
| | - Felipe Suarez
- University of Paris Cité, Paris, France; Imagine Institute, INSERM UMR 1163, Laboratory of Molecular Mechanisms of Hematologic Disorders and Therapeutic Implications, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France; Hematology Department, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | | | - Dalila Nouar
- Service d'Immunologie Clinique et d'Allergologie, Centre Hospitalier Régional Universitaire, Tours, France
| | - Dominique Debray
- Pediatric Liver Unit, National Reference Center for Rare Diseases, Biliary Atresia and Genetic Cholestasis, Inflammatory Biliary Diseases and Autoimmune Hepatitis, ERN Rare Liver, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Florence Lacaille
- Gastroenterology-Hepatology-Nutrition Unit, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Capucine Picard
- Study Center for Primary Immunodeficiencies, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France; Laboratory of Lymphocyte Activation and Susceptibility to EBV Infection, Imagine Institute, INSERM UMR 1163, Paris, France
| | - Philippe Pérot
- Pathogen Discovery Laboratory, Institut Pasteur, Université de Paris, Paris, France; OIE Collaborating Center for the Detection and Identification in Humans of Emerging Animal Pathogens, Institut Pasteur, Paris, France
| | - Béatrice Regnault
- Pathogen Discovery Laboratory, Institut Pasteur, Université de Paris, Paris, France; OIE Collaborating Center for the Detection and Identification in Humans of Emerging Animal Pathogens, Institut Pasteur, Paris, France
| | - Nicolas Da Rocha
- Pathogen Discovery Laboratory, Institut Pasteur, Université de Paris, Paris, France; OIE Collaborating Center for the Detection and Identification in Humans of Emerging Animal Pathogens, Institut Pasteur, Paris, France
| | - Camille de Cevins
- University of Paris Cité, Paris, France; Laboratory of Inflammatory Responses and Transcriptomic Networks in Diseases, Atip-Avenir Team, Imagine Institute, INSERM UMR 1163, Paris, France; Artificial Intelligence & Deep Analytics (AIDA) Group, Data & Data Science (DDS), Sanofi R&D, Chilly-Mazarin, France
| | - Laure Delage
- University of Paris Cité, Paris, France; Laboratory of Immunogenetics of Pediatric Autoimmune Diseases, Imagine Institute, INSERM UMR 1163, Paris, France
| | - Brieuc P Pérot
- University of Paris Cité, Paris, France; Laboratory of Inflammatory Responses and Transcriptomic Networks in Diseases, Atip-Avenir Team, Imagine Institute, INSERM UMR 1163, Paris, France
| | - Angélique Vinit
- Sorbonne Université, UMS037, PASS, Plateforme de Cytométrie de la Pitié-Salpêtrière CyPS, Paris, France
| | - Francesco Carbone
- University of Paris Cité, Paris, France; Labtech Single-Cell@Imagine, Imagine Institute, INSERM UMR 1163, Paris, France; Laboratory of Inflammatory Responses and Transcriptomic Networks in Diseases, Atip-Avenir Team, Imagine Institute, INSERM UMR 1163, Paris, France
| | - Camille Brunaud
- University of Paris Cité, Paris, France; Laboratory of Immunogenetics of Pediatric Autoimmune Diseases, Imagine Institute, INSERM UMR 1163, Paris, France
| | - Manon Marchais
- University of Paris Cité, Paris, France; Laboratory of Immunogenetics of Pediatric Autoimmune Diseases, Imagine Institute, INSERM UMR 1163, Paris, France
| | - Marie-Claude Stolzenberg
- University of Paris Cité, Paris, France; Laboratory of Immunogenetics of Pediatric Autoimmune Diseases, Imagine Institute, INSERM UMR 1163, Paris, France
| | - Vahid Asnafi
- University of Paris Cité, Paris, France; Laboratory of Onco-Haematology, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France; the Institut Necker-Enfants Malades (INEM), INSERM UMR 1151, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Thierry Molina
- University of Paris Cité, Paris, France; Pathology Department, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Frédéric Rieux-Laucat
- University of Paris Cité, Paris, France; Laboratory of Immunogenetics of Pediatric Autoimmune Diseases, Imagine Institute, INSERM UMR 1163, Paris, France
| | - Luigi D Notarangelo
- Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md
| | | | - Jean Philippe Jais
- University of Paris Cité, Paris, France; Laboratory of Biostatistics, University of Paris Cité, Paris, France
| | - Despina Moshous
- University of Paris Cité, Paris, France; Pediatric Hematology-Immunology and Rheumatology Unit, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France; Laboratory of Genome Dynamics in the Immune System, Equipe Labellisée Ligue contre le Cancer, Imagine Institute, INSERM UMR 1163, Paris, France
| | - Stephane Blanche
- University of Paris Cité, Paris, France; Pediatric Hematology-Immunology and Rheumatology Unit, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Harry Malech
- Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md
| | - Marc Eloit
- Pathogen Discovery Laboratory, Institut Pasteur, Université de Paris, Paris, France; OIE Collaborating Center for the Detection and Identification in Humans of Emerging Animal Pathogens, Institut Pasteur, Paris, France; Ecole Nationale Vétérinaire d'Alfort, Maisons-Alfort, France
| | - Marina Cavazzana
- University of Paris Cité, Paris, France; Laboratory of Onco-Haematology, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France; Laboratory of Human Lympho-Hematopoiesis, Imagine Institute, INSERM UMR 1163, Paris, France
| | - Alain Fischer
- Pediatric Hematology-Immunology and Rheumatology Unit, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France; Collège de France, Paris, France
| | - Mickaël M Ménager
- University of Paris Cité, Paris, France; Labtech Single-Cell@Imagine, Imagine Institute, INSERM UMR 1163, Paris, France; Laboratory of Inflammatory Responses and Transcriptomic Networks in Diseases, Atip-Avenir Team, Imagine Institute, INSERM UMR 1163, Paris, France
| | - Bénédicte Neven
- University of Paris Cité, Paris, France; Laboratory of Immunogenetics of Pediatric Autoimmune Diseases, Imagine Institute, INSERM UMR 1163, Paris, France; Pediatric Hematology-Immunology and Rheumatology Unit, Necker-Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France.
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18
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Zhou H, He Y, Xiong W, Jing S, Duan X, Huang Z, Nahal GS, Peng Y, Li M, Zhu Y, Ye Q. MSC based gene delivery methods and strategies improve the therapeutic efficacy of neurological diseases. Bioact Mater 2023; 23:409-437. [PMCID: PMC9713256 DOI: 10.1016/j.bioactmat.2022.11.007] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2022] [Revised: 11/08/2022] [Accepted: 11/13/2022] [Indexed: 12/05/2022] Open
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19
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Allen D, Kalter N, Rosenberg M, Hendel A. Homology-Directed-Repair-Based Genome Editing in HSPCs for the Treatment of Inborn Errors of Immunity and Blood Disorders. Pharmaceutics 2023; 15:1329. [PMID: 37242571 PMCID: PMC10220672 DOI: 10.3390/pharmaceutics15051329] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2023] [Revised: 04/13/2023] [Accepted: 04/19/2023] [Indexed: 05/28/2023] Open
Abstract
Genome engineering via targeted nucleases, specifically CRISPR-Cas9, has revolutionized the field of gene therapy research, providing a potential treatment for diseases of the blood and immune system. While numerous genome editing techniques have been used, CRISPR-Cas9 homology-directed repair (HDR)-mediated editing represents a promising method for the site-specific insertion of large transgenes for gene knock-in or gene correction. Alternative methods, such as lentiviral/gammaretroviral gene addition, gene knock-out via non-homologous end joining (NHEJ)-mediated editing, and base or prime editing, have shown great promise for clinical applications, yet all possess significant drawbacks when applied in the treatment of patients suffering from inborn errors of immunity or blood system disorders. This review aims to highlight the transformational benefits of HDR-mediated gene therapy and possible solutions for the existing problems holding the methodology back. Together, we aim to help bring HDR-based gene therapy in CD34+ hematopoietic stem progenitor cells (HSPCs) from the lab bench to the bedside.
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Affiliation(s)
| | | | | | - Ayal Hendel
- Institute of Nanotechnology and Advanced Materials, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel; (D.A.); (N.K.); (M.R.)
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20
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Castiello MC, Ferrari S, Villa A. Correcting inborn errors of immunity: From viral mediated gene addition to gene editing. Semin Immunol 2023; 66:101731. [PMID: 36863140 PMCID: PMC10109147 DOI: 10.1016/j.smim.2023.101731] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/03/2022] [Revised: 01/25/2023] [Accepted: 02/14/2023] [Indexed: 03/04/2023]
Abstract
Allogeneic hematopoietic stem cell transplantation is an effective treatment to cure inborn errors of immunity. Remarkable progress has been achieved thanks to the development and optimization of effective combination of advanced conditioning regimens and use of immunoablative/suppressive agents preventing rejection as well as graft versus host disease. Despite these tremendous advances, autologous hematopoietic stem/progenitor cell therapy based on ex vivo gene addition exploiting integrating γ-retro- or lenti-viral vectors, has demonstrated to be an innovative and safe therapeutic strategy providing proof of correction without the complications of the allogeneic approach. The recent advent of targeted gene editing able to precisely correct genomic variants in an intended locus of the genome, by introducing deletions, insertions, nucleotide substitutions or introducing a corrective cassette, is emerging in the clinical setting, further extending the therapeutic armamentarium and offering a cure to inherited immune defects not approachable by conventional gene addition. In this review, we will analyze the current state-of-the art of conventional gene therapy and innovative protocols of genome editing in various primary immunodeficiencies, describing preclinical models and clinical data obtained from different trials, highlighting potential advantages and limits of gene correction.
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Affiliation(s)
- Maria Carmina Castiello
- San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan 20132, Italy; Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche (IRGB-CNR), Milan, Italy
| | - Samuele Ferrari
- San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan 20132, Italy; Vita-Salute San Raffaele University, Milan 20132, Italy
| | - Anna Villa
- San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan 20132, Italy; Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche (IRGB-CNR), Milan, Italy.
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21
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Zhao Z, Grégoire C, Oliveira B, Chung K, Melenhorst JJ. Challenges and opportunities of CAR T cell therapies for CLL. Semin Hematol 2023; 60:25-33. [PMID: 37080707 DOI: 10.1053/j.seminhematol.2023.01.002] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2023] [Revised: 01/10/2023] [Accepted: 01/19/2023] [Indexed: 02/10/2023]
Abstract
Chimeric antigen receptor (CAR) T-cell therapies have transformed the treatment landscape of blood cancers. These engineered receptors which endow T cells with antibody-like target cell recognition combined with the typical T cell target cell lysis abilities. Introduced into the clinic in the 2010s, CAR T-cells have shown efficacy in chronic B lymphocytic leukemia (CLL), but a majority of patients do not achieve sustained remission. Here we discuss the current treatment landscape in CLL using small molecules and allogeneic stem cell transplantation, the niche CAR T-cells filled in this context, and what we have learned from biomarker and mechanistic studies. Several product parameters and improvements are introduced as examples of how the bedside-to-bench is translated into improved CAR T-cells for CLL. We hope to convey to our readers the crucial role translational medicine plays in transforming the treatment outcomes for patients with CLL and how this line of research is an essential component of modern medicine.
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22
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Mudde A, Booth C. Gene therapy for inborn error of immunity - current status and future perspectives. Curr Opin Allergy Clin Immunol 2023; 23:51-62. [PMID: 36539381 DOI: 10.1097/aci.0000000000000876] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
PURPOSE OF REVIEW Development of hematopoietic stem cell (HSC) gene therapy (GT) for inborn errors of immunity (IEIs) continues to progress rapidly. Although more patients are being treated with HSC GT based on viral vector mediated gene addition, gene editing techniques provide a promising new approach, in which transgene expression remains under the control of endogenous regulatory elements. RECENT FINDINGS Many gene therapy clinical trials are being conducted and evidence showing that HSC GT through viral vector mediated gene addition is a successful and safe curative treatment option for various IEIs is accumulating. Gene editing techniques for gene correction are, on the other hand, not in clinical use yet, despite rapid developments during the past decade. Current studies are focussing on improving rates of targeted integration, while preserving the primitive HSC population, which is essential for future clinical translation. SUMMARY As HSC GT is becoming available for more diseases, novel developments should focus on improving availability while reducing costs of the treatment. Continued follow up of treated patients is essential for providing information about long-term safety and efficacy. Editing techniques have great potential but need to be improved further before the translation to clinical studies can happen.
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Affiliation(s)
- Anne Mudde
- Molecular and Cellular Immunology Section, UCL Great Ormond Street Institute of Child Health
| | - Claire Booth
- Molecular and Cellular Immunology Section, UCL Great Ormond Street Institute of Child Health
- Department of Immunology and Gene Therapy, Great Ormond Street Hospital, London, UK
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23
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Abstract
There are more than 7,000 paediatric genetic diseases (PGDs) but less than 5% have treatment options. Treatment strategies targeting different levels of the biological process of the disease have led to optimal health outcomes in a subset of patients with PGDs, where treatment is available. In the past 3 decades, there has been rapid advancement in the development of novel therapies, including gene therapy, for many PGDs. The therapeutic success of treatment relies heavily on knowledge of the genetic basis and the disease mechanism. Specifically, gene therapy has been shown to be effective in various clinical trials, and indeed, these trials have led to regulatory approvals, paving the way for gene therapies for other types of PGDs. In this review, we provide an overview of the treatment strategies and focus on some of the recent advancements in therapeutics for PGDs.
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Affiliation(s)
- Ai Ling Koh
- Genetics Service, Department of Paediatrics, KK Women's and Children's Hospital, Singapore,SingHealth Duke-NUS Genomic Medicine Centre, Nanyang Technological University, Singapore,Duke-NUS Medical School, Nanyang Technological University, Singapore,Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore,Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Saumya Shekhar Jamuar
- Genetics Service, Department of Paediatrics, KK Women's and Children's Hospital, Singapore,SingHealth Duke-NUS Genomic Medicine Centre, Nanyang Technological University, Singapore,Duke-NUS Medical School, Nanyang Technological University, Singapore,Yong Loo Lin School of Medicine, National University of Singapore, Singapore,SingHealth Duke-NUS Institute of Precision Medicine, Singapore,Correspondence: Dr. Saumya Shekhar Jamuar, Senior Consultant, Genetics Service, Department of Paediatrics, KK Women's and Children's Hospital, 100 Bukit Timah Road, 229899, Singapore. E-mail:
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24
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Kuo CY, Kohn DB. Gene Therapy for Primary Immune Deficiency Diseases. Clin Immunol 2023. [DOI: 10.1016/b978-0-7020-8165-1.00091-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/09/2023]
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25
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Chakraborty A, Dharmaraj S, Truong N, Pearson RM. Excipient-Free Ionizable Polyester Nanoparticles for Lung-Selective and Innate Immune Cell Plasmid DNA and mRNA Transfection. ACS APPLIED MATERIALS & INTERFACES 2022; 14:56440-56453. [PMID: 36525379 PMCID: PMC9872050 DOI: 10.1021/acsami.2c14424] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/17/2023]
Abstract
Extrahepatic nucleic acid delivery using polymers typically requires the synthesis and purification of custom monomers, post-synthetic modifications, and incorporation of additional excipients to augment their stability, endosomal escape, and in vivo effectiveness. Here, we report the development of a single-component and excipient-free, polyester-based nucleic acid delivery nanoparticle platform comprising ionizable N-methyldiethanolamine (MDET) and various hydrophobic alkyl diols (Cp) that achieves lung-selective nucleic acid transfection in vivo. PolyMDET and polyMDET-Cp polyplexes displayed high serum and enzymatic stability, while delivering pDNA or mRNA to "hard-to-transfect" innate immune cells. PolyMDET-C4 and polyMDET-C6 mediated high protein expression in lung alveolar macrophages and dendritic cells without inducing tissue damage or systemic inflammatory responses. Improved strategies using readily available starting materials to produce a simple, excipient-free, non-viral nucleic acid delivery platform with lung-selective and innate immune cell tropism has the potential to expedite clinical deployment of polymer-based genetic medicines.
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Affiliation(s)
- Atanu Chakraborty
- Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 N. Pine Street, Baltimore, Maryland21201, United States
| | - Shruti Dharmaraj
- Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 N. Pine Street, Baltimore, Maryland21201, United States
| | - Nhu Truong
- Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 N. Pine Street, Baltimore, Maryland21201, United States
| | - Ryan M Pearson
- Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 N. Pine Street, Baltimore, Maryland21201, United States
- Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 W. Baltimore Street, Baltimore, Maryland21201, United States
- Program in Molecular Medicine, University of Maryland School of Medicine, 655 W. Baltimore Street, Baltimore, Maryland21201, United States
- Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, 22 S. Greene Street, Baltimore, Maryland21201, United States
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26
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Tsujimoto H, Osafune K. Current status and future directions of clinical applications using iPS cells-focus on Japan. FEBS J 2022; 289:7274-7291. [PMID: 34407307 DOI: 10.1111/febs.16162] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2021] [Revised: 08/04/2021] [Accepted: 08/17/2021] [Indexed: 01/13/2023]
Abstract
Regenerative medicine using iPS cell technologies has progressed remarkably in recent years. In this review, we summarize these technologies and their clinical application. First, we discuss progress in the establishment of iPS cells, including the HLA-homo iPS cell stock project in Japan and the advancement of low antigenic iPS cells using genome-editing technology. Then, we describe iPS cell-based therapies in or approaching clinical application, including those for ophthalmological, neurological, cardiac, hematological, cartilage, and metabolic diseases. Next, we introduce disease models generated from patient iPS cells and successfully used to identify therapeutic agents for intractable diseases. Clinical medicine using iPS cells has advanced safely and effectively by making full use of current scientific standards, but tests on cell safety need to be further developed and validated. The next decades will see the further spread of iPS cell technology-based regenerative medicine.
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Affiliation(s)
- Hiraku Tsujimoto
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Japan.,RegeNephro Co., Ltd., MIC bldg. Graduate School of Medicine, Kyoto University, Japan
| | - Kenji Osafune
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Japan.,Meiji University International Institute for Bio-Resource Research, Meiji University, Kanagawa, Japan
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27
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Hernandez JD, Hsieh EW. A great disturbance in the force: IL-2 receptor defects disrupt immune homeostasis. Curr Opin Pediatr 2022; 34:580-588. [PMID: 36165614 PMCID: PMC9633542 DOI: 10.1097/mop.0000000000001181] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
PURPOSE OF REVIEW The current review highlights how inborn errors of immunity (IEI) due to IL-2 receptor (IL-2R) subunit defects may result in children presenting with a wide variety of infectious and inflammatory presentations beyond typical X-linked severe combined immune deficiency (X-SCID) associated with IL-2Rγ. RECENT FINDINGS Newborn screening has made diagnosis of typical SCID presenting with severe infections less common. Instead, infants are typically diagnosed in the first days of life when they appear healthy. Although earlier diagnosis has improved clinical outcomes for X-SCID, atypical SCID or other IEI not detected on newborn screening may present with more limited infectious presentations and/or profound immune dysregulation. Early management to prevent/control infections and reduce inflammatory complications is important for optimal outcomes of definitive therapies. Hematopoietic stem cell transplant (HSCT) is curative for IL-2Rα, IL-2Rβ, and IL-2Rγ defects, but gene therapy may yield comparable results for X-SCID. SUMMARY Defects in IL-2R subunits present with infectious and inflammatory phenotypes that should raise clinician's concern for IEI. Immunophenotyping may support the suspicion for diagnosis, but ultimately genetic studies will confirm the diagnosis and enable family counseling. Management of infectious and inflammatory complications will determine the success of gene therapy or HSCT.
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Affiliation(s)
- Joseph D. Hernandez
- Department of Pediatrics, Division of Allergy, Immunology and Rheumatology, School of Medicine, Stanford University, Lucile Packard Children’s Hospital
| | - Elena W.Y. Hsieh
- Department of Pediatrics, Section of Allergy and Immunology, School of Medicine, University of Colorado, Children’s Hospital Colorado
- Department of Immunology and Microbiology, School of Medicine, University of Colorado
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28
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Sharma A, Farnia S, Otegbeye F, Rinkle A, Shah J, Shah NN, Gill S, Maus MV. Nomenclature for Cellular and Genetic Therapies: A Need for Standardization. Transplant Cell Ther 2022; 28:795-801. [PMID: 36058548 PMCID: PMC9729423 DOI: 10.1016/j.jtct.2022.08.029] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2022] [Revised: 08/25/2022] [Accepted: 08/26/2022] [Indexed: 12/24/2022]
Abstract
As the field of cellular and genetic therapies transitions from a scientific concept to a clinical reality, it has become evident that there are several conflicting or imprecise nomenclatures to describe these novel therapeutic products. The lack of uniformity and accuracy in the terminology often creates regulatory, educational, administrative, and billing quagmires. Standardization of the nomenclature for these therapeutic products is essential for creation of a harmonized regulatory and developmental framework, development of training paradigms and educational programs, equitable and rational decisions about accessibility, and consistency in the billing and coding structures used for reimbursement. Here we propose an updated framework as a foundation for categorizing these cell-based and genetically modified therapies.
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Affiliation(s)
- Akshay Sharma
- Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, Tennessee.
| | | | - Folashade Otegbeye
- Bone Marrow Transplantation and Immunotherapy Program, Clinical Research Division, Fred Hutchinson Cancer Center, Seattle, Washington
| | | | | | - Nirali N Shah
- Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
| | - Saar Gill
- Cell Therapy and Transplantation program, Division of Hematology-Oncology, Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania
| | - Marcela V Maus
- Cellular Immunotherapy Program and Cancer Center, Massachusetts General Hospital, Charlestown, Massachusetts; Harvard Medical School, Boston, Massachusetts
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29
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Fischer A. Gene therapy for inborn errors of immunity: past, present and future. Nat Rev Immunol 2022:10.1038/s41577-022-00800-6. [DOI: 10.1038/s41577-022-00800-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/26/2022] [Indexed: 11/27/2022]
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30
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Shabaneh TB, Moffett HF, Stull SM, Derezes T, Tait LJ, Park S, Riddell SR, Lajoie MJ. Safety switch optimization enhances antibody-mediated elimination of CAR T cells. FRONTIERS IN MOLECULAR MEDICINE 2022; 2:1026474. [PMID: 39086975 PMCID: PMC11285702 DOI: 10.3389/fmmed.2022.1026474] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 08/23/2022] [Accepted: 09/27/2022] [Indexed: 08/02/2024]
Abstract
Activation of a conditional safety switch has the potential to reverse serious toxicities arising from the administration of engineered cellular therapies, including chimeric antigen receptor (CAR) T cells. The functionally inert, non-immunogenic cell surface marker derived from human epidermal growth factor receptor (EGFRt) is a promising safety switch that has been used in multiple clinical constructs and can be targeted by cetuximab, a clinically available monoclonal antibody. However, this approach requires high and persistent cell surface expression of EGFRt to ensure that antibody-mediated depletion of engineered cells is rapid and complete. Here we show that incorporating a short juxtamembrane sequence into the EGFRt polypeptide enhances its expression on the surface of T cells and their susceptibility to antibody-dependent cellular cytotoxicity (ADCC). Incorporating this optimized variant (EGFRopt) into bicistronic and tricistronic CAR designs results in more rapid in vivo elimination of CAR T cells and robust termination of their effector activity compared to EGFRt. These studies establish EGFRopt as a superior safety switch for the development of next-generation cell-based therapeutics.
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Affiliation(s)
- Tamer B. Shabaneh
- Immunotherapy Integrated Research Center, Fred Hutchinson Cancer Center, Seattle, WA, United States
| | | | - Sylvia M. Stull
- Immunotherapy Integrated Research Center, Fred Hutchinson Cancer Center, Seattle, WA, United States
| | - Thomas Derezes
- Immunotherapy Integrated Research Center, Fred Hutchinson Cancer Center, Seattle, WA, United States
| | | | - Spencer Park
- Lyell Immunopharma, Inc., Seattle, WA, United States
| | - Stan R. Riddell
- Immunotherapy Integrated Research Center, Fred Hutchinson Cancer Center, Seattle, WA, United States
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31
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Falcon C, Smith L, Al-Obaidi M, Abu Zaanona M, Purvis K, Minagawa K, Athar M, Salzman D, Bhatia R, Goldman F, Di Stasi A. Combinatorial suicide gene strategies for the safety of cell therapies. Front Immunol 2022; 13:975233. [PMID: 36189285 PMCID: PMC9515659 DOI: 10.3389/fimmu.2022.975233] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2022] [Accepted: 08/29/2022] [Indexed: 11/13/2022] Open
Abstract
Gene-modified cellular therapies carry inherent risks of severe and potentially fatal adverse events, including the expansion of alloreactive cells or malignant transformation due to insertional mutagenesis. Strategies to mitigate uncontrolled proliferation of gene-modified cells include co-transfection of a suicide gene, such as the inducible caspase 9 safety switch (ΔiC9). However, the activation of the ΔiC9 fails to completely eliminate all gene-modified cells. Therefore, we tested a two suicide gene system used independently or together, with the goal of complete cell elimination. The first approach combined the ΔiC9 with an inducible caspase 8, ΔiC8, which lacks the endogenous prodomain. The rationale was to use a second caspase with an alternative and complementary mechanism of action. Jurkat cells co-transduced to co-express the ΔiC8, activatable by a BB homodimerizer, and the ΔiC9 activatable by the rapamycin analog sirolimus were used in a model to estimate the degree of inducible cell elimination. We found that both agents could activate each caspase independently, with enhanced elimination with superior reduction in cell regrowth of gene-modified cells when both systems were activated simultaneously. A second approach was employed in parallel, combining the ΔiC9 with the RQR8 compact suicide gene. RQR8 incorporates a CD20 mimotope, targeted by the anti-CD20 monoclonal antibody rituxan, and the QBend10, a ΔCD34 selectable marker. Likewise, enhanced cell elimination with superior reduction in cell regrowth was observed when both systems were activated together. A dose-titration effect was also noted utilizing the BB homodimerizer, whereas sirolimus remained very potent at minimal concentrations. Further in vivo studies are needed to validate these novel combination systems, which may play a role in future cancer therapies or regenerative medicine.
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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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Chetty K, Houghton BC, Booth C. Gene Therapy for Inborn Errors of Immunity. Hematol Oncol Clin North Am 2022; 36:813-827. [DOI: 10.1016/j.hoc.2022.03.010] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
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Mesenchymal stem cells: A living carrier for active tumor-targeted delivery. Adv Drug Deliv Rev 2022; 185:114300. [PMID: 35447165 DOI: 10.1016/j.addr.2022.114300] [Citation(s) in RCA: 23] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2021] [Revised: 03/22/2022] [Accepted: 04/12/2022] [Indexed: 12/16/2022]
Abstract
The strategy of using mesenchymal stem cells (MSCs) as a living carrier for active delivery of therapeutic agents targeting tumor sites has been attempted in a wide range of studies to validate the feasibility and efficacy for tumor treatment. This approach reveals powerful tumor targeting and tumor penetration. In addition, MSCs have been confirmed to actively participate in immunomodulation of the tumor microenvironment. Thus, MSCs are not inert delivery vehicles but have a strong impact on the fate of tumor cells. In this review, these active properties of MSCs are addressed to highlight the advantages and challenges of using MSCs for tumor-targeted delivery. In addition, some of the latest examples of using MSCs to carry a variety of anti-tumor agents for tumor-targeted therapy are summarized. Recent technologies to improve the performance and safety of this delivery strategy will be introduced. The advances, applications, and challenges summarized in this review will provide a general understanding of this promising strategy for actively delivering drugs to tumor tissues.
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Konturek-Ciesla A, Bryder D. Stem Cells, Hematopoiesis and Lineage Tracing: Transplantation-Centric Views and Beyond. Front Cell Dev Biol 2022; 10:903528. [PMID: 35573680 PMCID: PMC9091331 DOI: 10.3389/fcell.2022.903528] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2022] [Accepted: 04/12/2022] [Indexed: 12/26/2022] Open
Abstract
An appropriate production of mature blood cells, or hematopoiesis, is essential for organismal health and homeostasis. In this developmental cascade, hematopoietic stem cells (HSCs) differentiate into intermediate progenitor types, that subsequently give rise to the many distinct blood cell lineages. Here, we describe tools and methods that permit for temporal and native clonal-level HSC lineage tracing in the mouse, and that can now be combined with emerging single-cell molecular analyses. We integrate new insights derived from such experimental paradigms with past knowledge, which has predominantly been derived from transplantation-based approaches. Finally, we outline current knowledge and novel strategies derived from studies aimed to trace human HSC-derived hematopoiesis.
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Ravendran S, Hernández SS, König S, Bak RO. CRISPR/Cas-Based Gene Editing Strategies for DOCK8 Immunodeficiency Syndrome. Front Genome Ed 2022; 4:793010. [PMID: 35373187 PMCID: PMC8969908 DOI: 10.3389/fgeed.2022.793010] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2021] [Accepted: 02/14/2022] [Indexed: 12/17/2022] Open
Abstract
Defects in the DOCK8 gene causes combined immunodeficiency termed DOCK8 immunodeficiency syndrome (DIDS). DIDS previously belonged to the disease category of autosomal recessive hyper IgE syndrome (AR-HIES) but is now classified as a combined immunodeficiency (CID). This genetic disorder induces early onset of susceptibility to severe recurrent viral and bacterial infections, atopic diseases and malignancy resulting in high morbidity and mortality. This pathological state arises from impairment of actin polymerization and cytoskeletal rearrangement, which induces improper immune cell migration-, survival-, and effector functions. Owing to the severity of the disease, early allogenic hematopoietic stem cell transplantation is recommended even though it is associated with risk of unintended adverse effects, the need for compatible donors, and high expenses. So far, no alternative therapies have been developed, but the monogenic recessive nature of the disease suggests that gene therapy may be applied. The advent of the CRISPR/Cas gene editing system heralds a new era of possibilities in precision gene therapy, and positive results from clinical trials have already suggested that the tool may provide definitive cures for several genetic disorders. Here, we discuss the potential application of different CRISPR/Cas-mediated genetic therapies to correct the DOCK8 gene. Our findings encourage the pursuit of CRISPR/Cas-based gene editing approaches, which may constitute more precise, affordable, and low-risk definitive treatment options for DOCK8 deficiency.
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Affiliation(s)
| | | | | | - Rasmus O. Bak
- Department of Biomedicine, Aarhus University, Aarhus, Denmark
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Tucci F, Galimberti S, Naldini L, Valsecchi MG, Aiuti A. A systematic review and meta-analysis of gene therapy with hematopoietic stem and progenitor cells for monogenic disorders. Nat Commun 2022; 13:1315. [PMID: 35288539 PMCID: PMC8921234 DOI: 10.1038/s41467-022-28762-2] [Citation(s) in RCA: 77] [Impact Index Per Article: 25.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2021] [Accepted: 02/08/2022] [Indexed: 12/12/2022] Open
Abstract
Ex-vivo gene therapy (GT) with hematopoietic stem and progenitor cells (HSPCs) engineered with integrating vectors is a promising treatment for monogenic diseases, but lack of centralized databases is hampering an overall outcomes assessment. Here we aim to provide a comprehensive assessment of the short and long term safety of HSPC-GT from trials using different vector platforms. We review systematically the literature on HSPC-GT to describe survival, genotoxicity and engraftment of gene corrected cells. From 1995 to 2020, 55 trials for 14 diseases met inclusion criteria and 406 patients with primary immunodeficiencies (55.2%), metabolic diseases (17.0%), haemoglobinopathies (24.4%) and bone marrow failures (3.4%) were treated with gammaretroviral vector (γRV) (29.1%), self-inactivating γRV (2.2%) or lentiviral vectors (LV) (68.7%). The pooled overall incidence rate of death is 0.9 per 100 person-years of observation (PYO) (95% CI = 0.37-2.17). There are 21 genotoxic events out of 1504.02 PYO, which occurred in γRV trials (0.99 events per 100 PYO, 95% CI = 0.18-5.43) for primary immunodeficiencies. Pooled rate of engraftment is 86.7% (95% CI = 67.1-95.5%) for γRV and 98.7% (95% CI = 94.5-99.7%) for LV HSPC-GT (p = 0.005). Our analyses show stable reconstitution of haematopoiesis in most recipients with superior engraftment and safer profile in patients receiving LV-transduced HSPCs.
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Affiliation(s)
- Francesca Tucci
- Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Stefania Galimberti
- Bicocca Bioinformatics Biostatistics and Bioimaging B4 Center, School of Medicine and Surgery, University of Milano - Bicocca, Monza, Italy
| | - Luigi Naldini
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
- Vita-Salute San Raffaele University, Milan, Italy
| | - Maria Grazia Valsecchi
- Bicocca Bioinformatics Biostatistics and Bioimaging B4 Center, School of Medicine and Surgery, University of Milano - Bicocca, Monza, Italy
| | - Alessandro Aiuti
- Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy.
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy.
- Vita-Salute San Raffaele University, Milan, Italy.
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Sayed N, Allawadhi P, Khurana A, Singh V, Navik U, Pasumarthi SK, Khurana I, Banothu AK, Weiskirchen R, Bharani KK. Gene therapy: Comprehensive overview and therapeutic applications. Life Sci 2022; 294:120375. [PMID: 35123997 DOI: 10.1016/j.lfs.2022.120375] [Citation(s) in RCA: 112] [Impact Index Per Article: 37.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2021] [Revised: 01/24/2022] [Accepted: 01/31/2022] [Indexed: 02/07/2023]
Abstract
Gene therapy is the product of man's quest to eliminate diseases. Gene therapy has three facets namely, gene silencing using siRNA, shRNA and miRNA, gene replacement where the desired gene in the form of plasmids and viral vectors, are directly administered and finally gene editing based therapy where mutations are modified using specific nucleases such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short tandem repeats (CRISPR)/CRISPR-associated protein (Cas)-associated nucleases. Transfer of gene is either through transformation where under specific conditions the gene is directly taken up by the bacterial cells, transduction where a bacteriophage is used to transfer the genetic material and lastly transfection that involves forceful delivery of gene using either viral or non-viral vectors. The non-viral transfection methods are subdivided into physical, chemical and biological. The physical methods include electroporation, biolistic, microinjection, laser, elevated temperature, ultrasound and hydrodynamic gene transfer. The chemical methods utilize calcium- phosphate, DAE-dextran, liposomes and nanoparticles for transfection. The biological methods are increasingly using viruses for gene transfer, these viruses could either integrate within the genome of the host cell conferring a stable gene expression, whereas few other non-integrating viruses are episomal and their expression is diluted proportional to the cell division. So far, gene therapy has been wielded in a plethora of diseases. However, coherent and innocuous delivery of genes is among the major hurdles in the use of this promising therapy. Hence this review aims to highlight the current options available for gene transfer along with the advantages and limitations of every method.
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Affiliation(s)
- Nilofer Sayed
- Department of Pharmacy, Pravara Rural Education Society's (P.R.E.S.'s) College of Pharmacy, Shreemati Nathibai Damodar Thackersey (SNDT) Women's University, Nashik 400020, Maharashtra, India
| | - Prince Allawadhi
- Department of Biosciences and Bioengineering, Indian Institute of Technology (IIT), Roorkee, Roorkee, Uttarakhand 247667, India
| | - Amit Khurana
- Centre for Biomedical Engineering (CBME), Indian Institute of Technology (IIT) Delhi, Hauz Khas, New Delhi 110016, India; Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science (CVSc), PVNRTVU, Rajendranagar, Hyderabad 500030, Telangana, India; Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science (CVSc), PVNRTVU, Mamnoor, Warangal 506166, Telangana, India; Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH Aachen University Hospital, Pauwelsstr. 30, D-52074 Aachen, Germany.
| | - Vishakha Singh
- Department of Biosciences and Bioengineering, Indian Institute of Technology (IIT), Roorkee, Roorkee, Uttarakhand 247667, India
| | - Umashanker Navik
- Department of Pharmacology, Central University of Punjab, Ghudda, Bathinda 151401, Punjab, India
| | | | - Isha Khurana
- Department of Pharmaceutical Chemistry, University Institute of Pharmaceutical Sciences (UIPS), Panjab University, Chandigarh 160014, India
| | - Anil Kumar Banothu
- Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science (CVSc), PVNRTVU, Rajendranagar, Hyderabad 500030, Telangana, India
| | - Ralf Weiskirchen
- Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH Aachen University Hospital, Pauwelsstr. 30, D-52074 Aachen, Germany.
| | - Kala Kumar Bharani
- Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science (CVSc), PVNRTVU, Mamnoor, Warangal 506166, Telangana, India.
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Regenerative Medicine Application of Mesenchymal Stem Cells. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2022; 1387:25-42. [DOI: 10.1007/5584_2022_713] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/09/2022]
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Development and clinical translation of ex vivo gene therapy. Comput Struct Biotechnol J 2022; 20:2986-3003. [PMID: 35782737 PMCID: PMC9218169 DOI: 10.1016/j.csbj.2022.06.015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2022] [Revised: 06/07/2022] [Accepted: 06/07/2022] [Indexed: 11/27/2022] Open
Abstract
Retroviral gene therapy has emerged as a promising therapeutic modality for multiple inherited and acquired human diseases. The capability of delivering curative treatment or mediating therapeutic benefits for a long-term period following a single application fundamentally distinguishes this medical intervention from traditional medicine and various lentiviral/γ-retroviral vector-mediated gene therapy products have been approved for clinical use. Continued advances in retroviral vector engineering, genomic editing, synthetic biology and immunology will broaden the medical applications of gene therapy and improve the efficacy and safety of the treatments based on genetic correction and alteration. This review will summarize the advent and clinical translation of ex vivo gene therapy, with the focus on the milestones during the exploitation of genetically engineered hematopoietic stem cells (HSCs) tackling a variety of pathological conditions which led to marketing approval. Finally, current statue and future prospects of gene editing as an alternative therapeutic approach are also discussed.
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Abstract
Lentiviral vectors are the workhorses of modern cell biology. They can infect a wide variety of cells including non-dividing cells and stem cells. They integrate into the genome of infected cells leading to stable expression. It is easy to transduce 100% of the cells in a culture and possible to infect cells simultaneously with multiple vectors, greatly facilitating studies on malignant transformation. We present simple protocols to produce and titrate lentiviral vectors, infect mammary epithelial cells, and check for contamination with replication competent viruses.
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Affiliation(s)
- Richard Iggo
- INSERM U1218, Institut Bergonié, University of Bordeaux, Bordeaux, France.
- DRMCRL Lab, University of Adelaide Medical School, Adelaide, SA, Australia.
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A Nanoparticle-Conjugated Anti-TBK1 siRNA Induces Autophagy-Related Apoptosis and Enhances cGAS-STING Pathway in GBM Cells. EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE 2021; 2021:6521953. [PMID: 34931127 PMCID: PMC8684524 DOI: 10.1155/2021/6521953] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/07/2021] [Accepted: 11/05/2021] [Indexed: 11/30/2022]
Abstract
Background Gene therapy shows considerable clinical benefit in cancer therapy, in which single-stranded ribonucleic acid (siRNA) is a promising strategy in the treatment of glioblastoma (GBM). TANK-binding kinase 1 (TBK1) is critical in tumorigenesis and development, which lays a foundation for an ideal target for tumor therapy. However, the practical application of free siRNA is limited. It is urgent to develop novel strategies to deliver TBK1 siRNA to activate apoptosis and cGAS-STING pathway as a therapeutic strategy for GBM. Methods The expression and prognostic value of TBK1 were evaluated in the TCGA, CGGA, and GTEx databases. A novel gene delivery system was designed here via PEGylated reduced graphene oxide (rGO-PEG) to targeted delivery of anti-TBK1 siRNA efficiently. The efficacy of TBK1si/rGO-PEG was evaluated in GBM cells. The underlying pathways were explored by Western blot. Results TBK1 was highly expressed in glioma samples, and its high expression indicated poor prognoses in glioma patients. The rGO-PEG presented great efficiency in targeted delivery of TBK1si RNA into GBM cells with up to 97.1% transfection efficiency. TBK1si/rGO-PEG exhibited anti-GBM activities by inhibiting TBK1 and autophagy, as well as activating apoptosis and cGAS-STING pathway. Conclusion The rGO-PEG could be an efficient system facilitating the delivery of specific siRNA. TBK1si/rGO-PEG could be a novel strategy for the treatment of GBM.
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Genetic Modification of Mesenchymal Stem Cells for Neurological Disease Therapy: What Effects Does it Have on Phenotype/Cell Behavior, Determining Their Effectiveness? Mol Diagn Ther 2021; 24:683-702. [PMID: 32926348 DOI: 10.1007/s40291-020-00491-6] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Mesenchymal stem cells are a promising tool in regenerative medicine, and their functions can be enhanced through genetic modification. Recent advances in genetic engineering provide several methods that enable gene delivery to mesenchymal stem cells. However, it remains to be decided whether genetic modification of mesenchymal stem cells by vectors carrying reporter or therapeutic genes leads to adverse effects on morphology, phenotypic profiles, and viability of transplanted cells. In this regard, we focus on the description of genetic modification methods of mesenchymal stem cells, their effectiveness, and the impact on phenotype/cell behavior/proliferation and the differentiation ability of these cells in vitro and in vivo. Furthermore, we compare the main effects of genetically modified mesenchymal stem cells with native mesenchymal stem cells when applied in the therapy of neurological diseases.
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Morgan MA, Galla M, Grez M, Fehse B, Schambach A. Retroviral gene therapy in Germany with a view on previous experience and future perspectives. Gene Ther 2021; 28:494-512. [PMID: 33753908 PMCID: PMC8455336 DOI: 10.1038/s41434-021-00237-x] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2020] [Revised: 01/13/2021] [Accepted: 02/01/2021] [Indexed: 02/01/2023]
Abstract
Gene therapy can be used to restore cell function in monogenic disorders or to endow cells with new capabilities, such as improved killing of cancer cells, expression of suicide genes for controlled elimination of cell populations, or protection against chemotherapy or viral infection. While gene therapies were originally most often used to treat monogenic diseases and to improve hematopoietic stem cell transplantation outcome, the advent of genetically modified immune cell therapies, such as chimeric antigen receptor modified T cells, has contributed to the increased numbers of patients treated with gene and cell therapies. The advancement of gene therapy with integrating retroviral vectors continues to depend upon world-wide efforts. As the topic of this special issue is "Spotlight on Germany," the goal of this review is to provide an overview of contributions to this field made by German clinical and research institutions. Research groups in Germany made, and continue to make, important contributions to the development of gene therapy, including design of vectors and transduction protocols for improved cell modification, methods to assess gene therapy vector efficacy and safety (e.g., clonal imbalance, insertion sites), as well as in the design and conduction of clinical gene therapy trials.
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Affiliation(s)
- Michael A Morgan
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
- REBIRTH Research Center for Translational Regenerative Medicine, Hannover Medical School, Hannover, Germany
| | - Melanie Galla
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
- REBIRTH Research Center for Translational Regenerative Medicine, Hannover Medical School, Hannover, Germany
| | - Manuel Grez
- Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt, Germany
| | - Boris Fehse
- Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Axel Schambach
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
- REBIRTH Research Center for Translational Regenerative Medicine, Hannover Medical School, Hannover, Germany.
- Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.
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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: 22] [Impact Index Per Article: 5.5] [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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Baeten P, Van Zeebroeck L, Kleinewietfeld M, Hellings N, Broux B. Improving the Efficacy of Regulatory T Cell Therapy. Clin Rev Allergy Immunol 2021; 62:363-381. [PMID: 34224053 PMCID: PMC8256646 DOI: 10.1007/s12016-021-08866-1] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/31/2021] [Indexed: 12/11/2022]
Abstract
Autoimmunity is caused by an unbalanced immune system, giving rise to a variety of organ-specific to system disorders. Patients with autoimmune diseases are commonly treated with broad-acting immunomodulatory drugs, with the risk of severe side effects. Regulatory T cells (Tregs) have the inherent capacity to induce peripheral tolerance as well as tissue regeneration and are therefore a prime candidate to use as cell therapy in patients with autoimmune disorders. (Pre)clinical studies using Treg therapy have already established safety and feasibility, and some show clinical benefits. However, Tregs are known to be functionally impaired in autoimmune diseases. Therefore, ex vivo manipulation to boost and stably maintain their suppressive function is necessary when considering autologous transplantation. Similar to autoimmunity, severe coronavirus disease 2019 (COVID-19) is characterized by an exaggerated immune reaction and altered Treg responses. In light of this, Treg-based therapies are currently under investigation to treat severe COVID-19. This review provides a detailed overview of the current progress and clinical challenges of Treg therapy for autoimmune and hyperinflammatory diseases, with a focus on recent successes of ex vivo Treg manipulation.
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Affiliation(s)
- Paulien Baeten
- Neuro-Immune Connections and Repair Lab, Department of Immunology and Infection, Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium.,University MS Center, Campus Diepenbeek, Diepenbeek, Belgium
| | - Lauren Van Zeebroeck
- University MS Center, Campus Diepenbeek, Diepenbeek, Belgium.,VIB Laboratory of Translational Immunomodulation, Center for Inflammation Research (IRC), Department of Immunology and Infection, Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium
| | - Markus Kleinewietfeld
- University MS Center, Campus Diepenbeek, Diepenbeek, Belgium.,VIB Laboratory of Translational Immunomodulation, Center for Inflammation Research (IRC), Department of Immunology and Infection, Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium
| | - Niels Hellings
- Neuro-Immune Connections and Repair Lab, Department of Immunology and Infection, Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium.,University MS Center, Campus Diepenbeek, Diepenbeek, Belgium
| | - Bieke Broux
- Neuro-Immune Connections and Repair Lab, Department of Immunology and Infection, Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium. .,University MS Center, Campus Diepenbeek, Diepenbeek, Belgium. .,Department of Internal Medicine, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands.
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Ramos CV, Martins VC. Cell competition in hematopoietic cells: Quality control in homeostasis and its role in leukemia. Dev Biol 2021; 475:1-9. [DOI: 10.1016/j.ydbio.2021.02.013] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2020] [Revised: 02/19/2021] [Accepted: 02/23/2021] [Indexed: 12/24/2022]
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Galanopoulos M, Doukatas A, Gkeros F, Viazis N, Liatsos C. Room for improvement in the treatment of pancreatic cancer: Novel opportunities from gene targeted therapy. World J Gastroenterol 2021; 27:3568-3580. [PMID: 34239270 PMCID: PMC8240062 DOI: 10.3748/wjg.v27.i24.3568] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/16/2021] [Revised: 04/11/2021] [Accepted: 05/22/2021] [Indexed: 02/06/2023] Open
Abstract
Pancreatic cancer is one of the highest and in fact, unchanged mortality-associated tumor, with an exceptionally low survival rate due to its challenging diagnostic approach. So far, its treatment is based on a combination of approaches (such as surgical resection with or rarely without chemotherapeutic agents), but with finite limits. Thus, looking for additional space to improve pancreatic tumorigenesis therapeutic approach, research has focused on gene therapy with unexpectedly growing horizons not only for the treatment of inoperable pancreatic disease, but also for its early stages. In vivo gene delivery viral vectors, despite few disadvantages (possible immunogenicity, toxicity, mutagenicity, or high cost), could be one of the most efficient cancer gene therapeutic strategies for clinical application due to their superiority compared with other systems (ex vivo delivery strategies). Their dominance consists of simple preparation, easy operation and a wide range of functions. Adenoviruses are one of the most common used vectors, inducing strong immune as well as inflammatory reactions. Oncolytic virotherapy, using the above mentioned in vivo viral vectors, is one of the most promising non-pathogenic, highly-selective cytotoxic anti-cancer therapy using anti-cancer agents with high anti-tumor potency and strong oncolytic effect. There have been a variety of targeted therapeutic and pre-clinical strategies tested for gene therapy in pancreatic cancer such as gene-editing systems (e.g., clustered regularly interspaced palindromic repeats-Cas9), RNA interference technology (e.g., microRNAs, short hairpin RNA or small interfering RNA), adoptive immunotherapy and vaccination (e.g., chimeric antigen receptor T-cell therapy) with encouraging results.
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Affiliation(s)
- Michail Galanopoulos
- Department of Gastroenterology, Addenbrooke’s Hospital, Cambridge CB2 0QQ, United Kingdom
| | - Aris Doukatas
- Department of Pharmacy, National and Kapodistrian University of Athens, Athens GR 15772, Greece
| | - Filippos Gkeros
- Department of Gastroenterology, Evangelismos, Ophthalmiatreion Athinon and Polyclinic Hospitals, Athens 10676, Greece
| | - Nikos Viazis
- Department of Gastroenterology, Evangelismos, Ophthalmiatreion Athinon and Polyclinic Hospitals, Athens 10676, Greece
| | - Christos Liatsos
- Department of Gastroenterology, 401 General Military Hospital, Athens 11525, Greece
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Retrieval of vector integration sites from cell-free DNA. Nat Med 2021; 27:1458-1470. [PMID: 34140705 DOI: 10.1038/s41591-021-01389-4] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2020] [Accepted: 05/07/2021] [Indexed: 12/23/2022]
Abstract
Gene therapy (GT) has rapidly attracted renewed interest as a treatment for otherwise incurable diseases, with several GT products already on the market and many more entering clinical testing for selected indications. Clonal tracking techniques based on vector integration enable monitoring of the fate of engineered cells in the blood of patients receiving GT and allow assessment of the safety and efficacy of these procedures. However, owing to the limited number of cells that can be tested and the impracticality of studying cells residing in peripheral organs without performing invasive biopsies, this approach provides only a partial snapshot of the clonal repertoire and dynamics of genetically modified cells and reduces the predictive power as a safety readout. In this study, we developed liquid biopsy integration site sequencing, or LiBIS-seq, a polymerase chain reaction technique optimized to quantitatively retrieve vector integration sites from cell-free DNA released into the bloodstream by dying cells residing in several tissues. This approach enabled longitudinal monitoring of in vivo liver-directed GT and clonal tracking in patients receiving hematopoietic stem cell GT, improving our understanding of the clonal composition and turnover of genetically modified cells in solid tissues and, in contrast to conventional analyses based only on circulating blood cells, enabling earlier detection of vector-marked clones that are aberrantly expanding in peripheral tissues.
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Sipe CJ, Claudio Vázquez PN, Skeate JG, McIvor RS, Moriarity BS. Targeted genome editing for the correction or alleviation of primary Immunodeficiencies. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2021; 182:111-151. [PMID: 34175040 DOI: 10.1016/bs.pmbts.2021.03.001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Primary immunodeficiencies (PID) are a growing list of unique disorders that result in a failure of the innate/adaptive immune systems to fully respond to disease or infection. PIDs are classified into five broad categories; B cell disorders, combined B and T cell disorders, phagocytic disorders, complement disorders, and disorders with recurrent fevers and inflammation. Many of these disorders, such as X-SCID, WAS, and CGD lead to early death in children if intervention is not implemented. At present, the predominant method of curative therapy remains an allogeneic transplant from a healthy donor, however many complications and limitations exist with his therapy such as availability of donors, graft vs host disease, graft rejection, and infection. More recently, gene therapy using viral based complementation vectors have successfully been implemented to functionally correct patient cells in an autologous transplant, but these methods carry significant risks, including insertional mutagenesis, and provide non-physiological gene expression. For these reasons, gene-editing reagents such as targeted nucleases, base editors (BE), and prime editors (PE) are being explored. The BE and PE tools, sometimes referred to as digital editors, are of very high interest as they provide both enhanced molecular specificity and do not rely on DNA repair pathways after DSBs to change individual base pairs or directly replace DNA sequences responsible for pathogenic phenotypes. With this in mind the purpose of this chapter is to highlight some of the most common PIDs found within the human population, discuss successes and shortcomings of previous intervention strategies, and highlight how the next generation of gene-editing tools may be deployed to directly repair the underlying genetic causes of this class of disease.
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Affiliation(s)
- Christopher J Sipe
- Department of Pediatrics, University of Minnesota, Minneapolis, MN, United States; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States; Center for Genome Engineering, University of Minnesota, Minneapolis, MN, United States; Stem Cell Institute, University of Minnesota, Minneapolis, MN, United States; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, United States
| | - Patricia N Claudio Vázquez
- Department of Pediatrics, University of Minnesota, Minneapolis, MN, United States; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States; Center for Genome Engineering, University of Minnesota, Minneapolis, MN, United States; Stem Cell Institute, University of Minnesota, Minneapolis, MN, United States; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, United States
| | - Joseph G Skeate
- Department of Pediatrics, University of Minnesota, Minneapolis, MN, United States; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States; Center for Genome Engineering, University of Minnesota, Minneapolis, MN, United States; Stem Cell Institute, University of Minnesota, Minneapolis, MN, United States
| | - R Scott McIvor
- Department of Pediatrics, University of Minnesota, Minneapolis, MN, United States; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States; Center for Genome Engineering, University of Minnesota, Minneapolis, MN, United States; Stem Cell Institute, University of Minnesota, Minneapolis, MN, United States; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, United States
| | - Branden S Moriarity
- Department of Pediatrics, University of Minnesota, Minneapolis, MN, United States; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States; Center for Genome Engineering, University of Minnesota, Minneapolis, MN, United States; Stem Cell Institute, University of Minnesota, Minneapolis, MN, United States; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, United States.
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