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Stevens CS, Carmichael JC, Watkinson R, Kowdle S, Reis RA, Hamane K, Jang J, Park A, Pernet O, Khamaikawin W, Hong P, Thibault P, Gowlikar A, An DS, Lee B. A temperature-sensitive and less immunogenic Sendai virus for efficient gene editing. J Virol 2024; 98:e0083224. [PMID: 39494910 PMCID: PMC11650993 DOI: 10.1128/jvi.00832-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2024] [Accepted: 10/10/2024] [Indexed: 11/05/2024] Open
Abstract
The therapeutic potential of gene editing technologies hinges on the development of safe and effective delivery methods. In this study, we developed a temperature-sensitive and less immunogenic Sendai virus (ts SeV) as a novel delivery vector for CRISPR-Cas9 and for efficient gene editing in sensitive human cell types with limited induction of an innate immune response. ts SeV demonstrates high transduction efficiency in human CD34+ hematopoietic stem and progenitor cells (HSPCs) including transduction of the CD34+/CD38-/CD45RA-/CD90+(Thy1+)/CD49fhigh stem cell enriched subpopulation. The frequency of CCR5 editing exceeded 90% and bi-allelic CCR5 editing exceeded 70% resulting in significant inhibition of HIV-1 infection in primary human CD14+ monocytes. These results demonstrate the potential of the ts SeV platform as a safe, efficient, and flexible addition to the current gene-editing tool delivery methods, which may help further expand the possibilities in personalized medicine and the treatment of genetic disorders. IMPORTANCE Gene editing has the potential to be a powerful tool for the treatment of human diseases including HIV, β-thalassemias, and sickle cell disease. Recent advances have begun to overcome one of the major limiting factors of this technology, namely delivery of the CRISPR-Cas9 gene editing machinery, by utilizing viral vectors. However, gene editing therapies have yet to be implemented due to inherent risks associated with the DNA viral vectors typically used for delivery. As an alternative strategy, we have developed an RNA-based Sendai virus CRISPR-Cas9 delivery vector that does not integrate into the genome, is temperature sensitive, and does not induce a significant host interferon response. This recombinant SeV successfully delivered CRISPR-Cas9 in primary human CD14+ monocytes ex vivo resulting in a high level of CCR5 editing and inhibition of HIV infection.
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Affiliation(s)
- Christian S. Stevens
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Jillian C. Carmichael
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Ruth Watkinson
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Shreyas Kowdle
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Rebecca A. Reis
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Kory Hamane
- UCLA School of Nursing, Los Angeles, California, USA
- UCLA AIDS Institute, Los Angeles, California, USA
| | - Jason Jang
- UCLA School of Nursing, Los Angeles, California, USA
- UCLA AIDS Institute, Los Angeles, California, USA
| | - Arnold Park
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Olivier Pernet
- UCLA School of Nursing, Los Angeles, California, USA
- UCLA AIDS Institute, Los Angeles, California, USA
| | - Wannisa Khamaikawin
- UCLA School of Nursing, Los Angeles, California, USA
- UCLA AIDS Institute, Los Angeles, California, USA
| | - Patrick Hong
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Patricia Thibault
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Aditya Gowlikar
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
| | - Dong Sung An
- UCLA School of Nursing, Los Angeles, California, USA
- UCLA AIDS Institute, Los Angeles, California, USA
| | - Benhur Lee
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
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Stevens CS, Carmichael J, Watkinson R, Kowdle S, Reis RA, Hamane K, Jang J, Park A, Pernet O, Khamaikawin W, Hong P, Thibault P, Gowlikar A, An DS, Lee B. A temperature-sensitive and interferon-silent Sendai virus vector for CRISPR-Cas9 delivery and gene editing in primary human cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.05.03.592383. [PMID: 38746439 PMCID: PMC11092779 DOI: 10.1101/2024.05.03.592383] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/16/2024]
Abstract
The transformative potential of gene editing technologies hinges on the development of safe and effective delivery methods. In this study, we developed a temperature-sensitive and interferon-silent Sendai virus (ts SeV) as a novel delivery vector for CRISPR-Cas9 and for efficient gene editing in sensitive human cell types without inducing IFN responses. ts SeV demonstrates unprecedented transduction efficiency in human CD34+ hematopoietic stem and progenitor cells (HSPCs) including transduction of the CD34+/CD38-/CD45RA-/CD90+(Thy1+)/CD49fhigh stem cell enriched subpopulation. The frequency of CCR5 editing exceeded 90% and bi-allelic CCR5 editing exceeded 70% resulting in significant inhibition of HIV-1 infection in primary human CD14+ monocytes. These results demonstrate the potential of the ts SeV platform as a safe, efficient, and flexible addition to the current gene-editing tool delivery methods, which may help to further expand the possibilities in personalized medicine and the treatment of genetic disorders.
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Affiliation(s)
- Christian S Stevens
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Jillian Carmichael
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Ruth Watkinson
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Shreyas Kowdle
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Rebecca A Reis
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Kory Hamane
- UCLA School of Nursing, Los Angeles, California, 90095
- UCLA AIDS Institute, Los Angeles, California, 90095
| | - Jason Jang
- UCLA School of Nursing, Los Angeles, California, 90095
- UCLA AIDS Institute, Los Angeles, California, 90095
| | - Arnold Park
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Olivier Pernet
- UCLA School of Nursing, Los Angeles, California, 90095
- UCLA AIDS Institute, Los Angeles, California, 90095
| | - Wannisa Khamaikawin
- UCLA School of Nursing, Los Angeles, California, 90095
- UCLA AIDS Institute, Los Angeles, California, 90095
| | - Patrick Hong
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Patricia Thibault
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Aditya Gowlikar
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Dong Sung An
- UCLA School of Nursing, Los Angeles, California, 90095
- UCLA AIDS Institute, Los Angeles, California, 90095
| | - Benhur Lee
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
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3
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Chen Y, Li M, Wu Y. The occurrence and development of induced pluripotent stem cells. Front Genet 2024; 15:1389558. [PMID: 38699229 PMCID: PMC11063328 DOI: 10.3389/fgene.2024.1389558] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2024] [Accepted: 04/08/2024] [Indexed: 05/05/2024] Open
Abstract
The ectopic expression of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc (OSKM), known as "Yamanaka factors," can reprogram or stimulate the production of induced pluripotent stem cells (iPSCs). Although OSKM is still the gold standard, there are multiple ways to reprogram cells into iPSCs. In recent years, significant progress has been made in improving the efficiency of this technology. Ten years after the first report was published, human pluripotent stem cells have gradually been applied in clinical settings, including disease modeling, cell therapy, new drug development, and cell derivation. Here, we provide a review of the discovery of iPSCs and their applications in disease and development.
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Affiliation(s)
| | - Meng Li
- Department of Cardiology, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, China
| | - Yanqing Wu
- Department of Cardiology, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, China
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Grygoryev D, Ekstrom T, Manalo E, Link JM, Alshaikh A, Keith D, Allen-Petersen BL, Sheppard B, Morgan T, Soufi A, Sears RC, Kim J. Sendai virus is robust and consistent in delivering genes into human pancreatic cancer cells. Heliyon 2024; 10:e27221. [PMID: 38463758 PMCID: PMC10923719 DOI: 10.1016/j.heliyon.2024.e27221] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2023] [Revised: 02/22/2024] [Accepted: 02/26/2024] [Indexed: 03/12/2024] Open
Abstract
Background Pancreatic ductal adenocarcinoma (PDAC) is a highly intratumorally heterogeneous disease that includes several subtypes and is highly plastic. Effective gene delivery to all PDAC cells is essential for modulating gene expression and identifying potential gene-based therapeutic targets in PDAC. Most current gene delivery systems for pancreatic cells are optimized for islet or acinar cells. Lentiviral vectors are the current main gene delivery vectors for PDAC, but their transduction efficiencies vary depending on pancreatic cell type, and are especially poor for the classical subtype of PDAC cells from both primary tumors and cell lines. Methods We systemically compare transduction efficiencies of glycoprotein G of vesicular stomatitis virus (VSV-G)-pseudotyped lentiviral and Sendai viral vectors in human normal pancreatic ductal and PDAC cells. Results We find that the Sendai viral vector gives the most robust gene delivery efficiency regardless of PDAC cell type. Therefore, we propose using Sendai viral vectors to transduce ectopic genes into PDAC cells.
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Affiliation(s)
- Dmytro Grygoryev
- Cancer Early Detection Advanced Research Center at Knight Cancer Institute, Oregon Health & Science University School of Medicine, USA
| | - Taelor Ekstrom
- Cancer Early Detection Advanced Research Center at Knight Cancer Institute, Oregon Health & Science University School of Medicine, USA
| | - Elise Manalo
- Cancer Early Detection Advanced Research Center at Knight Cancer Institute, Oregon Health & Science University School of Medicine, USA
| | - Jason M. Link
- Department of Molecular and Medical Genetics, Oregon Health & Science University School of Medicine, USA
- Brenden-Colson Center for Pancreatic Care, Oregon Health & Science University School of Medicine, USA
| | - Amani Alshaikh
- The University of Edinburgh, Centre for Regenerative Medicine, Institute of Regeneration and Repair, Institute of Stem Cell Research, Edinburgh, UK
- King Abdulaziz City for Science and Technology, Health Sector (KACST), Riyadh, Saudi Arabia
| | - Dove Keith
- Brenden-Colson Center for Pancreatic Care, Oregon Health & Science University School of Medicine, USA
| | - Brittany L. Allen-Petersen
- Department of Molecular and Medical Genetics, Oregon Health & Science University School of Medicine, USA
- Brenden-Colson Center for Pancreatic Care, Oregon Health & Science University School of Medicine, USA
| | - Brett Sheppard
- Brenden-Colson Center for Pancreatic Care, Oregon Health & Science University School of Medicine, USA
- Department of Surgery, Oregon Health & Science University School of Medicine, USA
| | - Terry Morgan
- Cancer Early Detection Advanced Research Center at Knight Cancer Institute, Oregon Health & Science University School of Medicine, USA
- Department of Pathology, Oregon Health & Science University School of Medicine, USA
- Cancer Biology Research Program, Knight Cancer Institute, Oregon Health & Science University School of Medicine, Portland, OR, 97201, USA
| | - Abdenour Soufi
- The University of Edinburgh, Centre for Regenerative Medicine, Institute of Regeneration and Repair, Institute of Stem Cell Research, Edinburgh, UK
| | - Rosalie C. Sears
- Department of Molecular and Medical Genetics, Oregon Health & Science University School of Medicine, USA
- Brenden-Colson Center for Pancreatic Care, Oregon Health & Science University School of Medicine, USA
- Cancer Biology Research Program, Knight Cancer Institute, Oregon Health & Science University School of Medicine, Portland, OR, 97201, USA
| | - Jungsun Kim
- Cancer Early Detection Advanced Research Center at Knight Cancer Institute, Oregon Health & Science University School of Medicine, USA
- Department of Molecular and Medical Genetics, Oregon Health & Science University School of Medicine, USA
- Cancer Biology Research Program, Knight Cancer Institute, Oregon Health & Science University School of Medicine, Portland, OR, 97201, USA
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Schaerlaekens S, Jacobs L, Stobbelaar K, Cos P, Delputte P. All Eyes on the Prefusion-Stabilized F Construct, but Are We Missing the Potential of Alternative Targets for Respiratory Syncytial Virus Vaccine Design? Vaccines (Basel) 2024; 12:97. [PMID: 38250910 PMCID: PMC10819635 DOI: 10.3390/vaccines12010097] [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: 12/12/2023] [Revised: 01/12/2024] [Accepted: 01/13/2024] [Indexed: 01/23/2024] Open
Abstract
Respiratory Syncytial Virus (RSV) poses a significant global health concern as a major cause of lower respiratory tract infections (LRTIs). Over the last few years, substantial efforts have been directed towards developing vaccines and therapeutics to combat RSV, leading to a diverse landscape of vaccine candidates. Notably, two vaccines targeting the elderly and the first maternal vaccine have recently been approved. The majority of the vaccines and vaccine candidates rely solely on a prefusion-stabilized conformation known for its highly neutralizing epitopes. Although, so far, this antigen design appears to be successful for the elderly, our current understanding remains incomplete, requiring further improvement and refinement in this field. Pediatric vaccines still have a long journey ahead, and we must ensure that vaccines currently entering the market do not lose efficacy due to the emergence of mutations in RSV's circulating strains. This review will provide an overview of the current status of vaccine designs and what to focus on in the future. Further research into antigen design is essential, including the exploration of the potential of alternative RSV proteins to address these challenges and pave the way for the development of novel and effective vaccines, especially in the pediatric population.
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Affiliation(s)
- Sofie Schaerlaekens
- Laboratory for Microbiology, Parasitology and Hygiene, University of Antwerp (UA), Universiteitsplein 1 S.7, 2610 Antwerp, Belgium; (S.S.); (L.J.); (K.S.); (P.C.)
| | - Lotte Jacobs
- Laboratory for Microbiology, Parasitology and Hygiene, University of Antwerp (UA), Universiteitsplein 1 S.7, 2610 Antwerp, Belgium; (S.S.); (L.J.); (K.S.); (P.C.)
| | - Kim Stobbelaar
- Laboratory for Microbiology, Parasitology and Hygiene, University of Antwerp (UA), Universiteitsplein 1 S.7, 2610 Antwerp, Belgium; (S.S.); (L.J.); (K.S.); (P.C.)
- Pediatrics Department, Antwerp University Hospital (UZA), Wilrijkstraat 10, 2650 Edegem, Belgium
| | - Paul Cos
- Laboratory for Microbiology, Parasitology and Hygiene, University of Antwerp (UA), Universiteitsplein 1 S.7, 2610 Antwerp, Belgium; (S.S.); (L.J.); (K.S.); (P.C.)
- Infla-Med Centre of Excellence, University of Antwerp (UA), Universiteitsplein 1 S.7, 2610 Antwerp, Belgium
| | - Peter Delputte
- Laboratory for Microbiology, Parasitology and Hygiene, University of Antwerp (UA), Universiteitsplein 1 S.7, 2610 Antwerp, Belgium; (S.S.); (L.J.); (K.S.); (P.C.)
- Infla-Med Centre of Excellence, University of Antwerp (UA), Universiteitsplein 1 S.7, 2610 Antwerp, Belgium
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6
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Dhanjal DS, Singh R, Sharma V, Nepovimova E, Adam V, Kuca K, Chopra C. Advances in Genetic Reprogramming: Prospects from Developmental Biology to Regenerative Medicine. Curr Med Chem 2024; 31:1646-1690. [PMID: 37138422 DOI: 10.2174/0929867330666230503144619] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2022] [Revised: 03/13/2023] [Accepted: 03/16/2023] [Indexed: 05/05/2023]
Abstract
The foundations of cell reprogramming were laid by Yamanaka and co-workers, who showed that somatic cells can be reprogrammed into pluripotent cells (induced pluripotency). Since this discovery, the field of regenerative medicine has seen advancements. For example, because they can differentiate into multiple cell types, pluripotent stem cells are considered vital components in regenerative medicine aimed at the functional restoration of damaged tissue. Despite years of research, both replacement and restoration of failed organs/ tissues have remained elusive scientific feats. However, with the inception of cell engineering and nuclear reprogramming, useful solutions have been identified to counter the need for compatible and sustainable organs. By combining the science underlying genetic engineering and nuclear reprogramming with regenerative medicine, scientists have engineered cells to make gene and stem cell therapies applicable and effective. These approaches have enabled the targeting of various pathways to reprogramme cells, i.e., make them behave in beneficial ways in a patient-specific manner. Technological advancements have clearly supported the concept and realization of regenerative medicine. Genetic engineering is used for tissue engineering and nuclear reprogramming and has led to advances in regenerative medicine. Targeted therapies and replacement of traumatized , damaged, or aged organs can be realized through genetic engineering. Furthermore, the success of these therapies has been validated through thousands of clinical trials. Scientists are currently evaluating induced tissue-specific stem cells (iTSCs), which may lead to tumour-free applications of pluripotency induction. In this review, we present state-of-the-art genetic engineering that has been used in regenerative medicine. We also focus on ways that genetic engineering and nuclear reprogramming have transformed regenerative medicine and have become unique therapeutic niches.
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Affiliation(s)
- Daljeet Singh Dhanjal
- School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India
| | - Reena Singh
- School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India
| | - Varun Sharma
- Head of Bioinformatic Division, NMC Genetics India Pvt. Ltd., Gurugram, India
| | - Eugenie Nepovimova
- Department of Chemistry, Faculty of Science, University of Hradec Kralove, Hradec Kralove, 50003, Czech Republic
| | - Vojtech Adam
- Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, Brno, CZ 613 00, Czech Republic
- Central European Institute of Technology, Brno University of Technology, Purkynova 123, Brno, CZ-612 00, Czech Republic
| | - Kamil Kuca
- Department of Chemistry, Faculty of Science, University of Hradec Kralove, Hradec Kralove, 50003, Czech Republic
- Biomedical Research Center, University Hospital Hradec Kralove, Hradec Kralove, 50005, Czech Republic
| | - Chirag Chopra
- School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India
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Nath SC, Menendez L, Friedrich Ben-Nun I. Overcoming the Variability of iPSCs in the Manufacturing of Cell-Based Therapies. Int J Mol Sci 2023; 24:16929. [PMID: 38069252 PMCID: PMC10706975 DOI: 10.3390/ijms242316929] [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: 11/09/2023] [Revised: 11/26/2023] [Accepted: 11/27/2023] [Indexed: 12/18/2023] Open
Abstract
Various factors are known to contribute to the diversity of human induced pluripotent stem cells (hiPSCs). Among these are the donor's genetic background and family history, the somatic cell source, the iPSC reprogramming method, and the culture system of choice. Moreover, variability is seen even in iPSC clones, generated in a single reprogramming event, where the donor, somatic cell type, and reprogramming platform are the same. The diversity seen in iPSC lines often translates to epigenetic differences, as well as to differences in the expansion rate, iPSC line culture robustness, and their ability to differentiate into specific cell types. As such, the diversity of iPSCs presents a hurdle to standardizing iPSC-based cell therapy manufacturing. In this review, we will expand on the various factors that impact iPSC diversity and the strategies and tools that could be taken by the industry to overcome the differences amongst various iPSC lines, therefore enabling robust and reproducible iPSC-based cell therapy manufacturing processes.
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Affiliation(s)
- Suman C. Nath
- Cell Therapy Process Department, Lonza Inc., Houston, TX 77047, USA; (S.C.N.); (L.M.)
| | - Laura Menendez
- Cell Therapy Process Department, Lonza Inc., Houston, TX 77047, USA; (S.C.N.); (L.M.)
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Kwak G, Lee D, Suk JS. Advanced approaches to overcome biological barriers in respiratory and systemic routes of administration for enhanced nucleic acid delivery to the lung. Expert Opin Drug Deliv 2023; 20:1531-1552. [PMID: 37946533 PMCID: PMC10872418 DOI: 10.1080/17425247.2023.2282535] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2023] [Accepted: 11/08/2023] [Indexed: 11/12/2023]
Abstract
INTRODUCTION Numerous delivery strategies, primarily novel nucleic acid delivery carriers, have been developed and explored to enable therapeutically relevant lung gene therapy. However, its clinical translation is yet to be achieved despite over 30 years of efforts, which is attributed to the inability to overcome a series of biological barriers that hamper efficient nucleic acid transfer to target cells in the lung. AREAS COVERED This review is initiated with the fundamentals of nucleic acid therapy and a brief overview of previous and ongoing efforts on clinical translation of lung gene therapy. We then walk through the nature of biological barriers encountered by nucleic acid carriers administered via respiratory and/or systemic routes. Finally, we introduce advanced strategies developed to overcome those barriers to achieve therapeutically relevant nucleic acid delivery efficiency in the lung. EXPERT OPINION We are now stepping close to the clinical translation of lung gene therapy, thanks to the discovery of novel delivery strategies that overcome biological barriers via comprehensive preclinical studies. However, preclinical findings should be cautiously interpreted and validated to ultimately realize meaningful therapeutic outcomes with newly developed delivery strategies in humans. In particular, individual strategies should be selected, tailored, and implemented in a manner directly relevant to specific therapeutic applications and goals.
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Affiliation(s)
- Gijung Kwak
- Department of Neurosurgery and Medicine Institute for Neuroscience Discovery (UM-MIND), University of Maryland School of Medicine, Baltimore, MD, USA
- Center for Nanomedicine, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA
- Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Daiheon Lee
- Department of Neurosurgery and Medicine Institute for Neuroscience Discovery (UM-MIND), University of Maryland School of Medicine, Baltimore, MD, USA
- Center for Nanomedicine, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA
- Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Jung Soo Suk
- Department of Neurosurgery and Medicine Institute for Neuroscience Discovery (UM-MIND), University of Maryland School of Medicine, Baltimore, MD, USA
- Center for Nanomedicine, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA
- Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
- Department of Chemical & Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA
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9
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Morimoto S, Saeki K, Takeshita M, Hirano K, Shirakawa M, Yamada Y, Nakamura S, Ozawa F, Okano H. Intranasal Sendai virus-based SARS-CoV-2 vaccine using a mouse model. Genes Cells 2023; 28:29-41. [PMID: 36401755 DOI: 10.1111/gtc.12992] [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: 09/15/2022] [Revised: 10/26/2022] [Accepted: 11/15/2022] [Indexed: 11/21/2022]
Abstract
The coronavirus disease 2019 (COVID-19) epidemic remains worldwide. The usefulness of the intranasal vaccine and boost immunization against severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) has recently received much attention. We developed an intranasal SARS-CoV-2 vaccine by loading the receptor binding domain of the S protein (S-RBD) of SARS-CoV-2 as an antigen into an F-deficient Sendai virus vector. After the S-RBD-Fd antigen with trimer formation ability was intranasally administered to mice, S-RBD-specific IgM, IgG, IgA, and neutralizing antibody titers were increased in serum or bronchoalveolar lavage fluid for 12 weeks. Furthermore, in mice that received a booster dose at week 8, a marked increase in neutralizing antibodies in the serum and bronchoalveolar lavage fluid was observed at the final evaluation at week 12, which neutralized the pseudotyped lentivirus expressing the SARS-CoV-2 spike protein, indicating the usefulness of the Sendai virus-based SARS-CoV-2 intranasal vaccine.
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Affiliation(s)
- Satoru Morimoto
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan
| | | | - Masaru Takeshita
- Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan
| | | | | | | | - Shiho Nakamura
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan
| | - Fumiko Ozawa
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan
| | - Hideyuki Okano
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan
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10
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Sharp B, Rallabandi R, Devaux P. Advances in RNA Viral Vector Technology to Reprogram Somatic Cells: The Paramyxovirus Wave. Mol Diagn Ther 2022; 26:353-367. [PMID: 35763161 DOI: 10.1007/s40291-022-00599-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/16/2022] [Indexed: 11/24/2022]
Abstract
Ethical issues are a significant barrier to the use of embryonic stem cells in patients due to their origin: human embryos. To further the development of stem cells in a patient application, alternative sources of cells were sought. A process referred to as reprogramming was established to create induced pluripotent stem cells from somatic cells, resolving the ethical issues, and vectors were developed to deliver the reprogramming factors to generate induced pluripotent stem cells. Early viral vectors used integrating retroviruses and lentiviruses as delivery vehicles for the transcription factors required to initiate reprogramming. However, because of the inherent risk associated with vectors that integrate into the host genome, non-integrating approaches were explored. The development of non-integrating viral vectors offers a safer alternative, and these modern vectors are reliable, efficient, and easy to use to achieve induced pluripotent stem cells suitable for direct patient application in the growing field of individualized medicine. This review summarizes all the RNA viral vectors in the field of reprogramming with a special focus on the emerging delivery vectors based on non-integrating Paramyxoviruses, Sendai and measles viruses. We discuss their design and evolution towards being safe and efficient reprogramming vectors in generating induced pluripotent stem cells from somatic cells.
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Affiliation(s)
- Brenna Sharp
- Department of Molecular Medicine, Mayo Clinic, Rochester, MN, 55905, USA
| | - Ramya Rallabandi
- Virology and Gene Therapy Graduate Program, Mayo Clinic, Rochester, MN, USA.,Regenerative Sciences Program, Mayo Clinic, Rochester, MN, USA
| | - Patricia Devaux
- Department of Molecular Medicine, Mayo Clinic, Rochester, MN, 55905, USA. .,Virology and Gene Therapy Graduate Program, Mayo Clinic, Rochester, MN, USA. .,Regenerative Sciences Program, Mayo Clinic, Rochester, MN, USA.
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11
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Ohira M, Kikuchi E, Mizuta S, Yoshida N, Onodera M, Nakanishi M, Okuyama T, Mashima R. Production of therapeutic iduronate-2-sulfatase enzyme with a novel single-stranded RNA virus vector. Genes Cells 2021; 26:891-904. [PMID: 34480399 DOI: 10.1111/gtc.12894] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2021] [Revised: 08/04/2021] [Accepted: 08/20/2021] [Indexed: 11/28/2022]
Abstract
The Sendai virus vector has received a lot of attention due to its broad tropism for mammalian cells. As a result of efforts for genetic studies based on a mutant virus, we can now express more than 10 genes of up to 13.5 kilo nucleotides in a single vector with high protein expression efficiency. To prove this benefit, we examined the efficacy of the novel ribonucleic acid (RNA) virus vector harboring the human iduronate-2-sulfatase (IDS) gene with 1,653 base pairs, a causative gene for mucopolysaccharidosis type II, also known as a disorder of lysosomal storage disorders. As expected, this novel RNA vector with the human IDS gene exhibited its marked expression as determined by the expression of enhanced green fluorescent protein and IDS enzyme activity. While these cells exhibited a normal growth rate, the BHK-21 transformant cells stably expressing the human IDS gene persistently generated an active human IDS enzyme extracellularly. The human IDS protein produced failed to be incorporated into the lysosome when cells were pretreated with mannose-6-phosphate, demonstrating that this human IDS enzyme has potential for therapeutic use by cross-correction. These results suggest that our novel RNA vector may be applicable for further clinical settings.
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Affiliation(s)
- Mari Ohira
- Department of Clinical Laboratory Medicine, National Center for Child Health and Development, Tokyo, Japan
| | - Emika Kikuchi
- Department of Clinical Laboratory Medicine, National Center for Child Health and Development, Tokyo, Japan
| | | | | | - Masafumi Onodera
- Department of Human Genetics, National Research Institute for Child Health and Development, Tokyo, Japan
| | | | - Torayuki Okuyama
- Department of Clinical Laboratory Medicine, National Center for Child Health and Development, Tokyo, Japan
| | - Ryuichi Mashima
- Department of Clinical Laboratory Medicine, National Center for Child Health and Development, Tokyo, Japan
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12
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iPSC Preparation and Epigenetic Memory: Does the Tissue Origin Matter? Cells 2021; 10:cells10061470. [PMID: 34208270 PMCID: PMC8230744 DOI: 10.3390/cells10061470] [Citation(s) in RCA: 38] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2021] [Revised: 06/06/2021] [Accepted: 06/10/2021] [Indexed: 02/07/2023] Open
Abstract
The production of induced pluripotent stem cells (iPSCs) represent a breakthrough in regenerative medicine, providing new opportunities for understanding basic molecular mechanisms of human development and molecular aspects of degenerative diseases. In contrast to human embryonic stem cells (ESCs), iPSCs do not raise any ethical concerns regarding the onset of human personhood. Still, they present some technical issues related to immune rejection after transplantation and potential tumorigenicity, indicating that more steps forward must be completed to use iPSCs as a viable tool for in vivo tissue regeneration. On the other hand, cell source origin may be pivotal to iPSC generation since residual epigenetic memory could influence the iPSC phenotype and transplantation outcome. In this paper, we first review the impact of reprogramming methods and the choice of the tissue of origin on the epigenetic memory of the iPSCs or their differentiated cells. Next, we describe the importance of induction methods to determine the reprogramming efficiency and avoid integration in the host genome that could alter gene expression. Finally, we compare the significance of the tissue of origin and the inter-individual genetic variation modification that has been lightly evaluated so far, but which significantly impacts reprogramming.
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13
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Mercier J, Ruffin M, Corvol H, Guillot L. Gene Therapy: A Possible Alternative to CFTR Modulators? Front Pharmacol 2021; 12:648203. [PMID: 33967785 PMCID: PMC8097140 DOI: 10.3389/fphar.2021.648203] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2020] [Accepted: 02/24/2021] [Indexed: 12/12/2022] Open
Abstract
Cystic fibrosis (CF) is a rare genetic disease that affects several organs, but lung disease is the major cause of morbidity and mortality. The gene responsible for CF, the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene, has been discovered in 1989. Since then, gene therapy i.e., defective gene replacement by a functional one, remained the ultimate goal but unfortunately, it has not yet been achieved. However, patients care and symptomatic treatments considerably increased CF patients’ life expectancy ranging from 5 years old in the 1960s to 40 today. In the last decade, research works on CFTR protein structure and activity led to the development of new drugs which, by readdressing CFTR to the plasma membrane (correctors) or by enhancing its transport activity (potentiators), allow, alone or in combination, an improvement of CF patients’ lung function and quality of life. While expected, it is not yet known whether taking these drugs from an early age and for years will improve the quality of life of CF patients in the long term and further increase their life expectancy. Besides, these molecules are not available (specific variants of CFTR) or accessible (national health policies) for all patients and there is still no curative treatment. Another alternative that could benefit from new technologies, such as gene therapy, is therefore still attractive, although it is not yet offered to patients. Faced with the development of new CFTR correctors and potentiators, the question arises as to whether there is still a place for gene therapy and this is discussed in this perspective.
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Affiliation(s)
- J Mercier
- Sorbonne Université, Inserm, Centre de Recherche, Saint Antoine, F-75012, Paris, France
| | - M Ruffin
- Sorbonne Université, Inserm, Centre de Recherche, Saint Antoine, F-75012, Paris, France
| | - H Corvol
- Sorbonne Université, Inserm, Centre de Recherche, Saint Antoine, F-75012, Paris, France.,Pneumologie Pédiatrique, APHP, Hôpital Trousseau, Paris, France
| | - L Guillot
- Sorbonne Université, Inserm, Centre de Recherche, Saint Antoine, F-75012, Paris, France
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14
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Successes and Challenges: Inhaled Treatment Approaches Using Magnetic Nanoparticles in Cystic Fibrosis. MAGNETOCHEMISTRY 2020. [DOI: 10.3390/magnetochemistry6020025] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Magnetic nanoparticles have been largely applied to increase the efficacy of antibiotics due to passive accumulation provided by enhancing permeability and retention, which is essential for the treatment of lung infections. Recurring lung infections such as in the life-shortening genetic disease cystic fibrosis (CF) are a major problem. The recent advent of the CF modulator drug ivacaftor, alone or in combination with lumacaftor or tezacaftor, has enabled systemic treatment of the majority of patients. Magnetic nanoparticles (MNPs) show unique properties such as biocompatibility and biodegradability as well as magnetic and heat-medicated characteristics. These properties make them suitable to be used as drug carriers and hyperthermia-based agents. Hyperthermia is a promising approach for the thermal activation therapy of several diseases, including pulmonary diseases. The benefits of delivering CF drugs via inhalation using MNPs as drug carriers afford application of sufficient therapeutic dosages directly to the primary target site, while avoiding potential suboptimal pharmacokinetics/pharmacodynamics and minimizing the risks of systemic toxicity. This review explores the multidisciplinary approach of using MNPs as vehicles of drug delivery. Additionally, we highlight advantages such as increased drug concentration at disease site, minimized drug loss and the possibility of specific cell targeting, while addressing major challenges for this emerging field.
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15
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Al Abbar A, Ngai SC, Nograles N, Alhaji SY, Abdullah S. Induced Pluripotent Stem Cells: Reprogramming Platforms and Applications in Cell Replacement Therapy. Biores Open Access 2020; 9:121-136. [PMID: 32368414 PMCID: PMC7194323 DOI: 10.1089/biores.2019.0046] [Citation(s) in RCA: 51] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/27/2020] [Indexed: 12/15/2022] Open
Abstract
The generation of induced pluripotent stem cells (iPSCs) from differentiated mature cells is one of the most promising technologies in the field of regenerative medicine. The ability to generate patient-specific iPSCs offers an invaluable reservoir of pluripotent cells, which could be genetically engineered and differentiated into target cells to treat various genetic and degenerative diseases once transplanted, hence counteracting the risk of graft versus host disease. In this context, we review the scientific research streams that lead to the emergence of iPSCs, the roles of reprogramming factors in reprogramming to pluripotency, and the reprogramming strategies. As iPSCs serve tremendous correction potentials for various diseases, we highlight the successes and challenges of iPSCs in cell replacement therapy and the synergy of iPSCs and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing tools in therapeutics research.
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Affiliation(s)
- Akram Al Abbar
- Medical Genetics Laboratory, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia
| | - Siew Ching Ngai
- School of Biosciences, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Malaysia
| | - Nadine Nograles
- Newcastle University Medicine Malaysia, Educity, Iskandar Puteri, Johor, Malaysia
| | - Suleiman Yusuf Alhaji
- Medical Genetics Laboratory, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia
| | - Syahril Abdullah
- Medical Genetics Laboratory, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia
- UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, Serdang, Malaysia
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16
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Beyond cystic fibrosis transmembrane conductance regulator therapy: a perspective on gene therapy and small molecule treatment for cystic fibrosis. Gene Ther 2019; 26:354-362. [PMID: 31300729 DOI: 10.1038/s41434-019-0092-5] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2018] [Revised: 04/07/2019] [Accepted: 06/25/2019] [Indexed: 12/30/2022]
Abstract
Cystic fibrosis (CF) is a life-limiting disease caused by defective or deficient cystic fibrosis transmembrane conductance regulator (CFTR) activity. The recent advent of the FDA-approved CFTR modulator drug ivacaftor, alone or in combination with lumacaftor or tezacaftor, has enabled treatment of the majority of patients suffering from CF. Even before the identification of the CFTR gene, gene therapy was put forward as a viable treatment option for this genetic condition. However, initial enthusiasm has been hampered as CFTR gene delivery to the lungs has proven to be more challenging than expected. This review covers the contemporary clinical and scientific knowledge base for small molecule CFTR modulator drug therapy, gene delivery vectors and CRISPR/Cas9 gene editing and highlights the prospect of these technologies for future treatment options.
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17
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Sequential actions of immune effector cells induced by viral activation of dendritic cells to eliminate murine neuroblastoma. J Pediatr Surg 2018; 53:1615-1620. [PMID: 28941928 DOI: 10.1016/j.jpedsurg.2017.08.019] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/14/2017] [Revised: 07/16/2017] [Accepted: 08/22/2017] [Indexed: 11/24/2022]
Abstract
PURPOSE In preclinical trails, we reported the antitumor effect of dendritic cells activated with Sendai virus (rSeV/DC) combined with γ-irradiation against neuroblastoma. However, what kind of effector cells for the combined therapy were used to show the antitumor effect was unclear. In this study, we performed radiation and rSeV/DC therapy in vivo and examined the effector cells involved. METHODS Dendritic cells were cultured from bone marrow cells, activated with SeV and administered intratumorally at 106 weekly for 3weeks. Radiation was administered at 4Gy/time × 3 times. During the treatment, CD4+ and CD8+ cells and natural killer (NK) cells were removed by antibodies. RESULTS Complete remission of neuroblastoma was observed in 62.5% of individuals in the combined therapy group. By depleting the effector cells using antibodies, the tumor increased in size from an early stage of treatment in the CD4+ and NK cell-depleted group. In contrast, the tumor increased in size in the late stage of treatment in the CD8+ cell-depleted group. CONCLUSION The combination of radiation and rSeV/DC therapy induces different effector cells, depending on the time point during treatment. LEVEL OF EVIDENCE V.
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18
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Araki K, Suzuki H, Uno K, Tomifuji M, Shiotani A. Gene Therapy for Recurrent Laryngeal Nerve Injury. Genes (Basel) 2018; 9:E316. [PMID: 29941853 PMCID: PMC6071248 DOI: 10.3390/genes9070316] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2018] [Accepted: 06/20/2018] [Indexed: 11/23/2022] Open
Abstract
Recurrent laryngeal nerve (RLN) injury has considerable clinical implications, including voice and swallowing dysfunction, which may considerably impair the patient’s quality of life. Recovery of vocal fold movement is an essential novel treatment option for RLN injury. The potential of gene therapy for addressing this issue is highly promising. The target sites for RLN gene therapy are the central nervous system, nerve fibers, laryngeal muscles, and vocal cord mucosa. Gene transduction has been reported in each site using viral or non-viral methods. The major issues ensuing after RLN injury are loss of motoneurons in the nucleus ambiguus, degeneration and poor regeneration of nerve fibers and motor end plates, and laryngeal muscle atrophy. Gene therapy using neurotrophic factors has been assessed for most of these issues, and its efficacy has been reported. Another important matter for functional vocal fold movement recovery is misdirected regeneration, in which the wrong neurons may innervate other laryngeal muscles, where even if innervation is reestablished, proper motor function is not restored. Novel strategies involving gene therapy bear promise for overcoming this issue and further investigations are underway.
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Affiliation(s)
- Koji Araki
- Department of Otolaryngology-Head & Neck Surgery, National Defense Medical College, Saitama 3598513, Japan.
| | - Hiroshi Suzuki
- Department of Otolaryngology-Head & Neck Surgery, National Defense Medical College, Saitama 3598513, Japan.
| | - Kosuke Uno
- Department of Otolaryngology-Head & Neck Surgery, National Defense Medical College, Saitama 3598513, Japan.
| | - Masayuki Tomifuji
- Department of Otolaryngology-Head & Neck Surgery, National Defense Medical College, Saitama 3598513, Japan.
| | - Akihiro Shiotani
- Department of Otolaryngology-Head & Neck Surgery, National Defense Medical College, Saitama 3598513, Japan.
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19
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A Bond-Fluctuation Model of Translational Dynamics of Chain-like Particles through Mucosal Scaffolds. Biophys J 2018; 114:2732-2742. [PMID: 29874621 DOI: 10.1016/j.bpj.2018.04.031] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2017] [Revised: 03/24/2018] [Accepted: 04/18/2018] [Indexed: 11/22/2022] Open
Abstract
Mucus scaffolds represent one of the most common barriers in targeted drug delivery and can remarkably reduce the outcome of pharmacological therapies. An efficient transport of drug particles through a mucus barrier is a precondition for an efficient drug delivery. Understanding the transport mechanism is particularly important for treatment of disorders such as cystic fibrosis. These are characterized by an onset of high-density mucus scaffolds imposing an increased steric filtering. In this study, we employed the bond-fluctuation model to analyze the effect of steric interactions on slowing the translational dynamics of compound chain-like particles traversing through scaffolds of different configurations (regular isotropic and anisotropic versus irregular random). The model, which accounts for both the geometry-imposed steric interaction as well as the intrachain steric interaction between the chain subunits, yields a transient subdiffusive motional pattern persists between the short-time and long-time Gaussian diffusion limits. The motion is analyzed in terms of a mean-squared displacement, diffusion coefficient, and radius of gyration. With higher levels of restriction or larger particles, the subdiffusive motional regime persists longer. The study also demonstrates that an important feature of the motion is also geometry-induced chain accommodation. The presented model is generic and could also be applied to studying the translational dynamics of other particles with more complex architecture such as dendrites or chain-decorated nanoparticles.
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20
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Miyamoto K, Akiyama M, Tamura F, Isomi M, Yamakawa H, Sadahiro T, Muraoka N, Kojima H, Haginiwa S, Kurotsu S, Tani H, Wang L, Qian L, Inoue M, Ide Y, Kurokawa J, Yamamoto T, Seki T, Aeba R, Yamagishi H, Fukuda K, Ieda M. Direct In Vivo Reprogramming with Sendai Virus Vectors Improves Cardiac Function after Myocardial Infarction. Cell Stem Cell 2017; 22:91-103.e5. [PMID: 29276141 DOI: 10.1016/j.stem.2017.11.010] [Citation(s) in RCA: 126] [Impact Index Per Article: 15.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2017] [Revised: 05/31/2017] [Accepted: 11/07/2017] [Indexed: 12/11/2022]
Abstract
Direct cardiac reprogramming holds great promise for regenerative medicine. We previously generated directly reprogrammed induced cardiomyocyte-like cells (iCMs) by overexpression of Gata4, Mef2c, and Tbx5 (GMT) using retrovirus vectors. However, integrating vectors pose risks associated with insertional mutagenesis and disruption of gene expression and are inefficient. Here, we show that Sendai virus (SeV) vectors expressing cardiac reprogramming factors efficiently and rapidly reprogram both mouse and human fibroblasts into integration-free iCMs via robust transgene expression. SeV-GMT generated 100-fold more beating iCMs than retroviral-GMT and shortened the duration to induce beating cells from 30 to 10 days in mouse fibroblasts. In vivo lineage tracing revealed that the gene transfer of SeV-GMT was more efficient than retroviral-GMT in reprogramming resident cardiac fibroblasts into iCMs in mouse infarct hearts. Moreover, SeV-GMT improved cardiac function and reduced fibrosis after myocardial infarction. Thus, efficient, non-integrating SeV vectors may serve as a powerful system for cardiac regeneration.
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Affiliation(s)
- Kazutaka Miyamoto
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Mizuha Akiyama
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Fumiya Tamura
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Mari Isomi
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Hiroyuki Yamakawa
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Taketaro Sadahiro
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Naoto Muraoka
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Hidenori Kojima
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Sho Haginiwa
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Shota Kurotsu
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Hidenori Tani
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Li Wang
- Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Li Qian
- Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA
| | | | - Yoshinori Ide
- Pharmacological Evaluation Institute of Japan, Center for Pharmacological Science, 3-25-22-424 Tonomachi, Kawasaki-ku, Kawasaki-shi, Kanagawa 210-0821, Japan
| | - Junko Kurokawa
- Department of Bio-informational Pharmacology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka-shi, Shizuoka 422-8526, Japan
| | - Tsunehisa Yamamoto
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Tomohisa Seki
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Ryo Aeba
- Division of Cardiovascular Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Hiroyuki Yamagishi
- Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Keiichi Fukuda
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Masaki Ieda
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
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21
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Carvalho M, Sepodes B, Martins AP. Regulatory and Scientific Advancements in Gene Therapy: State-of-the-Art of Clinical Applications and of the Supporting European Regulatory Framework. Front Med (Lausanne) 2017; 4:182. [PMID: 29124055 PMCID: PMC5662580 DOI: 10.3389/fmed.2017.00182] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2017] [Accepted: 10/11/2017] [Indexed: 12/21/2022] Open
Abstract
Advanced therapy medicinal products (ATMPs) have a massive potential to address existing unmet medical needs. Specifically, gene therapy medicinal products (GTMPs) may potentially provide cure for several genetic diseases. In Europe, the ATMP regulation was fully implemented in 2009 and, at this point, the Committee for Advanced Therapies was created as a dedicated group of specialists to evaluate medicinal products requiring specific expertise in this area. To date, there are three authorized GTMPs, and the first one was approved in 2012. Broad research has been conducted in this field over the last few decades and different clinical applications are being investigated worldwide, using different strategies that range from direct gene replacement or addition to more complex pathways such as specific gene editing or RNA targeting. Important safety risks, limited efficacy, manufacturing hurdles, or ethical conflicts may represent challenges in the success of a candidate GTMP. During the development process, it is fundamental to take such aspects into account and establish overcoming strategies. This article reviews the current European legal framework of ATMPs, provides an overview of the clinical applications for approved and investigational GTMPs, and discusses critical challenges in the development of GTMPs.
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Affiliation(s)
- Marta Carvalho
- Faculdade de Farmácia, Research Institute for Medicines and Pharmaceutical Sciences (iMed.ULisboa), Universidade de Lisboa, Lisboa, Portugal
| | - Bruno Sepodes
- Faculdade de Farmácia, Research Institute for Medicines and Pharmaceutical Sciences (iMed.ULisboa), Universidade de Lisboa, Lisboa, Portugal
| | - Ana Paula Martins
- Faculdade de Farmácia, Research Institute for Medicines and Pharmaceutical Sciences (iMed.ULisboa), Universidade de Lisboa, Lisboa, Portugal
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22
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Medrano RF, Hunger A, Mendonça SA, Barbuto JAM, Strauss BE. Immunomodulatory and antitumor effects of type I interferons and their application in cancer therapy. Oncotarget 2017; 8:71249-71284. [PMID: 29050360 PMCID: PMC5642635 DOI: 10.18632/oncotarget.19531] [Citation(s) in RCA: 128] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2017] [Accepted: 07/12/2017] [Indexed: 02/07/2023] Open
Abstract
During the last decades, the pleiotropic antitumor functions exerted by type I interferons (IFNs) have become universally acknowledged, especially their role in mediating interactions between the tumor and the immune system. Indeed, type I IFNs are now appreciated as a critical component of dendritic cell (DC) driven T cell responses to cancer. Here we focus on IFN-α and IFN-β, and their antitumor effects, impact on immune responses and their use as therapeutic agents. IFN-α/β share many properties, including activation of the JAK-STAT signaling pathway and induction of a variety of cellular phenotypes. For example, type I IFNs drive not only the high maturation status of DCs, but also have a direct impact in cytotoxic T lymphocytes, NK cell activation, induction of tumor cell death and inhibition of angiogenesis. A variety of stimuli, including some standard cancer treatments, promote the expression of endogenous IFN-α/β, which then participates as a fundamental component of immunogenic cell death. Systemic treatment with recombinant protein has been used for the treatment of melanoma. The induction of endogenous IFN-α/β has been tested, including stimulation through pattern recognition receptors. Gene therapies involving IFN-α/β have also been described. Thus, harnessing type I IFNs as an effective tool for cancer therapy continues to be studied.
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Affiliation(s)
- Ruan F.V. Medrano
- Viral Vector Laboratory, Center for Translational Investigation in Oncology, Cancer Institute of São Paulo/LIM 24, University of São Paulo School of Medicine, São Paulo, Brazil
| | - Aline Hunger
- Viral Vector Laboratory, Center for Translational Investigation in Oncology, Cancer Institute of São Paulo/LIM 24, University of São Paulo School of Medicine, São Paulo, Brazil
| | - Samir Andrade Mendonça
- Viral Vector Laboratory, Center for Translational Investigation in Oncology, Cancer Institute of São Paulo/LIM 24, University of São Paulo School of Medicine, São Paulo, Brazil
| | - José Alexandre M. Barbuto
- Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
- Cell and Molecular Therapy Center, NUCEL-NETCEM, University of São Paulo, São Paulo, Brazil
| | - Bryan E. Strauss
- Viral Vector Laboratory, Center for Translational Investigation in Oncology, Cancer Institute of São Paulo/LIM 24, University of São Paulo School of Medicine, São Paulo, Brazil
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23
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Sondhi D, Stiles KM, De BP, Crystal RG. Genetic Modification of the Lung Directed Toward Treatment of Human Disease. Hum Gene Ther 2017; 28:3-84. [PMID: 27927014 DOI: 10.1089/hum.2016.152] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Genetic modification therapy is a promising therapeutic strategy for many diseases of the lung intractable to other treatments. Lung gene therapy has been the subject of numerous preclinical animal experiments and human clinical trials, for targets including genetic diseases such as cystic fibrosis and α1-antitrypsin deficiency, complex disorders such as asthma, allergy, and lung cancer, infections such as respiratory syncytial virus (RSV) and Pseudomonas, as well as pulmonary arterial hypertension, transplant rejection, and lung injury. A variety of viral and non-viral vectors have been employed to overcome the many physical barriers to gene transfer imposed by lung anatomy and natural defenses. Beyond the treatment of lung diseases, the lung has the potential to be used as a metabolic factory for generating proteins for delivery to the circulation for treatment of systemic diseases. Although much has been learned through a myriad of experiments about the development of genetic modification of the lung, more work is still needed to improve the delivery vehicles and to overcome challenges such as entry barriers, persistent expression, specific cell targeting, and circumventing host anti-vector responses.
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Affiliation(s)
- Dolan Sondhi
- Department of Genetic Medicine, Weill Cornell Medical College , New York, New York
| | - Katie M Stiles
- Department of Genetic Medicine, Weill Cornell Medical College , New York, New York
| | - Bishnu P De
- Department of Genetic Medicine, Weill Cornell Medical College , New York, New York
| | - Ronald G Crystal
- Department of Genetic Medicine, Weill Cornell Medical College , New York, New York
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24
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Wiegand MA, Gori-Savellini G, Gandolfo C, Papa G, Kaufmann C, Felder E, Ginori A, Disanto MG, Spina D, Cusi MG. A Respiratory Syncytial Virus Vaccine Vectored by a Stable Chimeric and Replication-Deficient Sendai Virus Protects Mice without Inducing Enhanced Disease. J Virol 2017; 91:e02298-16. [PMID: 28250126 PMCID: PMC5411584 DOI: 10.1128/jvi.02298-16] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2016] [Accepted: 02/21/2017] [Indexed: 11/20/2022] Open
Abstract
Respiratory syncytial virus (RSV) is a major cause of severe respiratory infections in children and elderly people, and no marketed vaccine exists. In this study, we generated and analyzed a subunit vaccine against RSV based on a novel genome replication-deficient Sendai virus (SeV) vector. We inserted the RSV F protein, known to be a genetically stable antigen, into our vector in a specific way to optimize the vaccine features. By exchanging the ectodomain of the SeV F protein for its counterpart from RSV, we created a chimeric vectored vaccine that contains the RSV F protein as an essential structural component. In this way, the antigen is actively expressed on the surfaces of vaccine particles in its prefusion conformation, and as recently reported for other vectored vaccines, the occurrence of silencing mutations of the transgene in the vaccine genome can be prevented. In addition, its active gene expression contributes to further stimulation of the immune response. In order to understand the best route of immunization, we compared vaccine efficacies after intranasal (i.n.) or intramuscular (i.m.) immunization of BALB/c mice. Via both routes, substantial RSV-specific immune responses were induced, consisting of serum IgG and neutralizing antibodies, as well as cytotoxic T cells. Moreover, i.n. immunization was also able to stimulate specific mucosal IgA in the upper and lower respiratory tract. In virus challenge experiments, animals were protected against RSV infection after both i.n. and i.m. immunization without inducing vaccine-enhanced disease. Above all, the replication-deficient SeV appeared to be safe and well tolerated.IMPORTANCE Respiratory syncytial virus (RSV) is a major cause of respiratory diseases in young children and elderly people worldwide. There is a great demand for a licensed vaccine. Promising existing vaccine approaches based on live-attenuated vaccines or viral vectors have suffered from unforeseen drawbacks related to immunogenicity and attenuation. We provide a novel RSV vaccine concept based on a genome replication-deficient Sendai vector that has many favorable vaccine characteristics. The specific vaccine design guarantees genetic stability of the transgene; furthermore, it supports a favorable presentation of the antigen, activating the adaptive response, features that other vectored vaccine approaches have often had difficulties with. Wide immunological and pathological analyses in mice confirmed the validity and efficacy of this approach after both parenteral and mucosal administration. Above all, this concept is suitable for initiating clinical studies, and it could also be applied to other infectious diseases.
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MESH Headings
- Animals
- Antibodies, Neutralizing/immunology
- Antibodies, Viral/immunology
- Female
- Genetic Vectors
- Immunization
- Immunoglobulin A/immunology
- Immunoglobulin G/blood
- Mice
- Mice, Inbred BALB C
- Respiratory Syncytial Virus Infections/prevention & control
- Respiratory Syncytial Virus Infections/virology
- Respiratory Syncytial Virus Vaccines/administration & dosage
- Respiratory Syncytial Virus Vaccines/chemistry
- Respiratory Syncytial Virus Vaccines/genetics
- Respiratory Syncytial Virus Vaccines/immunology
- Respiratory Syncytial Virus, Human/genetics
- Respiratory Syncytial Virus, Human/immunology
- Respiratory Syncytial Virus, Human/physiology
- Sendai virus/genetics
- Sendai virus/immunology
- Vaccines, Attenuated
- Vaccines, Subunit/administration & dosage
- Vaccines, Subunit/chemistry
- Vaccines, Subunit/genetics
- Vaccines, Subunit/immunology
- Viral Fusion Proteins/genetics
- Viral Fusion Proteins/immunology
- Virus Replication
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Affiliation(s)
| | - Gianni Gori-Savellini
- Department of Medical Biotechnologies, Microbiology Section, University of Siena, Siena, Italy
| | - Claudia Gandolfo
- Department of Medical Biotechnologies, Microbiology Section, University of Siena, Siena, Italy
| | - Guido Papa
- Department of Medical Biotechnologies, Microbiology Section, University of Siena, Siena, Italy
| | | | - Eva Felder
- AmVac Research GmbH, Martinsried, Germany
| | - Alessandro Ginori
- Department of Medical Sciences, Surgery and Neurosciences, University of Siena, Siena, Italy
| | - Maria Giulia Disanto
- Department of Medical Sciences, Surgery and Neurosciences, University of Siena, Siena, Italy
| | - Donatella Spina
- Department of Medical Sciences, Surgery and Neurosciences, University of Siena, Siena, Italy
| | - Maria Grazia Cusi
- Department of Medical Biotechnologies, Microbiology Section, University of Siena, Siena, Italy
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Transdifferentiation and reprogramming: Overview of the processes, their similarities and differences. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2017; 1864:1359-1369. [PMID: 28460880 DOI: 10.1016/j.bbamcr.2017.04.017] [Citation(s) in RCA: 55] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/08/2017] [Revised: 04/24/2017] [Accepted: 04/26/2017] [Indexed: 12/24/2022]
Abstract
Reprogramming, or generation of induced pluripotent stem (iPS) cells (functionally similar to embryonic stem cells or ES cells) by the use of transcription factors (typically: Oct3/4, Sox2, c-Myc, Klf4) called "Yamanaka factors" (OSKM), has revolutionized regenerative medicine. However, factors used to induce stemness are also overexpressed in cancer. Both, ES cells and iPS cells cause teratoma formation when injected to tissues. This raises a safety concern for therapies based on iPS derivates. Transdifferentiation (lineage reprogramming, or -conversion), is a process in which one mature, specialized cell type changes into another without entering a pluripotent state. This process involves an ectopic expression of transcription factors and/or other stimuli. Unlike in the case of reprogramming, tissues obtained by this method do not carry the risk of subsequent teratomagenesis.
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26
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Liu Y, Zheng Y, Li S, Xue H, Schmitt K, Hergenroeder GW, Wu J, Zhang Y, Kim DH, Cao Q. Human neural progenitors derived from integration-free iPSCs for SCI therapy. Stem Cell Res 2017; 19:55-64. [PMID: 28073086 PMCID: PMC5629634 DOI: 10.1016/j.scr.2017.01.004] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/17/2016] [Revised: 12/19/2016] [Accepted: 01/03/2017] [Indexed: 01/16/2023] Open
Abstract
As a potentially unlimited autologous cell source, patient induced pluripotent stem cells (iPSCs) provide great capability for tissue regeneration, particularly in spinal cord injury (SCI). However, despite significant progress made in translation of iPSC-derived neural progenitor cells (NPCs) to clinical settings, a few hurdles remain. Among them, non-invasive approach to obtain source cells in a timely manner, safer integration-free delivery of reprogramming factors, and purification of NPCs before transplantation are top priorities to overcome. In this study, we developed a safe and cost-effective pipeline to generate clinically relevant NPCs. We first isolated cells from patients' urine and reprogrammed them into iPSCs by non-integrating Sendai viral vectors, and carried out experiments on neural differentiation. NPCs were purified by A2B5, an antibody specifically recognizing a glycoganglioside on the cell surface of neural lineage cells, via fluorescence activated cell sorting. Upon further in vitro induction, NPCs were able to give rise to neurons, oligodendrocytes and astrocytes. To test the functionality of the A2B5+ NPCs, we grafted them into the contused mouse thoracic spinal cord. Eight weeks after transplantation, the grafted cells survived, integrated into the injured spinal cord, and differentiated into neurons and glia. Our specific focus on cell source, reprogramming, differentiation and purification method purposely addresses timing and safety issues of transplantation to SCI models. It is our belief that this work takes one step closer on using human iPSC derivatives to SCI clinical settings.
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Affiliation(s)
- Ying Liu
- Department of Neurosurgery, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA; Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA; The Senator Lloyd & B.A. Bentsen Center for Stroke Research, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA.
| | - Yiyan Zheng
- Department of Neurosurgery, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA; Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Shenglan Li
- Department of Neurosurgery, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA; Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Haipeng Xue
- Department of Neurosurgery, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA; Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Karl Schmitt
- Department of Neurosurgery, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Georgene W Hergenroeder
- Department of Neurosurgery, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Jiaqian Wu
- Department of Neurosurgery, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA; Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA; The Senator Lloyd & B.A. Bentsen Center for Stroke Research, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Yuanyuan Zhang
- Wake Forest Institute for Regenerative Medicine, Wake Forest Health Sciences, 391 Technology Way, Winston-Salem, NC 27101, USA
| | - Dong H Kim
- Department of Neurosurgery, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA; Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Qilin Cao
- Department of Neurosurgery, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA; Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA; The Senator Lloyd & B.A. Bentsen Center for Stroke Research, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA.
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28
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Nyombayire J, Anzala O, Gazzard B, Karita E, Bergin P, Hayes P, Kopycinski J, Omosa-Manyonyi G, Jackson A, Bizimana J, Farah B, Sayeed E, Parks CL, Inoue M, Hironaka T, Hara H, Shu T, Matano T, Dally L, Barin B, Park H, Gilmour J, Lombardo A, Excler JL, Fast P, Laufer DS, Cox JH. First-in-Human Evaluation of the Safety and Immunogenicity of an Intranasally Administered Replication-Competent Sendai Virus-Vectored HIV Type 1 Gag Vaccine: Induction of Potent T-Cell or Antibody Responses in Prime-Boost Regimens. J Infect Dis 2016; 215:95-104. [PMID: 28077588 PMCID: PMC5225252 DOI: 10.1093/infdis/jiw500] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2016] [Accepted: 10/13/2016] [Indexed: 11/22/2022] Open
Abstract
Background. We report the first-in-human safety and immunogenicity assessment of a prototype intranasally administered, replication-competent Sendai virus (SeV)–vectored, human immunodeficiency virus type 1 (HIV-1) vaccine. Methods. Sixty-five HIV-1–uninfected adults in Kenya, Rwanda, and the United Kingdom were assigned to receive 1 of 4 prime-boost regimens (administered at 0 and 4 months, respectively; ratio of vaccine to placebo recipients, 12:4): priming with a lower-dose SeV-Gag given intranasally, followed by boosting with an adenovirus 35–vectored vaccine encoding HIV-1 Gag, reverse transcriptase, integrase, and Nef (Ad35-GRIN) given intramuscularly (SLA); priming with a higher-dose SeV-Gag given intranasally, followed by boosting with Ad35-GRIN given intramuscularly (SHA); priming with Ad35-GRIN given intramuscularly, followed by boosting with a higher-dose SeV-Gag given intranasally (ASH); and priming and boosting with a higher-dose SeV-Gag given intranasally (SHSH). Results. All vaccine regimens were well tolerated. Gag-specific IFN-γ enzyme-linked immunospot–determined response rates and geometric mean responses were higher (96% and 248 spot-forming units, respectively) in groups primed with SeV-Gag and boosted with Ad35-GRIN (SLA and SHA) than those after a single dose of Ad35-GRIN (56% and 54 spot-forming units, respectively) or SeV-Gag (55% and 59 spot-forming units, respectively); responses persisted for ≥8 months after completion of the prime-boost regimen. Functional CD8+ T-cell responses with greater breadth, magnitude, and frequency in a viral inhibition assay were also seen in the SLA and SHA groups after Ad35-GRIN boost, compared with those who received either vaccine alone. SeV-Gag did not boost T-cell counts in the ASH group. In contrast, the highest Gag-specific antibody titers were seen in the ASH group. Mucosal antibody responses were sporadic. Conclusions. SeV-Gag primed functional, durable HIV-specific T-cell responses and boosted antibody responses. The prime-boost sequence appears to determine which arm of the immune response is stimulated. Clinical Trials Registration. NCT01705990.
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Affiliation(s)
| | - Omu Anzala
- Kenya AIDS Vaccine Initiative Institute of Clinical Research, Nairobi
| | - Brian Gazzard
- Chelsea and Westminster Healthcare NHS Foundation Trust
| | | | - Philip Bergin
- Human Immunology Laboratory, International AIDS Vaccine Initiative, London, United Kingdom
| | - Peter Hayes
- Human Immunology Laboratory, International AIDS Vaccine Initiative, London, United Kingdom
| | - Jakub Kopycinski
- Human Immunology Laboratory, International AIDS Vaccine Initiative, London, United Kingdom
| | | | - Akil Jackson
- Chelsea and Westminster Healthcare NHS Foundation Trust
| | | | - Bashir Farah
- Kenya AIDS Vaccine Initiative Institute of Clinical Research, Nairobi
| | - Eddy Sayeed
- International AIDS Vaccine Initiative, New York, New York
| | | | | | | | | | | | - Tetsuro Matano
- University of Tokyo.,National Institute of Infectious Diseases, Tokyo, Japan
| | - Len Dally
- Emmes Corporation, Rockville, Maryland
| | | | - Harriet Park
- International AIDS Vaccine Initiative, New York, New York
| | - Jill Gilmour
- Human Immunology Laboratory, International AIDS Vaccine Initiative, London, United Kingdom
| | | | | | - Patricia Fast
- International AIDS Vaccine Initiative, New York, New York
| | - Dagna S Laufer
- International AIDS Vaccine Initiative, New York, New York
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29
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Alton EWFW, Boyd AC, Davies JC, Gill DR, Griesenbach U, Harrison PT, Henig N, Higgins T, Hyde SC, Innes JA, Korman MSD. Genetic medicines for CF: Hype versus reality. Pediatr Pulmonol 2016; 51:S5-S17. [PMID: 27662105 DOI: 10.1002/ppul.23543] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/04/2016] [Revised: 06/14/2016] [Accepted: 06/15/2016] [Indexed: 12/19/2022]
Abstract
Since identification of the CFTR gene over 25 years ago, gene therapy for cystic fibrosis (CF) has been actively developed. More recently gene therapy has been joined by other forms of "genetic medicines" including mRNA delivery, as well as genome editing and mRNA repair-based strategies. Proof-of-concept that gene therapy can stabilize the progression of CF lung disease has recently been established in a Phase IIb trial. An early phase study to assess the safety and explore efficacy of CFTR mRNA repair is ongoing, while mRNA delivery and genome editing-based strategies are currently at the pre-clinical phase of development. This review has been written jointly by some of those involved in the various CF "genetic medicine" fields and will summarize the current state-of-the-art, as well as discuss future developments. Where applicable, it highlights common problems faced by each of the strategies, and also tries to highlight where a specific strategy may have an advantage on the pathway to clinical translation. We hope that this review will contribute to the ongoing discussion about the hype versus reality of genetic medicine-based treatment approaches in CF. Pediatr Pulmonol. 2016;51:S5-S17. © 2016 Wiley Periodicals, Inc.
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Affiliation(s)
- Eric W F W Alton
- UK Cystic Fibrosis Gene Therapy Consortium, Edinburgh, Oxford, London
| | | | - Jane C Davies
- UK Cystic Fibrosis Gene Therapy Consortium, Edinburgh, Oxford, London
| | - Deborah R Gill
- UK Cystic Fibrosis Gene Therapy Consortium, Edinburgh, Oxford, London
| | - Uta Griesenbach
- UK Cystic Fibrosis Gene Therapy Consortium, Edinburgh, Oxford, London.
| | - Patrick T Harrison
- Department of Physiology and BioSciences Institute, University College Cork, Cork, Ireland
| | | | - Tracy Higgins
- UK Cystic Fibrosis Gene Therapy Consortium, Edinburgh, Oxford, London
| | - Stephen C Hyde
- UK Cystic Fibrosis Gene Therapy Consortium, Edinburgh, Oxford, London
| | - J Alastair Innes
- UK Cystic Fibrosis Gene Therapy Consortium, Edinburgh, Oxford, London
| | - Michael S D Korman
- Department of Pediatrics I - Pediatric Infectiology and Immunology - Translational Genomics and Gene Therapy, University of Tübingen, Tübingen, Germany
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30
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Park A, Hong P, Won ST, Thibault PA, Vigant F, Oguntuyo KY, Taft JD, Lee B. Sendai virus, an RNA virus with no risk of genomic integration, delivers CRISPR/Cas9 for efficient gene editing. MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 2016; 3:16057. [PMID: 27606350 PMCID: PMC4996130 DOI: 10.1038/mtm.2016.57] [Citation(s) in RCA: 42] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/16/2016] [Revised: 07/01/2016] [Accepted: 07/03/2016] [Indexed: 12/19/2022]
Abstract
The advent of RNA-guided endonuclease (RGEN)-mediated gene editing, specifically via CRISPR/Cas9, has spurred intensive efforts to improve the efficiency of both RGEN delivery and targeted mutagenesis. The major viral vectors in use for delivery of Cas9 and its associated guide RNA, lentiviral and adeno-associated viral systems, have the potential for undesired random integration into the host genome. Here, we repurpose Sendai virus, an RNA virus with no viral DNA phase and that replicates solely in the cytoplasm, as a delivery system for efficient Cas9-mediated gene editing. The high efficiency of Sendai virus infection resulted in high rates of on-target mutagenesis in cell lines (75–98% at various endogenous and transgenic loci) and primary human monocytes (88% at the ccr5 locus) in the absence of any selection. In conjunction with extensive former work on Sendai virus as a promising gene therapy vector that can infect a wide range of cell types including hematopoietic stem cells, this proof-of-concept study opens the door to using Sendai virus as well as other related paramyxoviruses as versatile and efficient tools for gene editing.
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Affiliation(s)
- Arnold Park
- Department of Microbiology, Icahn School of Medicine at Mount Sinai , New York, New York, USA
| | - Patrick Hong
- Department of Microbiology, Icahn School of Medicine at Mount Sinai , New York, New York, USA
| | - Sohui T Won
- Department of Microbiology, Icahn School of Medicine at Mount Sinai , New York, New York, USA
| | - Patricia A Thibault
- Department of Microbiology, Icahn School of Medicine at Mount Sinai , New York, New York, USA
| | - Frederic Vigant
- Department of Microbiology, Icahn School of Medicine at Mount Sinai , New York, New York, USA
| | - Kasopefoluwa Y Oguntuyo
- Department of Microbiology, Icahn School of Medicine at Mount Sinai , New York, New York, USA
| | - Justin D Taft
- Department of Microbiology, Icahn School of Medicine at Mount Sinai , New York, New York, USA
| | - Benhur Lee
- Department of Microbiology, Icahn School of Medicine at Mount Sinai , New York, New York, USA
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31
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Matsumoto T, Tanaka M, Yoshiya K, Yoshiga R, Matsubara Y, Horiuchi-Yoshida K, Yonemitsu Y, Maehara Y. Improved quality of life in patients with no-option critical limb ischemia undergoing gene therapy with DVC1-0101. Sci Rep 2016; 6:30035. [PMID: 27418463 PMCID: PMC4945920 DOI: 10.1038/srep30035] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2016] [Accepted: 06/29/2016] [Indexed: 11/09/2022] Open
Abstract
Critical limb ischemia (CLI) has a poor prognosis and adversely affects patients' quality of life (QOL). Therapeutic angiogenesis may improve mobility, mortality, and QOL in CLI patients. However, the effectiveness of gene therapy on such patients' QOL is unknown. DVC1-0101, a non-transmissible recombinant Sendai virus vector expressing human fibroblast growth factor-2 gene, demonstrated safety and efficacy in a phase I/II study of CLI patients. We investigated the effects of DVC1-0101 on QOL in this cohort. QOL was assessed using the Short Form-36 health survey version 2 (SF-36) in 12 patients at pre-administration, 28 days, and 3, 6, and 12 months post-treatment. We examined differences between pre and post-administration QOL scores and correlations between QOL scores and vascular parameters. Patients demonstrated low baselines scores on every SF-36 dimension. Post-treatment scores showed significant improvements in physical functioning at 3 and 6 months (P < 0.05), role-physical at 3, 6, and 12 months (P < 0.05), bodily pain at 1, 3, 6, and 12 months (P < 0.05), vitality at 1, 6, and 12 months (P < 0.05), and physical component summary at 6 and 12 months (P < 0.05). DVC1-0101-based gene therapy may improve QOL in CLI patients over a 6-month period.
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Affiliation(s)
- Takuya Matsumoto
- Department of Surgery and Science, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Michiko Tanaka
- R&D Laboratory for Innovative Biotherapeutics Science, Graduate School of Pharmaceutical Sciences, Kyushu University, Rm. 601, Collaborative Research Station I, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Keiji Yoshiya
- Department of Surgery and Science, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Ryosuke Yoshiga
- Department of Surgery and Science, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Yutaka Matsubara
- Department of Surgery and Science, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Kumi Horiuchi-Yoshida
- R&D Laboratory for Innovative Biotherapeutics Science, Graduate School of Pharmaceutical Sciences, Kyushu University, Rm. 601, Collaborative Research Station I, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Yoshikazu Yonemitsu
- R&D Laboratory for Innovative Biotherapeutics Science, Graduate School of Pharmaceutical Sciences, Kyushu University, Rm. 601, Collaborative Research Station I, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Yoshihiko Maehara
- Department of Surgery and Science, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
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Schott JW, Morgan M, Galla M, Schambach A. Viral and Synthetic RNA Vector Technologies and Applications. Mol Ther 2016; 24:1513-27. [PMID: 27377044 DOI: 10.1038/mt.2016.143] [Citation(s) in RCA: 52] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2015] [Accepted: 06/30/2016] [Indexed: 12/21/2022] Open
Abstract
Use of RNA is an increasingly popular method to transiently deliver genetic information for cell manipulation in basic research and clinical therapy. In these settings, viral and nonviral RNA platforms are employed for delivery of small interfering RNA and protein-coding mRNA. Technological advances allowing RNA modification for increased stability, improved translation and reduced immunogenicity have led to increased use of nonviral synthetic RNA, which is delivered in naked form or upon formulation. Alternatively, highly efficient viral entry pathways are exploited to transfer genes of interest as RNA incorporated into viral particles. Current viral RNA transfer technologies are derived from Retroviruses, nonsegmented negative-strand RNA viruses or positive-stranded Alpha- and Flaviviruses. In retroviral particles, the genes of interest can either be incorporated directly into the viral RNA genome or as nonviral RNA. Nonsegmented negative-strand virus-, Alpha- and Flavivirus-derived vectors support prolonged expression windows through replication of viral RNA encoding genes of interest. Mixed technologies combining viral and nonviral components are also available. RNA transfer is ideal for all settings that do not require permanent transgene expression and excludes potentially detrimental DNA integration into the target cell genome. Thus, RNA-based technologies are successfully applied for reprogramming, transdifferentiation, gene editing, vaccination, tumor therapy, and gene therapy.
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Affiliation(s)
- Juliane W Schott
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany
| | - Michael Morgan
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany
| | - Melanie Galla
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany
| | - Axel Schambach
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany.,Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA
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Kim N, Duncan GA, Hanes J, Suk JS. Barriers to inhaled gene therapy of obstructive lung diseases: A review. J Control Release 2016; 240:465-488. [PMID: 27196742 DOI: 10.1016/j.jconrel.2016.05.031] [Citation(s) in RCA: 80] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2016] [Revised: 05/11/2016] [Accepted: 05/12/2016] [Indexed: 12/29/2022]
Abstract
Knowledge of genetic origins of obstructive lung diseases has made inhaled gene therapy an attractive alternative to the current standards of care that are limited to managing disease symptoms. Initial lung gene therapy clinical trials occurred in the early 1990s following the discovery of the genetic defect responsible for cystic fibrosis (CF), a monogenic disorder. However, despite over two decades of intensive effort, gene therapy has yet to help patients with CF or any other obstructive lung disease. The slow progress is due in part to poor understanding of the biological barriers to inhaled gene therapy. Encouragingly, clinical trials have shown that inhaled gene therapy with various viral vectors and non-viral gene vectors is well tolerated by patients, and continued research has provided valuable lessons and resources that may lead to future success of this therapeutic strategy. In this review, we first introduce representative obstructive lung diseases and examine limitations of currently available therapeutic options. We then review key components for successful execution of inhaled gene therapy, including gene delivery systems, primary physiological barriers and strategies to overcome them, and advances in preclinical disease models with which the most promising systems may be identified for human clinical trials.
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Affiliation(s)
- Namho Kim
- The Center for Nanomedicine, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA
| | - Gregg A Duncan
- The Center for Nanomedicine, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA
| | - Justin Hanes
- The Center for Nanomedicine, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA; Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Environmental and Health Sciences, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Oncology, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Neurosurgery, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Pharmacology and Molecular Sciences, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Jung Soo Suk
- The Center for Nanomedicine, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.
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Paul-Smith MC, Bell RV, Alton WE, Alton EW, Griesenbach U. Gene therapy for cystic fibrosis: recent progress and current aims. Expert Opin Orphan Drugs 2016. [DOI: 10.1080/21678707.2016.1180974] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Affiliation(s)
- Michael C. Paul-Smith
- Department of Gene Therapy and the UK Cystic Fibrosis Gene Therapy Consortium, Imperial College, London, UK
| | - Robyn V. Bell
- Department of Gene Therapy and the UK Cystic Fibrosis Gene Therapy Consortium, Imperial College, London, UK
| | - William E. Alton
- Department of Gene Therapy and the UK Cystic Fibrosis Gene Therapy Consortium, Imperial College, London, UK
| | - Eric W.F.W. Alton
- Department of Gene Therapy and the UK Cystic Fibrosis Gene Therapy Consortium, Imperial College, London, UK
| | - Uta Griesenbach
- Department of Gene Therapy and the UK Cystic Fibrosis Gene Therapy Consortium, Imperial College, London, UK
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Tanaka N, Araki K, Mizokami D, Miyagawa Y, Yamashita T, Tomifuji M, Ueda Y, Inoue M, Matsushita K, Nomura F, Shimada H, Shiotani A. Sendai virus-mediated gene transfer of the c-myc suppressor far-upstream element-binding protein-interacting repressor suppresses head and neck cancer. Gene Ther 2015; 22:297-304. [PMID: 25588744 DOI: 10.1038/gt.2014.123] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2014] [Revised: 10/03/2014] [Accepted: 11/20/2014] [Indexed: 01/25/2023]
Abstract
Far-upstream element-binding protein-interacting repressor (FIR) is a transcription factor that inhibits c-Myc expression and has been shown to have antitumor effects in some malignancies. Here, we evaluated the antitumor effects of FIR using fusion gene-deleted Sendai virus (SeV/ΔF) as a nontransmissible vector against head and neck squamous cell carcinoma (HNSCC). Using in vitro and in vivo xenograft mouse models, we observed efficient expression of green fluorescent protein (GFP) following transduction with the SeV/ΔF vector encoding GFP (GFP-SeV/ΔF) into HNSCC cells. In vitro and in vivo studies revealed that administration of the FIR-encoded SeV/ΔF (FIR-SeV/ΔF) vector exerted significant antitumor effects, suppressed c-Myc expression and induced apoptosis in HNSCC. Additionally, the antitumor effects of FIR or the expression of GFP following administration of the FIR- or GFP-SeV/ΔF vector, respectively, were dependent on the multiplicity of infection or titer. Furthermore, the SeV/ΔF vector itself had no cytotoxic effects. Therefore, the SeV/ΔF vector may be safe and useful for the treatment of HNSCC, allowing for high-titer SeV/ΔF vector administration for anticancer gene therapy. In addition, SeV/ΔF vector-mediated FIR gene therapy demonstrated effective tumor suppression in HNSCC, suggesting that this therapy may have the potential for clinical use as a novel strategy for HNSCC treatment.
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Affiliation(s)
- N Tanaka
- Department of Otorhinolaryngology - Head and Neck Surgery, National Defense Medical College, Saitama, Japan
| | - K Araki
- Department of Otorhinolaryngology - Head and Neck Surgery, National Defense Medical College, Saitama, Japan
| | - D Mizokami
- Department of Otorhinolaryngology - Head and Neck Surgery, National Defense Medical College, Saitama, Japan
| | - Y Miyagawa
- Department of Otorhinolaryngology - Head and Neck Surgery, National Defense Medical College, Saitama, Japan
| | - T Yamashita
- Department of Otorhinolaryngology - Head and Neck Surgery, National Defense Medical College, Saitama, Japan
| | - M Tomifuji
- Department of Otorhinolaryngology - Head and Neck Surgery, National Defense Medical College, Saitama, Japan
| | - Y Ueda
- Department of Gene Medicine, DNAVEC Corporation, Ibaraki, Japan
| | - M Inoue
- Department of Gene Medicine, DNAVEC Corporation, Ibaraki, Japan
| | - K Matsushita
- Department of Molecular Diagnosis and Division of Clinical Genetics and Proteomics, Chiba University Graduate School of Medicine, Chiba, Japan
| | - F Nomura
- Department of Molecular Diagnosis and Division of Clinical Genetics and Proteomics, Chiba University Graduate School of Medicine, Chiba, Japan
| | - H Shimada
- Department of Surgery, Toho University School of Medicine, Tokyo, Japan
| | - A Shiotani
- Department of Otorhinolaryngology - Head and Neck Surgery, National Defense Medical College, Saitama, Japan
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Mizokami D, Araki K, Tanaka N, Suzuki H, Tomifuji M, Yamashita T, Ueda Y, Shimada H, Matsushita K, Shiotani A. Gene therapy of c-myc suppressor FUSE-binding protein-interacting repressor by Sendai virus delivery prevents tracheal stenosis. PLoS One 2015; 10:e0116279. [PMID: 25569246 PMCID: PMC4287628 DOI: 10.1371/journal.pone.0116279] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2014] [Accepted: 12/04/2014] [Indexed: 02/06/2023] Open
Abstract
Acquired tracheal stenosis remains a challenging problem for otolaryngologists. The objective of this study was to determine whether the Sendai virus (SeV)-mediated c-myc suppressor, a far upstream element (FUSE)-binding protein (FBP)-interacting repressor (FIR), modulates wound healing of the airway mucosa, and whether it prevents tracheal stenosis in an animal model of induced mucosal injury. A fusion gene-deleted, non-transmissible SeV vector encoding FIR (FIR-SeV/ΔF) was prepared. Rats with scraped airway mucosae were administered FIR-SeV/ΔF through the tracheostoma. The pathological changes in the airway mucosa and in the tracheal lumen were assessed five days after scraping. Untreated animals showed hyperplasia of the airway epithelium and a thickened submucosal layer with extensive fibrosis, angiogenesis, and collagen deposition causing lumen stenosis. By contrast, the administration of FIR-SeV/ΔF decreased the degree of tracheal stenosis (P < 0.05) and improved the survival rate (P < 0.05). Immunohistochemical staining showed that c-Myc expression was downregulated in the tracheal basal cells of the FIR-SeV/ΔF-treated animals, suggesting that c-myc was suppressed by FIR-SeV/ΔF in the regenerating airway epithelium of the injured tracheal mucosa. The airway-targeted gene therapy of the c-myc suppressor FIR, using a recombinant SeV vector, prevented tracheal stenosis in a rat model of airway mucosal injury.
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Affiliation(s)
- Daisuke Mizokami
- Department of Otolaryngology, Head & Neck Surgery, National Defense Medical College, Tokorozawa, Saitama, Japan
| | - Koji Araki
- Department of Otolaryngology, Head & Neck Surgery, National Defense Medical College, Tokorozawa, Saitama, Japan
- * E-mail:
| | - Nobuaki Tanaka
- Department of Otolaryngology, Head & Neck Surgery, National Defense Medical College, Tokorozawa, Saitama, Japan
| | - Hiroshi Suzuki
- Department of Otolaryngology, Head & Neck Surgery, National Defense Medical College, Tokorozawa, Saitama, Japan
| | - Masayuki Tomifuji
- Department of Otolaryngology, Head & Neck Surgery, National Defense Medical College, Tokorozawa, Saitama, Japan
| | - Taku Yamashita
- Department of Otolaryngology, Head & Neck Surgery, National Defense Medical College, Tokorozawa, Saitama, Japan
| | | | - Hideaki Shimada
- Department of Surgery, Toho University School of Medicine, Ota-Ku, Tokyo, Japan
| | - Kazuyuki Matsushita
- Department of Molecular Diagnosis (F8), Chiba University Graduate School of Medicine, Chiba City, Chiba, Japan
| | - Akihiro Shiotani
- Department of Otolaryngology, Head & Neck Surgery, National Defense Medical College, Tokorozawa, Saitama, Japan
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Abstract
The advent of reverse genetic approaches to manipulate the genomes of both positive (+) and negative (-) sense RNA viruses allowed researchers to harness these genomes for basic research. Manipulation of positive sense RNA virus genomes occurred first largely because infectious RNA could be transcribed directly from cDNA versions of the RNA genomes. Manipulation of negative strand RNA virus genomes rapidly followed as more sophisticated approaches to provide RNA-dependent RNA polymerase complexes coupled with negative-strand RNA templates were developed. These advances have driven an explosion of RNA virus vaccine vector development. That is, development of approaches to exploit the basic replication and expression strategies of RNA viruses to produce vaccine antigens that have been engineered into their genomes. This study has led to significant preclinical testing of many RNA virus vectors against a wide range of pathogens as well as cancer targets. Multiple RNA virus vectors have advanced through preclinical testing to human clinical evaluation. This review will focus on RNA virus vectors designed to express heterologous genes that are packaged into viral particles and have progressed to clinical testing.
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Affiliation(s)
- Mark A Mogler
- Harrisvaccines, Inc., 1102 Southern Hills Drive, Suite 101, Ames, IA 50010, USA
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Hu K. All roads lead to induced pluripotent stem cells: the technologies of iPSC generation. Stem Cells Dev 2014; 23:1285-300. [PMID: 24524728 PMCID: PMC4046204 DOI: 10.1089/scd.2013.0620] [Citation(s) in RCA: 73] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2013] [Accepted: 02/12/2014] [Indexed: 12/26/2022] Open
Abstract
Generation of induced pluripotent stem cells (iPSCs) via the ectopic expression of reprogramming factors is a simple, advanced, yet often perplexing technology due to low efficiency, slow kinetics, and the use of numerous distinct systems for factor delivery. Scientists have used almost all available approaches for the delivery of reprogramming factors. Even the well-established retroviral vectors confuse some scientists due to different tropisms in use. The canonical virus-based reprogramming poses many problems, including insertional mutagenesis, residual expression and re-activation of reprogramming factors, uncontrolled silencing of transgenes, apoptosis, cell senescence, and strong immunogenicity. To eliminate or alleviate these problems, scientists have tried various other approaches for factor delivery and transgene removal. These include transient transfection, nonintegrating viral vectors, Cre-loxP excision of transgenes, excisable transposon, protein transduction, RNA transfection, microRNA transfection, RNA virion, RNA replicon, nonintegrating replicating episomal plasmids, minicircles, polycistron, and preintegration of inducible reprogramming factors. These alternative approaches have their own limitations. Even iPSCs generated with RNA approaches should be screened for possible transgene insertions mediated by active endogenous retroviruses in the human genome. Even experienced researchers may encounter difficulty in selecting and using these different technologies. This survey presents overviews of iPSC technologies with the intention to provide a quick yet comprehensive reference for both new and experienced reprogrammers.
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Affiliation(s)
- Kejin Hu
- Department of Biochemistry and Molecular Genetics, UAB Stem Cell Insitute, School of Medicine, University of Alabama at Birmingham , Birmingham, Alabama
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Sen D, Balakrishnan B, Jayandharan GR. Cellular unfolded protein response against viruses used in gene therapy. Front Microbiol 2014; 5:250. [PMID: 24904562 PMCID: PMC4033601 DOI: 10.3389/fmicb.2014.00250] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2014] [Accepted: 05/07/2014] [Indexed: 01/21/2023] Open
Abstract
Viruses are excellent vehicles for gene therapy due to their natural ability to infect and deliver the cargo to specific tissues with high efficiency. Although such vectors are usually "gutted" and are replication defective, they are subjected to clearance by the host cells by immune recognition and destruction. Unfolded protein response (UPR) is a naturally evolved cyto-protective signaling pathway which is triggered due to endoplasmic reticulum (ER) stress caused by accumulation of unfolded/misfolded proteins in its lumen. The UPR signaling consists of three signaling pathways, namely PKR-like ER kinase, activating transcription factor 6, and inositol-requiring protein-1. Once activated, UPR triggers the production of ER molecular chaperones and stress response proteins to help reduce the protein load within the ER. This occurs by degradation of the misfolded proteins and ensues in the arrest of protein translation machinery. If the burden of protein load in ER is beyond its processing capacity, UPR can activate pro-apoptotic pathways or autophagy leading to cell death. Viruses are naturally evolved in hijacking the host cellular translation machinery to generate a large amount of proteins. This phenomenon disrupts ER homeostasis and leads to ER stress. Alternatively, in the case of gutted vectors used in gene therapy, the excess load of recombinant vectors administered and encountered by the cell can trigger UPR. Thus, in the context of gene therapy, UPR becomes a major roadblock that can potentially trigger inflammatory responses against the vectors and reduce the efficiency of gene transfer.
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Affiliation(s)
- Dwaipayan Sen
- Department of Hematology, Christian Medical College Vellore, India
| | | | - Giridhara R Jayandharan
- Department of Hematology, Christian Medical College Vellore, India ; Centre for Stem Cell Research, Christian Medical College Vellore, India
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Abstract
The principal cause of morbidity and mortality in cystic fibrosis (CF) is pulmonary disease, so the focus of new treatments in this condition is primarily targeted at the lungs. Since the cloning of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene in 1989, there has been significant interest in the possibility of gene therapy as a treatment for CF. Early studies using viral vectors carrying a healthy CFTR plasmid highlighted the difficulties with overcoming the body's host defences. This article reviews the work on gene therapy in CF to date and describes the ongoing work of the UK CF Gene Therapy Consortium in investigating the potential of gene therapy as a treatment for patients with CF.
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Matsushita K, Shimada H, Ueda Y, Inoue M, Hasegawa M, Tomonaga T, Matsubara H, Nomura F. Non-transmissible Sendai virus vector encoding c-myc suppressor FBP-interacting repressor for cancer therapy. World J Gastroenterol 2014; 20:4316-4328. [PMID: 24764668 PMCID: PMC3989966 DOI: 10.3748/wjg.v20.i15.4316] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/22/2013] [Revised: 12/14/2013] [Accepted: 01/20/2014] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate a novel therapeutic strategy to target and suppress c-myc in human cancers using far up stream element (FUSE)-binding protein-interacting repressor (FIR).
METHODS: Endogenous c-Myc suppression and apoptosis induction by a transient FIR-expressing vector was examined in vivo via a HA-tagged FIR (HA-FIR) expression vector. A fusion gene-deficient, non-transmissible, Sendai virus (SeV) vector encoding FIR cDNA, SeV/dF/FIR, was prepared. SeV/dF/FIR was examined for its gene transduction efficiency, viral dose dependency of antitumor effect and apoptosis induction in HeLa (cervical squamous cell carcinoma) cells and SW480 (colon adenocarcinoma) cells. Antitumor efficacy in a mouse xenograft model was also examined. The molecular mechanism of the anti-tumor effect and c-Myc suppression by SeV/dF/FIR was examined using Spliceostatin A (SSA), a SAP155 inhibitor, or SAP155 siRNA which induce c-Myc by increasing FIR∆exon2 in HeLa cells.
RESULTS: FIR was found to repress c-myc transcription and in turn the overexpression of FIR drove apoptosis through c-myc suppression. Thus, FIR expressing vectors are potentially applicable for cancer therapy. FIR is alternatively spliced by SAP155 in cancer cells lacking the transcriptional repression domain within exon 2 (FIR∆exon2), counteracting FIR for c-Myc protein expression. Furthermore, FIR forms a complex with SAP155 and inhibits mutual well-established functions. Thus, both the valuable effects and side effects of exogenous FIR stimuli should be tested for future clinical application. SeV/dF/FIR, a cytoplasmic RNA virus, was successfully prepared and showed highly efficient gene transduction in in vivo experiments. Furthermore, in nude mouse tumor xenograft models, SeV/dF/FIR displayed high antitumor efficiency against human cancer cells. SeV/dF/FIR suppressed SSA-activated c-Myc. SAP155 siRNA, potentially produces FIR∆exon2, and led to c-Myc overexpression with phosphorylation at Ser62. HA-FIR suppressed endogenous c-Myc expression and induced apoptosis in HeLa and SW480 cells. A c-myc transcriptional suppressor FIR expressing SeV/dF/FIR showed high gene transduction efficiency with significant antitumor effects and apoptosis induction in HeLa and SW480 cells.
CONCLUSION: SeV/dF/FIR showed strong tumor growth suppression with no significant side effects in an animal xenograft model, thus SeV/dF/FIR is potentially applicable for future clinical cancer treatment.
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Nakanishi M, Otsu M. Development of Sendai virus vectors and their potential applications in gene therapy and regenerative medicine. Curr Gene Ther 2013; 12:410-6. [PMID: 22920683 PMCID: PMC3504922 DOI: 10.2174/156652312802762518] [Citation(s) in RCA: 85] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2012] [Revised: 08/15/2012] [Accepted: 08/16/2012] [Indexed: 01/14/2023]
Abstract
Gene delivery/expression vectors have been used as fundamental technologies in gene therapy since the 1980s. These technologies are also being applied in regenerative medicine as tools to reprogram cell genomes to a pluripotent state and to other cell lineages. Rapid progress in these new research areas and expectations for their translation into clinical applications have facilitated the development of more sophisticated gene delivery/expression technologies. Since its isolation in 1953 in Japan, Sendai virus (SeV) has been widely used as a research tool in cell biology and in industry, but the application of SeV as a recombinant viral vector has been investigated only recently. Recombinant SeV vectors have various unique characteristics, such as low pathogenicity, powerful capacity for gene expression and a wide host range. In addition, the cytoplasmic gene expression mediated by this vector is advantageous for applications, in that chromosomal integration of exogenous genes can be undesirable. In this review, we introduce a brief historical background on the development of recombinant SeV vectors and describe their current applications in gene therapy. We also describe the application of SeV vectors in advanced nuclear reprogramming and introduce a defective and persistent SeV vector (SeVdp) optimized for such reprogramming.
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Affiliation(s)
- Mahito Nakanishi
- Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Central 4, Tsukuba, Ibaraki, 305-8562, Japan.
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Bernal JA. RNA-based tools for nuclear reprogramming and lineage-conversion: towards clinical applications. J Cardiovasc Transl Res 2013; 6:956-68. [PMID: 23852582 PMCID: PMC3838600 DOI: 10.1007/s12265-013-9494-8] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/14/2013] [Accepted: 06/21/2013] [Indexed: 02/06/2023]
Abstract
The therapeutic potential of induced pluripotent stem cells (iPSCs) is well established. Safety concerns remain, however, and these have driven considerable efforts aimed at avoiding host genome alteration during the reprogramming process. At present, the tools used to generate human iPSCs include (1) DNA-based integrative and non-integrative methods and (2) DNA-free reprogramming technologies, including RNA-based approaches. Because of their combined efficiency and safety characteristics, RNA-based methods have emerged as the most promising tool for future iPSC-based regenerative medicine applications. Here, I will discuss novel recent advances in reprogramming technology, especially those utilizing the Sendai virus (SeV) and synthetic modified mRNA. In the future, these technologies may find utility in iPSC reprogramming for cellular lineage-conversion, and its subsequent use in cell-based therapies.
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Affiliation(s)
- Juan A Bernal
- Cardiovascular Development and Repair Department, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, 28029, Madrid, Spain,
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Wiegand M, Gori-Savellini G, Martorelli B, Bossow S, Neubert WJ, Cusi MG. Evaluation of a novel immunogenic vaccine platform based on a genome replication-deficient Sendai vector. Vaccine 2013; 31:3888-93. [PMID: 23831325 DOI: 10.1016/j.vaccine.2013.06.053] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2013] [Revised: 06/11/2013] [Accepted: 06/19/2013] [Indexed: 11/17/2022]
Abstract
We developed a novel vaccine platform based on a paramyxoviral, genome replication-deficient Sendai virus vector that can express heterologous genes inserted into the genome. To validate the novel approach in vivo, we generated a combined vaccine candidate against human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (PIV3). The present study compares two different methods of displaying heterologous antigens: (i) the RSV fusion (F) protein, encoded as a secretable version in an additional transcription unit, serves as an antigen only after being expressed in infected cells; (ii) PIV3 fusion (F) and hemagglutinin-neuraminidase (HN) genes, replacing Sendai counterparts in the vector genome, are also expressed as structural components on the surface of vaccine particles. The efficacy of this prototype vaccine was assessed in a mouse model after mucosal administration. The vaccine candidate was able to elicit specific mucosal, humoral and T cell-mediated immune responses against RSV and PIV3. However, PIV3 antigen display on the vaccine particles' surface induced higher antibody titers than the RSV antigen, being expressed only after cell infection. Consequently, this construct induced an adequate neutralizing antibody response only to PIV3. Finally, replicating virus particles were not detected in the lungs of immunized mice, confirming the genome stability and replication deficiency of this vaccine vector in vivo. Both factors can contribute substantially to the safety profile of vaccine candidates. In conclusion, this replication-deficient Sendai vector represents an efficient platform that can be used for vaccine developments against various viral pathogens.
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Affiliation(s)
- Marian Wiegand
- Department of Molecular Virology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
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45
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Mizokami D, Araki K, Tanaka N, Suzuki H, Tomifuji M, Yamashita T, Inoue M, Hasegawa M, Shiotani A. Sendai virus transgene in a novel gene therapy for laryngotracheal disease. Laryngoscope 2013; 123:1717-24. [DOI: 10.1002/lary.23917] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/05/2012] [Indexed: 12/14/2022]
Affiliation(s)
- Daisuke Mizokami
- Department of Otolaryngology-Head & Neck Surgery; National Defense Medical College; Tokorozawa; Saitama
| | - Koji Araki
- Department of Otolaryngology-Head & Neck Surgery; National Defense Medical College; Tokorozawa; Saitama
| | - Nobuaki Tanaka
- Department of Otolaryngology-Head & Neck Surgery; National Defense Medical College; Tokorozawa; Saitama
| | - Hiroshi Suzuki
- Department of Otolaryngology-Head & Neck Surgery; National Defense Medical College; Tokorozawa; Saitama
| | - Masayuki Tomifuji
- Department of Otolaryngology-Head & Neck Surgery; National Defense Medical College; Tokorozawa; Saitama
| | - Taku Yamashita
- Department of Otolaryngology-Head & Neck Surgery; National Defense Medical College; Tokorozawa; Saitama
| | | | | | - Akihiro Shiotani
- Department of Otolaryngology-Head & Neck Surgery; National Defense Medical College; Tokorozawa; Saitama
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Lu J, Liu H, Huang CTL, Chen H, Du Z, Liu Y, Sherafat MA, Zhang SC. Generation of integration-free and region-specific neural progenitors from primate fibroblasts. Cell Rep 2013; 3:1580-91. [PMID: 23643533 DOI: 10.1016/j.celrep.2013.04.004] [Citation(s) in RCA: 86] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2012] [Revised: 02/12/2013] [Accepted: 04/03/2013] [Indexed: 01/09/2023] Open
Abstract
Postnatal and adult human and monkey fibroblasts were infected with Sendai virus containing the Yamanaka factors for 24 hr, then they were cultured in a chemically defined medium containing leukemia inhibitory factor (LIF), transforming growth factor (TGF)-β inhibitor SB431542, and glycogen synthase kinase (GSK)-3β inhibitor CHIR99021 at 39°C for inactivation of the virus. Induced neural progenitor (iNP) colonies appeared as early as day 13 and can be expanded for >20 passages. Under the same defined condition, no induced pluripotent stem cell (iPSC) colonies formed at either 37°C or 39°C. The iNPs predominantly express hindbrain genes and differentiate into hindbrain neurons, and when caudalized, they produced an enriched population of spinal motor neurons. Following transplantation into the forebrain, the iNP-derived cells retained the hindbrain identity. The ability to generate defined, integration-free iNPs from adult primate fibroblasts under a defined condition with predictable fate choices will facilitate disease modeling and therapeutic development.
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Affiliation(s)
- Jianfeng Lu
- Department of Neuroscience, School of Medicine and Public Health, Waisman Center, University of Wisconsin, Madison, Madison, WI 53705, USA
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Yonemitsu Y, Matsumoto T, Itoh H, Okazaki J, Uchiyama M, Yoshida K, Onimaru M, Onohara T, Inoguchi H, Kyuragi R, Shimokawa M, Ban H, Tanaka M, Inoue M, Shu T, Hasegawa M, Nakanishi Y, Maehara Y. DVC1-0101 to treat peripheral arterial disease: a Phase I/IIa open-label dose-escalation clinical trial. Mol Ther 2013; 21:707-14. [PMID: 23319060 PMCID: PMC3589164 DOI: 10.1038/mt.2012.279] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2012] [Accepted: 12/05/2012] [Indexed: 11/08/2022] Open
Abstract
We here report the results of a Phase I/IIa open-label four dose-escalation clinical study assessing the safety, tolerability, and possible therapeutic efficacy of a single intramuscular administration of DVC1-0101, a new gene transfer vector based on a nontransmissible recombinant Sendai virus (rSeV) expressing the human fibroblast growth factor-2 (FGF-2) gene (rSeV/dF-hFGF2), in patients with peripheral arterial disease (PAD). Gene transfer was done in 12 limbs of 12 patients with rest pain, and three of them had ischemic ulcer(s). No cardiovascular or other serious adverse events (SAEs) caused by gene transfer were detected in the patients over a 6-month follow-up. No infectious viral particles, as assessed by hemagglutination activity, were detected in any patient during the study. No representative elevation of proinflammatory cytokines or plasma FGF-2 was seen. Significant and continuous improvements in Rutherford category, absolute claudication distance (ACD), and rest pain were observed (P < 0.05 to 0.01). To the best of our knowledge, this is the first clinical trial of the use of a gene transfer vector based on rSeV. The single intramuscular administration of DVC1-0101 to PAD patients was safe and well tolerated, and resulted in significant improvements of limb function. Larger pivotal studies are warranted as a next step.
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Affiliation(s)
- Yoshikazu Yonemitsu
- Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
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Griesenbach U, Inoue M, Meng C, Farley R, Chan M, Newman NK, Brum A, You J, Kerton A, Shoemark A, Boyd AC, Davies JC, Higgins TE, Gill DR, Hyde SC, Innes JA, Porteous DJ, Hasegawa M, Alton EWFW. Assessment of F/HN-pseudotyped lentivirus as a clinically relevant vector for lung gene therapy. Am J Respir Crit Care Med 2012; 186:846-56. [PMID: 22955314 PMCID: PMC3530223 DOI: 10.1164/rccm.201206-1056oc] [Citation(s) in RCA: 76] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2012] [Accepted: 08/21/2012] [Indexed: 02/05/2023] Open
Abstract
RATIONALE Ongoing efforts to improve pulmonary gene transfer thereby enabling gene therapy for the treatment of lung diseases, such as cystic fibrosis (CF), has led to the assessment of a lentiviral vector (simian immunodeficiency virus [SIV]) pseudotyped with the Sendai virus envelope proteins F and HN. OBJECTIVES To place this vector onto a translational pathway to the clinic by addressing some key milestones that have to be achieved. METHODS F/HN-SIV transduction efficiency, duration of expression, and toxicity were assessed in mice. In addition, F/HN-SIV was assessed in differentiated human air-liquid interface cultures, primary human nasal epithelial cells, and human and sheep lung slices. MEASUREMENTS AND MAIN RESULTS A single dose produces lung expression for the lifetime of the mouse (~2 yr). Only brief contact time is needed to achieve transduction. Repeated daily administration leads to a dose-related increase in gene expression. Repeated monthly administration to mouse lower airways is feasible without loss of gene expression. There is no evidence of chronic toxicity during a 2-year study period. F/HN-SIV leads to persistent gene expression in human differentiated airway cultures and human lung slices and transduces freshly obtained primary human airway epithelial cells. CONCLUSIONS The data support F/HN-pseudotyped SIV as a promising vector for pulmonary gene therapy for several diseases including CF. We are now undertaking the necessary refinements to progress this vector into clinical trials.
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Affiliation(s)
- Uta Griesenbach
- Department of Gene Therapy and
- The United Kingdom Cystic Fibrosis Gene Therapy Consortium, London, United Kingdom
| | | | - Cuixiang Meng
- Department of Gene Therapy and
- The United Kingdom Cystic Fibrosis Gene Therapy Consortium, London, United Kingdom
| | - Raymond Farley
- Department of Gene Therapy and
- The United Kingdom Cystic Fibrosis Gene Therapy Consortium, London, United Kingdom
| | - Mario Chan
- Department of Gene Therapy and
- The United Kingdom Cystic Fibrosis Gene Therapy Consortium, London, United Kingdom
| | - Nikki K. Newman
- Department of Gene Therapy and
- The United Kingdom Cystic Fibrosis Gene Therapy Consortium, London, United Kingdom
| | - Andrea Brum
- Department of Gene Therapy and
- The United Kingdom Cystic Fibrosis Gene Therapy Consortium, London, United Kingdom
| | - Jun You
- DNAVEC Corporation, Tsukuba, Japan
| | - Angela Kerton
- Central Biomedical Services, Imperial College London, London, United Kingdom
| | - Amelia Shoemark
- Paediatric Department, Royal Brompton Hospital, London, United Kingdom
| | - A. Christopher Boyd
- The United Kingdom Cystic Fibrosis Gene Therapy Consortium, London, United Kingdom
- Medical Genetics Section, Centre for Molecular Medicine, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom; and
| | - Jane C. Davies
- Department of Gene Therapy and
- The United Kingdom Cystic Fibrosis Gene Therapy Consortium, London, United Kingdom
| | - Tracy E. Higgins
- Department of Gene Therapy and
- The United Kingdom Cystic Fibrosis Gene Therapy Consortium, London, United Kingdom
| | - Deborah R. Gill
- The United Kingdom Cystic Fibrosis Gene Therapy Consortium, London, United Kingdom
- Gene Medicine Group, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom
| | - Stephen C. Hyde
- The United Kingdom Cystic Fibrosis Gene Therapy Consortium, London, United Kingdom
- Gene Medicine Group, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom
| | - J. Alastair Innes
- The United Kingdom Cystic Fibrosis Gene Therapy Consortium, London, United Kingdom
- Medical Genetics Section, Centre for Molecular Medicine, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom; and
| | - David J. Porteous
- The United Kingdom Cystic Fibrosis Gene Therapy Consortium, London, United Kingdom
- Medical Genetics Section, Centre for Molecular Medicine, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom; and
| | | | - Eric W. F. W. Alton
- Department of Gene Therapy and
- The United Kingdom Cystic Fibrosis Gene Therapy Consortium, London, United Kingdom
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Generation of induced pluripotent stem cells from human nasal epithelial cells using a Sendai virus vector. PLoS One 2012; 7:e42855. [PMID: 22912751 PMCID: PMC3418281 DOI: 10.1371/journal.pone.0042855] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2012] [Accepted: 07/12/2012] [Indexed: 11/21/2022] Open
Abstract
The generation of induced pluripotent stem cells (iPSCs) by introducing reprogramming factors into somatic cells is a promising method for stem cell therapy in regenerative medicine. Therefore, it is desirable to develop a minimally invasive simple method to create iPSCs. In this study, we generated human nasal epithelial cells (HNECs)-derived iPSCs by gene transduction with Sendai virus (SeV) vectors. HNECs can be obtained from subjects in a noninvasive manner, without anesthesia or biopsy. In addition, SeV carries no risk of altering the host genome, which provides an additional level of safety during generation of human iPSCs. The multiplicity of SeV infection ranged from 3 to 4, and the reprogramming efficiency of HNECs was 0.08–0.10%. iPSCs derived from HNECs had global gene expression profiles and epigenetic states consistent with those of human embryonic stem cells. The ease with which HNECs can be obtained, together with their robust reprogramming characteristics, will provide opportunities to investigate disease pathogenesis and molecular mechanisms in vitro, using cells with particular genotypes.
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50
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Generation of human-induced pluripotent stem cells by a nonintegrating RNA Sendai virus vector in feeder-free or xeno-free conditions. Stem Cells Int 2012; 2012:564612. [PMID: 22550511 PMCID: PMC3328201 DOI: 10.1155/2012/564612] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2011] [Accepted: 01/04/2012] [Indexed: 01/19/2023] Open
Abstract
The generation of induced pluripotent stem cells (iPSCs) from somatic cells has enabled the possibility of providing unprecedented access to patient-specific iPSC cells for drug screening, disease modeling, and cell therapy applications. However, a major obstacle to the use of iPSC for therapeutic applications is the potential of genomic modifications caused by insertion of viral transgenes in the cellular genome. A second concern is that reprogramming often requires the use of animal feeder layers and reagents that contain animal origin products, which hinder the generation of clinical-grade iPSCs. Here, we report the generation of iPSCs by an RNA Sendai virus vector that does not integrate into the cells genome, providing transgene-free iPSC line. In addition, reprogramming can be performed in feeder-free condition with StemPro hESC SFM medium and in xeno-free (XF) conditions. Generation of an integrant-free iPSCs generated in xeno-free media should facilitate the safe downstream applications of iPSC-based cell therapies.
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