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Lussana F, Magnani CF, Galimberti S, Gritti G, Gaipa G, Belotti D, Cabiati B, Napolitano S, Ferrari S, Moretti A, Buracchi C, Borleri GM, Rambaldi B, Rizzuto G, Grassi A, Paganessi M, Meli C, Tettamanti S, Risca G, Pais G, Spinozzi G, Benedicenti F, Cazzaniga G, Capelli C, Gotti E, Introna M, Golay J, Montini E, Balduzzi A, Valsecchi MG, Dastoli G, Rambaldi A, Biondi A. Donor-derived CARCIK-CD19 cells engineered with Sleeping Beauty transposon in acute lymphoblastic leukemia relapsed after allogeneic transplantation. Blood Cancer J 2025; 15:54. [PMID: 40180925 PMCID: PMC11968829 DOI: 10.1038/s41408-025-01260-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2025] [Revised: 02/26/2025] [Accepted: 03/19/2025] [Indexed: 04/05/2025] Open
Abstract
Non-viral engineering can ease CAR-T cell production and reduce regulatory and cost requirements. We utilized Sleeping Beauty transposon to engineer donor-derived anti-CD19.CD28.OX40.CD3zeta T cells differentiated in cytokine-induced killer (CARCIK-CD19) for B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (alloHSCT). We report the results of CARCIK-CD19 observed in 36 patients (4 children and 32 adults) treated according to the final recommended dose. Cytokine release syndrome of grade 2 or lower occurred in 15 patients, ICANS grade 2 in 1 patient, and late-onset peripheral neurotoxicity of grade 3 in 2 patients. GVHD never occurred after treatment with allogeneic CARCIK-CD19. Complete remission was achieved by 30 out of 36 patients (83.3%), with MRD negativity in 89% of responders. With a median follow-up of 2.2 years, the 1-year overall survival was 57.0%, and event-free survival was 32.0%. The median duration of response at 1 year was 38.6%. CAR-T cells expanded rapidly after infusion and remained detectable for over 2 years. Integration site analysis after infusion showed a high clonal diversity. These data demonstrated that SB-engineered CAR-T cells are safe and induce durable remission in heavily pretreated patients with BCP-ALL relapsed after alloHSCT. Trial registration: The phase 1/2 and phase II trials are registered at www.clinicaltrials.gov as NCT#03389035 and NCT#05252403.
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Affiliation(s)
- Federico Lussana
- Hematology and Bone Marrow Transplant Unit, Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy
- Department of Oncology and Hematology, University of Milan, Milan, Italy
| | - Chiara F Magnani
- Tettamanti Center, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
- Department of Medical Oncology and Hematology, University Hospital Zurich and University of Zurich, Zurich, Switzerland
| | - Stefania Galimberti
- Department of Medicine and Surgery, Bicocca Bioinformatics, Biostatistics and Bioimaging Centre B4, University of Milano-Bicocca, Milan, Italy
- Biostatistics and Clinical Epidemiology, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
| | - Giuseppe Gritti
- Hematology and Bone Marrow Transplant Unit, Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy
| | - Giuseppe Gaipa
- Tettamanti Center, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
- Laboratorio di Terapia Cellulare e Genica Stefano Verri, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
| | - Daniela Belotti
- Tettamanti Center, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
- Laboratorio di Terapia Cellulare e Genica Stefano Verri, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
| | - Benedetta Cabiati
- Tettamanti Center, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
- Laboratorio di Terapia Cellulare e Genica Stefano Verri, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
| | - Sara Napolitano
- Department of Pediatrics, University of Milano-Bicocca, Fondazione MBBM, Monza, Italy
| | - Silvia Ferrari
- Hematology and Bone Marrow Transplant Unit, Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy
| | - Alex Moretti
- Tettamanti Center, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
- Department of Pediatrics, University of Milano-Bicocca, Fondazione MBBM, Monza, Italy
| | - Chiara Buracchi
- Tettamanti Center, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
| | - Gian Maria Borleri
- Hematology and Bone Marrow Transplant Unit, Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy
| | - Benedetta Rambaldi
- Hematology and Bone Marrow Transplant Unit, Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy
| | - Giuliana Rizzuto
- Hematology and Bone Marrow Transplant Unit, Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy
- Department of Oncology and Hematology, University of Milan, Milan, Italy
| | - Anna Grassi
- Hematology and Bone Marrow Transplant Unit, Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy
| | - Muriel Paganessi
- Hematology and Bone Marrow Transplant Unit, Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy
| | - Cristian Meli
- Hematology and Bone Marrow Transplant Unit, Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy
| | - Sarah Tettamanti
- Tettamanti Center, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
| | - Giulia Risca
- Biostatistics and Clinical Epidemiology, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
| | - Giulia Pais
- San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Giulio Spinozzi
- San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Fabrizio Benedicenti
- San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Giovanni Cazzaniga
- Tettamanti Center, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
| | - Chiara Capelli
- USS Centro di Terapia Cellulare "G. Lanzani", Bergamo, Italy
| | - Elisa Gotti
- USS Centro di Terapia Cellulare "G. Lanzani", Bergamo, Italy
| | - Martino Introna
- Hematology and Bone Marrow Transplant Unit, Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy
- USS Centro di Terapia Cellulare "G. Lanzani", Bergamo, Italy
| | - Josée Golay
- Hematology and Bone Marrow Transplant Unit, Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy
- USS Centro di Terapia Cellulare "G. Lanzani", Bergamo, Italy
| | - Eugenio Montini
- San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Adriana Balduzzi
- Department of Pediatrics, University of Milano-Bicocca, Fondazione MBBM, Monza, Italy
| | - Maria Grazia Valsecchi
- Department of Medicine and Surgery, Bicocca Bioinformatics, Biostatistics and Bioimaging Centre B4, University of Milano-Bicocca, Milan, Italy
- Biostatistics and Clinical Epidemiology, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
| | - Giuseppe Dastoli
- Tettamanti Center, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
| | - Alessandro Rambaldi
- Hematology and Bone Marrow Transplant Unit, Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy
- Department of Oncology and Hematology, University of Milan, Milan, Italy
| | - Andrea Biondi
- Tettamanti Center, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy.
- Department of Pediatrics, University of Milano-Bicocca, Fondazione MBBM, Monza, Italy.
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Zhou Z, Chen Y, Ba Y, Xu H, Zuo A, Liu S, Zhang Y, Weng S, Ren Y, Luo P, Cheng Q, Zuo L, Zhu S, Zhou X, Zhang C, Chen Y, Han X, Pan T, Liu Z. Revolutionising Cancer Immunotherapy: Advancements and Prospects in Non-Viral CAR-NK Cell Engineering. Cell Prolif 2025; 58:e13791. [PMID: 39731215 PMCID: PMC11969250 DOI: 10.1111/cpr.13791] [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: 05/22/2024] [Revised: 10/14/2024] [Accepted: 11/28/2024] [Indexed: 12/29/2024] Open
Abstract
The recent advancements in cancer immunotherapy have spotlighted the potential of natural killer (NK) cells, particularly chimeric antigen receptor (CAR)-transduced NK cells. These cells, pivotal in innate immunity, offer a rapid and potent response against cancer cells and pathogens without the need for prior sensitization or recognition of peptide antigens. Although NK cell genetic modification is evolving, the viral transduction method continues to be inefficient and fraught with risks, often resulting in cytotoxic outcomes and the possibility of insertional mutagenesis. Consequently, there has been a surge in the development of non-viral transfection technologies to overcome these challenges in NK cell engineering. Non-viral approaches for CAR-NK cell generation are becoming increasingly essential. Cutting-edge techniques such as trogocytosis, electroporation, lipid nanoparticle (LNP) delivery, clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) gene editing and transposons not only enhance the efficiency and safety of CAR-NK cell engineering but also open new avenues for novel therapeutic possibilities. Additionally, the infusion of technologies already successful in CAR T-cell therapy into the CAR-NK paradigm holds immense potential for further advancements. In this review, we present an overview of the potential of NK cells in cancer immunotherapies, as well as non-viral transfection technologies for engineering NK cells.
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Affiliation(s)
- Zhaokai Zhou
- Department of Interventional RadiologyThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouChina
- Department of UrologyThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouChina
| | - Yifeng Chen
- The First Affiliated Hospital of Zhengzhou UniversityZhengzhouChina
| | - Yuhao Ba
- Department of Interventional RadiologyThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouChina
| | - Hui Xu
- Department of Interventional RadiologyThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouChina
| | - Anning Zuo
- Department of Interventional RadiologyThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouChina
| | - Shutong Liu
- Department of Interventional RadiologyThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouChina
| | - Yuyuan Zhang
- Department of Interventional RadiologyThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouChina
| | - Siyuan Weng
- Department of Interventional RadiologyThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouChina
| | - Yuqing Ren
- Department of Respiratory and Critical Care MedicineThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouChina
| | - Peng Luo
- The Department of OncologyZhujiang Hospital, Southern Medical UniversityGuangzhouChina
| | - Quan Cheng
- Department of NeurosurgeryXiangya Hospital, Central South UniversityChangshaChina
| | - Lulu Zuo
- Center of Reproductive MedicineThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouChina
| | - Shanshan Zhu
- Department of GastroenterologyThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouChina
| | - Xing Zhou
- Department of Pediatric SurgeryThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouChina
| | - Chuhan Zhang
- Department of OncologyThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouChina
| | - Yukang Chen
- The First Affiliated Hospital of Zhengzhou UniversityZhengzhouChina
| | - Xinwei Han
- Department of Interventional RadiologyThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouChina
- Interventional Institute of Zhengzhou UniversityZhengzhouChina
- Interventional Treatment and Clinical Research Center of Henan ProvinceZhengzhouChina
| | - Teng Pan
- Longgang District Maternity & Child Healthcare Hospital of Shenzhen City (Longgang Maternity and Child Institute of Shantou University Medical College)ShenzhenChina
| | - Zaoqu Liu
- Department of Interventional RadiologyThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouChina
- Interventional Institute of Zhengzhou UniversityZhengzhouChina
- Interventional Treatment and Clinical Research Center of Henan ProvinceZhengzhouChina
- Institute of Basic Medical SciencesChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
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3
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Boros BD, Gachechiladze MA, Guo J, Galloway DA, Mueller SM, Shabsovich M, Yen A, Cammack AJ, Shen T, Mitra RD, Dougherty JD, Miller TM. Prior epigenetic status predicts future susceptibility to seizures in mice. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.03.20.644199. [PMID: 40166300 PMCID: PMC11957114 DOI: 10.1101/2025.03.20.644199] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 04/02/2025]
Abstract
Wide variation of responses to identical stimuli presented to genetically inbred mice suggests the hypothesis that stochastic epigenetic variation during neurodevelopment can mediate such phenotypic differences. However, this hypothesis is largely untested since capturing pre-existing molecular states requires non-destructive, longitudinal recording. Therefore, we tested the potential of Calling Cards (CC) to record transient neuronal enhancer activity during postnatal development, and thereby associate epigenetic variation with a subsequent phenotypic presentation - degree of seizure response to the pro-convulsant pentylenetetrazol. We show that recorded differences in epigenetics at 243 loci predict a severe vs. mild response, and that these are enriched near genes associated with human epilepsy. We also validated pharmacologically a seizure-modifying role for two novel genes, Htr1f and Let7c. This proof-of-principle supports using CC broadly to discover predisposition loci for other neuropsychiatric traits and behaviors. Finally, as, human disease is also influenced by non-inherited factors, similar epigenetic predispositions are possible in humans.
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Affiliation(s)
- Benjamin D. Boros
- Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110
| | - Mariam A. Gachechiladze
- Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO 63110
| | - Juanru Guo
- Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110
| | - Dylan A. Galloway
- Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110
| | - Shayna M. Mueller
- Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO 63110
| | - Mark Shabsovich
- Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110
| | - Allen Yen
- Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO 63110
| | - Alexander J. Cammack
- Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110
| | - Tao Shen
- Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110
| | - Robi D. Mitra
- Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110
| | - Joseph D. Dougherty
- Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO 63110
- Intellectual and Developmental Disabilities Research Center, Washington University School of Medicine, St. Louis, MO 63110
| | - Timothy M. Miller
- Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110
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4
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Diez B, Calviño C, Fernández-García M, Rodríguez-Márquez P, Rodríguez-Díaz S, Martínez-Turillas R, Ceballos C, Illarramendi J, Serrano-López J, Miskey C, Navarro-Bailón A, López-Corral L, Llamas P, Redondo M, Sánchez-Guijo F, Rifon J, Alfonso-Piérola A, Ivics Z, Inogés S, López-Díaz de Cerio A, Yanez R, Bueren JA, Rodríguez-Madoz JR, Prosper F. Generation and GMP scale-up of human CAR-T cells using non-viral Sleeping Beauty transposons for B cell malignances. Mol Ther Methods Clin Dev 2025; 33:101425. [PMID: 40034423 PMCID: PMC11874549 DOI: 10.1016/j.omtm.2025.101425] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2024] [Accepted: 01/28/2025] [Indexed: 03/05/2025]
Abstract
Most CAR-T therapies rely on genetic T cell engineering with integrating viral vectors that, although effective, are associated with prohibitive costs. Here we have generated TranspoCART19 cells, a fully functional 4-1BB second-generation CAR-T cell product targeting CD19, fused to a truncated version of the human EGFR (hEGFRt) as reporter gene and safety switch, based on the Sleeping Beauty transposon delivery system. Our manufacturing protocol allowed generation of TranspoCART19 cells under GMP conditions, showing similar in vitro and in vivo antitumoral efficacy than conventional CAR-T cells generated with lentiviral vectors. Additionally, membrane expression of hEGFRt facilitated in vivo CAR-T cell elimination after cetuximab administration. Safety analyses showed that TranspoCART19 cells presented low vector copy numbers and close-to-random vector integration profiles. Moreover, final TranspoCART19 products lacked non-integrated genomic material used for the generation of CAR-T cells and were free from transposase protein. In vivo biodistribution analyses revealed that TranspoCART19 cells were mainly present in hematopoietic organs with no gender bias. Altogether, this study provides a cost-effective, GMP-compliant manufacturing process for the generation of CAR-T cells using non-viral vectors. These results have supported the approval of a clinical trial to evaluate TranspoCART19 cells in patients with relapsed/refractory lymphoma (NCT06378190) that is currently ongoing.
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Affiliation(s)
- Begoña Diez
- Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energeticas Medioambientales y Tecnologicas (CIEMAT), Madrid, Spain
- Advanced Therapies Unit, IIS Fundación Jimenez Diaz, Universidad Autonoma de Madrid, Madrid, Spain
- Biomedical Research Network Center on Rare Diseases (CIBERER), Madrid, Spain
| | - Cristina Calviño
- Hematology and Cell Therapy Department, Clinica Universidad de Navarra (CUN), IdiSNA, Pamplona, Spain
| | - María Fernández-García
- Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energeticas Medioambientales y Tecnologicas (CIEMAT), Madrid, Spain
- Advanced Therapies Unit, IIS Fundación Jimenez Diaz, Universidad Autonoma de Madrid, Madrid, Spain
- Biomedical Research Network Center on Rare Diseases (CIBERER), Madrid, Spain
| | - Paula Rodríguez-Márquez
- Hemato-Oncology Program, Cancer Division, Cima Universidad de Navarra, IdiSNA, Pamplona, Spain
| | - Saray Rodríguez-Díaz
- Hemato-Oncology Program, Cancer Division, Cima Universidad de Navarra, IdiSNA, Pamplona, Spain
| | - Rebeca Martínez-Turillas
- Hemato-Oncology Program, Cancer Division, Cima Universidad de Navarra, IdiSNA, Pamplona, Spain
- Biomedical Research Network Center on Cancer (CIBERONC), Madrid, Spain
| | - Candela Ceballos
- Servicio de Hematología, Hospital Universitario de Navarra, IdiSNA, Pamplona, Spain
| | - Jorge Illarramendi
- Servicio de Hematología, Hospital Universitario de Navarra, IdiSNA, Pamplona, Spain
| | | | - Csaba Miskey
- Transposition and Genome Engineering, Division of Hematology, Cell and Gene Therapy, Paul Ehrlich Institute, Langen, Germany
| | - Almudena Navarro-Bailón
- Biomedical Research Network Center on Cancer (CIBERONC), Madrid, Spain
- Hematology Department, IBSAL-University Hospital of Salamanca, Salamanca, Spain
- Center for Cancer Research (CIC-IBMCC) and Department of Medicine, University of Salamanca, Salamanca, Spain
| | - Lucía López-Corral
- Biomedical Research Network Center on Cancer (CIBERONC), Madrid, Spain
- Hematology Department, IBSAL-University Hospital of Salamanca, Salamanca, Spain
- Center for Cancer Research (CIC-IBMCC) and Department of Medicine, University of Salamanca, Salamanca, Spain
| | - Pilar Llamas
- Experimental Hematology Lab, IIS-Fundacion Jimenez Díaz, UAM, Madrid, Spain
- Hematology Department, Fundacion Jiménez Diaz University Hospital, Madrid, Spain
| | - Margarita Redondo
- Servicio de Hematología, Hospital Universitario de Navarra, IdiSNA, Pamplona, Spain
| | - Fermín Sánchez-Guijo
- Biomedical Research Network Center on Cancer (CIBERONC), Madrid, Spain
- Hematology Department, IBSAL-University Hospital of Salamanca, Salamanca, Spain
- Center for Cancer Research (CIC-IBMCC) and Department of Medicine, University of Salamanca, Salamanca, Spain
| | - Jose Rifon
- Hematology and Cell Therapy Department, Clinica Universidad de Navarra (CUN), IdiSNA, Pamplona, Spain
- Cancer Center Clinica Universidad de Navarra (CCUN), Pamplona, Spain
- Biomedical Research Network Center on Cancer (CIBERONC), Madrid, Spain
| | - Ana Alfonso-Piérola
- Hematology and Cell Therapy Department, Clinica Universidad de Navarra (CUN), IdiSNA, Pamplona, Spain
- Cancer Center Clinica Universidad de Navarra (CCUN), Pamplona, Spain
- Biomedical Research Network Center on Cancer (CIBERONC), Madrid, Spain
| | - Zoltán Ivics
- Transposition and Genome Engineering, Division of Hematology, Cell and Gene Therapy, Paul Ehrlich Institute, Langen, Germany
| | - Susana Inogés
- Hematology and Cell Therapy Department, Clinica Universidad de Navarra (CUN), IdiSNA, Pamplona, Spain
- Cancer Center Clinica Universidad de Navarra (CCUN), Pamplona, Spain
- Biomedical Research Network Center on Cancer (CIBERONC), Madrid, Spain
| | - Ascensión López-Díaz de Cerio
- Hematology and Cell Therapy Department, Clinica Universidad de Navarra (CUN), IdiSNA, Pamplona, Spain
- Cancer Center Clinica Universidad de Navarra (CCUN), Pamplona, Spain
- Biomedical Research Network Center on Cancer (CIBERONC), Madrid, Spain
| | - Rosa Yanez
- Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energeticas Medioambientales y Tecnologicas (CIEMAT), Madrid, Spain
- Advanced Therapies Unit, IIS Fundación Jimenez Diaz, Universidad Autonoma de Madrid, Madrid, Spain
- Biomedical Research Network Center on Rare Diseases (CIBERER), Madrid, Spain
| | - Juan A. Bueren
- Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energeticas Medioambientales y Tecnologicas (CIEMAT), Madrid, Spain
- Advanced Therapies Unit, IIS Fundación Jimenez Diaz, Universidad Autonoma de Madrid, Madrid, Spain
- Biomedical Research Network Center on Rare Diseases (CIBERER), Madrid, Spain
| | - Juan R. Rodríguez-Madoz
- Hemato-Oncology Program, Cancer Division, Cima Universidad de Navarra, IdiSNA, Pamplona, Spain
- Cancer Center Clinica Universidad de Navarra (CCUN), Pamplona, Spain
- Biomedical Research Network Center on Cancer (CIBERONC), Madrid, Spain
| | - Felipe Prosper
- Hematology and Cell Therapy Department, Clinica Universidad de Navarra (CUN), IdiSNA, Pamplona, Spain
- Hemato-Oncology Program, Cancer Division, Cima Universidad de Navarra, IdiSNA, Pamplona, Spain
- Cancer Center Clinica Universidad de Navarra (CCUN), Pamplona, Spain
- Biomedical Research Network Center on Cancer (CIBERONC), Madrid, Spain
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5
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Elsworth B, Ye S, Dass S, Tennessen JA, Sultana Q, Thommen BT, Paul AS, Kanjee U, Grüring C, Ferreira MU, Gubbels MJ, Zarringhalam K, Duraisingh MT. The essential genome of Plasmodium knowlesi reveals determinants of antimalarial susceptibility. Science 2025; 387:eadq6241. [PMID: 39913579 DOI: 10.1126/science.adq6241] [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: 06/03/2024] [Accepted: 12/05/2024] [Indexed: 02/09/2025]
Abstract
Measures to combat the parasites that cause malaria have become compromised because of reliance on a small arsenal of drugs and emerging drug resistance. We conducted a transposon mutagenesis screen in the primate malaria parasite Plasmodium knowlesi, producing the most complete classification of gene essentiality in any Plasmodium spp. to date, with the resolution to define truncatable genes. We found conservation in the druggable genome between Plasmodium spp. and divergences in mitochondrial metabolism. Perturbation analyses with the frontline antimalarial artemisinin revealed modulators that both increase and decrease drug susceptibility. Our findings aid prioritization of drug and vaccine targets for the Plasmodium vivax clade and reveal mechanisms of resistance that can inform therapeutic development.
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Affiliation(s)
- Brendan Elsworth
- Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA
- Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA
| | - Sida Ye
- Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA
- Department of Mathematics, University of Massachusetts Boston, Boston, MA, USA
- Center for Personalized Cancer Therapy, University of Massachusetts Boston, Boston, MA, USA
| | - Sheena Dass
- Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | - Jacob A Tennessen
- Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | - Qudseen Sultana
- Center for Personalized Cancer Therapy, University of Massachusetts Boston, Boston, MA, USA
- Department of Computer Science, University of Massachusetts Boston, Boston, MA, USA
| | - Basil T Thommen
- Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | - Aditya S Paul
- Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | - Usheer Kanjee
- Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | - Christof Grüring
- Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | - Marcelo U Ferreira
- Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
- Global Health and Tropical Medicine, Institute of Hygiene and Tropical Medicine, Nova University of Lisbon, Lisbon, Portugal
| | | | - Kourosh Zarringhalam
- Department of Mathematics, University of Massachusetts Boston, Boston, MA, USA
- Center for Personalized Cancer Therapy, University of Massachusetts Boston, Boston, MA, USA
| | - Manoj T Duraisingh
- Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA
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6
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Maurer K, Jacobson CA. T-cell neoplasias and secondary malignancies after CAR-T cell therapy: current knowledge, risk factors, and implications from CAR-T engineering strategies. Leuk Lymphoma 2025:1-9. [PMID: 39894954 DOI: 10.1080/10428194.2025.2460043] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2024] [Revised: 01/23/2025] [Accepted: 01/24/2025] [Indexed: 02/04/2025]
Abstract
Widespread use of CAR-T cell therapies for treatment of B cell malignancies has resulted in a frameshift in treatment strategies and improved patient outcomes since the first CAR-T product was FDA approved in 2017. Currently over 30,000 patients have been treated with approved CAR-T cell products, with many more likely to be treated in future, both as standard of care therapy as well as on clinical trials. As more patients are treated, development of rare complications has begun to emerge, and the incidence of second primary malignancies after CAR-T cell therapy is evolving from a hypothetical to a realized concern. Furthermore, in November 2023, the FDA issued a warning regarding the potential for CAR-T cell-derived T cell neoplasias to arise as a result of CAR-T manufacturing. Here we review patient risk factors for development of second primary malignancies including T cell neoplasias, CAR-T engineering strategies that may increase this risk, and the current body of literature surrounding incidence of second primary malignancies and case reports of T cell neoplasias arising after CAR-T cell therapy.
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Affiliation(s)
- Katie Maurer
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
| | - Caron A Jacobson
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
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7
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Lenser M, Ngo HG, Sarrafha L, Rajendra Y. Evaluation of two transposases for improving expression of recombinant proteins in Chinese hamster ovary cell stable pools by co-transfection and supertransfection approaches. Biotechnol Prog 2025; 41:e3496. [PMID: 39016635 DOI: 10.1002/btpr.3496] [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: 03/26/2024] [Revised: 06/13/2024] [Accepted: 07/09/2024] [Indexed: 07/18/2024]
Abstract
Transposons are genetic elements capable of cutting and pasting genes of interest via the action of a transposase and offer many advantages over random or targeted integration of DNA in the creation of Chinese hamster ovary (CHO) cell lines for recombinant protein expression. Unique transposases have different recognition sites, allowing multiple transposases to be co-transfected together. They also allow for supertransfection (transfection on a previously transfected pool or cell line) with a second transposase to integrate additional copies of the same gene or an additional gene without disruption of the previously integrated DNA which to our knowledge has not been previously described in literature. Two fluorescent proteins, EGFP and tagRFP657, were either co-transfected or supertransfected into CHO cells using two unique transposases and showed high expression efficiency with similar expression levels (measured as mean fluorescence intensity), regardless of whether the genes were co-transfected or supertransfected onto an existing stable pool. Additionally, dual selection of the genes, both in the absence of L-glutamine and the presence of puromycin, led to higher expression levels than single selection alone. These results demonstrate that supertransfection using unique transposases could be a useful strategy for increasing titers of existing cell lines or for overexpressing helper (non-therapeutic) genes to improve expression and/or product quality of existing pools and cell lines, potentially saving significant time and resources.
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Affiliation(s)
- Melina Lenser
- Bioprocess Development, Technical Operations, Denali Therapeutics, Inc., South San Francisco, California, USA
| | - Hanh Giai Ngo
- Bioprocess Development, Technical Operations, Denali Therapeutics, Inc., South San Francisco, California, USA
| | - Lily Sarrafha
- Discovery Biology, Denali Therapeutics, Inc., South San Francisco, California, USA
| | - Yashas Rajendra
- Bioprocess Development, Technical Operations, Denali Therapeutics, Inc., South San Francisco, California, USA
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8
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Gehrke L, Gonçalves VDR, Andrae D, Rasko T, Ho P, Einsele H, Hudecek M, Friedel SR. Current Non-Viral-Based Strategies to Manufacture CAR-T Cells. Int J Mol Sci 2024; 25:13685. [PMID: 39769449 PMCID: PMC11728233 DOI: 10.3390/ijms252413685] [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: 11/19/2024] [Revised: 12/12/2024] [Accepted: 12/14/2024] [Indexed: 01/16/2025] Open
Abstract
The successful application of CAR-T cells in the treatment of hematologic malignancies has fundamentally changed cancer therapy. With increasing numbers of registered CAR-T cell clinical trials, efforts are being made to streamline and reduce the costs of CAR-T cell manufacturing while improving their safety. To date, all approved CAR-T cell products have relied on viral-based gene delivery and genomic integration methods. While viral vectors offer high transfection efficiencies, concerns regarding potential malignant transformation coupled with costly and time-consuming vector manufacturing are constant drivers in the search for cheaper, easier-to-use, safer, and more efficient alternatives. In this review, we examine different non-viral gene transfer methods as alternatives for CAR-T cell production, their advantages and disadvantages, and examples of their applications. Transposon-based gene transfer methods lead to stable but non-targeted gene integration, are easy to handle, and achieve high gene transfer rates. Programmable endonucleases allow targeted integration, reducing the potential risk of integration-mediated malignant transformation of CAR-T cells. Non-integrating CAR-encoding vectors avoid this risk completely and achieve only transient CAR expression. With these promising alternative techniques for gene transfer, all avenues are open to fully exploiting the potential of next-generation CAR-T cell therapy and applying it in a wide range of applications.
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Affiliation(s)
- Leon Gehrke
- Medizinische Klinik und Poliklinik II und Lehrstuhl für Zelluläre Immuntherapie, Universitätsklinikum Würzburg, 97080 Würzburg, Germany
| | - Vasco Dos Reis Gonçalves
- Medizinische Klinik und Poliklinik II und Lehrstuhl für Zelluläre Immuntherapie, Universitätsklinikum Würzburg, 97080 Würzburg, Germany
| | - Dominik Andrae
- Medizinische Klinik und Poliklinik II und Lehrstuhl für Zelluläre Immuntherapie, Universitätsklinikum Würzburg, 97080 Würzburg, Germany
| | - Tamas Rasko
- Medizinische Klinik und Poliklinik II und Lehrstuhl für Zelluläre Immuntherapie, Universitätsklinikum Würzburg, 97080 Würzburg, Germany
| | - Patrick Ho
- Medizinische Klinik und Poliklinik II und Lehrstuhl für Zelluläre Immuntherapie, Universitätsklinikum Würzburg, 97080 Würzburg, Germany
| | - Hermann Einsele
- Medizinische Klinik und Poliklinik II und Lehrstuhl für Zelluläre Immuntherapie, Universitätsklinikum Würzburg, 97080 Würzburg, Germany
| | - Michael Hudecek
- Medizinische Klinik und Poliklinik II und Lehrstuhl für Zelluläre Immuntherapie, Universitätsklinikum Würzburg, 97080 Würzburg, Germany
- Fraunhofer-Institut für Zelltherapie und Immunologie, Außenstelle Zelluläre Immuntherapie, 97070 Würzburg, Germany
| | - Sabrina R. Friedel
- Medizinische Klinik und Poliklinik II und Lehrstuhl für Zelluläre Immuntherapie, Universitätsklinikum Würzburg, 97080 Würzburg, Germany
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9
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Garbayo E, El Moukhtari SH, Rodríguez-Nogales C, Agirre X, Rodriguez-Madoz JR, Rodriguez-Marquez P, Prósper F, Couvreur P, Blanco-Prieto MJ. RNA-loaded nanoparticles for the treatment of hematological cancers. Adv Drug Deliv Rev 2024; 214:115448. [PMID: 39303823 DOI: 10.1016/j.addr.2024.115448] [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: 02/19/2024] [Revised: 06/07/2024] [Accepted: 09/08/2024] [Indexed: 09/22/2024]
Abstract
Hematological cancers encompass a diverse group of malignancies affecting the blood, bone marrow, lymph nodes, and spleen. These disorders present unique challenges due to their complex etiology and varied clinical manifestations. Despite significant advancements in understanding and treating hematological malignancies, innovative therapeutic approaches are continually sought to enhance patient outcomes. This review highlights the application of RNA nanoparticles (RNA-NPs) in the treatment of hematological cancers. We delve into detailed discussions on in vitro and preclinical studies involving RNA-NPs for adult patients, as well as the application of RNA-NPs in pediatric hematological cancer. The review also addresses ongoing clinical trials involving RNA-NPs and explores the emerging field of CAR-T therapy engineered by RNA-NPs. Finally, we discuss the challenges still faced in translating RNA-NP research to clinics.
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Affiliation(s)
- Elisa Garbayo
- Department of Pharmaceutical Sciences, School of Pharmacy and Nutrition, Universidad de Navarra, C/Irunlarrea 1, 31008 Pamplona, Spain; Instituto de Investigación Sanitaria de Navarra, IdiSNA, C/Irunlarrea 3, 31008 Pamplona, Spain; Cancer Center Clinica Universidad de Navarra (CCUN). Avenida Pio XII 36, 31008 Pamplona, Spain
| | - Souhaila H El Moukhtari
- Department of Pharmaceutical Sciences, School of Pharmacy and Nutrition, Universidad de Navarra, C/Irunlarrea 1, 31008 Pamplona, Spain; Instituto de Investigación Sanitaria de Navarra, IdiSNA, C/Irunlarrea 3, 31008 Pamplona, Spain
| | - Carlos Rodríguez-Nogales
- Department of Pharmaceutical Sciences, School of Pharmacy and Nutrition, Universidad de Navarra, C/Irunlarrea 1, 31008 Pamplona, Spain; Instituto de Investigación Sanitaria de Navarra, IdiSNA, C/Irunlarrea 3, 31008 Pamplona, Spain; Cancer Center Clinica Universidad de Navarra (CCUN). Avenida Pio XII 36, 31008 Pamplona, Spain
| | - Xabier Agirre
- Instituto de Investigación Sanitaria de Navarra, IdiSNA, C/Irunlarrea 3, 31008 Pamplona, Spain; Cancer Center Clinica Universidad de Navarra (CCUN). Avenida Pio XII 36, 31008 Pamplona, Spain; Hemato-Oncology Program, Center for Applied Medical Research (CIMA), University of Navarra, Avenida Pío XII 55, 31008 Pamplona, Spain; Centro de Investigación Biomédica en Red Cáncer (CIBERONC), 28029 Madrid, Spain
| | - Juan R Rodriguez-Madoz
- Instituto de Investigación Sanitaria de Navarra, IdiSNA, C/Irunlarrea 3, 31008 Pamplona, Spain; Cancer Center Clinica Universidad de Navarra (CCUN). Avenida Pio XII 36, 31008 Pamplona, Spain; Hemato-Oncology Program, Center for Applied Medical Research (CIMA), University of Navarra, Avenida Pío XII 55, 31008 Pamplona, Spain; Centro de Investigación Biomédica en Red Cáncer (CIBERONC), 28029 Madrid, Spain
| | - Paula Rodriguez-Marquez
- Instituto de Investigación Sanitaria de Navarra, IdiSNA, C/Irunlarrea 3, 31008 Pamplona, Spain; Cancer Center Clinica Universidad de Navarra (CCUN). Avenida Pio XII 36, 31008 Pamplona, Spain; Hemato-Oncology Program, Center for Applied Medical Research (CIMA), University of Navarra, Avenida Pío XII 55, 31008 Pamplona, Spain; Centro de Investigación Biomédica en Red Cáncer (CIBERONC), 28029 Madrid, Spain
| | - Felipe Prósper
- Instituto de Investigación Sanitaria de Navarra, IdiSNA, C/Irunlarrea 3, 31008 Pamplona, Spain; Cancer Center Clinica Universidad de Navarra (CCUN). Avenida Pio XII 36, 31008 Pamplona, Spain; Hemato-Oncology Program, Center for Applied Medical Research (CIMA), University of Navarra, Avenida Pío XII 55, 31008 Pamplona, Spain; Centro de Investigación Biomédica en Red Cáncer (CIBERONC), 28029 Madrid, Spain; Departmento de Hematología and CCUN, Clínica Universidad de Navarra, University of Navarra, Avenida Pío XII 36, 31008 Pamplona, Spain
| | - Patrick Couvreur
- Institut Galien Paris-Sud, UMR CNRS 8612, Université Paris-Saclay, Orsay Cedex, France.
| | - María J Blanco-Prieto
- Department of Pharmaceutical Sciences, School of Pharmacy and Nutrition, Universidad de Navarra, C/Irunlarrea 1, 31008 Pamplona, Spain; Instituto de Investigación Sanitaria de Navarra, IdiSNA, C/Irunlarrea 3, 31008 Pamplona, Spain; Cancer Center Clinica Universidad de Navarra (CCUN). Avenida Pio XII 36, 31008 Pamplona, Spain.
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10
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Sandoval-Villegas N, Ivics Z. The best of both worlds: AAV-mediated gene transfer empowered by LNP delivery of Sleeping Beauty transposase for durable transgene expression in vivo. Mol Ther 2024; 32:3211-3214. [PMID: 39326408 PMCID: PMC11489527 DOI: 10.1016/j.ymthe.2024.09.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2024] [Revised: 09/04/2024] [Accepted: 09/04/2024] [Indexed: 09/28/2024] Open
Affiliation(s)
| | - Zoltán Ivics
- Institute of Clinical Immunology, University of Leipzig, Leipzig, Germany; Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.
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11
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Tridgett M, Mulet M, Johny SP, Ababi M, Raghunath M, Fustinoni C, Galabova B, Fernández-Díaz C, Mikalajūnaitė I, Tomás HA, Kucej M, Dunajová L, Zgrundo Z, Page E, McCall L, Parker-Manuel R, Payne T, Peckett M, Kent J, Holland L, Asatryan R, Montgomery L, Chow TL, Beveridge R, Salkauskaite I, Alam MT, Hollard D, Dowding S, Gabriel HB, Branciaroli C, Cawood R, Valenti W, Chang D, Patrício MI, Liu Q. Lentiviral vector packaging and producer cell lines yield titers equivalent to the industry-standard four-plasmid process. Mol Ther Methods Clin Dev 2024; 32:101315. [PMID: 39282073 PMCID: PMC11401174 DOI: 10.1016/j.omtm.2024.101315] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2024] [Accepted: 08/05/2024] [Indexed: 09/18/2024]
Abstract
Lentiviral vector (LVV)-mediated cell and gene therapies have the potential to cure diseases that currently require lifelong intervention. However, the requirement for plasmid transfection hinders large-scale LVV manufacture. Moreover, large-scale plasmid production, testing, and transfection contribute to operational risk and the high cost associated with this therapeutic modality. Thus, we developed LVV packaging and producer cell lines, which reduce or eliminate the need for plasmid transfection during LVV manufacture. To develop a packaging cell line, lentiviral packaging genes were stably integrated by random integration of linearized plasmid DNA. Then, to develop EGFP- and anti-CD19 chimeric antigen receptor-encoding producer cell lines, transfer plasmids were integrated by transposase-mediated integration. Single-cell isolation and testing were performed to isolate the top-performing clonal packaging and producer cell lines. Production of LVVs that encode various cargo genes revealed consistency in the production performance of the packaging and producer cell lines compared to the industry-standard four-plasmid transfection method. By reducing or eliminating the requirement for plasmid transfection, while achieving production performance consistent with the current industry standard, the packaging and producer cell lines developed here can reduce costs and operational risks of LVV manufacture, thus increasing patient access to LVV-mediated cell and gene therapies.
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Affiliation(s)
- Matthew Tridgett
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Marie Mulet
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Sherin Parokkaran Johny
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Maria Ababi
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Meenakshi Raghunath
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Chloé Fustinoni
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Boryana Galabova
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Cristina Fernández-Díaz
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Iveta Mikalajūnaitė
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Hélio A Tomás
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Marek Kucej
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Lucia Dunajová
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Zofia Zgrundo
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Emma Page
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Lorna McCall
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Richard Parker-Manuel
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Tom Payne
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Matthew Peckett
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Jade Kent
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Louise Holland
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Robert Asatryan
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Louise Montgomery
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Tsz Lung Chow
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Ryan Beveridge
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Ieva Salkauskaite
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Mohine T Alam
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Daniel Hollard
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Sarah Dowding
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Heloísa Berti Gabriel
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Corinne Branciaroli
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Ryan Cawood
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Weimin Valenti
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
- WuXi Advanced Therapies, 4701 League Island Blvd, Philadelphia, PA 19112, USA
| | - David Chang
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
- WuXi Advanced Therapies, 4701 League Island Blvd, Philadelphia, PA 19112, USA
| | - Maria I Patrício
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
| | - Qian Liu
- OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford, Oxfordshire OX4 4HG, UK
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12
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Diaby M, Wu H, Gao B, Shi S, Wang B, Wang S, Wang Y, Wu Z, Chen C, Wang X, Song C. A Naturally Active Spy Transposon Discovered from the Insect Genome of Colletes gigas as a Promising Novel Gene Transfer Tool. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2024; 11:e2400969. [PMID: 38774947 PMCID: PMC11304231 DOI: 10.1002/advs.202400969] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/31/2024] [Revised: 04/09/2024] [Indexed: 08/09/2024]
Abstract
Novel active DNA transposons, such as Spy transposons from the PHIS superfamily, are identified through bioinformatics in this study. The native transposases cgSpy and cvSpy displayed transposition activities of approximately 85% and 35% compared to the hyperactive piggyBac transposase (hyPB). The cgSpy transposon showed unique characteristics, including a lack of overproduction inhibition and reduced efficiency for insertion sizes between 3.1 to 8.5 kb. Integration preferences of cgSpy are found in genes and regulatory regions, making it suitable for genetic manipulation. Evaluation in T-cell engineering demonstrated that cgSpy-mediated chimeric antigen receptor (CAR) modification is comparable to the PB system, indicating its potential utility in cell therapy. This study unveils the promising application of the active native transposase, Spy, from Colletes gigas, as a valuable tool for genetic engineering, particularly in T-cell manipulation.
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Affiliation(s)
- Mohamed Diaby
- College of Animal Science & TechnologyYangzhou UniversityYangzhouJiangsu225009China
| | - Han Wu
- School of Basic Medical SciencesShenzhen University Medical SchoolShenzhen UniversityShenzhenGuangdong518055China
| | - Bo Gao
- College of Animal Science & TechnologyYangzhou UniversityYangzhouJiangsu225009China
| | - Shasha Shi
- College of Animal Science & TechnologyYangzhou UniversityYangzhouJiangsu225009China
| | - Bingqing Wang
- College of Animal Science & TechnologyYangzhou UniversityYangzhouJiangsu225009China
| | - Saisai Wang
- College of Animal Science & TechnologyYangzhou UniversityYangzhouJiangsu225009China
| | - Yali Wang
- College of Animal Science & TechnologyYangzhou UniversityYangzhouJiangsu225009China
| | - Zherui Wu
- School of Basic Medical SciencesShenzhen University Medical SchoolShenzhen UniversityShenzhenGuangdong518055China
| | - Cai Chen
- College of Animal Science & TechnologyYangzhou UniversityYangzhouJiangsu225009China
| | - Xiaoyan Wang
- College of Animal Science & TechnologyYangzhou UniversityYangzhouJiangsu225009China
| | - Chengyi Song
- College of Animal Science & TechnologyYangzhou UniversityYangzhouJiangsu225009China
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13
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Taghdiri M, Mussolino C. Viral and Non-Viral Systems to Deliver Gene Therapeutics to Clinical Targets. Int J Mol Sci 2024; 25:7333. [PMID: 39000440 PMCID: PMC11242246 DOI: 10.3390/ijms25137333] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2024] [Revised: 06/10/2024] [Accepted: 06/24/2024] [Indexed: 07/16/2024] Open
Abstract
Clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized the field of gene therapy as it has enabled precise genome editing with unprecedented accuracy and efficiency, paving the way for clinical applications to treat otherwise incurable genetic disorders. Typically, precise genome editing requires the delivery of multiple components to the target cells that, depending on the editing platform used, may include messenger RNA (mRNA), protein complexes, and DNA fragments. For clinical purposes, these have to be efficiently delivered into transplantable cells, such as primary T lymphocytes or hematopoietic stem and progenitor cells that are typically sensitive to exogenous substances. This challenge has limited the broad applicability of precise gene therapy applications to those strategies for which efficient delivery methods are available. Electroporation-based methodologies have been generally applied for gene editing applications, but procedure-associated toxicity has represented a major burden. With the advent of novel and less disruptive methodologies to deliver genetic cargo to transplantable cells, it is now possible to safely and efficiently deliver multiple components for precise genome editing, thus expanding the applicability of these strategies. In this review, we describe the different delivery systems available for genome editing components, including viral and non-viral systems, highlighting their advantages, limitations, and recent clinical applications. Recent improvements to these delivery methods to achieve cell specificity represent a critical development that may enable in vivo targeting in the future and will certainly play a pivotal role in the gene therapy field.
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Affiliation(s)
- Maryam Taghdiri
- Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, 79106 Freiburg, Germany
- Center for Chronic Immunodeficiency (CCI), Medical Center-University of Freiburg, 79106 Freiburg, Germany
- Ph.D. Program, Faculty of Biology, University of Freiburg, 79106 Freiburg, Germany
| | - Claudio Mussolino
- Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, 79106 Freiburg, Germany
- Center for Chronic Immunodeficiency (CCI), Medical Center-University of Freiburg, 79106 Freiburg, Germany
- Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany
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14
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Bexte T, Botezatu L, Miskey C, Gierschek F, Moter A, Wendel P, Reindl LM, Campe J, Villena-Ossa JF, Gebel V, Stein K, Cathomen T, Cremer A, Wels WS, Hudecek M, Ivics Z, Ullrich E. Engineering of potent CAR NK cells using non-viral Sleeping Beauty transposition from minimalistic DNA vectors. Mol Ther 2024; 32:2357-2372. [PMID: 38751112 PMCID: PMC11287004 DOI: 10.1016/j.ymthe.2024.05.022] [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: 01/23/2024] [Revised: 03/25/2024] [Accepted: 05/09/2024] [Indexed: 06/06/2024] Open
Abstract
Natural killer (NK) cells have high intrinsic cytotoxic capacity, and clinical trials have demonstrated their safety and efficacy for adoptive cancer therapy. Expression of chimeric antigen receptors (CARs) enhances NK cell target specificity, with these cells applicable as off-the-shelf products generated from allogeneic donors. Here, we present for the first time an innovative approach for CAR NK cell engineering employing a non-viral Sleeping Beauty (SB) transposon/transposase-based system and minimized DNA vectors termed minicircles. SB-modified peripheral blood-derived primary NK cells displayed high and stable CAR expression and more frequent vector integration into genomic safe harbors than lentiviral vectors. Importantly, SB-generated CAR NK cells demonstrated enhanced cytotoxicity compared with non-transfected NK cells. A strong antileukemic potential was confirmed using established acute lymphocytic leukemia cells and patient-derived primary acute B cell leukemia and lymphoma samples as targets in vitro and in vivo in a xenograft leukemia mouse model. Our data suggest that the SB-transposon system is an efficient, safe, and cost-effective approach to non-viral engineering of highly functional CAR NK cells, which may be suitable for cancer immunotherapy of leukemia as well as many other malignancies.
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Affiliation(s)
- Tobias Bexte
- Goethe University, Department of Pediatrics, Experimental Immunology and Cell Therapy, Frankfurt am Main, Germany; Frankfurt Cancer Institute (FCI), Goethe University, Frankfurt am Main, Germany; University Cancer Center (UCT) Frankfurt, Frankfurt, Germany; Mildred Scheel Career Center (MSNZ), Hospital of the Goethe University Frankfurt, Frankfurt, Germany; Institute for Transfusion Medicine and Immunohematology, German Red Cross Blood Service Baden-Württemberg - Hesse, Frankfurt, Germany
| | - Lacramioara Botezatu
- Research Centre, Division of Hematology, Gene and Cell Therapy, Paul-Ehrlich-Institut, Langen, Germany; German Cancer Consortium (DKTK), partner site Heidelberg, Heidelberg, Germany
| | - Csaba Miskey
- Research Centre, Division of Hematology, Gene and Cell Therapy, Paul-Ehrlich-Institut, Langen, Germany
| | - Fenja Gierschek
- Goethe University, Department of Pediatrics, Experimental Immunology and Cell Therapy, Frankfurt am Main, Germany; Frankfurt Cancer Institute (FCI), Goethe University, Frankfurt am Main, Germany
| | - Alina Moter
- Goethe University, Department of Pediatrics, Experimental Immunology and Cell Therapy, Frankfurt am Main, Germany; Frankfurt Cancer Institute (FCI), Goethe University, Frankfurt am Main, Germany
| | - Philipp Wendel
- Goethe University, Department of Pediatrics, Experimental Immunology and Cell Therapy, Frankfurt am Main, Germany; Frankfurt Cancer Institute (FCI), Goethe University, Frankfurt am Main, Germany; German Cancer Consortium (DKTK), partner site Frankfurt/Mainz and German Cancer Research Center (DKFZ), Heidelberg, Germany; Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, Germany
| | - Lisa Marie Reindl
- Goethe University, Department of Pediatrics, Experimental Immunology and Cell Therapy, Frankfurt am Main, Germany; Frankfurt Cancer Institute (FCI), Goethe University, Frankfurt am Main, Germany
| | - Julia Campe
- Goethe University, Department of Pediatrics, Experimental Immunology and Cell Therapy, Frankfurt am Main, Germany; Frankfurt Cancer Institute (FCI), Goethe University, Frankfurt am Main, Germany
| | - Jose Francisco Villena-Ossa
- Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany; Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, Germany
| | - Veronika Gebel
- Goethe University, Department of Pediatrics, Experimental Immunology and Cell Therapy, Frankfurt am Main, Germany; Frankfurt Cancer Institute (FCI), Goethe University, Frankfurt am Main, Germany; University Cancer Center (UCT) Frankfurt, Frankfurt, Germany; Mildred Scheel Career Center (MSNZ), Hospital of the Goethe University Frankfurt, Frankfurt, Germany
| | - Katja Stein
- Goethe University, Department of Pediatrics, Experimental Immunology and Cell Therapy, Frankfurt am Main, Germany; Frankfurt Cancer Institute (FCI), Goethe University, Frankfurt am Main, Germany; University Cancer Center (UCT) Frankfurt, Frankfurt, Germany
| | - Toni Cathomen
- Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany; Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany; German Cancer Consortium (DKTK), partner site Freiburg, Freiburg, Germany
| | - Anjali Cremer
- Frankfurt Cancer Institute (FCI), Goethe University, Frankfurt am Main, Germany; University Cancer Center (UCT) Frankfurt, Frankfurt, Germany; Mildred Scheel Career Center (MSNZ), Hospital of the Goethe University Frankfurt, Frankfurt, Germany; German Cancer Consortium (DKTK), partner site Frankfurt/Mainz and German Cancer Research Center (DKFZ), Heidelberg, Germany; Department of Hematology/Oncology, University Hospital Frankfurt, Frankfurt am Main, Germany
| | - Winfried S Wels
- Frankfurt Cancer Institute (FCI), Goethe University, Frankfurt am Main, Germany; German Cancer Consortium (DKTK), partner site Frankfurt/Mainz and German Cancer Research Center (DKFZ), Heidelberg, Germany; Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany
| | - Michael Hudecek
- Department of Medicine II, Chaire in Cellular Immunotherapy, University Hospital Würzburg, Würzburg, Germany; Fraunhofer Institute for Cell Therapy and Immunology, Cellular Immunotherapy Branch Site Würzburg, Würzburg, Germany
| | - Zoltán Ivics
- Research Centre, Division of Hematology, Gene and Cell Therapy, Paul-Ehrlich-Institut, Langen, Germany; German Cancer Consortium (DKTK), partner site Heidelberg, Heidelberg, Germany
| | - Evelyn Ullrich
- Goethe University, Department of Pediatrics, Experimental Immunology and Cell Therapy, Frankfurt am Main, Germany; Frankfurt Cancer Institute (FCI), Goethe University, Frankfurt am Main, Germany; University Cancer Center (UCT) Frankfurt, Frankfurt, Germany; Mildred Scheel Career Center (MSNZ), Hospital of the Goethe University Frankfurt, Frankfurt, Germany; German Cancer Consortium (DKTK), partner site Frankfurt/Mainz and German Cancer Research Center (DKFZ), Heidelberg, Germany.
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15
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Metanat Y, Viktor P, Amajd A, Kaur I, Hamed AM, Abed Al-Abadi NK, Alwan NH, Chaitanya MVNL, Lakshmaiya N, Ghildiyal P, Khalaf OM, Ciongradi CI, Sârbu I. The paths toward non-viral CAR-T cell manufacturing: A comprehensive review of state-of-the-art methods. Life Sci 2024; 348:122683. [PMID: 38702027 DOI: 10.1016/j.lfs.2024.122683] [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: 01/24/2024] [Revised: 04/11/2024] [Accepted: 04/28/2024] [Indexed: 05/06/2024]
Abstract
Although CAR-T cell therapy has emerged as a game-changer in cancer immunotherapy several bottlenecks limit its widespread use as a front-line therapy. Current protocols for the production of CAR-T cells rely mainly on the use of lentiviral/retroviral vectors. Nevertheless, according to the safety concerns around the use of viral vectors, there are several regulatory hurdles to their clinical use. Large-scale production of viral vectors under "Current Good Manufacturing Practice" (cGMP) involves rigorous quality control assessments and regulatory requirements that impose exorbitant costs on suppliers and as a result, lead to a significant increase in the cost of treatment. Pursuing an efficient non-viral method for genetic modification of immune cells is a hot topic in cell-based gene therapy. This study aims to investigate the current state-of-the-art in non-viral methods of CAR-T cell manufacturing. In the first part of this study, after reviewing the advantages and disadvantages of the clinical use of viral vectors, different non-viral vectors and the path of their clinical translation are discussed. These vectors include transposons (sleeping beauty, piggyBac, Tol2, and Tc Buster), programmable nucleases (ZFNs, TALENs, and CRISPR/Cas9), mRNA, plasmids, minicircles, and nanoplasmids. Afterward, various methods for efficient delivery of non-viral vectors into the cells are reviewed.
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Affiliation(s)
- Yekta Metanat
- Faculty of Medicine, Zahedan University of Medical Sciences, Sistan and Baluchestan Province, Iran
| | - Patrik Viktor
- Óbuda University, Karoly Keleti faculty, Tavaszmező u. 15-17, H-1084 Budapest, Hungary
| | - Ayesha Amajd
- Faculty of Transport and Aviation Engineering, Silesian University of Technology, Krasińskiego 8 Street, 40-019 Katowice, Poland
| | - Irwanjot Kaur
- Department of Biotechnology and Genetics, Jain (Deemed-to-be) University, Bangalore, Karnataka, India; Department of Allied Healthcare and Sciences, Vivekananda Global University, Jaipur, Rajasthan-303012, India
| | | | | | | | - M V N L Chaitanya
- School of pharmaceutical sciences, Lovely Professional University, Jalandhar-Delhi G.T. Road, Phagwara, Punjab - 144411, India
| | | | - Pallavi Ghildiyal
- Uttaranchal Institute of Pharmaceutical Sciences, Uttaranchal University, Dehradun, India
| | | | - Carmen Iulia Ciongradi
- 2nd Department of Surgery-Pediatric Surgery and Orthopedics, "Grigore T. Popa" University of Medicine and Pharmacy, 700115 Iași, Romania.
| | - Ioan Sârbu
- 2nd Department of Surgery-Pediatric Surgery and Orthopedics, "Grigore T. Popa" University of Medicine and Pharmacy, 700115 Iași, Romania.
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16
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Rossi M, Breman E. Engineering strategies to safely drive CAR T-cells into the future. Front Immunol 2024; 15:1411393. [PMID: 38962002 PMCID: PMC11219585 DOI: 10.3389/fimmu.2024.1411393] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2024] [Accepted: 05/27/2024] [Indexed: 07/05/2024] Open
Abstract
Chimeric antigen receptor (CAR) T-cell therapy has proven a breakthrough in cancer treatment in the last decade, giving unprecedented results against hematological malignancies. All approved CAR T-cell products, as well as many being assessed in clinical trials, are generated using viral vectors to deploy the exogenous genetic material into T-cells. Viral vectors have a long-standing clinical history in gene delivery, and thus underwent iterations of optimization to improve their efficiency and safety. Nonetheless, their capacity to integrate semi-randomly into the host genome makes them potentially oncogenic via insertional mutagenesis and dysregulation of key cellular genes. Secondary cancers following CAR T-cell administration appear to be a rare adverse event. However several cases documented in the last few years put the spotlight on this issue, which might have been underestimated so far, given the relatively recent deployment of CAR T-cell therapies. Furthermore, the initial successes obtained in hematological malignancies have not yet been replicated in solid tumors. It is now clear that further enhancements are needed to allow CAR T-cells to increase long-term persistence, overcome exhaustion and cope with the immunosuppressive tumor microenvironment. To this aim, a variety of genomic engineering strategies are under evaluation, most relying on CRISPR/Cas9 or other gene editing technologies. These approaches are liable to introduce unintended, irreversible genomic alterations in the product cells. In the first part of this review, we will discuss the viral and non-viral approaches used for the generation of CAR T-cells, whereas in the second part we will focus on gene editing and non-gene editing T-cell engineering, with particular regard to advantages, limitations, and safety. Finally, we will critically analyze the different gene deployment and genomic engineering combinations, delineating strategies with a superior safety profile for the production of next-generation CAR T-cell.
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17
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Bexte T, Ullrich E. Empowering virus-free CAR immune cell therapies. Mol Ther 2024; 32:1609-1611. [PMID: 38795701 PMCID: PMC11184381 DOI: 10.1016/j.ymthe.2024.05.023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2024] [Revised: 05/09/2024] [Accepted: 05/09/2024] [Indexed: 05/28/2024] Open
Affiliation(s)
- Tobias Bexte
- Goethe University, Department of Pediatrics, Experimental Immunology and Cell Therapy, Frankfurt am Main, Germany; Goethe University, Frankfurt Cancer Institute, Frankfurt am Main, Germany; University Cancer Center (UCT), Frankfurt, Germany; Institute for Transfusion Medicine and Immunohematology, German Red Cross Blood Service Baden-Württemberg - Hesse, Frankfurt, Germany
| | - Evelyn Ullrich
- Goethe University, Department of Pediatrics, Experimental Immunology and Cell Therapy, Frankfurt am Main, Germany; Goethe University, Frankfurt Cancer Institute, Frankfurt am Main, Germany; University Cancer Center (UCT), Frankfurt, Germany; German Cancer Consortium (DKTK) Partner Site Frankfurt/Mainz, Frankfurt am Main, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany.
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18
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Skeate JG, Pomeroy EJ, Slipek NJ, Jones BJ, Wick BJ, Chang JW, Lahr WS, Stelljes EM, Patrinostro X, Barnes B, Zarecki T, Krueger JB, Bridge JE, Robbins GM, McCormick MD, Leerar JR, Wenzel KT, Hornberger KM, Walker K, Smedley D, Largaespada DA, Otto N, Webber BR, Moriarity BS. Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies. Mol Ther 2024; 32:1817-1834. [PMID: 38627969 PMCID: PMC11184336 DOI: 10.1016/j.ymthe.2024.04.024] [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: 08/31/2023] [Revised: 03/06/2024] [Accepted: 04/12/2024] [Indexed: 06/09/2024] Open
Abstract
Cellular therapies for the treatment of human diseases, such as chimeric antigen receptor (CAR) T and natural killer (NK) cells have shown remarkable clinical efficacy in treating hematological malignancies; however, current methods mainly utilize viral vectors that are limited by their cargo size capacities, high cost, and long timelines for production of clinical reagent. Delivery of genetic cargo via DNA transposon engineering is a more timely and cost-effective approach, yet has been held back by less efficient integration rates. Here, we report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure-guided and in vitro evolution approaches that achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. Our proof-of-principle TcB-M engineering of CAR-NK and CAR-T cells shows low integrated vector copy number, a safe insertion site profile, robust in vitro function, and improves survival in a Burkitt lymphoma xenograft model in vivo. Overall, TcB-M is a versatile, safe, efficient and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition.
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Affiliation(s)
- Joseph G Skeate
- Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
| | - Emily J Pomeroy
- Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
| | - Nicholas J Slipek
- Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
| | | | - Bryce J Wick
- Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
| | - Jae-Woong Chang
- Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
| | - Walker S Lahr
- Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
| | - Erin M Stelljes
- Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
| | | | | | | | - Joshua B Krueger
- Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
| | - Jacob E Bridge
- Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
| | - Gabrielle M Robbins
- Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
| | - Madeline D McCormick
- Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
| | | | | | | | | | | | - David A Largaespada
- Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
| | - Neil Otto
- Bio-Techne, Minneapolis, MN 55413, USA
| | - Beau R Webber
- Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
| | - Branden S Moriarity
- Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
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19
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Shao W, Yao Y, Yang L, Li X, Ge T, Zheng Y, Zhu Q, Ge S, Gu X, Jia R, Song X, Zhuang A. Novel insights into TCR-T cell therapy in solid neoplasms: optimizing adoptive immunotherapy. Exp Hematol Oncol 2024; 13:37. [PMID: 38570883 PMCID: PMC10988985 DOI: 10.1186/s40164-024-00504-8] [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: 12/08/2023] [Accepted: 03/21/2024] [Indexed: 04/05/2024] Open
Abstract
Adoptive immunotherapy in the T cell landscape exhibits efficacy in cancer treatment. Over the past few decades, genetically modified T cells, particularly chimeric antigen receptor T cells, have enabled remarkable strides in the treatment of hematological malignancies. Besides, extensive exploration of multiple antigens for the treatment of solid tumors has led to clinical interest in the potential of T cells expressing the engineered T cell receptor (TCR). TCR-T cells possess the capacity to recognize intracellular antigen families and maintain the intrinsic properties of TCRs in terms of affinity to target epitopes and signal transduction. Recent research has provided critical insight into their capability and therapeutic targets for multiple refractory solid tumors, but also exposes some challenges for durable efficacy. In this review, we describe the screening and identification of available tumor antigens, and the acquisition and optimization of TCRs for TCR-T cell therapy. Furthermore, we summarize the complete flow from laboratory to clinical applications of TCR-T cells. Last, we emerge future prospects for improving therapeutic efficacy in cancer world with combination therapies or TCR-T derived products. In conclusion, this review depicts our current understanding of TCR-T cell therapy in solid neoplasms, and provides new perspectives for expanding its clinical applications and improving therapeutic efficacy.
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Affiliation(s)
- Weihuan Shao
- Department of Ophthalmology, Ninth People's Hospital, Shanghai JiaoTong University School of Medicine, 639 Zhi Zao Ju Road, Shanghai Ninth People's Hospital, Shanghai, 200011, People's Republic of China
- Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, 200011, People's Republic of China
| | - Yiran Yao
- Department of Ophthalmology, Ninth People's Hospital, Shanghai JiaoTong University School of Medicine, 639 Zhi Zao Ju Road, Shanghai Ninth People's Hospital, Shanghai, 200011, People's Republic of China
- Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, 200011, People's Republic of China
| | - Ludi Yang
- Department of Ophthalmology, Ninth People's Hospital, Shanghai JiaoTong University School of Medicine, 639 Zhi Zao Ju Road, Shanghai Ninth People's Hospital, Shanghai, 200011, People's Republic of China
- Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, 200011, People's Republic of China
| | - Xiaoran Li
- Department of Ophthalmology, Ninth People's Hospital, Shanghai JiaoTong University School of Medicine, 639 Zhi Zao Ju Road, Shanghai Ninth People's Hospital, Shanghai, 200011, People's Republic of China
- Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, 200011, People's Republic of China
| | - Tongxin Ge
- Department of Ophthalmology, Ninth People's Hospital, Shanghai JiaoTong University School of Medicine, 639 Zhi Zao Ju Road, Shanghai Ninth People's Hospital, Shanghai, 200011, People's Republic of China
- Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, 200011, People's Republic of China
| | - Yue Zheng
- Department of Ophthalmology, Ninth People's Hospital, Shanghai JiaoTong University School of Medicine, 639 Zhi Zao Ju Road, Shanghai Ninth People's Hospital, Shanghai, 200011, People's Republic of China
- Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, 200011, People's Republic of China
| | - Qiuyi Zhu
- Department of Ophthalmology, Ninth People's Hospital, Shanghai JiaoTong University School of Medicine, 639 Zhi Zao Ju Road, Shanghai Ninth People's Hospital, Shanghai, 200011, People's Republic of China
- Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, 200011, People's Republic of China
| | - Shengfang Ge
- Department of Ophthalmology, Ninth People's Hospital, Shanghai JiaoTong University School of Medicine, 639 Zhi Zao Ju Road, Shanghai Ninth People's Hospital, Shanghai, 200011, People's Republic of China
- Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, 200011, People's Republic of China
| | - Xiang Gu
- Department of Ophthalmology, Ninth People's Hospital, Shanghai JiaoTong University School of Medicine, 639 Zhi Zao Ju Road, Shanghai Ninth People's Hospital, Shanghai, 200011, People's Republic of China
- Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, 200011, People's Republic of China
| | - Renbing Jia
- Department of Ophthalmology, Ninth People's Hospital, Shanghai JiaoTong University School of Medicine, 639 Zhi Zao Ju Road, Shanghai Ninth People's Hospital, Shanghai, 200011, People's Republic of China.
- Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, 200011, People's Republic of China.
| | - Xin Song
- Department of Ophthalmology, Ninth People's Hospital, Shanghai JiaoTong University School of Medicine, 639 Zhi Zao Ju Road, Shanghai Ninth People's Hospital, Shanghai, 200011, People's Republic of China.
- Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, 200011, People's Republic of China.
| | - Ai Zhuang
- Department of Ophthalmology, Ninth People's Hospital, Shanghai JiaoTong University School of Medicine, 639 Zhi Zao Ju Road, Shanghai Ninth People's Hospital, Shanghai, 200011, People's Republic of China.
- Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, 200011, People's Republic of China.
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20
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Tian J, Tong D, Li Z, Wang E, Yu Y, Lv H, Hu Z, Sun F, Wang G, He M, Xia T. Mage transposon: a novel gene delivery system for mammalian cells. Nucleic Acids Res 2024; 52:2724-2739. [PMID: 38300794 PMCID: PMC10954464 DOI: 10.1093/nar/gkae048] [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: 09/14/2023] [Revised: 01/10/2024] [Accepted: 01/22/2024] [Indexed: 02/03/2024] Open
Abstract
Transposons, as non-viral integration vectors, provide a secure and efficient method for stable gene delivery. In this study, we have discovered Mage (MG), a novel member of the piggyBac(PB) family, which exhibits strong transposability in a variety of mammalian cells and primary T cells. The wild-type MG showed a weaker insertion preference for near genes, transcription start sites (TSS), CpG islands, and DNaseI hypersensitive sites in comparison to PB, approaching the random insertion pattern. Utilizing in silico virtual screening and feasible combinatorial mutagenesis in vitro, we effectively produced the hyperactive MG transposase (hyMagease). This variant boasts a transposition rate 60% greater than its native counterpart without significantly altering its insertion pattern. Furthermore, we applied the hyMagease to efficiently deliver chimeric antigen receptor (CAR) into T cells, leading to stable high-level expression and inducing significant anti-tumor effects both in vitro and in xenograft mice models. These findings provide a compelling tool for gene transfer research, emphasizing its potential and prospects in the domains of genetic engineering and gene therapy.
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Affiliation(s)
- Jinghan Tian
- Institute of Pathology, Department of Pathology, School of Basic Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China
| | - Doudou Tong
- Institute of Pathology, Department of Pathology, School of Basic Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China
| | | | - Erqiang Wang
- Institute of Pathology, Department of Pathology, School of Basic Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China
| | - Yifei Yu
- Elongevity Inc, Wuhan, Hubei 430000, China
| | - Hangya Lv
- Elongevity Inc, Wuhan, Hubei 430000, China
| | - Zhendan Hu
- Elongevity Inc, Wuhan, Hubei 430000, China
| | - Fang Sun
- Elongevity Inc, Wuhan, Hubei 430000, China
| | - Guoping Wang
- Institute of Pathology, Department of Pathology, School of Basic Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China
| | - Min He
- Elongevity Inc, Wuhan, Hubei 430000, China
| | - Tian Xia
- Institute of Pathology, Department of Pathology, School of Basic Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China
- School of Artificial Intelligence and Automation, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China
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21
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Marie C, Scherman D. Antibiotic-Free Gene Vectors: A 25-Year Journey to Clinical Trials. Genes (Basel) 2024; 15:261. [PMID: 38540320 PMCID: PMC10970329 DOI: 10.3390/genes15030261] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2024] [Revised: 02/07/2024] [Accepted: 02/15/2024] [Indexed: 06/15/2024] Open
Abstract
Until very recently, the major use, for gene therapy, specifically of linear or circular DNA, such as plasmids, was as ancillary products for viral vectors' production or as a genetic template for mRNA production. Thanks to targeted and more efficient physical or chemical delivery techniques and to the refinement of their structure, non-viral plasmid DNA are now under intensive consideration as pharmaceutical drugs. Plasmids traditionally carry an antibiotic resistance gene for providing the selection pressure necessary for maintenance in a bacterial host. Nearly a dozen different antibiotic-free gene vectors have now been developed and are currently assessed in preclinical assays and phase I/II clinical trials. Their reduced size leads to increased transfection efficiency and prolonged transgene expression. In addition, associating non-viral gene vectors and DNA transposons, which mediate transgene integration into the host genome, circumvents plasmid dilution in dividing eukaryotic cells which generate a loss of the therapeutic gene. Combining these novel molecular tools allowed a significantly higher yield of genetically engineered T and Natural Killer cells for adoptive immunotherapies due to a reduced cytotoxicity and increased transposition rate. This review describes the main progresses accomplished for safer, more efficient and cost-effective gene and cell therapies using non-viral approaches and antibiotic-free gene vectors.
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Affiliation(s)
- Corinne Marie
- Université Paris Cité, CNRS, Inserm, UTCBS, 75006 Paris, France;
- Chimie ParisTech, Université PSL, 75005 Paris, France
| | - Daniel Scherman
- Université Paris Cité, CNRS, Inserm, UTCBS, 75006 Paris, France;
- Fondation Maladies Rares, 75014 Paris, France
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22
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Ayala Ceja M, Khericha M, Harris CM, Puig-Saus C, Chen YY. CAR-T cell manufacturing: Major process parameters and next-generation strategies. J Exp Med 2024; 221:e20230903. [PMID: 38226974 PMCID: PMC10791545 DOI: 10.1084/jem.20230903] [Citation(s) in RCA: 57] [Impact Index Per Article: 57.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2023] [Revised: 12/02/2023] [Accepted: 12/14/2023] [Indexed: 01/17/2024] Open
Abstract
Chimeric antigen receptor (CAR)-T cell therapies have demonstrated strong curative potential and become a critical component in the array of B-cell malignancy treatments. Successful deployment of CAR-T cell therapies to treat hematologic and solid cancers, as well as other indications such as autoimmune diseases, is dependent on effective CAR-T cell manufacturing that impacts not only product safety and efficacy but also overall accessibility to patients in need. In this review, we discuss the major process parameters of autologous CAR-T cell manufacturing, as well as regulatory considerations and ongoing developments that will enable the next generation of CAR-T cell therapies.
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Affiliation(s)
- Melanie Ayala Ceja
- Department of Microbiology, Immunology, and Molecular Genetics, University of California−Los Angeles, Los Angeles, CA, USA
| | - Mobina Khericha
- Department of Microbiology, Immunology, and Molecular Genetics, University of California−Los Angeles, Los Angeles, CA, USA
| | - Caitlin M. Harris
- Department of Microbiology, Immunology, and Molecular Genetics, University of California−Los Angeles, Los Angeles, CA, USA
| | - Cristina Puig-Saus
- Department of Medicine, University of California−Los Angeles, Los Angeles, CA, USA
- Jonsson Comprehensive Cancer Center, University of California−Los Angeles, Los Angeles, CA, USA
- Parker Institute for Cancer Immunotherapy Center at University of California−Los Angeles, Los Angeles, CA, USA
| | - Yvonne Y. Chen
- Department of Microbiology, Immunology, and Molecular Genetics, University of California−Los Angeles, Los Angeles, CA, USA
- Jonsson Comprehensive Cancer Center, University of California−Los Angeles, Los Angeles, CA, USA
- Parker Institute for Cancer Immunotherapy Center at University of California−Los Angeles, Los Angeles, CA, USA
- Department of Chemical and Biomolecular Engineering, University of California−Los Angeles, Los Angeles, CA, USA
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23
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Colina AS, Shah V, Shah RK, Kozlik T, Dash RK, Terhune S, Zamora AE. Current advances in experimental and computational approaches to enhance CAR T cell manufacturing protocols and improve clinical efficacy. FRONTIERS IN MOLECULAR MEDICINE 2024; 4:1310002. [PMID: 39086435 PMCID: PMC11285593 DOI: 10.3389/fmmed.2024.1310002] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/09/2023] [Accepted: 01/08/2024] [Indexed: 08/02/2024]
Abstract
Since the FDA's approval of chimeric antigen receptor (CAR) T cells in 2017, significant improvements have been made in the design of chimeric antigen receptor constructs and in the manufacturing of CAR T cell therapies resulting in increased in vivo CAR T cell persistence and improved clinical outcome in certain hematological malignancies. Despite the remarkable clinical response seen in some patients, challenges remain in achieving durable long-term tumor-free survival, reducing therapy associated malignancies and toxicities, and expanding on the types of cancers that can be treated with this therapeutic modality. Careful analysis of the biological factors demarcating efficacious from suboptimal CAR T cell responses will be of paramount importance to address these shortcomings. With the ever-expanding toolbox of experimental approaches, single-cell technologies, and computational resources, there is renowned interest in discovering new ways to streamline the development and validation of new CAR T cell products. Better and more accurate prognostic and predictive models can be developed to help guide and inform clinical decision making by incorporating these approaches into translational and clinical workflows. In this review, we provide a brief overview of recent advancements in CAR T cell manufacturing and describe the strategies used to selectively expand specific phenotypic subsets. Additionally, we review experimental approaches to assess CAR T cell functionality and summarize current in silico methods which have the potential to improve CAR T cell manufacturing and predict clinical outcomes.
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Affiliation(s)
- Alfredo S. Colina
- Department of Microbiology & Immunology, Medical College of Wisconsin, Milwaukee, WI, United States
| | - Viren Shah
- Department of Biomedical Engineering, Medical College of Wisconsin and Marquette University, Milwaukee, WI, United States
| | - Ravi K. Shah
- Department of Medicine, Medical College of Wisconsin, Milwaukee, WI, United States
| | - Tanya Kozlik
- Department of Microbiology & Immunology, Medical College of Wisconsin, Milwaukee, WI, United States
| | - Ranjan K. Dash
- Department of Biomedical Engineering, Medical College of Wisconsin and Marquette University, Milwaukee, WI, United States
| | - Scott Terhune
- Department of Microbiology & Immunology, Medical College of Wisconsin, Milwaukee, WI, United States
- Department of Biomedical Engineering, Medical College of Wisconsin and Marquette University, Milwaukee, WI, United States
| | - Anthony E. Zamora
- Department of Microbiology & Immunology, Medical College of Wisconsin, Milwaukee, WI, United States
- Department of Medicine, Medical College of Wisconsin, Milwaukee, WI, United States
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24
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Guo J, Zhang W, Chen X, Yen A, Chen L, Shively CA, Li D, Wang T, Dougherty JD, Mitra RD. Pycallingcards: an integrated environment for visualizing, analyzing, and interpreting Calling Cards data. Bioinformatics 2024; 40:btae070. [PMID: 38323623 PMCID: PMC10881108 DOI: 10.1093/bioinformatics/btae070] [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: 07/14/2023] [Revised: 12/25/2023] [Accepted: 02/05/2024] [Indexed: 02/08/2024] Open
Abstract
MOTIVATION Unraveling the transcriptional programs that control how cells divide, differentiate, and respond to their environments requires a precise understanding of transcription factors' (TFs) DNA-binding activities. Calling cards (CC) technology uses transposons to capture transient TF binding events at one instant in time and then read them out at a later time. This methodology can also be used to simultaneously measure TF binding and mRNA expression from single-cell CC and to record and integrate TF binding events across time in any cell type of interest without the need for purification. Despite these advantages, there has been a lack of dedicated bioinformatics tools for the detailed analysis of CC data. RESULTS We introduce Pycallingcards, a comprehensive Python module specifically designed for the analysis of single-cell and bulk CC data across multiple species. Pycallingcards introduces two innovative peak callers, CCcaller and MACCs, enhancing the accuracy and speed of pinpointing TF binding sites from CC data. Pycallingcards offers a fully integrated environment for data visualization, motif finding, and comparative analysis with RNA-seq and ChIP-seq datasets. To illustrate its practical application, we have reanalyzed previously published mouse cortex and glioblastoma datasets. This analysis revealed novel cell-type-specific binding sites and potential sex-linked TF regulators, furthering our understanding of TF binding and gene expression relationships. Thus, Pycallingcards, with its user-friendly design and seamless interface with the Python data science ecosystem, stands as a critical tool for advancing the analysis of TF functions via CC data. AVAILABILITY AND IMPLEMENTATION Pycallingcards can be accessed on the GitHub repository: https://github.com/The-Mitra-Lab/pycallingcards.
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Affiliation(s)
- Juanru Guo
- Department of Genetics, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
- Edison Family Center for Genome Sciences and Systems Biology, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
| | - Wenjin Zhang
- Department of Genetics, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
- Edison Family Center for Genome Sciences and Systems Biology, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
| | - Xuhua Chen
- Department of Genetics, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
- Edison Family Center for Genome Sciences and Systems Biology, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
| | - Allen Yen
- Department of Genetics, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
- Department of Psychiatry, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
| | - Lucy Chen
- Department of Genetics, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
- Edison Family Center for Genome Sciences and Systems Biology, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
| | - Christian A Shively
- Department of Genetics, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
- Edison Family Center for Genome Sciences and Systems Biology, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
| | - Daofeng Li
- Department of Genetics, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
- Edison Family Center for Genome Sciences and Systems Biology, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
| | - Ting Wang
- Department of Genetics, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
- Edison Family Center for Genome Sciences and Systems Biology, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
- McDonnell Genome Institute, , Washington University in St. Louis School of Medicine, Saint Louis, MO, 63110, United States
| | - Joseph D Dougherty
- Department of Genetics, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
- Department of Psychiatry, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
- Intellectual and Developmental Disabilities Research Center, Washington University School of Medicine, Saint Louis, MO 63108, United States
| | - Robi D Mitra
- Department of Genetics, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
- Edison Family Center for Genome Sciences and Systems Biology, Washington University in St. Louis School of Medicine, Saint Louis, MO 63110, United States
- McDonnell Genome Institute, , Washington University in St. Louis School of Medicine, Saint Louis, MO, 63110, United States
- Intellectual and Developmental Disabilities Research Center, Washington University School of Medicine, Saint Louis, MO 63108, United States
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25
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Bexte T, Reindl LM, Ullrich E. Nonviral technologies can pave the way for CAR-NK cell therapy. J Leukoc Biol 2023; 114:475-486. [PMID: 37403203 DOI: 10.1093/jleuko/qiad074] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2022] [Revised: 05/25/2023] [Accepted: 06/22/2023] [Indexed: 07/06/2023] Open
Abstract
Natural killer cells are a promising platform for cancer immunotherapy. Natural killer cells have high intrinsic killing capability, and the insertion of a chimeric antigen receptor can further enhance their antitumor potential. In first-in-human trials, chimeric antigen receptor-natural killer cells demonstrated strong clinical activity without therapy-induced side effects. The applicability of natural killer cells as an "off-the-shelf" product makes them highly attractive for gene-engineered cell therapies. Traditionally, viral transduction has been used for gene editing; however, the use of viral vectors remains a safety concern and is associated with high costs and regulatory requirements. Here, we review the current landscape of nonviral approaches for chimeric antigen receptor-natural killer cell generation. This includes transfection of vector particles and electroporation of mRNA and DNA vectors, resulting in transient modification and chimeric antigen receptor expression. In addition, using nonviral transposon technologies, natural killer cells can be stably modified ensuring long-lasting chimeric antigen receptor expression. Finally, we discuss CRISPR/Cas9 tools to edit key genes for natural killer cell functionality.
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Affiliation(s)
- Tobias Bexte
- Goethe University Frankfurt, Department of Pediatrics, Experimental Immunology & Cell Therapy, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany
- Frankfurt Cancer Institute, Goethe University, Paul-Ehrlich-Straße 42-44, 60596 Frankfurt am Main, Germany
- University Cancer Center (UCT), Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany
- Mildred Scheel Career Center (MSNZ), Hospital of the Goethe University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany
| | - Lisa Marie Reindl
- Goethe University Frankfurt, Department of Pediatrics, Experimental Immunology & Cell Therapy, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany
- Frankfurt Cancer Institute, Goethe University, Paul-Ehrlich-Straße 42-44, 60596 Frankfurt am Main, Germany
| | - Evelyn Ullrich
- Goethe University Frankfurt, Department of Pediatrics, Experimental Immunology & Cell Therapy, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany
- Frankfurt Cancer Institute, Goethe University, Paul-Ehrlich-Straße 42-44, 60596 Frankfurt am Main, Germany
- University Cancer Center (UCT), Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany
- Mildred Scheel Career Center (MSNZ), Hospital of the Goethe University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany
- German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ) partner site Frankfurt/Mainz; Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany
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26
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Kovač A, Miskey C, Ivics Z. Sleeping Beauty Transposon Insertions into Nucleolar DNA by an Engineered Transposase Localized in the Nucleolus. Int J Mol Sci 2023; 24:14978. [PMID: 37834425 PMCID: PMC10573994 DOI: 10.3390/ijms241914978] [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: 06/06/2023] [Revised: 09/22/2023] [Accepted: 09/28/2023] [Indexed: 10/15/2023] Open
Abstract
Transposons are nature's gene delivery vehicles that can be harnessed for experimental and therapeutic purposes. The Sleeping Beauty (SB) transposon shows efficient transposition and long-term transgene expression in human cells, and is currently under clinical development for gene therapy. SB transposition occurs into the human genome in a random manner, which carries a risk of potential genotoxic effects associated with transposon integration. Here, we evaluated an experimental strategy to manipulate SB's target site distribution by preferentially compartmentalizing the SB transposase to the nucleolus, which contains repetitive ribosomal RNA (rRNA) genes. We generated a fusion protein composed of the nucleolar protein nucleophosmin (B23) and the SB100X transposase, which was found to retain almost full transposition activity as compared to unfused transposase and to be predominantly localized to nucleoli in transfected human cells. Analysis of transposon integration sites generated by B23-SB100X revealed a significant enrichment into the p-arms of chromosomes containing nucleolus organizing regions (NORs), with preferential integration into the p13 and p11.2 cytobands directly neighboring the NORs. This bias in the integration pattern was accompanied by an enrichment of insertions into nucleolus-associated chromatin domains (NADs) at the periphery of nucleolar DNA and into lamina-associated domains (LADs). Finally, sub-nuclear targeting of the transposase resulted in preferential integration into chromosomal domains associated with the Upstream Binding Transcription Factor (UBTF) that plays a critical role in the transcription of 47S rDNA gene repeats of the NORs by RNA Pol I. Future modifications of this technology may allow the development of methods for specific gene insertion for precision genetic engineering.
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Affiliation(s)
| | | | - Zoltán Ivics
- Transposition and Genome Engineering, Research Centre of the Division of Hematology, Gene and Cell Therapy, Paul Ehrlich Institute, Paul Ehrlich Str. 51–59, D-63225 Langen, Germany; (A.K.); (C.M.)
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27
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Kim J, Park M, Baek G, Kim JI, Kwon E, Kang BC, Kim JI, Kang HJ. Tagmentation-based analysis reveals the clonal behavior of CAR-T cells in association with lentivector integration sites. Mol Ther Oncolytics 2023; 30:1-13. [PMID: 37360944 PMCID: PMC10285042 DOI: 10.1016/j.omto.2023.05.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2022] [Accepted: 05/12/2023] [Indexed: 06/28/2023] Open
Abstract
Integration site (IS) analysis is essential in ensuring safety and efficacy of gene therapies when integrating vectors are used. Although clinical trials of gene therapy are rapidly increasing, current methods have limited use in clinical settings because of their lengthy protocols. Here, we describe a novel genome-wide IS analysis method, "detection of the integration sites in a time-efficient manner, quantifying clonal size using tagmentation sequencing" (DIStinct-seq). In DIStinct-seq, a bead-linked Tn5 transposome is used, allowing the sequencing library to be prepared within a single day. We validated the quantification performance of DIStinct-seq for measuring clonal size with clones of known IS. Using ex vivo chimeric antigen receptor (CAR)-T cells, we revealed the characteristics of lentiviral IS. We then applied it to CAR-T cells collected at various times from tumor-engrafted mice, detecting 1,034-6,233 IS. Notably, we observed that the highly expanded clones had a higher integration frequency in the transcription units and vice versa in genomic safe harbors (GSH). Also, in GSH, persistent clones had more frequent IS. Together with these findings, the new IS analysis method will help to improve the safety and efficacy of gene therapies.
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Affiliation(s)
- Jaeryuk Kim
- Genomic Medicine Institute, Medical Research Center, Seoul National University, Seoul, Republic of Korea
- Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, Republic of Korea
- Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Miyoung Park
- Seoul National University Cancer Research Institute, Seoul, Republic of Korea
- Department of Pediatrics, Seoul National University College of Medicine, Seoul, Republic of Korea
- Seoul National University Children’s Hospital, Seoul, Republic of Korea
| | - Gyungwon Baek
- Seoul National University Cancer Research Institute, Seoul, Republic of Korea
- Department of Pediatrics, Seoul National University College of Medicine, Seoul, Republic of Korea
- Seoul National University Children’s Hospital, Seoul, Republic of Korea
| | - Joo-Il Kim
- Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea
- Department of Experimental Animal Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea
| | - Euna Kwon
- Department of Experimental Animal Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea
| | - Byeong-Cheol Kang
- Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea
- Department of Experimental Animal Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea
- Biomedical Center for Animal Resource and Development; Seoul National University College of Medicine, Seoul, Republic of Korea
- Designed Animal Resource Center, Institute of GreenBio Science Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea
| | - Jong-Il Kim
- Genomic Medicine Institute, Medical Research Center, Seoul National University, Seoul, Republic of Korea
- Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, Republic of Korea
- Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea
- Seoul National University Cancer Research Institute, Seoul, Republic of Korea
| | - Hyoung Jin Kang
- Seoul National University Cancer Research Institute, Seoul, Republic of Korea
- Department of Pediatrics, Seoul National University College of Medicine, Seoul, Republic of Korea
- Seoul National University Children’s Hospital, Seoul, Republic of Korea
- Wide River Institute of Immunology, Hongcheon, Republic of Korea
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28
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Calviño C, Ceballos C, Alfonso A, Jauregui P, Calleja-Cervantes ME, San Martin-Uriz P, Rodriguez-Marquez P, Martin-Mallo A, Iglesias E, Abizanda G, Rodriguez-Diaz S, Martinez-Turrillas R, Illarramendi J, Viguria MC, Redondo M, Rifon J, Villar S, Lasarte JJ, Inoges S, Lopez-Diaz de Cerio A, Hernaez M, Prosper F, Rodriguez-Madoz JR. Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia. Front Immunol 2023; 14:1270843. [PMID: 37795087 PMCID: PMC10546312 DOI: 10.3389/fimmu.2023.1270843] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2023] [Accepted: 08/28/2023] [Indexed: 10/06/2023] Open
Abstract
Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased in vivo antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using Sleeping Beauty transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes (HLA-IKO/TCRKO CAR-T cells) for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile to non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, the production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-IKO/TCRKO CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allows the generation of HLA-IKO/TCRKO CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment.
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MESH Headings
- Animals
- Humans
- Aged
- Receptors, Antigen, T-Cell/genetics
- Receptors, Antigen, T-Cell/metabolism
- T-Lymphocytes/metabolism
- Leukemia, Myeloid, Acute/genetics
- Leukemia, Myeloid, Acute/therapy
- Leukemia, Myeloid, Acute/metabolism
- Immunotherapy, Adoptive/methods
- Disease Models, Animal
- Hematopoietic Stem Cell Transplantation
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Affiliation(s)
- Cristina Calviño
- Hematology and Cell Therapy Department, Clinica Universidad de Navarra, IdiSNA, Pamplona, Spain
| | - Candela Ceballos
- Hematology Department, Hospital Universitario de Navarra, IdiSNA, Pamplona, Spain
| | - Ana Alfonso
- Hematology and Cell Therapy Department, Clinica Universidad de Navarra, IdiSNA, Pamplona, Spain
- Centro de Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, Spain
| | - Patricia Jauregui
- Hematology and Cell Therapy Department, Clinica Universidad de Navarra, IdiSNA, Pamplona, Spain
| | - Maria E. Calleja-Cervantes
- Hemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, Spain
- Computational Biology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, Spain
| | | | - Paula Rodriguez-Marquez
- Centro de Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, Spain
- Hemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, Spain
| | - Angel Martin-Mallo
- Hemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, Spain
| | - Elena Iglesias
- Hemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, Spain
| | - Gloria Abizanda
- Hemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, Spain
| | | | - Rebeca Martinez-Turrillas
- Centro de Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, Spain
- Hemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, Spain
| | - Jorge Illarramendi
- Hematology Department, Hospital Universitario de Navarra, IdiSNA, Pamplona, Spain
| | - Maria C. Viguria
- Hematology Department, Hospital Universitario de Navarra, IdiSNA, Pamplona, Spain
| | - Margarita Redondo
- Hematology Department, Hospital Universitario de Navarra, IdiSNA, Pamplona, Spain
| | - Jose Rifon
- Hematology and Cell Therapy Department, Clinica Universidad de Navarra, IdiSNA, Pamplona, Spain
- Centro de Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, Spain
| | - Sara Villar
- Hematology and Cell Therapy Department, Clinica Universidad de Navarra, IdiSNA, Pamplona, Spain
| | - Juan J. Lasarte
- Immunology and Immunotherapy Program, Cima Universidad de Navarra, IdiSNA, Pamplona, Spain
- Cancer Center Clinica Universidad de Navarra (CCUN), Pamplona, Spain
| | - Susana Inoges
- Hematology and Cell Therapy Department, Clinica Universidad de Navarra, IdiSNA, Pamplona, Spain
- Centro de Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, Spain
- Cancer Center Clinica Universidad de Navarra (CCUN), Pamplona, Spain
- Immunology and Immunotherapy Department, Clinica Universidad de Navarra, Pamplona, Spain
| | - Ascension Lopez-Diaz de Cerio
- Hematology and Cell Therapy Department, Clinica Universidad de Navarra, IdiSNA, Pamplona, Spain
- Centro de Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, Spain
- Cancer Center Clinica Universidad de Navarra (CCUN), Pamplona, Spain
- Immunology and Immunotherapy Department, Clinica Universidad de Navarra, Pamplona, Spain
| | - Mikel Hernaez
- Centro de Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, Spain
- Computational Biology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, Spain
- Cancer Center Clinica Universidad de Navarra (CCUN), Pamplona, Spain
- Data Science and Artificial Intelligence Institute (DATAI), Universidad de Navarra, Pamplona, Spain
| | - Felipe Prosper
- Hematology and Cell Therapy Department, Clinica Universidad de Navarra, IdiSNA, Pamplona, Spain
- Centro de Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, Spain
- Hemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, Spain
- Cancer Center Clinica Universidad de Navarra (CCUN), Pamplona, Spain
| | - Juan R. Rodriguez-Madoz
- Centro de Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, Spain
- Hemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, Spain
- Cancer Center Clinica Universidad de Navarra (CCUN), Pamplona, Spain
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29
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Kasahara Y, Tamamura S, Hiyama G, Takagi M, Nakamichi K, Doi Y, Semba K, Watanabe S, Ishikawa K. Tyrosine Kinase Inhibitor Profiling Using Multiple Forskolin-Responsive Reporter Cells. Int J Mol Sci 2023; 24:13863. [PMID: 37762164 PMCID: PMC10530646 DOI: 10.3390/ijms241813863] [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: 07/14/2023] [Revised: 08/29/2023] [Accepted: 09/07/2023] [Indexed: 09/29/2023] Open
Abstract
We have developed a highly sensitive promoter trap vector system using transposons to generate reporter cells with high efficiency. Using an EGFP/luciferase reporter cell clone responsive to forskolin, which is thought to activate adenylate cyclase, isolated from human chronic myelogenous leukemia cell line K562, we found several compounds unexpectedly caused reporter responses. These included tyrosine kinase inhibitors such as dasatinib and cerdulatinib, which were seemingly unrelated to the forskolin-reactive pathway. To investigate whether any other clones of forskolin-responsive cells would show the same response, nine additional forskolin-responsive clones, each with a unique integration site, were generated and quantitatively evaluated by luciferase assay. The results showed that each clone represented different response patterns to the reactive compounds. Also, it became clear that each of the reactive compounds could be profiled as a unique pattern by the 10 reporter clones. When other TKIs, mainly bcr-abl inhibitors, were evaluated using a more focused set of five reporter clones, they also showed unique profiling. Among them, dasatinib and bosutinib, and imatinib and bafetinib showed homologous profiling. The tyrosine kinase inhibitors mentioned above are approved as anticancer agents, and the system could be used for similarity evaluation, efficacy prediction, etc., in the development of new anticancer agents.
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Affiliation(s)
- Yamato Kasahara
- Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan; (Y.K.); (K.N.); (Y.D.); (K.S.)
| | - Sakura Tamamura
- Japan Biological Informatics Consortium (JBiC), 2-45 Aomi, Koto-ku, Tokyo 135-8073, Japan;
| | - Gen Hiyama
- Translational Research Center, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan; (G.H.); (M.T.); (S.W.)
| | - Motoki Takagi
- Translational Research Center, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan; (G.H.); (M.T.); (S.W.)
| | - Kazuya Nakamichi
- Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan; (Y.K.); (K.N.); (Y.D.); (K.S.)
| | - Yuta Doi
- Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan; (Y.K.); (K.N.); (Y.D.); (K.S.)
| | - Kentaro Semba
- Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan; (Y.K.); (K.N.); (Y.D.); (K.S.)
- Translational Research Center, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan; (G.H.); (M.T.); (S.W.)
| | - Shinya Watanabe
- Translational Research Center, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan; (G.H.); (M.T.); (S.W.)
| | - Kosuke Ishikawa
- Japan Biological Informatics Consortium (JBiC), 2-45 Aomi, Koto-ku, Tokyo 135-8073, Japan;
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30
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Schulz TF, Freise A, Stein SC. Kaposi sarcoma-associated herpesvirus latency-associated nuclear antigen: more than a key mediator of viral persistence. Curr Opin Virol 2023; 61:101336. [PMID: 37331160 DOI: 10.1016/j.coviro.2023.101336] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2022] [Revised: 05/03/2023] [Accepted: 05/22/2023] [Indexed: 06/20/2023]
Abstract
Kaposi sarcoma-associated herpesvirus (KSHV), or human herpesvirus-8, is an oncogenic herpesvirus. Its latency-associated nuclear antigen (LANA) is essential for the persistence of KSHV in latently infected cells. LANA mediates replication of the latent viral genome during the S phase of a dividing cell and partitions episomes to daughter cells by attaching them to mitotic chromosomes. It also mediates the establishment of latency in newly infected cells through epigenetic mechanisms and suppresses the activation of the productive replication cycle. Furthermore, LANA promotes the proliferation of infected cell by acting as a transcriptional regulator and by modulating the cellular proteome through the recruitment of several cellular ubiquitin ligases. Finally, LANA interferes with the innate and adaptive immune system to facilitate the immune escape of infected cells.
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Affiliation(s)
- Thomas F Schulz
- Institute of Virology, Hannover Medical School, Germany; Cluster of Excellence 2155 RESIST, Germany; German Center for Infection Research, Hannover-Braunschweig Site, Germany.
| | - Anika Freise
- Institute of Virology, Hannover Medical School, Germany
| | - Saskia C Stein
- Institute of Virology, Hannover Medical School, Germany; Cluster of Excellence 2155 RESIST, Germany
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31
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Harmening N, Johnen S, Izsvák Z, Ivics Z, Kropp M, Bascuas T, Walter P, Kreis A, Pajic B, Thumann G. Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells. Biomolecules 2023; 13:biom13040658. [PMID: 37189405 DOI: 10.3390/biom13040658] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2023] [Revised: 03/31/2023] [Accepted: 04/04/2023] [Indexed: 05/17/2023] Open
Abstract
Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal neovascularization (CNV), which leads to retinal pigment epithelial (RPE) cell and photoreceptor degeneration and blindness if untreated. Since blood vessel growth is mediated by endothelial cell growth factors, including vascular endothelial growth factor (VEGF), treatment consists of repeated, often monthly, intravitreal injections of anti-angiogenic biopharmaceuticals. Frequent injections are costly and present logistic difficulties; therefore, our laboratories are developing a cell-based gene therapy based on autologous RPE cells transfected ex vivo with the pigment epithelium derived factor (PEDF), which is the most potent natural antagonist of VEGF. Gene delivery and long-term expression of the transgene are enabled by the use of the non-viral Sleeping Beauty (SB100X) transposon system that is introduced into the cells by electroporation. The transposase may have a cytotoxic effect and a low risk of remobilization of the transposon if supplied in the form of DNA. Here, we investigated the use of the SB100X transposase delivered as mRNA and showed that ARPE-19 cells as well as primary human RPE cells were successfully transfected with the Venus or the PEDF gene, followed by stable transgene expression. In human RPE cells, secretion of recombinant PEDF could be detected in cell culture up to one year. Non-viral ex vivo transfection using SB100X-mRNA in combination with electroporation increases the biosafety of our gene therapeutic approach to treat nvAMD while ensuring high transfection efficiency and long-term transgene expression in RPE cells.
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Affiliation(s)
- Nina Harmening
- Experimental Ophthalmology, University of Geneva, 1205 Geneva, Switzerland
- Department of Ophthalmology, University Hospitals of Geneva, 1205 Geneva, Switzerland
| | - Sandra Johnen
- Department of Ophthalmology, University Hospital RWTH Aachen, 52074 Aachen, Germany
| | - Zsuzsanna Izsvák
- Max Delbrück Center for Molecular Medicine in the Helmholtz Association, 13125 Berlin, Germany
| | - Zoltan Ivics
- Division of Medical Biotechnology, Paul-Ehrlich-Institute, 63225 Langen, Germany
| | - Martina Kropp
- Experimental Ophthalmology, University of Geneva, 1205 Geneva, Switzerland
- Department of Ophthalmology, University Hospitals of Geneva, 1205 Geneva, Switzerland
| | - Thais Bascuas
- Experimental Ophthalmology, University of Geneva, 1205 Geneva, Switzerland
- Department of Ophthalmology, University Hospitals of Geneva, 1205 Geneva, Switzerland
| | - Peter Walter
- Department of Ophthalmology, University Hospital RWTH Aachen, 52074 Aachen, Germany
| | - Andreas Kreis
- Experimental Ophthalmology, University of Geneva, 1205 Geneva, Switzerland
- Department of Ophthalmology, University Hospitals of Geneva, 1205 Geneva, Switzerland
| | - Bojan Pajic
- Experimental Ophthalmology, University of Geneva, 1205 Geneva, Switzerland
- Department of Ophthalmology, University Hospitals of Geneva, 1205 Geneva, Switzerland
- Eye Clinic ORASIS, Swiss Eye Research Foundation, 5734 Reinach, Switzerland
- Faculty of Sciences, Department of Physics, University of Novi Sad, Trg Dositeja Obradovica 4, 21000 Novi Sad, Serbia
- Faculty of Medicine of the Military Medical Academy, University of Defense, 11000 Belgrade, Serbia
| | - Gabriele Thumann
- Experimental Ophthalmology, University of Geneva, 1205 Geneva, Switzerland
- Department of Ophthalmology, University Hospitals of Geneva, 1205 Geneva, Switzerland
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32
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Fischer A, Lersch R, de Andrade Krätzig N, Strong A, Friedrich MJ, Weber J, Engleitner T, Öllinger R, Yen HY, Kohlhofer U, Gonzalez-Menendez I, Sailer D, Kogan L, Lahnalampi M, Laukkanen S, Kaltenbacher T, Klement C, Rezaei M, Ammon T, Montero JJ, Schneider G, Mayerle J, Heikenwälder M, Schmidt-Supprian M, Quintanilla-Martinez L, Steiger K, Liu P, Cadiñanos J, Vassiliou GS, Saur D, Lohi O, Heinäniemi M, Conte N, Bradley A, Rad L, Rad R. In vivo interrogation of regulatory genomes reveals extensive quasi-insufficiency in cancer evolution. CELL GENOMICS 2023; 3:100276. [PMID: 36950387 PMCID: PMC10025556 DOI: 10.1016/j.xgen.2023.100276] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/10/2022] [Revised: 09/05/2022] [Accepted: 02/08/2023] [Indexed: 03/10/2023]
Abstract
In contrast to mono- or biallelic loss of tumor-suppressor function, effects of discrete gene dysregulations, as caused by non-coding (epi)genome alterations, are poorly understood. Here, by perturbing the regulatory genome in mice, we uncover pervasive roles of subtle gene expression variation in cancer evolution. Genome-wide screens characterizing 1,450 tumors revealed that such quasi-insufficiency is extensive across entities and displays diverse context dependencies, such as distinct cell-of-origin associations in T-ALL subtypes. We compile catalogs of non-coding regions linked to quasi-insufficiency, show their enrichment with human cancer risk variants, and provide functional insights by engineering regulatory alterations in mice. As such, kilo-/megabase deletions in a Bcl11b-linked non-coding region triggered aggressive malignancies, with allele-specific tumor spectra reflecting gradual gene dysregulations through modular and cell-type-specific enhancer activities. Our study constitutes a first survey toward a systems-level understanding of quasi-insufficiency in cancer and gives multifaceted insights into tumor evolution and the tissue-specific effects of non-coding mutations.
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Affiliation(s)
- Anja Fischer
- Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675 Munich, Germany
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technische Universität München, 81675 Munich, Germany
- German Cancer Consortium (DKTK), Heidelberg, Germany
| | - Robert Lersch
- Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675 Munich, Germany
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technische Universität München, 81675 Munich, Germany
| | - Niklas de Andrade Krätzig
- Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675 Munich, Germany
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technische Universität München, 81675 Munich, Germany
| | - Alexander Strong
- The Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge, UK
| | - Mathias J. Friedrich
- Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675 Munich, Germany
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technische Universität München, 81675 Munich, Germany
- Department of Medicine II, Klinikum rechts der Isar, School of Medicine, Technische Universität München, 81675 Munich, Germany
| | - Julia Weber
- Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675 Munich, Germany
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technische Universität München, 81675 Munich, Germany
| | - Thomas Engleitner
- Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675 Munich, Germany
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technische Universität München, 81675 Munich, Germany
| | - Rupert Öllinger
- Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675 Munich, Germany
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technische Universität München, 81675 Munich, Germany
| | - Hsi-Yu Yen
- German Cancer Consortium (DKTK), Heidelberg, Germany
- Comparative Experimental Pathology, School of Medicine, Technische Universität München, 81675 Munich, Germany
| | - Ursula Kohlhofer
- Institute of Pathology and Comprehensive Cancer Center, Eberhard Karls Universität Tübingen, 72076 Tübingen, Germany
| | - Irene Gonzalez-Menendez
- Institute of Pathology and Comprehensive Cancer Center, Eberhard Karls Universität Tübingen, 72076 Tübingen, Germany
| | - David Sailer
- Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675 Munich, Germany
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technische Universität München, 81675 Munich, Germany
| | - Liz Kogan
- Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675 Munich, Germany
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technische Universität München, 81675 Munich, Germany
| | - Mari Lahnalampi
- Institute of Biomedicine, School of Medicine, University of Eastern Finland, Kuopio, Finland
| | - Saara Laukkanen
- Faculty of Medicine and Health Technology, Tampere Center for Child, Adolescent and Maternal Health Research and Tays Cancer Center, Tampere University, Tampere, Finland
| | - Thorsten Kaltenbacher
- Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675 Munich, Germany
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technische Universität München, 81675 Munich, Germany
| | - Christine Klement
- Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675 Munich, Germany
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technische Universität München, 81675 Munich, Germany
| | - Majdaddin Rezaei
- Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675 Munich, Germany
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technische Universität München, 81675 Munich, Germany
| | - Tim Ammon
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technische Universität München, 81675 Munich, Germany
- Institute of Experimental Hematology, TUM School of Medicine, Technical University of Munich, 81675 Munich, Germany
| | - Juan J. Montero
- Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675 Munich, Germany
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technische Universität München, 81675 Munich, Germany
| | - Günter Schneider
- Department of Medicine II, Klinikum rechts der Isar, School of Medicine, Technische Universität München, 81675 Munich, Germany
- Department of General, Visceral and Pediatric Surgery, University Medical Center Göttingen, 37075 Göttingen, Germany
| | - Julia Mayerle
- Medical Department II, University Hospital, LMU Munich, Munich, Germany
| | - Mathias Heikenwälder
- German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
- Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
| | - Marc Schmidt-Supprian
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technische Universität München, 81675 Munich, Germany
- German Cancer Consortium (DKTK), Heidelberg, Germany
- Institute of Experimental Hematology, TUM School of Medicine, Technical University of Munich, 81675 Munich, Germany
| | - Leticia Quintanilla-Martinez
- Institute of Pathology and Comprehensive Cancer Center, Eberhard Karls Universität Tübingen, 72076 Tübingen, Germany
| | - Katja Steiger
- German Cancer Consortium (DKTK), Heidelberg, Germany
- Comparative Experimental Pathology, School of Medicine, Technische Universität München, 81675 Munich, Germany
| | - Pentao Liu
- The Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge, UK
- Li Ka Shing Faculty of Medicine, Stem Cell and Regenerative Medicine Consortium, School of Biomedical Sciences, University of Hong Kong, Hong Kong, China
| | - Juan Cadiñanos
- Instituto de Medicina Oncológica y Molecular de Asturias (IMOMA), 33193 Oviedo, Spain
| | - George S. Vassiliou
- The Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge, UK
- Wellcome Trust-MRC Stem Cell Institute, Cambridge Biomedical Campus, University of Cambridge, Cambridge CB2 0XY, UK
- Department of Haematology, Cambridge University Hospitals NHS Trust, Cambridge CB2 0PT, UK
| | - Dieter Saur
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technische Universität München, 81675 Munich, Germany
- Department of Medicine II, Klinikum rechts der Isar, School of Medicine, Technische Universität München, 81675 Munich, Germany
- Institute for Experimental Cancer Therapy, School of Medicine, Technische Universität München, 81675 Munich, Germany
| | - Olli Lohi
- Faculty of Medicine and Health Technology, Tampere Center for Child, Adolescent and Maternal Health Research and Tays Cancer Center, Tampere University, Tampere, Finland
| | - Merja Heinäniemi
- Institute of Biomedicine, School of Medicine, University of Eastern Finland, Kuopio, Finland
| | - Nathalie Conte
- The Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge, UK
| | - Allan Bradley
- The Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge, UK
- Cambridge Institute of Therapeutic Immunology & Infectious Disease (CITIID), University of Cambridge, Puddicombe Way, Cambridge CB2 0AW, UK
| | - Lena Rad
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technische Universität München, 81675 Munich, Germany
- Institute for Experimental Cancer Therapy, School of Medicine, Technische Universität München, 81675 Munich, Germany
| | - Roland Rad
- Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675 Munich, Germany
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technische Universität München, 81675 Munich, Germany
- German Cancer Consortium (DKTK), Heidelberg, Germany
- Department of Medicine II, Klinikum rechts der Isar, School of Medicine, Technische Universität München, 81675 Munich, Germany
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33
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Bredthauer C, Fischer A, Ahari AJ, Cao X, Weber J, Rad L, Rad R, Wachutka L, Gagneur J. Transmicron: accurate prediction of insertion probabilities improves detection of cancer driver genes from transposon mutagenesis screens. Nucleic Acids Res 2023; 51:e21. [PMID: 36617985 PMCID: PMC9976929 DOI: 10.1093/nar/gkac1215] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2022] [Revised: 11/06/2022] [Accepted: 12/17/2022] [Indexed: 01/10/2023] Open
Abstract
Transposon screens are powerful in vivo assays used to identify loci driving carcinogenesis. These loci are identified as Common Insertion Sites (CISs), i.e. regions with more transposon insertions than expected by chance. However, the identification of CISs is affected by biases in the insertion behaviour of transposon systems. Here, we introduce Transmicron, a novel method that differs from previous methods by (i) modelling neutral insertion rates based on chromatin accessibility, transcriptional activity and sequence context and (ii) estimating oncogenic selection for each genomic region using Poisson regression to model insertion counts while controlling for neutral insertion rates. To assess the benefits of our approach, we generated a dataset applying two different transposon systems under comparable conditions. Benchmarking for enrichment of known cancer genes showed improved performance of Transmicron against state-of-the-art methods. Modelling neutral insertion rates allowed for better control of false positives and stronger agreement of the results between transposon systems. Moreover, using Poisson regression to consider intra-sample and inter-sample information proved beneficial in small and moderately-sized datasets. Transmicron is open-source and freely available. Overall, this study contributes to the understanding of transposon biology and introduces a novel approach to use this knowledge for discovering cancer driver genes.
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Affiliation(s)
- Carl Bredthauer
- TUM School of Computation, Information and Technology, Technical University of Munich, 81675 Munich, Germany.,Institute of Molecular Oncology and Functional Genomics, TUM School of Medicine, Technical University of Munich, 81675 Munich, Germany.,Center for Translational Cancer Research (TranslaTUM), TUM School of Medicine, Technical University of Munich, 81675 Munich, Germany.,Computational Health Center, Helmholtz Zentrum Munich, Neuherberg, Germany
| | - Anja Fischer
- Institute of Molecular Oncology and Functional Genomics, TUM School of Medicine, Technical University of Munich, 81675 Munich, Germany.,Center for Translational Cancer Research (TranslaTUM), TUM School of Medicine, Technical University of Munich, 81675 Munich, Germany
| | - Ata Jadid Ahari
- TUM School of Computation, Information and Technology, Technical University of Munich, 81675 Munich, Germany
| | - Xueqi Cao
- TUM School of Computation, Information and Technology, Technical University of Munich, 81675 Munich, Germany.,Graduate School of Quantitative Biosciences (QBM), Ludwig-Maximilians-Universität München, 81377 Munich, Germany
| | - Julia Weber
- Institute of Molecular Oncology and Functional Genomics, TUM School of Medicine, Technical University of Munich, 81675 Munich, Germany.,Center for Translational Cancer Research (TranslaTUM), TUM School of Medicine, Technical University of Munich, 81675 Munich, Germany
| | - Lena Rad
- Center for Translational Cancer Research (TranslaTUM), TUM School of Medicine, Technical University of Munich, 81675 Munich, Germany.,Institute for Experimental Cancer Therapy, TUM School of Medicine, Technical University of Munich, 81675 Munich, Germany
| | - Roland Rad
- Institute of Molecular Oncology and Functional Genomics, TUM School of Medicine, Technical University of Munich, 81675 Munich, Germany.,Center for Translational Cancer Research (TranslaTUM), TUM School of Medicine, Technical University of Munich, 81675 Munich, Germany.,German Cancer Consortium (DKTK), 69120 Heidelberg, Germany.,Department of Medicine II, Klinikum rechts der Isar, TUM School of Medicine, Technical University of Munich, 81675 Munich, Germany
| | - Leonhard Wachutka
- TUM School of Computation, Information and Technology, Technical University of Munich, 81675 Munich, Germany
| | - Julien Gagneur
- TUM School of Computation, Information and Technology, Technical University of Munich, 81675 Munich, Germany.,Computational Health Center, Helmholtz Zentrum Munich, Neuherberg, Germany.,Institute of Human Genetics, TUM School of Medicine, Technical University of Munich, 81675 Munich, Germany
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34
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Wang S, Gao B, Miskey C, Guan Z, Sang Y, Chen C, Wang X, Ivics Z, Song C. Passer, a highly active transposon from a fish genome, as a potential new robust genetic manipulation tool. Nucleic Acids Res 2023; 51:1843-1858. [PMID: 36688327 PMCID: PMC9976928 DOI: 10.1093/nar/gkad005] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2022] [Revised: 12/26/2022] [Accepted: 01/03/2023] [Indexed: 01/24/2023] Open
Abstract
The discovery of new, active DNA transposons can expand the range of genetic tools and provide more options for genomic manipulation. In this study, a bioinformatics analysis suggested that Passer (PS) transposons, which are members of the pogo superfamily, show signs of recent and current activity in animals and may be active in some species. Cell-based transposition assays revealed that the native PS transposases from Gasterosteus aculeatus and Danio rerio displayed very high activity in human cells relative to the Sleeping Beauty transposon. A typical overproduction inhibition phenomenon was observed for PS, and transposition capacity was decreased by ∼12% with each kilobase increase in the insertion size. Furthermore, PS exhibited a pronounced integration preference for genes and their transcriptional regulatory regions. We further show that two domesticated human proteins derived from PS transposases have lost their transposition activity. Overall, PS may represent an alternative with a potentially efficient genetic manipulation tool for transgenesis and mutagenesis applications.
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Affiliation(s)
- Saisai Wang
- College of Animal Science & Technology, Yangzhou University, Yangzhou, Jiangsu, 225009, China
| | - Bo Gao
- College of Animal Science & Technology, Yangzhou University, Yangzhou, Jiangsu, 225009, China
| | - Csaba Miskey
- Division of Medical Biotechnology, Paul Ehrlich Institute, D-63225 Langen, Germany
| | - Zhongxia Guan
- College of Animal Science & Technology, Yangzhou University, Yangzhou, Jiangsu, 225009, China
| | - Yatong Sang
- College of Animal Science & Technology, Yangzhou University, Yangzhou, Jiangsu, 225009, China
| | - Cai Chen
- College of Animal Science & Technology, Yangzhou University, Yangzhou, Jiangsu, 225009, China
| | - Xiaoyan Wang
- College of Animal Science & Technology, Yangzhou University, Yangzhou, Jiangsu, 225009, China
| | - Zoltán Ivics
- Division of Medical Biotechnology, Paul Ehrlich Institute, D-63225 Langen, Germany
| | - Chengyi Song
- College of Animal Science & Technology, Yangzhou University, Yangzhou, Jiangsu, 225009, China
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35
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Penkov D, Zubkova E, Parfyonova Y. Tn5 DNA Transposase in Multi-Omics Research. Methods Protoc 2023; 6:mps6020024. [PMID: 36961044 PMCID: PMC10037646 DOI: 10.3390/mps6020024] [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: 12/15/2022] [Revised: 02/21/2023] [Accepted: 02/23/2023] [Indexed: 03/06/2023] Open
Abstract
Tn5 transposase use in biotechnology has substantially advanced the sequencing applications of genome-wide analysis of cells. This is mainly due to the ability of Tn5 transposase to efficiently transpose DNA essentially randomly into any target DNA without the aid of other factors. This concise review is focused on the advances in Tn5 applications in multi-omics technologies, genome-wide profiling, and Tn5 hybrid molecule creation. The possibilities of other transposase uses are also discussed.
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Affiliation(s)
- Dmitry Penkov
- IRCCS San Raffaele Hospital, 20132 Milan, Italy
- National Medical Research Centre of Cardiology Named after E. I. Chazov, 121552 Moscow, Russia
| | - Ekaterina Zubkova
- National Medical Research Centre of Cardiology Named after E. I. Chazov, 121552 Moscow, Russia
| | - Yelena Parfyonova
- National Medical Research Centre of Cardiology Named after E. I. Chazov, 121552 Moscow, Russia
- Faculty of Medicine, Lomonosov Moscow State University, 119991 Moscow, Russia
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36
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Luo W, Hickman AB, Genzor P, Ghirlando R, Furman C, Menshikh A, Haase A, Dyda F, Wilson M. Transposase N-terminal phosphorylation and asymmetric transposon ends inhibit piggyBac transposition in mammalian cells. Nucleic Acids Res 2022; 50:13128-13142. [PMID: 36537219 PMCID: PMC9825180 DOI: 10.1093/nar/gkac1191] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2022] [Revised: 11/09/2022] [Accepted: 12/07/2022] [Indexed: 12/24/2022] Open
Abstract
DNA transposon systems are widely used in mammalian cells for genetic modification experiments, but their regulation remains poorly understood. We used biochemical and cell-based assays together with AlphaFold modeling and rational protein redesign to evaluate aspects of piggyBac transposition including the previously unexplained role of the transposase N-terminus and the need for asymmetric transposon ends for cellular activity. We found that phosphorylation at predicted casein kinase II sites in the transposase N-terminus inhibits transposition, most likely by preventing transposase-DNA interactions. Deletion of the region containing these sites releases inhibition thereby enhancing activity. We also found that the N-terminal domain promotes transposase dimerization in the absence of transposon DNA. When the N-terminus is deleted, the transposase gains the ability to carry out transposition using symmetric transposon left ends. This novel activity is also conferred by appending a second C-terminal domain. When combined, these modifications together result in a transposase that is highly active when symmetric transposon ends are used. Our results demonstrate that transposase N-terminal phosphorylation and the requirement for asymmetric transposon ends both negatively regulate piggyBac transposition in mammalian cells. These novel insights into the mechanism and structure of the piggyBac transposase expand its potential use for genomic applications.
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Affiliation(s)
- Wentian Luo
- Department of Medicine, Division and Nephrology and Hypertension, Vanderbilt University Medical Center, Nashville, TN 37232, USA
| | - Alison B Hickman
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Pavol Genzor
- Laboratory of Cellular and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Rodolfo Ghirlando
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Christopher M Furman
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Anna Menshikh
- Department of Medicine, Division and Nephrology and Hypertension, Vanderbilt University Medical Center, Nashville, TN 37232, USA
| | - Astrid Haase
- Laboratory of Cellular and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Fred Dyda
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Matthew H Wilson
- Department of Medicine, Division and Nephrology and Hypertension, Vanderbilt University Medical Center, Nashville, TN 37232, USA
- Department of Veterans Affairs, Nashville, TN 37212, USA
- Departments of Pharmacology and Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232, USA
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Mattern L, Otten K, Miskey C, Fuest M, Izsvák Z, Ivics Z, Walter P, Thumann G, Johnen S. Molecular and Functional Characterization of BDNF-Overexpressing Human Retinal Pigment Epithelial Cells Established by Sleeping Beauty Transposon-Mediated Gene Transfer. Int J Mol Sci 2022; 23:12982. [PMID: 36361771 PMCID: PMC9656812 DOI: 10.3390/ijms232112982] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2022] [Revised: 09/30/2022] [Accepted: 10/25/2022] [Indexed: 04/12/2024] Open
Abstract
More and more patients suffer from multifactorial neurodegenerative diseases, such as age-related macular degeneration (AMD). However, their pathological mechanisms are still poorly understood, which complicates the development of effective therapies. To improve treatment of multifactorial diseases, cell-based gene therapy can be used to increase the expression of therapeutic factors. To date, there is no approved therapy for dry AMD, including late-stage geographic atrophy. We present a treatment option for dry AMD that transfers the brain-derived neurotrophic factor (BDNF) gene into retinal pigment epithelial (RPE) cells by electroporation using the plasmid-based Sleeping Beauty (SB) transposon system. ARPE-19 cells and primary human RPE cells were co-transfected with two plasmids encoding the SB100X transposase and the transposon carrying a BDNF transcription cassette. We demonstrated efficient expression and secretion of BDNF in both RPE cell types, which were further increased in ARPE-19 cell cultures exposed to hydrogen peroxide. BDNF-transfected cells exhibited lower apoptosis rates and stimulated neurite outgrowth in human SH-SY5Y cells. This study is an important step in the development of a cell-based BDNF gene therapy that could be applied as an advanced therapy medicinal product to treat dry AMD or other degenerative retinal diseases.
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Affiliation(s)
- Larissa Mattern
- Department of Ophthalmology, University Hospital RWTH Aachen, 52074 Aachen, Germany
| | - Katrin Otten
- Department of Ophthalmology, University Hospital RWTH Aachen, 52074 Aachen, Germany
| | - Csaba Miskey
- Division of Medical Biotechnology, Paul-Ehrlich-Institute, 63225 Langen, Germany
| | - Matthias Fuest
- Department of Ophthalmology, University Hospital RWTH Aachen, 52074 Aachen, Germany
| | - Zsuzsanna Izsvák
- Max Delbrück Center for Molecular Medicine in the Helmholtz Association, 13125 Berlin, Germany
| | - Zoltán Ivics
- Division of Medical Biotechnology, Paul-Ehrlich-Institute, 63225 Langen, Germany
| | - Peter Walter
- Department of Ophthalmology, University Hospital RWTH Aachen, 52074 Aachen, Germany
| | - Gabriele Thumann
- Department of Ophthalmology, University Hospitals of Geneva, 1205 Geneva, Switzerland
- Experimental Ophthalmology, University of Geneva, 1205 Geneva, Switzerland
| | - Sandra Johnen
- Department of Ophthalmology, University Hospital RWTH Aachen, 52074 Aachen, Germany
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38
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Episomes and Transposases-Utilities to Maintain Transgene Expression from Nonviral Vectors. Genes (Basel) 2022; 13:genes13101872. [PMID: 36292757 PMCID: PMC9601623 DOI: 10.3390/genes13101872] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2022] [Revised: 10/07/2022] [Accepted: 10/14/2022] [Indexed: 11/04/2022] Open
Abstract
The efficient delivery and stable transgene expression are critical for applications in gene therapy. While carefully selected and engineered viral vectors allowed for remarkable clinical successes, they still bear significant safety risks. Thus, nonviral vectors are a sound alternative and avoid genotoxicity and adverse immunological reactions. Nonviral vector systems have been extensively studied and refined during the last decades. Emerging knowledge of the epigenetic regulation of replication and spatial chromatin organisation, as well as new technologies, such as Crispr/Cas, were employed to enhance the performance of different nonviral vector systems. Thus, nonviral vectors are in focus and hold some promising perspectives for future applications in gene therapy. This review addresses three prominent nonviral vector systems: the Sleeping Beauty transposase, S/MAR-based episomes, and viral plasmid replicon-based EBV vectors. Exemplarily, we review different utilities, modifications, and new concepts that were pursued to overcome limitations regarding stable transgene expression and mitotic stability. New insights into the nuclear localisation of nonviral vector molecules and the potential consequences thereof are highlighted. Finally, we discuss the remaining limitations and provide an outlook on possible future developments in nonviral vector technology.
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Cas-CLOVER is a novel high-fidelity nuclease for safe and robust generation of TSCM-enriched allogeneic CAR-T cells. MOLECULAR THERAPY - NUCLEIC ACIDS 2022; 29:979-995. [PMID: 36189080 PMCID: PMC9481872 DOI: 10.1016/j.omtn.2022.06.003] [Citation(s) in RCA: 21] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/10/2021] [Accepted: 06/08/2022] [Indexed: 12/26/2022]
Abstract
The use of T cells from healthy donors for allogeneic chimeric antigen receptor T (CAR-T) cell cancer therapy is attractive because healthy donor T cells can produce versatile off-the-shelf CAR-T treatments. To maximize safety and durability of allogeneic products, the endogenous T cell receptor and major histocompatibility complex class I molecules are often removed via knockout of T cell receptor beta constant (TRBC) (or T cell receptor alpha constant [TRAC]) and B2M, respectively. However, gene editing tools (e.g., CRISPR-Cas9) can display poor fidelity, which may result in dangerous off-target mutations. Additionally, many gene editing technologies require T cell activation, resulting in a low percentage of desirable stem cell memory T cells (TSCM). We characterize an RNA-guided endonuclease, called Cas-CLOVER, consisting of the Clo051 nuclease domain fused with catalytically dead Cas9. In primary T cells from multiple donors, we find that Cas-CLOVER is a high-fidelity site-specific nuclease, with low off-target activity. Notably, Cas-CLOVER yields efficient multiplexed gene editing in resting T cells. In conjunction with the piggyBac transposon for delivery of a CAR transgene against the B cell maturation antigen (BCMA), we produce allogeneic CAR-T cells composed of high percentages of TSCM cells and possessing potent in vivo anti-tumor cytotoxicity.
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40
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Lock D, Monjezi R, Brandes C, Bates S, Lennartz S, Teppert K, Gehrke L, Karasakalidou-Seidt R, Lukic T, Schmeer M, Schleef M, Werchau N, Eyrich M, Assenmacher M, Kaiser A, Prommersberger S, Schaser T, Hudecek M. Automated, scaled, transposon-based production of CAR T cells. J Immunother Cancer 2022; 10:jitc-2022-005189. [PMID: 36096530 PMCID: PMC9472140 DOI: 10.1136/jitc-2022-005189] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/23/2022] [Indexed: 11/04/2022] Open
Abstract
BACKGROUND There is an increasing demand for chimeric antigen receptor (CAR) T cell products from patients and care givers. Here, we established an automated manufacturing process for CAR T cells on the CliniMACS Prodigy platform that is scaled to provide therapeutic doses and achieves gene-transfer with virus-free Sleeping Beauty (SB) transposition. METHODS We used an advanced CliniMACS Prodigy that is connected to an electroporator unit and performed a series of small-scale development and large-scale confirmation runs with primary human T cells. Transposition was accomplished with minicircle (MC) DNA-encoded SB100X transposase and pT2 transposon encoding a CD19 CAR. RESULTS We defined a bi-pulse electroporation shock with bi-directional and unidirectional electric field, respectively, that permitted efficient MC insertion and maintained a high frequency of viable T cells. In three large scale runs, 2E8 T cells were enriched from leukapheresis product, activated, gene-engineered and expanded to yield up to 3.5E9 total T cells/1.4E9 CAR-modified T cells within 12 days (CAR-modified T cells: 28.8%±12.3%). The resulting cell product contained highly pure T cells (97.3±1.6%) with balanced CD4/CD8 ratio and a high frequency of T cells with central memory phenotype (87.5%±10.4%). The transposon copy number was 7.0, 9.4 and 6.8 in runs #1-3, respectively, and gene analyses showed a balanced expression of activation/exhaustion markers. The CD19 CAR T cell product conferred potent anti-lymphoma reactivity in pre-clinical models. Notably, the operator hands-on-time was substantially reduced compared with conventional non-automated CAR T cell manufacturing campaigns. CONCLUSIONS We report on the first automated transposon-based manufacturing process for CAR T cells that is ready for formal validation and use in clinical manufacturing campaigns. This process and platform have the potential to facilitate access of patients to CAR T cell therapy and to accelerate scaled, multiplexed manufacturing both in the academic and industry setting.
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Affiliation(s)
- Dominik Lock
- Miltenyi Biotec BV & Co KG, Bergisch Gladbach, Germany
| | - Razieh Monjezi
- Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany
| | | | - Stephan Bates
- Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany
| | | | - Karin Teppert
- Miltenyi Biotec BV & Co KG, Bergisch Gladbach, Germany
| | - Leon Gehrke
- Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany
| | | | - Teodora Lukic
- Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany
| | | | | | - Niels Werchau
- Miltenyi Biotec BV & Co KG, Bergisch Gladbach, Germany
| | - Matthias Eyrich
- Universitätskinderklinik, Universitätsklinikum Würzburg, Würzburg, Germany
| | | | - Andrew Kaiser
- Miltenyi Biotec BV & Co KG, Bergisch Gladbach, Germany
| | | | | | - Michael Hudecek
- Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany
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41
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Ishikawa K, Tamamura S, Takahashi N, Takagi M, Semba K, Watanabe S. Isolation of Reporter Cells That Respond to Vitamin A and/or D Using a piggyBac Transposon Promoter-Trapping Vector System. Int J Mol Sci 2022; 23:9366. [PMID: 36012634 PMCID: PMC9409033 DOI: 10.3390/ijms23169366] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2022] [Revised: 08/15/2022] [Accepted: 08/18/2022] [Indexed: 11/16/2022] Open
Abstract
Previously, we established a highly sensitive promoter-trapping vector system using the piggyBac transposon for the efficient isolation of reporter cells. Herein, we examine whether this screening system can be applied to obtain vitamin-responsive cells. As a result, one and two reporter cells that responded to bexarotene (vitamin A) and calcitriol (vitamin D), respectively, were isolated from 4.7 × 106 seeded HeLaS3 cells. 5' RACE analyses identified the well-known CYP24A1 gene as a calcitriol-responsive gene, as well as two new bexarotene- or calcitriol-responsive genes, BDKRB2 and TSKU, respectively. TSKU, interestingly, also responded to bexarotene. Endogenous levels of the TSKU and BDKRB2 transcripts displayed only slight changes and were not detected in the comprehensive analyses performed to date. Dose-response analyses of BDKRB2 and TSKU reporter cells in parallel revealed a differential profile in response to each vitamin A agonist, suggesting a bioanalyzer. The present study demonstrates that producing multiple reporter cells by a type of random screening can efficiently identify novel genes with unusual characteristics and be used for the profiling of the properties of vitamin compounds. Similar approaches to the method shown here may be useful for identifying new markers and for the analysis or diagnosis of nutrients, toxins, metabolites, etc.
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Affiliation(s)
- Kosuke Ishikawa
- Japan Biological Informatics Consortium (JBiC), 2-45 Aomi, Koto-ku, Tokyo 135-8073, Japan
| | - Sakura Tamamura
- Japan Biological Informatics Consortium (JBiC), 2-45 Aomi, Koto-ku, Tokyo 135-8073, Japan
| | - Nobuhito Takahashi
- Medical-Industrial Translational Research Center, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan
| | - Motoki Takagi
- Medical-Industrial Translational Research Center, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan
| | - Kentaro Semba
- Medical-Industrial Translational Research Center, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan
- Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan
| | - Shinya Watanabe
- Medical-Industrial Translational Research Center, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan
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42
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Baldassarri S, Benati D, D’Alessio F, Patrizi C, Cattin E, Gentile M, Raggioli A, Recchia A. Engineered Sleeping Beauty Transposon as Efficient System to Optimize Chimp Adenoviral Production. Int J Mol Sci 2022; 23:ijms23147538. [PMID: 35886882 PMCID: PMC9316264 DOI: 10.3390/ijms23147538] [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: 03/09/2022] [Revised: 06/01/2022] [Accepted: 07/01/2022] [Indexed: 11/16/2022] Open
Abstract
Sleeping Beauty (SB) is the first DNA transposon employed for efficient transposition in vertebrate cells, opening new applications for genetic engineering and gene therapies. A transposon-based gene delivery system holds the favourable features of non-viral vectors and an attractive safety profile. Here, we employed SB to engineer HEK293 cells for optimizing the production of a chimpanzee Adenovector (chAd) belonging to the Human Mastadenovirus C species. To date, chAd vectors are employed in several clinical settings for infectious diseases, last but not least COVID-19. A robust, efficient and quick viral vector production could advance the clinical application of chAd vectors. To this aim, we firstly swapped the hAd5 E1 with chAd-C E1 gene by using the CRISPR/Cas9 system. We demonstrated that in the absence of human Ad5 E1, chimp Ad-C E1 gene did not support HEK293 survival. To improve chAd-C vector production, we engineered HEK293 cells to stably express the chAd-C precursor terminal protein (ch.pTP), which plays a crucial role in chimpanzee Adenoviral DNA replication. The results indicate that exogenous ch.pTP expression significantly ameliorate the packaging and amplification of recombinant chAd-C vectors thus, the engineered HEK293ch.pTP cells could represent a superior packaging cell line for the production of these vectors.
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Affiliation(s)
- Samantha Baldassarri
- Centre for Regenerative Medicine, Department of Life Sciences, University of Modena and Reggio Emilia, 41121 Modena, Italy; (S.B.); (D.B.); (C.P.); (E.C.)
| | - Daniela Benati
- Centre for Regenerative Medicine, Department of Life Sciences, University of Modena and Reggio Emilia, 41121 Modena, Italy; (S.B.); (D.B.); (C.P.); (E.C.)
| | - Federica D’Alessio
- ReiThera S.r.l., 00128 Rome, Italy; (F.D.); (M.G.); (A.R.)
- Department of Molecular Medicine and Medical Biotechnologies, University of Naples “Federico II”, 80138 Naples, Italy
| | - Clarissa Patrizi
- Centre for Regenerative Medicine, Department of Life Sciences, University of Modena and Reggio Emilia, 41121 Modena, Italy; (S.B.); (D.B.); (C.P.); (E.C.)
| | - Eleonora Cattin
- Centre for Regenerative Medicine, Department of Life Sciences, University of Modena and Reggio Emilia, 41121 Modena, Italy; (S.B.); (D.B.); (C.P.); (E.C.)
| | | | | | - Alessandra Recchia
- Centre for Regenerative Medicine, Department of Life Sciences, University of Modena and Reggio Emilia, 41121 Modena, Italy; (S.B.); (D.B.); (C.P.); (E.C.)
- Correspondence:
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43
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Moretti A, Ponzo M, Nicolette CA, Tcherepanova IY, Biondi A, Magnani CF. The Past, Present, and Future of Non-Viral CAR T Cells. Front Immunol 2022; 13:867013. [PMID: 35757746 PMCID: PMC9218214 DOI: 10.3389/fimmu.2022.867013] [Citation(s) in RCA: 68] [Impact Index Per Article: 22.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2022] [Accepted: 04/28/2022] [Indexed: 12/14/2022] Open
Abstract
Adoptive transfer of chimeric antigen receptor (CAR) T lymphocytes is a powerful technology that has revolutionized the way we conceive immunotherapy. The impressive clinical results of complete and prolonged response in refractory and relapsed diseases have shifted the landscape of treatment for hematological malignancies, particularly those of lymphoid origin, and opens up new possibilities for the treatment of solid neoplasms. However, the widening use of cell therapy is hampered by the accessibility to viral vectors that are commonly used for T cell transfection. In the era of messenger RNA (mRNA) vaccines and CRISPR/Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) precise genome editing, novel and virus-free methods for T cell engineering are emerging as a more versatile, flexible, and sustainable alternative for next-generation CAR T cell manufacturing. Here, we discuss how the use of non-viral vectors can address some of the limitations of the viral methods of gene transfer and allow us to deliver genetic information in a stable, effective and straightforward manner. In particular, we address the main transposon systems such as Sleeping Beauty (SB) and piggyBac (PB), the utilization of mRNA, and innovative approaches of nanotechnology like Lipid-based and Polymer-based DNA nanocarriers and nanovectors. We also describe the most relevant preclinical data that have recently led to the use of non-viral gene therapy in emerging clinical trials, and the related safety and efficacy aspects. We will also provide practical considerations for future trials to enable successful and safe cell therapy with non-viral methods for CAR T cell generation.
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Affiliation(s)
- Alex Moretti
- Tettamanti Research Center, Department of Pediatrics, University of Milano-Bicocca/Fondazione Monza e Brianza per il Bambino e la sua Mamma (MBBM), Monza, Italy
| | - Marianna Ponzo
- Tettamanti Research Center, Department of Pediatrics, University of Milano-Bicocca/Fondazione Monza e Brianza per il Bambino e la sua Mamma (MBBM), Monza, Italy
| | | | | | - Andrea Biondi
- Tettamanti Research Center, Department of Pediatrics, University of Milano-Bicocca/Fondazione Monza e Brianza per il Bambino e la sua Mamma (MBBM), Monza, Italy
- Department of Pediatrics, University of Milano - Bicocca, Milan, Italy
- Clinica Pediatrica, University of Milano - Bicocca/Fondazione MBBM, Monza, Italy
| | - Chiara F. Magnani
- Tettamanti Research Center, Department of Pediatrics, University of Milano-Bicocca/Fondazione Monza e Brianza per il Bambino e la sua Mamma (MBBM), Monza, Italy
- Department of Medical Oncology and Hematology, University Hospital Zurich and University of Zurich, Zurich, Switzerland
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44
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Characterizing piggyBat-a transposase for genetic modification of T cells. Mol Ther Methods Clin Dev 2022; 25:250-263. [PMID: 35474955 PMCID: PMC9018555 DOI: 10.1016/j.omtm.2022.03.012] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2021] [Accepted: 03/17/2022] [Indexed: 11/21/2022]
Abstract
Chimeric antigen receptor (CAR) T cells targeting CD19 have demonstrated remarkable efficacy in the treatment of B cell malignancies. Current CAR T cell manufacturing protocols are complex and costly due to their reliance on viral vectors. Non-viral systems of genetic modification, such as with transposase and transposon systems, offer a potential streamlined alternative for CAR T cell manufacture and are currently being evaluated in clinical trials. In this study, we utilized the previously described transposase from the little brown bat, designated piggyBat, for production of CD19-specific CAR T cells. PiggyBat demonstrates efficient CAR transgene delivery, with a relatively low variability in integration copy number across a range of manufacturing conditions as well as a similar integration site profile to super-piggyBac transposon and viral vectors. PiggyBat-generated CAR T cells demonstrate CD19-specific cytotoxic efficacy in vitro and in vivo. These data demonstrate that alternative, naturally occurring DNA transposons can be efficiently re-tooled to be exploited in real-world applications.
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45
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Wei M, Mi CL, Jing CQ, Wang TY. Progress of Transposon Vector System for Production of Recombinant Therapeutic Proteins in Mammalian Cells. Front Bioeng Biotechnol 2022; 10:879222. [PMID: 35600890 PMCID: PMC9114503 DOI: 10.3389/fbioe.2022.879222] [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: 02/19/2022] [Accepted: 04/04/2022] [Indexed: 11/13/2022] Open
Abstract
In recent years, mammalian cells have become the primary host cells for the production of recombinant therapeutic proteins (RTPs). Despite that the expression of RTPs in mammalian cells can be improved by directly optimizing or engineering the expression vectors, it is still influenced by the low stability and efficiency of gene integration. Transposons are mobile genetic elements that can be inserted and cleaved within the genome and can change their inserting position. The transposon vector system can be applied to establish a stable pool of cells with high efficiency in RTPs production through facilitating the integration of gene of interest into transcriptionally active sites under screening pressure. Here, the structure and optimization of transposon vector system and its application in expressing RTPs at high level in mammalian cells are reviewed.
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Affiliation(s)
- Mian Wei
- School of Life Science and Technology, Xinxiang Medical University, Xinxiang, China
- International Joint Research Laboratory for Recombinant Pharmaceutical Protein Expression System of Henan, Xinxiang, China
| | - Chun-Liu Mi
- International Joint Research Laboratory for Recombinant Pharmaceutical Protein Expression System of Henan, Xinxiang, China
| | - Chang-Qin Jing
- School of Life Science and Technology, Xinxiang Medical University, Xinxiang, China
- *Correspondence: Chang-Qin Jing, ; Tian-Yun Wang,
| | - Tian-Yun Wang
- International Joint Research Laboratory for Recombinant Pharmaceutical Protein Expression System of Henan, Xinxiang, China
- *Correspondence: Chang-Qin Jing, ; Tian-Yun Wang,
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46
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Poorebrahim M, Quiros-Fernandez I, Fakhr E, Cid-Arregui A. Generation of CAR-T cells using lentiviral vectors. Methods Cell Biol 2022; 167:39-69. [PMID: 35152998 DOI: 10.1016/bs.mcb.2021.07.001] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Cancer immunotherapy is nowadays largely focused on the development of therapeutic antibodies and chimeric antigen receptors (CARs). Two CARs targeting CD19 have been approved recently for the treatment of some hematological malignancies. This demonstrates the capability of engineered CAR T cells in generating effective tumor responses. Furthermore, several hundred ongoing clinical trials are exploring the feasibility of CAR-based approaches to target tumor-associated antigens in solid tumors. However, there still remain significant challenges and limitations in the design and production of CAR-modified T cells that need to be addressed, such as more effective transduction methods, expression and exhaustion issues, reliable in vitro and in vivo characterization methods, etc. Here we describe current techniques for generating CAR T cells using lentiviral vectors as well as detailed protocols for their functional characterization.
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Affiliation(s)
- Mansour Poorebrahim
- Targeted Tumor Vaccines Group, Clinical Cooperation Unit Applied Tumor Immunity, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Isaac Quiros-Fernandez
- Targeted Tumor Vaccines Group, Clinical Cooperation Unit Applied Tumor Immunity, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Elham Fakhr
- Targeted Tumor Vaccines Group, Clinical Cooperation Unit Applied Tumor Immunity, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Angel Cid-Arregui
- Targeted Tumor Vaccines Group, Clinical Cooperation Unit Applied Tumor Immunity, German Cancer Research Center (DKFZ), Heidelberg, Germany.
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47
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Wong DP, Roy NK, Zhang K, Anukanth A, Asthana A, Shirkey-Son NJ, Dunmire S, Jones BJ, Lahr WS, Webber BR, Moriarity BS, Caimi P, Parameswaran R. A BAFF ligand-based CAR-T cell targeting three receptors and multiple B cell cancers. Nat Commun 2022; 13:217. [PMID: 35017485 PMCID: PMC8752722 DOI: 10.1038/s41467-021-27853-w] [Citation(s) in RCA: 47] [Impact Index Per Article: 15.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2021] [Accepted: 12/20/2021] [Indexed: 12/21/2022] Open
Abstract
B cell-activating factor (BAFF) binds the three receptors BAFF-R, BCMA, and TACI, predominantly expressed on mature B cells. Almost all B cell cancers are reported to express at least one of these receptors. Here we develop a BAFF ligand-based chimeric antigen receptor (CAR) and generate BAFF CAR-T cells using a non-viral gene delivery method. We show that BAFF CAR-T cells bind specifically to each of the three BAFF receptors and are effective at killing multiple B cell cancers, including mantle cell lymphoma (MCL), multiple myeloma (MM), and acute lymphoblastic leukemia (ALL), in vitro and in vivo using different xenograft models. Co-culture of BAFF CAR-T cells with these tumor cells results in induction of activation marker CD69, degranulation marker CD107a, and multiple proinflammatory cytokines. In summary, we report a ligand-based BAFF CAR-T capable of binding three different receptors, minimizing the potential for antigen escape in the treatment of B cell cancers.
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MESH Headings
- Animals
- Antigens, CD/genetics
- Antigens, CD/immunology
- Antigens, Differentiation, T-Lymphocyte/genetics
- Antigens, Differentiation, T-Lymphocyte/immunology
- B-Cell Activating Factor/genetics
- B-Cell Activating Factor/immunology
- B-Cell Activation Factor Receptor/genetics
- B-Cell Activation Factor Receptor/immunology
- B-Cell Maturation Antigen/genetics
- B-Cell Maturation Antigen/immunology
- B-Lymphocytes/immunology
- B-Lymphocytes/pathology
- Cell Line, Tumor
- Coculture Techniques
- Cytotoxicity, Immunologic
- Female
- Gene Expression Regulation, Neoplastic
- Humans
- Lectins, C-Type/genetics
- Lectins, C-Type/immunology
- Lymphocyte Activation
- Lymphoma, Mantle-Cell/genetics
- Lymphoma, Mantle-Cell/immunology
- Lymphoma, Mantle-Cell/pathology
- Lymphoma, Mantle-Cell/therapy
- Lysosomal-Associated Membrane Protein 1/genetics
- Lysosomal-Associated Membrane Protein 1/immunology
- Male
- Mice
- Multiple Myeloma/genetics
- Multiple Myeloma/immunology
- Multiple Myeloma/pathology
- Multiple Myeloma/therapy
- Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics
- Precursor Cell Lymphoblastic Leukemia-Lymphoma/immunology
- Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology
- Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy
- Protein Binding
- Receptors, Chimeric Antigen/genetics
- Receptors, Chimeric Antigen/immunology
- Signal Transduction
- T-Lymphocytes/immunology
- T-Lymphocytes/transplantation
- Transmembrane Activator and CAML Interactor Protein/genetics
- Transmembrane Activator and CAML Interactor Protein/immunology
- Xenograft Model Antitumor Assays
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Affiliation(s)
- Derek P Wong
- Department of Pathology, Case Western Reserve University, Cleveland, OH, USA
| | - Nand K Roy
- Division of Hematology/Oncology, Department of Medicine, Case Western Reserve University, Cleveland, OH, USA
| | - Keman Zhang
- Division of Hematology/Oncology, Department of Medicine, Case Western Reserve University, Cleveland, OH, USA
| | - Anusha Anukanth
- Division of Pediatric Hematology/Oncology, Angie Fowler AYA Cancer Institute, UH Rainbow Babies & Children's Hospital, Cleveland, OH, USA
| | - Abhishek Asthana
- Division of Hematology/Oncology, Department of Medicine, Case Western Reserve University, Cleveland, OH, USA
| | | | | | | | - Walker S Lahr
- Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA
- Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA
- Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA
- Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
| | - Beau R Webber
- Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA
- Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA
- Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA
- Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
| | - Branden S Moriarity
- Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA
- Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA
- Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA
- Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
| | - Paolo Caimi
- Division of Hematology/Oncology, Department of Medicine, Case Western Reserve University, Cleveland, OH, USA
- The Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA
| | - Reshmi Parameswaran
- Division of Hematology/Oncology, Department of Medicine, Case Western Reserve University, Cleveland, OH, USA.
- The Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA.
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48
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Miskey C, Kesselring L, Querques I, Abrusán G, Barabas O, Ivics Z. OUP accepted manuscript. Nucleic Acids Res 2022; 50:2807-2825. [PMID: 35188569 PMCID: PMC8934666 DOI: 10.1093/nar/gkac092] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2021] [Revised: 01/24/2022] [Accepted: 02/08/2022] [Indexed: 11/14/2022] Open
Abstract
The Sleeping Beauty (SB) transposon system is a popular tool for genome engineering, but random integration into the genome carries a certain genotoxic risk in therapeutic applications. Here we investigate the role of amino acids H187, P247 and K248 in target site selection of the SB transposase. Structural modeling implicates these three amino acids located in positions analogous to amino acids with established functions in target site selection in retroviral integrases and transposases. Saturation mutagenesis of these residues in the SB transposase yielded variants with altered target site selection properties. Transposon integration profiling of several mutants reveals increased specificity of integrations into palindromic AT repeat target sequences in genomic regions characterized by high DNA bendability. The H187V and K248R mutants redirect integrations away from exons, transcriptional regulatory elements and nucleosomal DNA in the human genome, suggesting enhanced safety and thus utility of these SB variants in gene therapy applications.
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Affiliation(s)
| | | | - Irma Querques
- Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg 69117, Germany
- Department of Biochemistry, University of Zurich, Zurich 8057, Switzerland
| | - György Abrusán
- Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged 6726, Hungary
| | - Orsolya Barabas
- Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg 69117, Germany
- Department of Molecular Biology, University of Geneva, Geneva 1211, Switzerland
| | - Zoltán Ivics
- To whom correspondence should be addressed. Tel: +49 6103 77 6000; Fax: +49 6103 77 1280;
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BACH2 is a putative T-cell lymphoma tumor suppressor that may play a role in product-derived CAR T-cell lymphomas. Blood 2021; 138:2731-2733. [PMID: 34499707 PMCID: PMC8703361 DOI: 10.1182/blood.2021012641] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2020] [Accepted: 09/01/2021] [Indexed: 12/25/2022] Open
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50
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Bire S, Dusserre Y, Bigot Y, Mermod N. PiggyBac transposase and transposon derivatives for gene transfer targeting the ribosomal DNA loci of CHO cells. J Biotechnol 2021; 341:103-112. [PMID: 34560160 DOI: 10.1016/j.jbiotec.2021.09.011] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2021] [Revised: 08/24/2021] [Accepted: 09/15/2021] [Indexed: 11/17/2022]
Abstract
Integrative non-viral vectors such as transposons engineered to mediate targeted gene transfer into safe harbor sites in the genome may be a promising approach for the production of therapeutic proteins or for gene therapy in an efficient and secure way. In this context, we designed and evaluated two strategies for targeting the nuclear ribosomal DNA (rDNA) loci. One approach relied on the co-location of the transposase and transposon near transcriptionally active rDNA copies using a nucleolar localization signal (NoLS). Another one consisted of targeting the 18S-coding region in the rDNA loci using a NoLS-FokI-dCas9 endonuclease to perform targeted transgene knock-in. We show that integration into the rDNA of Chinese hamster ovary (CHO) cells can be achieved at a high frequency using the piggyBac transposon system, indicating that the rDNA is highly accessible for transposition. Consistently, rDNA-targeted transposition events were most frequently obtained when both the piggyBac transposon DNA and the transposase were nucleoli-targeted, yielding cells displaying stable and homogeneous expression of the transgene. This approach thus provides an alternative strategy to improve targeted transgene delivery and protein expression using CHO cells.
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Affiliation(s)
- Solenne Bire
- Institute of Biotechnology and Department of Fundamental Microbiology, Center for Biotechnology UNIL-EPFL, University of Lausanne, Lausanne, Switzerland
| | - Yves Dusserre
- Institute of Biotechnology and Department of Fundamental Microbiology, Center for Biotechnology UNIL-EPFL, University of Lausanne, Lausanne, Switzerland
| | - Yves Bigot
- UMR INRAE 0085, CNRS 7247, Physiologie de la Reproduction et des Comportements, Centre INRAE Val de Loire, 37380 Nouzilly, France
| | - Nicolas Mermod
- Institute of Biotechnology and Department of Fundamental Microbiology, Center for Biotechnology UNIL-EPFL, University of Lausanne, Lausanne, Switzerland.
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