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1
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Li C, Tian J, Liu N, Song D, Steer CJ, Han Q, Song G. MicroRNA-206 as a potential cholesterol-lowering drug is superior to statins in mice. J Lipid Res 2024; 65:100576. [PMID: 38866328 PMCID: PMC11292365 DOI: 10.1016/j.jlr.2024.100576] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2023] [Revised: 05/24/2024] [Accepted: 05/28/2024] [Indexed: 06/14/2024] Open
Abstract
Hypercholesterolemia is frequently intertwined with hepatosteatosis, hypertriglyceridemia, and hyperglycemia. This study is designed to assess the therapeutic efficacy of miR-206 in contrast to statins in the context of managing hypercholesterolemia in mice. We previously showed that miR-206 is a potent inhibitor of de novo lipogenesis (DNL), cholesterol synthesis, and gluconeogenesis in mice. Given that these processes occur within hepatocytes, we employed a mini-circle (MC) system to deliver miR-206 specifically to hepatocytes (designated as MC-miR-206). A single intravenous injection of MC-miR-206 maintained high levels of miR-206 in the liver for at least two weeks, thereby maintaining suppression of hepatic DNL, cholesterol synthesis, and gluconeogenesis. MC-miR-206 significantly reduced DNA damage, endoplasmic reticulum and oxidative stress, and hepatic toxicity. Therapeutically, both MC-miR-206 and statins significantly reduced total serum cholesterol and triglycerides as well as LDL cholesterol and VLDL cholesterol in mice maintained on the normal chow and high-fat high-cholesterol diet. MC-miR-206 reduced liver weight, hepatic triglycerides and cholesterol, and blood glucose, while statins slightly increased hepatic cholesterol and blood glucose and failed to affect levels of liver weight and hepatic triglycerides. Mechanistically, miR-206 alleviated hypercholesterolemia by inhibiting hepatic cholesterol synthesis, while statins increased HMGCR activity, hepatic cholesterol synthesis, and fecal-neutral steroid excretion. MiR-206 facilitates the regression of hypercholesterolemia, hypertriglyceridemia, hyperglycemia, and hepatosteatosis. MiR-206 outperforms statins by reducing hyperglycemia, hepatic cholesterol levels, and hepatic toxicity.
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Affiliation(s)
- Chao Li
- Department of Cardiology, The First Hospital of Shanxi Medical University, Taiyuan City, China; The First College of Clinical Medicine, Shanxi Medical University, Taiyuan City, China
| | - Jing Tian
- Department of Cardiology, The First Hospital of Shanxi Medical University, Taiyuan City, China
| | - Ningning Liu
- Department of Medicine, University of Minnesota, Minneapolis, MN, USA
| | - David Song
- Department of Medicine, University of Minnesota, Minneapolis, MN, USA
| | - Clifford J Steer
- Department of Medicine, University of Minnesota, Minneapolis, MN, USA
| | - Qinghua Han
- Department of Cardiology, The First Hospital of Shanxi Medical University, Taiyuan City, China.
| | - Guisheng Song
- Department of Medicine, University of Minnesota, Minneapolis, MN, USA.
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2
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Wang C, Yuan F. A comprehensive comparison of DNA and RNA vaccines. Adv Drug Deliv Rev 2024; 210:115340. [PMID: 38810703 PMCID: PMC11181159 DOI: 10.1016/j.addr.2024.115340] [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: 03/28/2024] [Revised: 05/06/2024] [Accepted: 05/18/2024] [Indexed: 05/31/2024]
Abstract
Nucleic acid technology has revolutionized vaccine development, enabling rapid design and production of RNA and DNA vaccines for prevention and treatment of diseases. The successful deployment of mRNA and plasmid DNA vaccines against COVID-19 has further validated the technology. At present, mRNA platform is prevailing due to its higher efficacy, while DNA platform is undergoing rapid evolution because it possesses unique advantages that can potentially overcome the problems associated with the mRNA platform. To help understand the recent performances of the two vaccine platforms and recognize their clinical potentials in the future, this review compares the advantages and drawbacks of mRNA and DNA vaccines that are currently known in the literature, in terms of development timeline, financial cost, ease of distribution, efficacy, safety, and regulatory approval of products. Additionally, the review discusses the ongoing clinical trials, strategies for improvement, and alternative designs of RNA and DNA platforms for vaccination.
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Affiliation(s)
- Chunxi Wang
- Department of Biomedical Engineering, Duke University, Durham, NC 27705, United States
| | - Fan Yuan
- Department of Biomedical Engineering, Duke University, Durham, NC 27705, United States.
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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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4
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Espuche B, Moya SE, Calderón M. Nanogels: Smart tools to enlarge the therapeutic window of gene therapy. Int J Pharm 2024; 653:123864. [PMID: 38309484 DOI: 10.1016/j.ijpharm.2024.123864] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2023] [Revised: 01/09/2024] [Accepted: 01/25/2024] [Indexed: 02/05/2024]
Abstract
Gene therapy can potentially treat a great number of diseases, from cancer to rare genetic disorders. Very recently, the development and emergency approval of nucleic acid-based COVID-19 vaccines confirmed its strength and versatility. However, gene therapy encounters limitations due to the lack of suitable carriers to vectorize therapeutic genetic material inside target cells. Nanogels are highly hydrated nano-size crosslinked polymeric networks that have been used in many biomedical applications, from drug delivery to tissue engineering and diagnostics. Due to their easy production, tunability, and swelling properties they have called the attention as promising vectors for gene delivery. In this review, nanogels are discussed as vectors for nucleic acid delivery aiming to enlarge gene therapy's therapeutic window. Recent works highlighting the optimization of inherent transfection efficiency and biocompatibility are reviewed here. The importance of the monomer choice, along with the internal structure, surface decoration, and responsive features are outlined for the different transfection modalities. The possible sources of toxicological endpoints in nanogels are analyzed, and the strategies to limit them are compared. Finally, perspectives are discussed to identify the remining challenges for the nanogels before their translation to the market as transfection agents.
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Affiliation(s)
- Bruno Espuche
- Center for Cooperative Research in Biomaterials (CIC biomaGUNE), Basque Research and Technology Alliance (BRTA), Paseo de Miramon 194, 20014 Donostia-San Sebastián, Spain; POLYMAT, Applied Chemistry Department, Faculty of Chemistry, University of the Basque Country UPV/EHU, Paseo Manuel de Lardizabal 3, 20018 Donostia-San Sebastián, Spain
| | - Sergio E Moya
- Center for Cooperative Research in Biomaterials (CIC biomaGUNE), Basque Research and Technology Alliance (BRTA), Paseo de Miramon 194, 20014 Donostia-San Sebastián, Spain.
| | - Marcelo Calderón
- POLYMAT, Applied Chemistry Department, Faculty of Chemistry, University of the Basque Country UPV/EHU, Paseo Manuel de Lardizabal 3, 20018 Donostia-San Sebastián, Spain; IKERBASQUE, Basque Foundation for Science, Plaza Euskadi 5, 48009 Bilbao, Spain.
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5
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Guan X, Pei Y, Song J. DNA-Based Nonviral Gene Therapy─Challenging but Promising. Mol Pharm 2024; 21:427-453. [PMID: 38198640 DOI: 10.1021/acs.molpharmaceut.3c00907] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2024]
Abstract
Over the past decades, significant progress has been made in utilizing nucleic acids, including DNA and RNA molecules, for therapeutic purposes. For DNA molecules, although various DNA delivery systems have been established, viral vector systems are the go-to choice for large-scale commercial applications. However, viral systems have certain disadvantages such as immune response, limited payload capacity, insertional mutagenesis and pre-existing immunity. In contrast, nonviral systems are less immunogenic, not size limited, safer, and easier for manufacturing compared with viral systems. What's more, nonviral DNA vectors have demonstrated their capacity to mediate specific protein expression in vivo for diverse therapeutic objectives containing a wide range of diseases such as cancer, rare diseases, neurodegenerative diseases, and infectious diseases, yielding promising therapeutic outcomes. However, exogenous plasmid DNA is prone to degrade and has poor immunogenicity in vivo. Thus, various strategies have been developed: (i) designing novel plasmids with special structures, (ii) optimizing plasmid sequences for higher expression, and (iii) developing more efficient nonviral DNA delivery systems. Based on these strategies, many interesting clinical results have been reported. This Review discusses the development of DNA-based nonviral gene therapy, including novel plasmids, nonviral delivery systems, clinical advances, and prospects. These developments hold great potential for enhancing the efficacy and safety of nonviral gene therapy and expanding its applications in the treatment of various diseases.
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Affiliation(s)
- Xiaocai Guan
- Institute of Nano Biomedicine and Engineering, Department of Instrument Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China
| | - Yufeng Pei
- Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, The Cancer Hospital of the University of Chinese Academy of Sciences, Hangzhou 310022, China
| | - Jie Song
- Institute of Nano Biomedicine and Engineering, Department of Instrument Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China
- Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, The Cancer Hospital of the University of Chinese Academy of Sciences, Hangzhou 310022, China
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6
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Sufian MA, Ilies MA. Lipid-based nucleic acid therapeutics with in vivo efficacy. WILEY INTERDISCIPLINARY REVIEWS. NANOMEDICINE AND NANOBIOTECHNOLOGY 2023; 15:e1856. [PMID: 36180107 PMCID: PMC10023279 DOI: 10.1002/wnan.1856] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/27/2022] [Revised: 07/22/2022] [Accepted: 08/30/2022] [Indexed: 03/09/2023]
Abstract
Synthetic vectors for therapeutic nucleic acid delivery are currently competing significantly with their viral counter parts due to their reduced immunogenicity, large payload capacity, and ease of manufacture under GMP-compliant norms. The approval of Onpattro, a lipid-based siRNA therapeutic, and the proven clinical success of two lipid-based COVID-19 vaccines from Pfizer-BioNTech, and Moderna heralded the specific advantages of lipid-based systems among all other synthetic nucleic acid carriers. Lipid-based systems with diverse payloads-plasmid DNA (pDNA), antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), and messenger RNA (mRNA)-are now becoming a mature technology, with growing impact in the clinic. Research over four decades identified the key factors determining the therapeutic success of these multi-component systems. Here, we discuss the main nucleic acid-based technologies, presenting their mechanism of action, delivery barriers facing them, the structural properties of the payload as well as the component lipids that regulate physicochemical properties, pharmacokinetics and biodistribution, efficacy, and toxicity of the resultant nanoparticles. We further detail on the formulation parameters, evolution of the manufacturing techniques that generate reproducible and scalable outputs, and key manufacturing aspects that enable control over physicochemical properties of the resultant particles. Preclinical applications of some of these formulations that were successfully translated from in vitro studies to animal models are subsequently discussed. Finally, clinical success and failure of these systems starting from 1993 to present are highlighted, in a holistic literature review focused on lipid-based nucleic acid delivery systems. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials.
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Affiliation(s)
- Md Abu Sufian
- Department of Pharmaceutical Sciences and Moulder Center for Drug Discovery Research, School of Pharmacy, Temple University, 3307 North Broad Street, Philadelphia, PA 19140, USA
| | - Marc A. Ilies
- Department of Pharmaceutical Sciences and Moulder Center for Drug Discovery Research, School of Pharmacy, Temple University, 3307 North Broad Street, Philadelphia, PA 19140, USA
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7
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Talebnia F, Pushparajah D, Chandrasekaran S, Hersch SJ, Nafissi N, Slavcev R. Application of an electro elution system for direct purification of linear covalently closed DNA fragments. J Chromatogr B Analyt Technol Biomed Life Sci 2023; 1218:123622. [PMID: 36842293 DOI: 10.1016/j.jchromb.2023.123622] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2022] [Revised: 01/23/2023] [Accepted: 01/25/2023] [Indexed: 02/10/2023]
Abstract
Gene therapy is a powerful treatment modality. Non-viral gene therapy vectors power one arm of this important approach, due to their enhanced safety profile compared to their viral counterparts. New non-viral approaches continue to be developed, but purification can bottleneck the scaleup and cost-effectiveness and quality of some of these advanced vectors. We require more advanced purification and separation techniques compared to conventional methods to maximize resolution in a scalable manner. The Prep Cell system is a continuous electro elution system that contains a circular gel casting tube where DNA mixtures can be run through and subsequently migrate into an elution chamber, to be eluted by a peristaltic pump. This DNA separation and purification process confers advantages over other conventional methods, including i) the elimination of multiple downstream purification process requirements; ii) its ability to be applied in mid-scale settings, and iii), its high-resolution power. In this study, we assessed the ability of this Prep Cell Model 491 system to purify a novel type of non-viral linear covalently closed (LCC) DNA minivector (ministring DNA) from its precursor parent plasmid DNA and process by-product DNA species by analyzing for effective separation via agarose gel electrophoresis, recovery yield, single enzyme digestion, and quality control assessments. Overall, effective separation and resolution of mini-DNA vectors was obtained using the Prep Cell system, conferring its potential to be applied towards mid-scale purification of DNA vectors for a variety of research, and eventually, clinical applications.
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Affiliation(s)
- F Talebnia
- Mediphage Bioceuticals, Inc. 661 University Avenue, Suite 1300, MaRS West Tower, Toronto, ON M5G0B7, Canada
| | - D Pushparajah
- University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada
| | - S Chandrasekaran
- Mediphage Bioceuticals, Inc. 661 University Avenue, Suite 1300, MaRS West Tower, Toronto, ON M5G0B7, Canada
| | - S J Hersch
- Mediphage Bioceuticals, Inc. 661 University Avenue, Suite 1300, MaRS West Tower, Toronto, ON M5G0B7, Canada
| | - N Nafissi
- Mediphage Bioceuticals, Inc. 661 University Avenue, Suite 1300, MaRS West Tower, Toronto, ON M5G0B7, Canada.
| | - R Slavcev
- Mediphage Bioceuticals, Inc. 661 University Avenue, Suite 1300, MaRS West Tower, Toronto, ON M5G0B7, Canada; University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada.
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8
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Lock D, Monjezi R, Brandes C, Bates S, Lennartz S, Teppert K, Gehrke L, Karasakalidou-Seidt R, Lukic T, Schmeer M, Schleef M, Werchau N, Eyrich M, Assenmacher M, Kaiser A, Prommersberger S, Schaser T, Hudecek M. Automated, scaled, transposon-based production of CAR T cells. J Immunother Cancer 2022; 10:jitc-2022-005189. [PMID: 36096530 PMCID: PMC9472140 DOI: 10.1136/jitc-2022-005189] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/23/2022] [Indexed: 11/04/2022] Open
Abstract
BACKGROUND There is an increasing demand for chimeric antigen receptor (CAR) T cell products from patients and care givers. Here, we established an automated manufacturing process for CAR T cells on the CliniMACS Prodigy platform that is scaled to provide therapeutic doses and achieves gene-transfer with virus-free Sleeping Beauty (SB) transposition. METHODS We used an advanced CliniMACS Prodigy that is connected to an electroporator unit and performed a series of small-scale development and large-scale confirmation runs with primary human T cells. Transposition was accomplished with minicircle (MC) DNA-encoded SB100X transposase and pT2 transposon encoding a CD19 CAR. RESULTS We defined a bi-pulse electroporation shock with bi-directional and unidirectional electric field, respectively, that permitted efficient MC insertion and maintained a high frequency of viable T cells. In three large scale runs, 2E8 T cells were enriched from leukapheresis product, activated, gene-engineered and expanded to yield up to 3.5E9 total T cells/1.4E9 CAR-modified T cells within 12 days (CAR-modified T cells: 28.8%±12.3%). The resulting cell product contained highly pure T cells (97.3±1.6%) with balanced CD4/CD8 ratio and a high frequency of T cells with central memory phenotype (87.5%±10.4%). The transposon copy number was 7.0, 9.4 and 6.8 in runs #1-3, respectively, and gene analyses showed a balanced expression of activation/exhaustion markers. The CD19 CAR T cell product conferred potent anti-lymphoma reactivity in pre-clinical models. Notably, the operator hands-on-time was substantially reduced compared with conventional non-automated CAR T cell manufacturing campaigns. CONCLUSIONS We report on the first automated transposon-based manufacturing process for CAR T cells that is ready for formal validation and use in clinical manufacturing campaigns. This process and platform have the potential to facilitate access of patients to CAR T cell therapy and to accelerate scaled, multiplexed manufacturing both in the academic and industry setting.
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Affiliation(s)
- Dominik Lock
- Miltenyi Biotec BV & Co KG, Bergisch Gladbach, Germany
| | - Razieh Monjezi
- Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany
| | | | - Stephan Bates
- Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany
| | | | - Karin Teppert
- Miltenyi Biotec BV & Co KG, Bergisch Gladbach, Germany
| | - Leon Gehrke
- Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany
| | | | - Teodora Lukic
- Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany
| | | | | | - Niels Werchau
- Miltenyi Biotec BV & Co KG, Bergisch Gladbach, Germany
| | - Matthias Eyrich
- Universitätskinderklinik, Universitätsklinikum Würzburg, Würzburg, Germany
| | | | - Andrew Kaiser
- Miltenyi Biotec BV & Co KG, Bergisch Gladbach, Germany
| | | | | | - Michael Hudecek
- Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany
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9
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Prommersberger S, Monjezi R, Botezatu L, Miskey C, Amberger M, Mestermann K, Hudecek M, Ivics Z. Generation of CAR-T Cells with Sleeping Beauty Transposon Gene Transfer. Methods Mol Biol 2022; 2521:41-66. [PMID: 35732992 DOI: 10.1007/978-1-0716-2441-8_3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/15/2023]
Abstract
Human T lymphocytes that transgenically express a chimeric antigen receptor (CAR) have proven efficacy and safety in gene- and cell-based immunotherapy of certain hematological cancers. Appropriate gene vectors and methods of genetic engineering are required for therapeutic cell products to be biologically potent and their manufacturing to be economically viable. Transposon-based gene transfer satisfies these needs, and is currently being evaluated in clinical trials. In this protocol we describe the basic Sleeping Beauty (SB) transposon vector components required for stable gene integration in human cells, with special emphasis on minicircle DNA vectors and the use of synthetic mRNA. We provide a protocol for functional validation of the vector components in cultured human cell lines on the basis of fluorescent reporter gene expression. Finally, we provide a protocol for CAR-T cell engineering and describe assays that address transgene expression, biological potency and genomic vector copy numbers in polyclonal cell populations. Because transposons allow virus-free gene transfer with naked nucleic acids, the protocol can be adopted by any laboratory equipped with biological safety level S1 facilities.
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Affiliation(s)
| | - Razieh Monjezi
- Department of Internal Medicine II, University Hospital of Würzburg, Würzburg, Germany
| | | | - Csaba Miskey
- Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany
| | | | - Katrin Mestermann
- Department of Internal Medicine II, University Hospital of Würzburg, Würzburg, Germany
| | - Michael Hudecek
- Department of Internal Medicine II, University Hospital of Würzburg, Würzburg, Germany
| | - Zoltán Ivics
- Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany.
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10
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Ramirez GA, Gasmi M. Manufacturing of Viral Gene Therapies. Int Ophthalmol Clin 2021; 61:91-112. [PMID: 34196319 DOI: 10.1097/iio.0000000000000362] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
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11
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Eusébio D, Almeida AM, Alves JM, Maia CJ, Queiroz JA, Sousa F, Sousa Â. The Performance of Minicircle DNA Versus Parental Plasmid in p53 Gene Delivery Into HPV-18-Infected Cervical Cancer Cells. Nucleic Acid Ther 2020; 31:82-91. [PMID: 33252302 DOI: 10.1089/nat.2020.0904] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Minicircle DNA (mcDNA) has been suggested as a vanguard technology for gene therapy, consisting of a nonviral DNA vector devoid of prokaryotic sequences. Unlike conventional plasmid DNA (pDNA), this small vector is able to sustain high expression rates throughout time. Thus, this work describes the construction, production, and purification of mcDNA-p53 and its precursor parental plasmid (PP)-p53 for a comparative study of both DNA vectors in the growth suppression of human papillomavirus (HPV)-18-infected cervical cancer cells. First, live cell imaging and fluorescence microscopy studies allowed to understand that mcDNA-p53 vector was able to enter cell nuclei more rapidly than PP-p53 vector, leading to a transfection efficiency of 68% against 34%, respectively. Then, p53 transcripts and protein expression assessment revealed that both vectors were able to induce transcription and the target protein expression. However, the mcDNA-p53 vector performance stood out, by demonstrating higher p53 expression levels (91.65 ± 2.82 U/mL vs. 74.75 ± 4.44 U/mL). After assuring the safety of both vectors by viability studies, such potential was confirmed by proliferation and apoptosis assays. These studies confirmed the mcDNA-p53 vector function toward cell cycle arrest and apoptosis in HPV-18-infected cervical cancer cells. Altogether, these results suggest that the mcDNA vector has a more promising and efficient role as a DNA vector than conventional pDNA, opening new investigation lines for cervical cancer treatment in the future.
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Affiliation(s)
- Dalinda Eusébio
- CICS-UBI-Health Science Research Centre, University of Beira Interior, Covilhã, Portugal
| | - Ana Margarida Almeida
- CICS-UBI-Health Science Research Centre, University of Beira Interior, Covilhã, Portugal
| | - Joel Marques Alves
- CICS-UBI-Health Science Research Centre, University of Beira Interior, Covilhã, Portugal
| | - Cláudio Jorge Maia
- CICS-UBI-Health Science Research Centre, University of Beira Interior, Covilhã, Portugal
| | - João António Queiroz
- CICS-UBI-Health Science Research Centre, University of Beira Interior, Covilhã, Portugal
| | - Fani Sousa
- CICS-UBI-Health Science Research Centre, University of Beira Interior, Covilhã, Portugal
| | - Ângela Sousa
- CICS-UBI-Health Science Research Centre, University of Beira Interior, Covilhã, Portugal
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12
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Almeida AM, Eusébio D, Queiroz JA, Sousa F, Sousa Â. Minicircle DNA Vaccine Purification and E7 Antigen Expression Assessment. Methods Mol Biol 2020; 2197:207-222. [PMID: 32827139 DOI: 10.1007/978-1-0716-0872-2_11] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/09/2023]
Abstract
Human papillomavirus (HPV ) has been extensively associated with the development of cervical cancer due to the expression of oncoproteins like E7. This protein can interfere with pRB tumor suppressor activity, enabling the uncontrolled proliferation of abnormal cells. DNA vaccines are known as the third-generation vaccines, providing the ability of targeting viral infections such as HPV in a preventive and therapeutic way. Although current strategies make use of plasmid DNA (pDNA) as the vector of choice to be used as a DNA vaccine, minicircle DNA (mcDNA) has been proving its added value as a non-viral DNA vector by demonstrating higher expression efficiency and increased biosafety than the pDNA. However, due to its innovative profile, few methodologies have been explored and implemented for the manufacture of this molecule. This chapter describes the detailed procedures for the production, extraction, and purification of supercoiled E7-mcDNA vaccine, by using size-exclusion chromatography to obtain mcDNA with a purity degree which meets the regulatory agency criteria. Then, the assessment of E7 antigen expression through immunocytochemistry is also described.
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Affiliation(s)
- Ana M Almeida
- CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal
| | - Dalinda Eusébio
- CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal
| | - João A Queiroz
- CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal
| | - Fani Sousa
- CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal
| | - Ângela Sousa
- CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal.
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13
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Nawaz W, Xu S, Li Y, Huang B, Wu X, Wu Z. Nanotechnology and immunoengineering: How nanotechnology can boost CAR-T therapy. Acta Biomater 2020; 109:21-36. [PMID: 32294554 DOI: 10.1016/j.actbio.2020.04.015] [Citation(s) in RCA: 42] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2019] [Revised: 03/29/2020] [Accepted: 04/07/2020] [Indexed: 12/13/2022]
Abstract
Chimeric antigen receptor (CAR) therapy has achieved remarkable clinical efficacy against hematological cancers and has been approved by FDA for treatment of B-cell tumors. However, the complex manufacturing process and limited success in solid tumors hamper its widespread applications, thus prompting the development of new strategies for overcoming the abovementioned hurdles. In the last decade, nanotechnology has provided sustainable strategies for improving cancer immunotherapy through vaccine development and delivery of immunomodulatory drugs. Nanotechnology can boost CAR-T therapy and may overcome the existing challenges by emerging as a carrier for CAR-T therapy or in combination with CAR-T, it may inhibit solid tumors more effectively than conventional approaches. The revealing of cellular mechanisms, barriers and potential strategies that could be used to manipulate and/or modify cells would enable unprecedented advances in nanotechnology for biologics delivery. This review outlines the journey and barriers of nanoparticles (NPs) across the cell. Subsequently, the approaches to tackle the barriers and strategies to modulate NPs as a carrier for CAR-T therapy are discussed. Finally, the role of NPs in CAR-T therapy and the potential challenges are summarized. This review aims to provide the readers with a detailed overview of NP-based CAR-T therapy research and distil this information into an accessible form conducive to design desired CAR-T therapy using NP approach. STATEMENT OF SIGNIFICANCE: Chimeric antigen receptor (CAR) T-cell therapy is the most vibrant field in immuno-oncology today, with enormous benefits to patients with B-cell malignancies. However, a rapid and straightforward procedure for CAR-T generation is an exigent need to broaden its therapeutic avenue. Nanotechnology has emerged as a novel alternative approach for CAR-T generation. To the best of our knowledge, this is the first in-depth review that briefly highlights the various aspects of nanotechnology in CAR-T therapy, including the strategies to brand NPs as an effective carrier for CAR cargo, its potential advantages, challenges, and future roadmap. It provides readers with a detailed overview of NP-based CAR-T therapy research, and researchers would be able to distill this information into an accessible form conducive to design the desired CAR therapy using the nanotechnology approach.
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Almeida AM, Queiroz JA, Sousa F, Sousa Â. Minicircle DNA: The Future for DNA-Based Vectors? Trends Biotechnol 2020; 38:1047-1051. [PMID: 32409109 DOI: 10.1016/j.tibtech.2020.04.008] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2020] [Revised: 04/15/2020] [Accepted: 04/16/2020] [Indexed: 01/16/2023]
Abstract
Minicircle DNA (mcDNA) is a smaller and safer version of non-viral DNA vectors that results from a cutting-edge in vivo recombination process to excise prokaryotic sequences from plasmid DNA (pDNA). Considering the molecule's potential and increasing interest as a non-viral DNA-based therapeutic, biomanufacturing methodologies need to be improved, especially in downstream processing.
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Affiliation(s)
- Ana Margarida Almeida
- CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal
| | - João António Queiroz
- CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal
| | - Fani Sousa
- CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal
| | - Ângela Sousa
- CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
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15
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Zimmermann A, Hercher D, Regner B, Frischer A, Sperger S, Redl H, Hacobian A. Evaluation of BMP-2 Minicircle DNA for Enhanced Bone Engineering and Regeneration. Curr Gene Ther 2020; 20:55-63. [PMID: 32338217 DOI: 10.2174/1566523220666200427121350] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2020] [Revised: 04/06/2020] [Accepted: 04/08/2020] [Indexed: 12/12/2022]
Abstract
BACKGROUND To date, the significant osteoinductive potential of bone morphogenetic protein 2 (BMP-2) non-viral gene therapy cannot be fully exploited therapeutically. This is mainly due to weak gene delivery and brief expression peaks restricting the therapeutic effect. OBJECTIVE Our objective was to test the application of minicircle DNA, allowing prolonged expression potential. It offers notable advantages over conventional plasmid DNA. The lack of bacterial sequences and the resulting reduction in size, enables safe usage and improved performance for tissue regeneration. METHODS We inserted an optimized BMP-2 gene cassette with minicircle plasmid technology. BMP-2 minicircle plasmids were produced in E. coli yielding plasmids lacking bacterial backbone elements. Comparative studies of these BMP-2 minicircles and conventional BMP-2 plasmids were performed in vitro in cell systems, including bone marrow derived stem cells. Tests performed included gene expression profiles and cell differentiation assays. RESULTS A C2C12 cell line transfected with the BMP-2-Advanced minicircle showed significantly elevated expression of osteocalcin, alkaline phosphatase (ALP) activity, and BMP-2 protein amount when compared to cells transfected with conventional BMP-2-Advanced plasmid. Furthermore, the plasmids show suitability for stem cell approaches by showing significantly higher levels of ALP activity and mineralization when introduced into human bone marrow stem cells (BMSCs). CONCLUSION We have designed a highly bioactive BMP-2 minicircle plasmid with the potential to fulfil clinical requirements for non-viral gene therapy in the field of bone regeneration.
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Affiliation(s)
- Alice Zimmermann
- Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
| | - David Hercher
- Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
| | - Benedikt Regner
- Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
| | - Amelie Frischer
- Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
| | - Simon Sperger
- Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
| | - Heinz Redl
- Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
| | - Ara Hacobian
- Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
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Barnea-Cramer AO, Singh M, Fischer D, De Silva S, McClements ME, Barnard AR, MacLaren RE. Repair of Retinal Degeneration following Ex Vivo Minicircle DNA Gene Therapy and Transplantation of Corrected Photoreceptor Progenitors. Mol Ther 2020; 28:830-844. [PMID: 32027843 DOI: 10.1016/j.ymthe.2020.01.023] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2019] [Revised: 01/07/2020] [Accepted: 01/07/2020] [Indexed: 10/25/2022] Open
Abstract
The authors describe retinal reconstruction and restoration of visual function in heritably blind mice missing the rhodopsin gene using a novel method of ex vivo gene therapy and cell transplantation. Photoreceptor precursors with the same chromosomal genetic mutation were treated ex vivo using minicircle DNA, a non-viral technique that does not present the packaging limitations of adeno-associated virus (AAV) vectors. Following transplantation, genetically modified cells reconstructed a functional retina and supported vision in blind mice harboring the same founder gene mutation. Gene delivery by minicircles showed comparable long-term efficiency to AAV in delivering the missing gene, representing the first non-viral system for robust treatment of photoreceptors. This important proof-of-concept finding provides an innovative convergence of cell and gene therapies for the treatment of hereditary neurodegenerative disease and may be applied in future studies toward ex vivo correction of patient-specific cells to provide an autologous source of tissue to replace lost photoreceptors in inherited retinal blindness. This is the first report using minicircles in photoreceptor progenitors and the first to transplant corrected photoreceptor precursors to restore vision in blind animals.
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Affiliation(s)
| | - Mandeep Singh
- Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, UK; Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
| | - Dominik Fischer
- Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, UK; University Eye Hospital and Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tuebingen, Tuebingen, Germany
| | - Samantha De Silva
- Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, UK; Oxford University Hospitals NHS Foundation Trust, NIHR Oxford Biomedical Research Centre, Oxford, UK
| | | | - Alun R Barnard
- Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, UK
| | - Robert E MacLaren
- Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, UK; Oxford University Hospitals NHS Foundation Trust, NIHR Oxford Biomedical Research Centre, Oxford, UK.
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Zeng M, Alshehri F, Zhou D, Lara-Sáez I, Wang X, Li X, A S, Xu Q, Zhang J, Wang W. Efficient and Robust Highly Branched Poly(β-amino ester)/Minicircle COL7A1 Polymeric Nanoparticles for Gene Delivery to Recessive Dystrophic Epidermolysis Bullosa Keratinocytes. ACS APPLIED MATERIALS & INTERFACES 2019; 11:30661-30672. [PMID: 31390173 DOI: 10.1021/acsami.9b13135] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/28/2023]
Abstract
Recessive dystrophic epidermolysis bullosa (RDEB) is a severe congenital skin fragility disease caused by COL7A1 mutations that result in type VII collagen (C7) deficiency. Herein, we report a synergistic polyplex system that can efficiently restore C7 expression in RDEB keratinocytes. A highly branched multifunctional poly(β-amino ester) (HPAE), termed as HC32-122, was optimized systematically as the high-performance gene delivery vector for keratinocytes, achieving much higher transfection capability than polyethylenimine, SuperFect, and Lipofectamine 2000 without inducing obvious cytotoxicity. Concurrently, a 12 kb length minicircle DNA encoding ∼9 kb full-length COL7A1 (MCC7) devoid of bacterial sequence was biosynthesized as the therapeutic gene. Combining the highly potent polymer and the miniaturized gene structure, HC32-122/MCC7 polyplexes achieve 96.4% cellular uptake efficiency, 4019-fold COL7A1 mRNA enhancement, and robust recombinant C7 expression. Structure-property investigations reveal that HC32-122 can effectively condense MCC7 to form small, uniform, compact, and positively charged spherical nanoparticles with high DNA release flexibility. Moreover, formulation study shows that sucrose is conductive to lyophilized HC32-122/DNA polyplexes for maintaining the transfection capability. Direct frozen polyplexes can maintain full gene transfection capability after one-year storage. High efficiency, biocompatibility, facile manipulation, and long-term stability make the HC32-122/MCC7 system a promising bench-to-bed candidate for treating the debilitating RDEB.
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Affiliation(s)
- Ming Zeng
- Charles Institute of Dermatology , University College Dublin , Dublin D04 V1W8 , Ireland
- Department of Dermatology , The First Affiliated Hospital of Anhui Medical University , Hefei 230022 , China
| | - Fatma Alshehri
- Charles Institute of Dermatology , University College Dublin , Dublin D04 V1W8 , Ireland
- Princess Nourah bint Abdulrahman University , Riyadh 11671 , Saudi Arabia
| | - Dezhong Zhou
- Charles Institute of Dermatology , University College Dublin , Dublin D04 V1W8 , Ireland
- School of Chemical Engineering and Technology , Xi'an Jiaotong University , Xi'an , Shaanxi 710049 , China
| | - Irene Lara-Sáez
- Charles Institute of Dermatology , University College Dublin , Dublin D04 V1W8 , Ireland
| | - Xi Wang
- Charles Institute of Dermatology , University College Dublin , Dublin D04 V1W8 , Ireland
| | - Xiaolin Li
- Charles Institute of Dermatology , University College Dublin , Dublin D04 V1W8 , Ireland
| | - Sigen A
- Charles Institute of Dermatology , University College Dublin , Dublin D04 V1W8 , Ireland
| | - Qian Xu
- Charles Institute of Dermatology , University College Dublin , Dublin D04 V1W8 , Ireland
| | - Jing Zhang
- Charles Institute of Dermatology , University College Dublin , Dublin D04 V1W8 , Ireland
| | - Wenxin Wang
- Charles Institute of Dermatology , University College Dublin , Dublin D04 V1W8 , Ireland
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Almeida AM, Eusébio D, Queiroz JA, Sousa F, Sousa A. The use of size-exclusion chromatography in the isolation of supercoiled minicircle DNA from Escherichia coli lysate. J Chromatogr A 2019; 1609:460444. [PMID: 31455515 DOI: 10.1016/j.chroma.2019.460444] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2019] [Revised: 08/07/2019] [Accepted: 08/08/2019] [Indexed: 11/13/2022]
Abstract
Minicircle DNA (mcDNA) is the new cutting-edge technology which researchers have been exploring for gene therapy and DNA vaccination. Although it presents enormous advantages in comparison to conventional plasmid DNA regarding bioactivity and safety, its challenging isolation from parental plasmid and miniplasmid has been setting back its launching in biomedical sciences. In this work, it is demonstrated the use of a simple size exclusion chromatographic method for the isolation of supercoiled mcDNA. Sephacryl S-1000 SF matrix was explored under different conditions (flow, peak fractionation volume and sample loading) to achieve the best performance and retrieve a mcDNA sample devoid of other bacterial contaminants or plasmid species resultant from the recombination process. This isolation methodology resulted in 66.7% of mcDNA recovery with 98.1% of purity. In addition, to show the robustness of the method, the potential of using this matrix for the isolation of a larger mcDNA was also evaluated. Upon adjusting the flow or the column volume, the larger mcDNA molecule was also successfully isolated. Overall, a simple and effective strategy has been established for the isolation of supercoiled mcDNA, underlining the potential of size exclusion chromatography in mcDNA separation.
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Affiliation(s)
- A M Almeida
- CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal
| | - D Eusébio
- CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal
| | - J A Queiroz
- CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal
| | - F Sousa
- CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
| | - A Sousa
- CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal
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Han D, Wang Y, Chen J, Zhang J, Yu P, Zhang R, Li S, Tao B, Wang Y, Qiu Y, Xu M, Gao E, Cao F. Activation of melatonin receptor 2 but not melatonin receptor 1 mediates melatonin-conferred cardioprotection against myocardial ischemia/reperfusion injury. J Pineal Res 2019; 67:e12571. [PMID: 30903623 DOI: 10.1111/jpi.12571] [Citation(s) in RCA: 59] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/29/2018] [Revised: 03/01/2019] [Accepted: 03/17/2019] [Indexed: 12/14/2022]
Abstract
Accumulated pieces of evidence have proved the beneficial effects of melatonin on myocardial ischemia/reperfusion (MI/R) injury, and these effects were largely dependent on melatonin membrane receptor activation. In humans and other mammals, there are two types of melatonin receptors, including the melatonin receptor 1 (MT1, melatonin receptor 1a or MTNR1A) and melatonin receptor 1 (MT2, melatonin receptor 1b or MTNR1B) receptor subtypes. However, which receptor mediates melatonin-conferred cardioprotection remains unclear. In this study, we employed both loss-of-function and gain-of-function approaches to reveal the answer. Mice (wild-type; MT1 or MT2 silencing by in vivo minicircle vector; and those overexpressing MT1 or MT2 by in vivo AAV9 vector) were exposed to MI/R injury. Both MT1 and MT2 were present in wild-type myocardium. MT2, but not MT1, was essentially upregulated after MI/R Melatonin administration significantly reduced myocardial injury and improved cardiac function after MI/R Mechanistically, melatonin treatment suppressed MI/R-initiated myocardial oxidative stress and nitrative stress, alleviated endoplasmic reticulum stress and mitochondrial injury, and inhibited myocardial apoptosis. These beneficial actions of melatonin were absent in MT2-silenced heart, but not the MT1 subtype. Furthermore, AAV9-mediated cardiomyocyte-specific overexpression of MT2, but not MT1, mitigated MI/R injury and improved cardiac dysfunction, which was accompanied by significant amelioration of oxidative stress, endoplasmic reticulum stress, and mitochondrial dysfunction. Mechanistically, MT2 protected primary cardiomyocytes against hypoxia/reoxygenation injury via MT2/Notch1/Hes1/RORα signaling. Our study presents the first direct evidence that the MT2 subtype, but not MT1, is a novel endogenous cardiac protective receptor against MI/R injury. Medications specifically targeting MT2 may hold promise in fighting ischemic heart disease.
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MESH Headings
- Animals
- Apoptosis
- Disease Models, Animal
- Endoplasmic Reticulum Stress/genetics
- Humans
- Male
- Mice
- Myocardial Reperfusion Injury/genetics
- Myocardial Reperfusion Injury/metabolism
- Myocardial Reperfusion Injury/pathology
- Myocardial Reperfusion Injury/prevention & control
- Myocardium/metabolism
- Myocardium/pathology
- Myocytes, Cardiac/metabolism
- Myocytes, Cardiac/pathology
- Oxidative Stress/genetics
- Receptor, Melatonin, MT1/genetics
- Receptor, Melatonin, MT1/metabolism
- Receptor, Melatonin, MT2/genetics
- Receptor, Melatonin, MT2/metabolism
- Signal Transduction
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Affiliation(s)
- Dong Han
- Department of Cardiology, National Clinical Research Center for Geriatric Diseases, 2nd Medical Center, Chinese PLA General Hospital, Beijing, China
- Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
| | - Yongjun Wang
- Department of Cardiovascular Surgery, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Jiangwei Chen
- Department of Cardiology, National Clinical Research Center for Geriatric Diseases, 2nd Medical Center, Chinese PLA General Hospital, Beijing, China
- Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
| | - Jibin Zhang
- Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
| | - Peng Yu
- Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
| | - Ran Zhang
- Department of Cardiology, National Clinical Research Center for Geriatric Diseases, 2nd Medical Center, Chinese PLA General Hospital, Beijing, China
| | - Shuang Li
- Department of Cardiology, Chengdu Military General Hospital, Chengdu, China
| | - Bo Tao
- Department of Cardiology, National Clinical Research Center for Geriatric Diseases, 2nd Medical Center, Chinese PLA General Hospital, Beijing, China
| | - Yabin Wang
- Department of Cardiology, National Clinical Research Center for Geriatric Diseases, 2nd Medical Center, Chinese PLA General Hospital, Beijing, China
| | - Ya Qiu
- Department of Cardiology, National Clinical Research Center for Geriatric Diseases, 2nd Medical Center, Chinese PLA General Hospital, Beijing, China
| | - Mengqi Xu
- Department of Cardiology, National Clinical Research Center for Geriatric Diseases, 2nd Medical Center, Chinese PLA General Hospital, Beijing, China
| | - Erhe Gao
- Center for Translational Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania
| | - Feng Cao
- Department of Cardiology, National Clinical Research Center for Geriatric Diseases, 2nd Medical Center, Chinese PLA General Hospital, Beijing, China
- Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
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Zhang T, Duan J, Zhang L, Li Z, Steer CJ, Yan G, Song G. LXRα Promotes Hepatosteatosis in Part Through Activation of MicroRNA-378 Transcription and Inhibition of Ppargc1β Expression. Hepatology 2019; 69:1488-1503. [PMID: 30281809 PMCID: PMC6519356 DOI: 10.1002/hep.30301] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/02/2018] [Accepted: 09/24/2018] [Indexed: 12/21/2022]
Abstract
Nonalcoholic fatty liver disease (NAFLD) is a major risk factor of many end-stage liver diseases. Alterations in microRNA expression have been reported in patients with NAFLD. However, the transcriptional mechanism(s) of dysregulated microRNAs under the state of NAFLD is poorly described, and microRNAs that regulate the pathogenesis of NAFLD synergistically with their regulators remain unknown. Here we report that microRNA-378 expression is significantly increased in fatty livers of mice and patients with NAFLD. Although microRNA-378 locates within the intron of Ppargc1β (peroxisome proliferator-activated receptor γ coactivator 1-beta), there was a significant uncoupling of Ppargc1β mRNA and microRNA-378 levels in both sources of fatty livers. Further studies identified a full-length primary transcript of microRNA-378. LXRα (liver X receptor alpha) functioned as a transcription activator of microRNA-378 and a repressor of Ppargc1β transcription. It is known that miR-378 is an inhibitor of fatty acid oxidation (FAO) and the function of Ppargc1β is opposite to that of miR-378. GW3965 treatment (LXRα agonist) of murine hepatocytes and mice increased microRNA-378 and reduced Ppargc1β, which subsequently impaired FAO and aggravated hepatosteatosis. In contrast, additional treatment of miR-378 inhibitor or Ppargc1β, which knocked down increased miR-378 or recovered expression of Ppargc1β, offset the effects of GW3965. Liver-specific ablation of Lxrα led to decreased miR-378 and increased Ppargc1β, which subsequently improved FAO and reduced hepatosteatosis. Conclusion: Our findings indicated that miR-378 possesses its own transcription machinery, which challenges the well-established dogma that miR-378 transcription is controlled by the promoter of Ppargc1β. LXRα selectively activates transcription of miR-378 and inhibits expression of Ppargc1β, which synergistically impairs FAO. In addition to lipogenesis, impaired FAO by miR-378 in part contributes to LXRα-induced hepatosteatosis.
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Affiliation(s)
- Tianpeng Zhang
- Department of MedicineUniversity of Minnesota Medical SchoolMinneapolisMinnesota
| | - Jiangyan Duan
- School of Life ScienceShanxi Normal UniversityLinfen CityChina
| | - Lei Zhang
- Department of Emergency SurgeryUnion Hospital, Tongji Medical College, Huazhong University of Science and TechnologyWuhanChina
| | - Zhuoyu Li
- Institute of BiotechnologyShanxi UniversityTaiyuanChina
| | - Clifford J. Steer
- Department of MedicineUniversity of Minnesota Medical SchoolMinneapolisMinnesota
- Department of Genetics, Cell Biology and DevelopmentUniversity of MinnesotaMinneapolisMinnesota
| | - Guiqin Yan
- School of Life ScienceShanxi Normal UniversityLinfen CityChina
| | - Guisheng Song
- Department of MedicineUniversity of Minnesota Medical SchoolMinneapolisMinnesota
- School of Life ScienceShanxi Normal UniversityLinfen CityChina
- Institute of BiotechnologyShanxi UniversityTaiyuanChina
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Zhang T, Hu J, Wang X, Zhao X, Li Z, Niu J, Steer CJ, Zheng G, Song G. MicroRNA-378 promotes hepatic inflammation and fibrosis via modulation of the NF-κB-TNFα pathway. J Hepatol 2019; 70:87-96. [PMID: 30218679 PMCID: PMC6554744 DOI: 10.1016/j.jhep.2018.08.026] [Citation(s) in RCA: 142] [Impact Index Per Article: 23.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/03/2018] [Revised: 07/25/2018] [Accepted: 08/31/2018] [Indexed: 12/25/2022]
Abstract
BACKGROUND & AIMS The progression of hepatosteatosis to non-alcoholic steatohepatitis (NASH) is a critical step in the pathogenesis of hepatocellular cancer. However, the underlying mechanism(s) for this progression is essentially unknown. This study was designed to determine the role of miR-378 in regulating NASH progression. METHODS We used immunohistochemistry, luciferase assays and immunoblotting to study the role of miR-378 in modulating an inflammatory pathway. Wild-type mice kept on a high-fat diet (HFD) were injected with miR-378 inhibitors or a mini-circle expression system containing miR-378, to study loss and gain-of functions of miR-378. RESULTS MiR-378 expression is increased in livers of dietary obese mice and patients with NASH. Further studies revealed that miR-378 directly targeted Prkag2 that encodes AMP-activated protein kinase γ 2 (AMPKγ2). AMPK signaling negatively regulates the NF-κB-TNFα inflammatory axis by increasing deacetylase activity of sirtuin 1. By targeting Prkag2, miR-378 reduced sirtuin 1 activity and facilitated an inflammatory pathway involving NF-κB-TNFα. In contrast, miR-378 knockdown induced expression of Prkag2, increased sirtuin 1 activity and blocked the NF-κB-TNFα axis. Additionally, knockdown of increased Prkag2 offset the inhibitory effects of miR-378 inhibitor on the NF-κB-TNFα axis, suggesting that AMPK signaling mediates the role of miR-378 in facilitating this inflammatory pathway. Liver-specific expression of miR-378 triggered the development of NASH and fibrosis by activating TNFα signaling. Ablation of TNFα in miR-378-treated mice impaired the ability of miR-378 to facilitate hepatic inflammation and fibrosis, suggesting that TNFα signaling is required for miR-378 to promote NASH. CONCLUSION MiR-378 plays a key role in the development of hepatic inflammation and fibrosis by positively regulating the NF-κB-TNFα axis. MiR-378 is a potential therapeutic target for the treatment of NASH. LAY SUMMARY The recent epidemic of obesity has been associated with a sharp rise in the incidence of non-alcoholic fatty liver disease (NAFLD). However, the underlying mechanism(s) remains poorly described and effective therapeutic approaches against NAFLD are lacking. The results establish that microRNA-378 facilitates the development of hepatic inflammation and fibrosis and suggests the therapeutic potential of microRNA-378 inhibitor for the treatment of NAFLD.
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Affiliation(s)
- Tianpeng Zhang
- Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA
| | - Junjie Hu
- Key Laboratory of Chinese Medicine Resource and Compound Prescription, Ministry of Education, Hubei University of Chinese Medicine, Wuhan, Hubei Province 430065, China
| | - Xiaomei Wang
- institute for Translational Medicine, Jilin University, Changchun, Jilin Province 130021, China
| | | | - Zhuoyu Li
- institute of Biotechnology, Shanxi University, Taiyuan, Shanxi Province 030006, China
| | - Junqi Niu
- Key Laboratory of Chinese Medicine Resource and Compound Prescription, Ministry of Education, Hubei University of Chinese Medicine, Wuhan, Hubei Province 430065, China
| | - Clifford J. Steer
- Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA,Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA
| | - Guohua Zheng
- Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA.
| | - Guisheng Song
- Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA; Key Laboratory of Chinese Medicine Resource and Compound Prescription, Ministry of Education, Hubei University of Chinese Medicine, Wuhan, Hubei Province 430065, China; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA.
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Zhang T, Zhao X, Steer CJ, Yan G, Song G. A negative feedback loop between microRNA-378 and Nrf1 promotes the development of hepatosteatosis in mice treated with a high fat diet. Metabolism 2018; 85:183-191. [PMID: 29625129 PMCID: PMC6062470 DOI: 10.1016/j.metabol.2018.03.023] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/05/2018] [Revised: 03/02/2018] [Accepted: 03/24/2018] [Indexed: 01/22/2023]
Abstract
BACKGROUNDS The incidence of nonalcoholic fatty liver disease (NAFLD) is rapidly increasing due to the prevalence of obesity. NAFLD is a major risk factor of hepatocellular carcinoma (HCC). Even with successful surgical removal, the presence of NAFLD is associated with an increased recurrence of HCC. Despite the extensive study of NAFLD, its underlying mechanism(s) remains essentially unknown and there are no FDA-approved drugs for its treatment. Alterations in microRNA (miR) expression have been observed in human fatty livers. However, regulatory mechanism(s) of miRNA biogenesis and their role in regulating the development of NAFLD is poorly described. METHODS We used immunohistochemistry, luciferase assays and immunoblotting to study the regulatory mechanism of miR-378 biogenesis. Wild-type mice kept on a high fat diet (HFD) were injected with miR-378 inhibitors or a mini-circle expression system containing miR-378 to study loss and gain-of functions of miR-378. RESULTS miR-378 was significantly increased in fatty livers of dietary obese mice and human hepatoma HepG2 cells with accumulated lipid. Further studies identified NRF1 (Nuclear receptor factor 1), a key regulator of fatty acid oxidation (FAO), as a direct target of miR-378. Overexpression of miR-378 impaired FAO and promoted lipid accumulation in murine hepatoma Hepa1-6 cells. In contrast, knockdown of miR-378 using its ASO (anti-sense oligo) improved FAO and reduced intracellular lipid content in Hepa1-6 cells. Liver-specific expression of miR-378 impaired FAO, which subsequently promoted the development of hepatosteatosis. Antagonizing miR-378 via injecting miR-378-ASO into HFD-treated mice led to increased expression of Nrf1, improved FAO and decreased hepatosteatosis. Additional knockdown of up-regulated Nrf1 offset the effects of miR-378-ASO, suggesting that Nrf1 mediated the inhibitory effect of miR-378-ASO on hepatosteatosis. Furthermore, Nrf1 was identified as a transcriptional repressor of miR-378. Ablation of Nrf1 using its shRNA in livers led to increased miR-378, which subsequently resulted in reduced FAO and elevated hepatic lipid content. CONCLUSIONS These findings identified a negative feedback loop between miR-378 and Nrf1 that promotes the pathogenesis of hepatosteatosis, and suggests the use of miR-378 as a potential therapeutic target for NAFLD.
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Affiliation(s)
- Tianpeng Zhang
- Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA
| | | | - Clifford J Steer
- Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA
| | - Guiqin Yan
- College of Life and Biological Sciences, Shanxi Normal University, Linfen City 041000, China.
| | - Guisheng Song
- Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA; McLab, South San Francisco, CA 94080, USA; College of Life and Biological Sciences, Shanxi Normal University, Linfen City 041000, China.
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23
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Yu H, Pan HM, Trau D, Patzel V. Capsule-like Safe Genetic Vectors-Cell-Penetrating Core-Shell Particles Selectively Release Functional Small RNA and Entrap Its Encoding DNA. ACS APPLIED MATERIALS & INTERFACES 2018; 10:21113-21124. [PMID: 29869496 DOI: 10.1021/acsami.8b04294] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/08/2023]
Abstract
The breakthrough of genetic therapy is set back by the lack of suitable genetic vector systems. We present the development of permeability-tunable, capsule-like, polymeric, micron-sized, core-shell particles for delivery of recombinant nucleic acids into target cells. These particles were demonstrated to effectively release rod-shaped small hairpin RNA and to selectively retain the RNA-encoding DNA template, which was designed to form a bulky tripartite structure. Thus, they can serve as delivery vectors preloaded with cargo RNA or alternatively as RNA-producing micro-bioreactors. The internalization of particles by human tissue culture cells inversely correlated with particle size and with the cell to particle ratio, although at a higher than stoichiometric excess of particles over cells, cell viability was impaired. Among primary human peripheral blood mononuclear cells, up to 50% of the monocytes displayed positive uptake of particles. Finally, these particles efficiently delivered siRNA into HEK293T cells triggering functional knockdown of the target gene lamin A/C. Particle-mediated knockdown was superior to that observed after conventional siRNA delivery via lipofection. Core-shell particles protect encapsulated nucleic acids from degradation and target cell genomes from direct contact with recombinant DNA, thus representing a promising delivery vector system that can be explored for genetic therapy and vaccination.
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Affiliation(s)
- Han Yu
- Department of Microbiology & Immunology, Yong Loo Lin School of Medicine , National University of Singapore , 5 Science Drive 2 , 117545 , Singapore
- School of Biological Sciences , Nanyang Technological University , 61 Biopolis Drive , 138673 , Singapore
| | - Houwen Matthew Pan
- Department of Biomedical Engineering , National University of Singapore , 4 Engineering Drive 3 , 117583 , Singapore
- School of Chemical and Biomedical Engineering , Nanyang Technological University , 70 Nanyang Drive , 637457 , Singapore
| | - Dieter Trau
- Department of Biomedical Engineering , National University of Singapore , 4 Engineering Drive 3 , 117583 , Singapore
| | - Volker Patzel
- Department of Microbiology & Immunology, Yong Loo Lin School of Medicine , National University of Singapore , 5 Science Drive 2 , 117545 , Singapore
- Department of Medicine , Addenbrooke's Hospital, University of Cambridge , Cambridge CB2 0QQ , U.K
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Togashi R, Tanaka H, Nakamura S, Yokota H, Tange K, Nakai Y, Yoshioka H, Harashima H, Akita H. A hepatic pDNA delivery system based on an intracellular environment sensitive vitamin E-scaffold lipid-like material with the aid of an anti-inflammatory drug. J Control Release 2018; 279:262-270. [PMID: 29673647 DOI: 10.1016/j.jconrel.2018.04.022] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2017] [Revised: 03/29/2018] [Accepted: 04/11/2018] [Indexed: 12/16/2022]
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25
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Holstein M, Mesa-Nuñez C, Miskey C, Almarza E, Poletti V, Schmeer M, Grueso E, Ordóñez Flores JC, Kobelt D, Walther W, Aneja MK, Geiger J, Bonig HB, Izsvák Z, Schleef M, Rudolph C, Mavilio F, Bueren JA, Guenechea G, Ivics Z. Efficient Non-viral Gene Delivery into Human Hematopoietic Stem Cells by Minicircle Sleeping Beauty Transposon Vectors. Mol Ther 2018; 26:1137-1153. [PMID: 29503198 PMCID: PMC6079369 DOI: 10.1016/j.ymthe.2018.01.012] [Citation(s) in RCA: 50] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2017] [Revised: 01/08/2018] [Accepted: 01/12/2018] [Indexed: 12/26/2022] Open
Abstract
The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34+ cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is ∼20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34+ cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34+ cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4–8 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol.
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Affiliation(s)
- Marta Holstein
- Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany
| | - Cristina Mesa-Nuñez
- Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIEMAT/CIBERER), Madrid, Spain; Advanced Therapies Unit, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM) Madrid, Spain
| | - Csaba Miskey
- Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany
| | - Elena Almarza
- Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIEMAT/CIBERER), Madrid, Spain; Advanced Therapies Unit, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM) Madrid, Spain
| | | | | | - Esther Grueso
- Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany
| | - Juan Carlos Ordóñez Flores
- Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany
| | - Dennis Kobelt
- Translational Oncology, Experimental and Clinical Research Center, Charité University Medicine, Berlin, Germany
| | - Wolfgang Walther
- Translational Oncology, Experimental and Clinical Research Center, Charité University Medicine, Berlin, Germany
| | | | | | - Halvard B Bonig
- Department of Transfusion Medicine and Immunohematology, Johann-Wolfgang-Goethe Universität, Frankfurt, Germany
| | - Zsuzsanna Izsvák
- Mobile DNA, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany
| | | | - Carsten Rudolph
- ethris GmbH, Planegg, Germany; Department of Pediatrics, Ludwig Maximilian University, Munich, Germany
| | - Fulvio Mavilio
- Genethon, Evry, France; Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
| | - Juan A Bueren
- Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIEMAT/CIBERER), Madrid, Spain; Advanced Therapies Unit, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM) Madrid, Spain
| | - Guillermo Guenechea
- Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIEMAT/CIBERER), Madrid, Spain; Advanced Therapies Unit, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM) Madrid, Spain
| | - Zoltán Ivics
- Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany.
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26
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Poddar S, Loh PS, Ooi ZH, Osman F, Eul J, Patzel V. RNA Structure Design Improves Activity and Specificity of trans-Splicing-Triggered Cell Death in a Suicide Gene Therapy Approach. MOLECULAR THERAPY-NUCLEIC ACIDS 2018; 11:41-56. [PMID: 29858076 PMCID: PMC5849863 DOI: 10.1016/j.omtn.2018.01.006] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/28/2017] [Revised: 01/17/2018] [Accepted: 01/17/2018] [Indexed: 01/20/2023]
Abstract
Spliceosome-mediated RNA trans-splicing enables correction or labeling of pre-mRNA, but therapeutic applications are hampered by issues related to the activity and target specificity of trans-splicing RNA (tsRNA). We employed computational RNA structure design to improve both on-target activity and specificity of tsRNA in a herpes simplex virus thymidine kinase/ganciclovir suicide gene therapy approach targeting alpha fetoprotein (AFP), a marker of hepatocellular carcinoma (HCC) or human papillomavirus type 16 (HPV-16) pre-mRNA. While unstructured, mismatched target binding domains significantly improved 3′ exon replacement (3’ER), 5′ exon replacement (5’ER) correlated with the thermodynamic stability of the tsRNA 3′ end. Alternative on-target trans-splicing was found to be a prevalent event. The specificity of trans-splicing with the intended target splice site was improved 10-fold by designing tsRNA that harbors secondary target binding domains shielding alternative on-target and blinding off-target splicing events. Such rationally designed suicide RNAs efficiently triggered death of HPV-16-transduced or hepatoblastoma-derived human tissue culture cells without evidence for off-target cell killing. Highest cell death activities were observed with novel dual-targeting tsRNAs programmed for trans-splicing toward AFP and a second HCC pre-mRNA biomarker. Our observations suggest trans-splicing represents a promising approach to suicide gene therapy.
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Affiliation(s)
- Sushmita Poddar
- Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4, Level 5, 5 Science Drive 2, Singapore 117597, Singapore
| | - Pei She Loh
- Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4, Level 5, 5 Science Drive 2, Singapore 117597, Singapore
| | - Zi Hao Ooi
- Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4, Level 5, 5 Science Drive 2, Singapore 117597, Singapore
| | - Farhana Osman
- Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4, Level 5, 5 Science Drive 2, Singapore 117597, Singapore
| | - Joachim Eul
- INEIDFO GmbH, Weserstrasse 23, 12045 Berlin, Germany
| | - Volker Patzel
- Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4, Level 5, 5 Science Drive 2, Singapore 117597, Singapore; Department of Medicine, Division of Infectious Diseases, University of Cambridge, Addenbrooke's Hospital, Level 5, Hills Road, Cambridge CB2 0QQ, UK.
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27
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Hacobian A, Hercher D. Pushing the Right Buttons: Improving Efficacy of Therapeutic DNA Vectors. TISSUE ENGINEERING PART B-REVIEWS 2017; 24:226-239. [PMID: 29264951 DOI: 10.1089/ten.teb.2017.0353] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Gene therapy represents a potent therapeutical application for regenerative medicine. So far, viral and nonviral approaches suffer from major drawbacks hindering efficient gene therapeutic applicability: the immunogenicity of viral systems on the one hand, and the low gene transfer efficiency of nonviral systems on the other hand. Therefore, there is a high demand for improvements of therapeutical systems at several levels. This review summarizes different DNA vector modifications to enhance biological efficacy and efficiency of therapeutical vectors, aiming for low toxicity, high specificity, and biological efficacy-the cornerstones for successful translation of gene therapy into the clinic. We aim to provide a step-by-step instruction to optimize their vectors to achieve the desired outcome of gene therapy. Our review provides the means to either construct a potent gene therapeutic vector de novo or to specifically address a bottleneck in the chain of events mandatory for therapeutic success. Although most of the introduced techniques can be translated into different areas, this review primarily addresses improvements for applications in transient gene therapy in the field of tissue engineering.
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Affiliation(s)
- Ara Hacobian
- Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Department of Molecular Biology, AUVA Research Center, The Austrian Cluster for Tissue Regeneration , Vienna, Austria
| | - David Hercher
- Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Department of Molecular Biology, AUVA Research Center, The Austrian Cluster for Tissue Regeneration , Vienna, Austria
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28
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Wu H, Tao J, Li X, Zhang T, Zhao L, Wang Y, Zhang L, Xiong J, Zeng Z, Zhan N, Steer CJ, Che L, Dong M, Wang X, Niu J, Li Z, Yan G, Chen X, Song G. MicroRNA-206 prevents the pathogenesis of hepatocellular carcinoma by modulating expression of met proto-oncogene and cyclin-dependent kinase 6 in mice. Hepatology 2017; 66:1952-1967. [PMID: 28714063 PMCID: PMC5696004 DOI: 10.1002/hep.29374] [Citation(s) in RCA: 63] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/21/2016] [Revised: 05/15/2017] [Accepted: 07/05/2017] [Indexed: 12/22/2022]
Abstract
UNLABELLED Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide, and therapeutic agents for this malignancy are lacking. MicroRNAs play critical roles in carcinogenesis and present tremendous therapeutic potential. Here, we report that microRNA-206 is a robust tumor suppressor that plays important roles in the development of HCC by regulating cell-cycle progression and the cMet signaling pathway. MicroRNA-206 was underexpressed in livers of two HCC mouse models, human individuals bearing HCC, and human HCC cell lines. Combining bioinformatic prediction and molecular and cellular approaches, we identified cMET (Met proto-oncogene), cyclin D1 (CCND1), and cyclin-dependent kinase 6 (CDK6) as functional targets of microRNA-206. By inhibiting expression of cMET, CCND1, and CDK6, microRNA-206 delayed cell-cycle progression, induced apoptosis, and impaired proliferation of three distinct human HCC cell lines. Systemic administration of microRNA-206 completely prevented HCC development in both cMyc and V-Akt murine thymoma viral oncogene homolog 1/neuroblastoma RAS viral oncogene homolog (AKT/Ras) HCC mice, whereas 100% of control mice died from lethal tumor burdens. Conversely, reintroduction of cMet or Cdk6 into livers of cMyc and AKT/Ras HCC mice recovered growth of HCC inhibited by microRNA-206. These results strongly suggested that cMet and Cdk6 were two functional targets that mediated the inhibitory effect of microRNA-206 on the development of HCC. MicroRNA-206 overexpression demonstrated a profound therapeutic effect on HCC in xenograft and cMyc HCC mice. CONCLUSION In summary, this study defines a potentially critical role of microRNA-206 in preventing the growth of HCC and suggests its use as a potential therapeutic strategy for this malignancy. (Hepatology 2017;66:1952-1967).
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Affiliation(s)
- Heng Wu
- Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA
| | - Junyan Tao
- Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California 94143, USA,School of Pharmacy, Hubei University of Chinese Medicine, Wuhan, Hubei Province 430060, China
| | - Xiaolei Li
- Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California 94143, USA
| | - Tianpeng Zhang
- Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA
| | - Lei Zhao
- Department of Infectious Diseases, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province 430022, China
| | - Yao Wang
- School of First Clinical Medicine, Hubei University of Chinese Medicine, Wuhan, Hubei Province 430060, China
| | - Lei Zhang
- Department of Emergency Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province 430022, China
| | - Jun Xiong
- Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province 430022, China
| | - Zhi Zeng
- Department of Pathology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province 430060, China
| | - Na Zhan
- Department of Pathology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province 430060, China
| | - Clifford J. Steer
- Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA,Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota 55455, USA
| | - Li Che
- Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California 94143, USA
| | - Mingjie Dong
- Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California 94143, USA
| | - Xiaomei Wang
- Institute for Translational Medicine, The First Hospital, Jilin University, Changchun, Jilin Province 130021, China
| | - Junqi Niu
- Institute for Translational Medicine, The First Hospital, Jilin University, Changchun, Jilin Province 130021, China
| | - Zhuoyu Li
- Institute of Biotechnology, Shanxi University, Taiyuan, Shanxi Province 030006, China
| | - Guiqing Yan
- Colleges of Life Science, Shanxi Normal University, 1 Gongyuan Street, Linfen City, Shanxi Province 041004, China
| | - Xin Chen
- Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California 94143, USA,To whom correspondence should be addressed: Guisheng Song, Ph.D., Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Minnesota Medical School, 406 Harvard Street SE, MMC36, Minneapolis MN 55455, ; Xin Chen, Ph.D., Departments of Bioengineering and Therapeutic Sciences, University of California San Francisco, 513 Parnassus Avenue, S816, San Francisco CA 94143:
| | - Guisheng Song
- Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA,Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota 55455, USA,Colleges of Life Science, Shanxi Normal University, 1 Gongyuan Street, Linfen City, Shanxi Province 041004, China,To whom correspondence should be addressed: Guisheng Song, Ph.D., Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Minnesota Medical School, 406 Harvard Street SE, MMC36, Minneapolis MN 55455, ; Xin Chen, Ph.D., Departments of Bioengineering and Therapeutic Sciences, University of California San Francisco, 513 Parnassus Avenue, S816, San Francisco CA 94143:
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29
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Baruteau J, Waddington SN, Alexander IE, Gissen P. Gene therapy for monogenic liver diseases: clinical successes, current challenges and future prospects. J Inherit Metab Dis 2017; 40:497-517. [PMID: 28567541 PMCID: PMC5500673 DOI: 10.1007/s10545-017-0053-3] [Citation(s) in RCA: 77] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/12/2017] [Revised: 04/27/2017] [Accepted: 04/28/2017] [Indexed: 02/08/2023]
Abstract
Over the last decade, pioneering liver-directed gene therapy trials for haemophilia B have achieved sustained clinical improvement after a single systemic injection of adeno-associated virus (AAV) derived vectors encoding the human factor IX cDNA. These trials demonstrate the potential of AAV technology to provide long-lasting clinical benefit in the treatment of monogenic liver disorders. Indeed, with more than ten ongoing or planned clinical trials for haemophilia A and B and dozens of trials planned for other inherited genetic/metabolic liver diseases, clinical translation is expanding rapidly. Gene therapy is likely to become an option for routine care of a subset of severe inherited genetic/metabolic liver diseases in the relatively near term. In this review, we aim to summarise the milestones in the development of gene therapy, present the different vector tools and their clinical applications for liver-directed gene therapy. AAV-derived vectors are emerging as the leading candidates for clinical translation of gene delivery to the liver. Therefore, we focus on clinical applications of AAV vectors in providing the most recent update on clinical outcomes of completed and ongoing gene therapy trials and comment on the current challenges that the field is facing for large-scale clinical translation. There is clearly an urgent need for more efficient therapies in many severe monogenic liver disorders, which will require careful risk-benefit analysis for each indication, especially in paediatrics.
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Affiliation(s)
- Julien Baruteau
- Genetics and Genomic Medicine Programme, Great Ormond Street Institute of Child Health, University College London, London, UK.
- Metabolic Medicine Department, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK.
- Gene Transfer Technology Group, Institute for Women's Health, University College London, London, UK.
| | - Simon N Waddington
- Gene Transfer Technology Group, Institute for Women's Health, University College London, London, UK
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Ian E Alexander
- Gene Therapy Research Unit, The Children's Hospital at Westmead and Children's Medical Research Institute, Westmead, Australia
- Discipline of Child and Adolescent Health, University of Sydney, Sydney, Australia
| | - Paul Gissen
- Genetics and Genomic Medicine Programme, Great Ormond Street Institute of Child Health, University College London, London, UK
- Metabolic Medicine Department, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK
- MRC Laboratory for Molecular Cell Biology, University College London, London, UK
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30
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Delaney AM, Adams CF, Fernandes AR, Al-Shakli AF, Sen J, Carwardine DR, Granger N, Chari DM. A fusion of minicircle DNA and nanoparticle delivery technologies facilitates therapeutic genetic engineering of autologous canine olfactory mucosal cells. NANOSCALE 2017; 9:8560-8566. [PMID: 28613324 DOI: 10.1039/c7nr00811b] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/07/2023]
Abstract
Olfactory ensheathing cells (OECs) promote axonal regeneration and improve locomotor function when transplanted into the injured spinal cord. A recent clinical trial demonstrated improved motor function in domestic dogs with spinal injury following autologous OEC transplantation. Their utility in canines offers promise for human translation, as dogs are comparable to humans in terms of clinical management and genetic/environmental variation. Moreover, the autologous, minimally invasive derivation of OECs makes them viable for human spinal injury investigation. Genetic engineering of transplant populations may augment their therapeutic potential, but relies heavily on viral methods which have several drawbacks for clinical translation. We present here the first proof that magnetic particles deployed with applied magnetic fields and advanced DNA minicircle vectors can safely bioengineer OECs to secrete a key neurotrophic factor, with an efficiency approaching that of viral vectors. We suggest that our alternative approach offers high translational potential for the delivery of augmented clinical cell therapies.
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Affiliation(s)
- Alexander M Delaney
- Cellular and Neural Engineering Group, Institute for Science and Technology in Medicine, Keele University, Keele, Staffordshire ST5 5BG, UK.
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31
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Hollevoet K, Declerck PJ. State of play and clinical prospects of antibody gene transfer. J Transl Med 2017; 15:131. [PMID: 28592330 PMCID: PMC5463339 DOI: 10.1186/s12967-017-1234-4] [Citation(s) in RCA: 47] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2017] [Accepted: 05/31/2017] [Indexed: 12/31/2022] Open
Abstract
Recombinant monoclonal antibodies (mAbs) are one of today's most successful therapeutic classes in inflammatory diseases and oncology. A wider accessibility and implementation, however, is hampered by the high product cost and prolonged need for frequent administration. The surge in more effective mAb combination therapies further adds to the costs and risk of toxicity. To address these issues, antibody gene transfer seeks to administer to patients the mAb-encoding nucleotide sequence, rather than the mAb protein. This allows the body to produce its own medicine in a cost- and labor-effective manner, for a prolonged period of time. Expressed mAbs can be secreted systemically or locally, depending on the production site. The current review outlines the state of play and clinical prospects of antibody gene transfer, thereby highlighting recent innovations, opportunities and remaining hurdles. Different expression platforms and a multitude of administration sites have been pursued. Viral vector-mediated mAb expression thereby made the most significant strides. Therapeutic proof of concept has been demonstrated in mice and non-human primates, and intramuscular vectored mAb therapy is under clinical evaluation. However, viral vectors face limitations, particularly in terms of immunogenicity. In recent years, naked DNA has gained ground as an alternative. Attained serum mAb titers in mice, however, remain far below those obtained with viral vectors, and robust pharmacokinetic data in larger animals is limited. The broad translatability of DNA-based antibody therapy remains uncertain, despite ongoing evaluation in patients. RNA presents another emerging platform for antibody gene transfer. Early reports in mice show that mRNA may be able to rival with viral vectors in terms of generated serum mAb titers, although expression appears more short-lived. Overall, substantial progress has been made in the clinical translation of antibody gene transfer. While challenges persist, clinical prospects are amplified by ongoing innovations and the versatility of antibody gene transfer. Clinical introduction can be expedited by selecting the platform approach currently best suited for the mAb or disease of interest. Innovations in expression platform, administration and antibody technology are expected to further improve overall safety and efficacy, and unlock the vast clinical potential of antibody gene transfer.
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Affiliation(s)
- Kevin Hollevoet
- Laboratory for Therapeutic and Diagnostic Antibodies, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven - University of Leuven, Campus Gasthuisberg O&N 2, P.B. 820, Herestraat 49, 3000 Leuven, Belgium
| | - Paul J. Declerck
- Laboratory for Therapeutic and Diagnostic Antibodies, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven - University of Leuven, Campus Gasthuisberg O&N 2, P.B. 820, Herestraat 49, 3000 Leuven, Belgium
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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: 66] [Impact Index Per Article: 8.3] [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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Minicircle Mediated Gene Delivery to Canine and Equine Mesenchymal Stem Cells. Int J Mol Sci 2017; 18:ijms18040819. [PMID: 28417917 PMCID: PMC5412403 DOI: 10.3390/ijms18040819] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2017] [Revised: 04/03/2017] [Accepted: 04/10/2017] [Indexed: 12/31/2022] Open
Abstract
Gene-directed tissue repair offers the clinician, human or veterinary, the chance to enhance cartilage regeneration and repair at a molecular level. Non-viral plasmid vectors have key biosafety advantages over viral vector systems for regenerative therapies due to their episomal integration however, conventional non-viral vectors can suffer from low transfection efficiency. Our objective was to identify and validate in vitro a novel non-viral gene expression vector that could be utilized for ex vivo and in vivo delivery to stromal-derived mesenchymal stem cells (MSCs). Minicircle plasmid DNA vector containing green fluorescent protein (GFP) was generated and transfected into adipose-derived MSCs from three species: canine, equine and rodent and transfection efficiency was determined. Both canine and rat cells showed transfection efficiencies of approximately 40% using minicircle vectors with equine cells exhibiting lower transfection efficiency. A Sox9-expressing minicircle vector was generated and transfected into canine MSCs. Successful transfection of the minicircle-Sox9 vector was confirmed in canine cells by Sox9 immunostaining. This study demonstrate the application and efficacy of a novel non-viral expression vector in canine and equine MSCs. Minicircle vectors have potential use in gene-directed regenerative therapies in non-rodent animal models for treatment of cartilage injury and repair.
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Wu J, Tan X, Lin J, Yuan L, Chen J, Qiu L, Huang W. Minicircle-oriP-miR-31 as a Novel EBNA1-Specific miRNA Therapy Approach for Nasopharyngeal Carcinoma. Hum Gene Ther 2016; 28:415-427. [PMID: 28042945 DOI: 10.1089/hum.2016.136] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
MicroRNAs (miRNAs) are important post-transcriptional regulators that control cancer development and progression. However, the application of miRNA therapy in cancer has been hampered by a lack of an efficient and targeted delivery system. In our previous studies, an oriP promoter-based minicircle system successfully mediated targeted foreign gene expression in EBNA1-positive nasopharyngeal carcinoma (NPC). However, it remains to be evaluated whether this system can be applied for tumor miRNA therapy. miR-31-5p, a tumor suppressive miRNA involved in the tumorigenesis of EBV-positive NPC, was selected as the therapeutic miRNA to be transferred. In this work, we constructed a novel EBNA1-specific miRNA expression system, minicircle-oriP-miR-31. The results indicated that mc-oriP-miR-31 mediated selective miR-31-5p expression in EBNA1-positive NPC cells. Both the proliferation and migration of EBNA1-positive NPC cell lines were inhibited by mc-oriP-miR-31 treatment in vitro. Furthermore, mc-oriP-miR-31 treatment inhibited xenograft growth and lung metastasis in vivo. We also identified WDR5 as a novel miR-31-5p target. Knockdown of WDR5 inhibited NPC cell proliferation and migration and was associated with downregulation of Notch1. Reintroduction of WDR5 partially abrogated the suppressive effects induced by miR-31-5p. In conclusion, we demonstrate for the first time that targeted expression of miR-31-5p using a nonviral minicircle vector can serve as a novel approach for tumor miRNA therapy. Moreover, WDR5 may be a promising therapeutic target for NPC treatment.
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Affiliation(s)
- Jiangxue Wu
- 1 State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center , Guangzhou, China
- 2 Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center , Guangzhou, China
| | - Xin Tan
- 1 State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center , Guangzhou, China
- 2 Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center , Guangzhou, China
| | - Jiaxin Lin
- 1 State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center , Guangzhou, China
- 2 Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center , Guangzhou, China
| | - Luping Yuan
- 1 State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center , Guangzhou, China
- 2 Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center , Guangzhou, China
| | - Jiemin Chen
- 1 State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center , Guangzhou, China
- 2 Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center , Guangzhou, China
| | - Lin Qiu
- 1 State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center , Guangzhou, China
- 2 Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center , Guangzhou, China
| | - Wenlin Huang
- 1 State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center , Guangzhou, China
- 2 Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center , Guangzhou, China
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Affiliation(s)
- Philip J Brooks
- 1 Division of Clinical Innovation and Office of Rare Diseases Research, National Center for Advancing Translational Sciences (NCATS), National Institutes of Health , Bethesda, Maryland
| | - N Nora Yang
- 2 Division of Preclinical Innovation, National Center for Advancing Translational Sciences (NCATS), National Institutes of Health , Bethesda, Maryland
| | - Christopher P Austin
- 3 Office of the Director, National Center for Advancing Translational Sciences (NCATS), National Institutes of Health , Bethesda, Maryland
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Part I: Minicircle vector technology limits DNA size restrictions on ex vivo gene delivery using nanoparticle vectors: Overcoming a translational barrier in neural stem cell therapy. J Control Release 2016; 238:289-299. [DOI: 10.1016/j.jconrel.2016.06.024] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2016] [Revised: 04/24/2016] [Accepted: 06/13/2016] [Indexed: 12/13/2022]
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Advanced Design of Dumbbell-shaped Genetic Minimal Vectors Improves Non-coding and Coding RNA Expression. Mol Ther 2016; 24:1581-91. [PMID: 27357627 DOI: 10.1038/mt.2016.138] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2015] [Accepted: 06/21/2016] [Indexed: 12/27/2022] Open
Abstract
Dumbbell-shaped DNA minimal vectors lacking nontherapeutic genes and bacterial sequences are considered a stable, safe alternative to viral, nonviral, and naked plasmid-based gene-transfer systems. We investigated novel molecular features of dumbbell vectors aiming to reduce vector size and to improve the expression of noncoding or coding RNA. We minimized small hairpin RNA (shRNA) or microRNA (miRNA) expressing dumbbell vectors in size down to 130 bp generating the smallest genetic expression vectors reported. This was achieved by using a minimal H1 promoter with integrated transcriptional terminator transcribing the RNA hairpin structure around the dumbbell loop. Such vectors were generated with high conversion yields using a novel protocol. Minimized shRNA-expressing dumbbells showed accelerated kinetics of delivery and transcription leading to enhanced gene silencing in human tissue culture cells. In primary human T cells, minimized miRNA-expressing dumbbells revealed higher stability and triggered stronger target gene suppression as compared with plasmids and miRNA mimics. Dumbbell-driven gene expression was enhanced up to 56- or 160-fold by implementation of an intron and the SV40 enhancer compared with control dumbbells or plasmids. Advanced dumbbell vectors may represent one option to close the gap between durable expression that is achievable with integrating viral vectors and short-term effects triggered by naked RNA.
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Part II: Functional delivery of a neurotherapeutic gene to neural stem cells using minicircle DNA and nanoparticles: Translational advantages for regenerative neurology. J Control Release 2016; 238:300-310. [PMID: 27369863 DOI: 10.1016/j.jconrel.2016.06.039] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2016] [Revised: 05/21/2016] [Accepted: 06/27/2016] [Indexed: 12/18/2022]
Abstract
Both neurotrophin-based therapy and neural stem cell (NSC)-based strategies have progressed to clinical trials for treatment of neurological diseases and injuries. Brain-derived neurotrophic factor (BDNF) in particular can confer neuroprotective and neuro-regenerative effects in preclinical studies, complementing the cell replacement benefits of NSCs. Therefore, combining both approaches by genetically-engineering NSCs to express BDNF is an attractive approach to achieve combinatorial therapy for complex neural injuries. Current genetic engineering approaches almost exclusively employ viral vectors for gene delivery to NSCs though safety and scalability pose major concerns for clinical translation and applicability. Magnetofection, a non-viral gene transfer approach deploying magnetic nanoparticles and DNA with magnetic fields offers a safe alternative but significant improvements are required to enhance its clinical application for delivery of large sized therapeutic plasmids. Here, we demonstrate for the first time the feasibility of using minicircles with magnetofection technology to safely engineer NSCs to overexpress BDNF. Primary mouse NSCs overexpressing BDNF generated increased daughter neuronal cell numbers post-differentiation, with accelerated maturation over a four-week period. Based on our findings we highlight the clinical potential of minicircle/magnetofection technology for therapeutic delivery of key neurotrophic agents.
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Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors. Leukemia 2016; 31:186-194. [PMID: 27491640 DOI: 10.1038/leu.2016.180] [Citation(s) in RCA: 183] [Impact Index Per Article: 20.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2016] [Revised: 06/08/2016] [Accepted: 06/10/2016] [Indexed: 12/28/2022]
Abstract
Immunotherapy with T cell modified with gamma-retroviral or lentiviral (LV) vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in clinical trials. However, the potential for insertional mutagenesis and genotoxicity of viral vectors is a safety concern, and their cost and regulatory demands a roadblock for rapid and broad clinical translation. Here, we demonstrate that CAR T cells can be engineered through non-viral Sleeping Beauty (SB) transposition of CAR genes from minimalistic DNA vectors called minicircles (MCs). We analyzed genomic distribution of SB and LV integrations and show that a significantly higher proportion of MC-derived CAR transposons compared with LV integrants had occurred outside of highly expressed and cancer-related genes into genomic safe harbor loci that are not expected to cause mutagenesis or genotoxicity. CD19-CAR T cells engineered with our enhanced SB approach conferred potent reactivity in vitro and eradicated lymphoma in a xenograft model in vivo. Intriguingly, electroporation of SB MCs is substantially more effective and less toxic compared with conventional plasmids, and enables cost-effective rapid preparation of therapeutic CAR T-cell doses. This approach sets a new standard in advanced cellular and gene therapy and will accelerate and increase the availability of CAR T-cell therapy to treat hematologic malignancies.
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40
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Šimčíková M, Alves CPA, Brito L, Prather KLJ, Prazeres DMF, Monteiro GA. Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase. Appl Microbiol Biotechnol 2016; 100:6725-6737. [DOI: 10.1007/s00253-016-7565-x] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2016] [Revised: 04/08/2016] [Accepted: 04/16/2016] [Indexed: 01/10/2023]
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Bazzani RP, Pringle IA, Connolly MM, Davies LA, Sumner-Jones SG, Schleef M, Hyde SC, Gill DR. Transgene sequences free of CG dinucleotides lead to high level, long-term expression in the lung independent of plasmid backbone design. Biomaterials 2016; 93:20-26. [PMID: 27061267 DOI: 10.1016/j.biomaterials.2016.03.029] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2016] [Revised: 03/17/2016] [Accepted: 03/17/2016] [Indexed: 12/13/2022]
Abstract
Non-viral aerosol gene therapy offers great potential for treating chronic lung diseases of the airways such as cystic fibrosis (CF). Early clinical trials showed that transgene expression in the airways was transient whereas maximal duration of transgene expression is essential in order to minimise the frequency of aerosol treatments. Improved vector design, such as careful selection of the promoter/enhancer, can lead to more persistent levels of transgene expression, but multiple factors affect expression in vivo. Following aerosol delivery to the lungs of mice, we measured reporter gene expression from a CpG-free luciferase transgene cassette in the context of both a plasmid and minicircle vector configuration and showed that the vector backbone had no effect on expression. Transgene activity was affected by the vector backbone however, when a similar, but sub-optimal CpG-containing transgene was used, suggesting that aspects of the plasmid backbone had a negative impact on transgene expression. Similar studies were performed in Toll-like receptor-9 (TLR9) knockout mice to investigate a potential role for the TLR9 signalling pathway in detecting CpGs in the vector sequence. Even in the absence of TLR9, persistent expression could only be achieved with a CpG-free transgene. Together, these data indicate that in order to achieve high levels of persistent expression in vivo, a CpG-free transgene cassette is required.
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Affiliation(s)
- Reto P Bazzani
- Gene Medicine Research Group, NDCLS, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, OX3 9DU, UK; The UK Cystic Fibrosis Gene Therapy Consortium, UK
| | - Ian A Pringle
- Gene Medicine Research Group, NDCLS, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, OX3 9DU, UK; The UK Cystic Fibrosis Gene Therapy Consortium, UK
| | - Mary M Connolly
- Gene Medicine Research Group, NDCLS, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, OX3 9DU, UK; The UK Cystic Fibrosis Gene Therapy Consortium, UK
| | - Lee A Davies
- Gene Medicine Research Group, NDCLS, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, OX3 9DU, UK; The UK Cystic Fibrosis Gene Therapy Consortium, UK
| | - Stephanie G Sumner-Jones
- Gene Medicine Research Group, NDCLS, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, OX3 9DU, UK; The UK Cystic Fibrosis Gene Therapy Consortium, UK
| | - Martin Schleef
- PlasmidFactory, Meisenstraße 96, D-33607 Bielefeld, Germany
| | - Stephen C Hyde
- Gene Medicine Research Group, NDCLS, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, OX3 9DU, UK; The UK Cystic Fibrosis Gene Therapy Consortium, UK
| | - Deborah R Gill
- Gene Medicine Research Group, NDCLS, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, OX3 9DU, UK; The UK Cystic Fibrosis Gene Therapy Consortium, UK.
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Foldvari M, Chen DW, Nafissi N, Calderon D, Narsineni L, Rafiee A. Non-viral gene therapy: Gains and challenges of non-invasive administration methods. J Control Release 2015; 240:165-190. [PMID: 26686079 DOI: 10.1016/j.jconrel.2015.12.012] [Citation(s) in RCA: 172] [Impact Index Per Article: 17.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2015] [Revised: 11/26/2015] [Accepted: 12/09/2015] [Indexed: 12/20/2022]
Abstract
Gene therapy is becoming an influential part of the rapidly increasing armamentarium of biopharmaceuticals for improving health and combating diseases. Currently, three gene therapy treatments are approved by regulatory agencies. While these treatments utilize viral vectors, non-viral alternative technologies are also being developed to improve the safety profile and manufacturability of gene carrier formulations. We present an overview of gene-based therapies focusing on non-viral gene delivery systems and the genetic therapeutic tools that will further revolutionize medical treatment with primary focus on the range and development of non-invasive delivery systems for dermal, transdermal, ocular and pulmonary administrations and perspectives on other administration methods such as intranasal, oral, buccal, vaginal, rectal and otic delivery.
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Affiliation(s)
- Marianna Foldvari
- School of Pharmacy, Waterloo Institute of Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada; Center for Bioengineering and Biotechnology, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada.
| | - Ding Wen Chen
- School of Pharmacy, Waterloo Institute of Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada; Center for Bioengineering and Biotechnology, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada
| | - Nafiseh Nafissi
- School of Pharmacy, Waterloo Institute of Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada; Center for Bioengineering and Biotechnology, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada
| | - Daniella Calderon
- School of Pharmacy, Waterloo Institute of Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada; Center for Bioengineering and Biotechnology, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada
| | - Lokesh Narsineni
- School of Pharmacy, Waterloo Institute of Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada; Center for Bioengineering and Biotechnology, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada
| | - Amirreza Rafiee
- School of Pharmacy, Waterloo Institute of Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada; Center for Bioengineering and Biotechnology, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada
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Nafissi N, Foldvari M. Neuroprotective therapies in glaucoma: II. Genetic nanotechnology tools. Front Neurosci 2015; 9:355. [PMID: 26528114 PMCID: PMC4604245 DOI: 10.3389/fnins.2015.00355] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2015] [Accepted: 09/17/2015] [Indexed: 01/01/2023] Open
Abstract
Neurotrophic factor genome engineering could have many potential applications not only in the deeper understanding of neurodegenerative disorders but also in improved therapeutics. The fields of nanomedicine, regenerative medicine, and gene/cell-based therapy have been revolutionized by the development of safer and efficient non-viral technologies for gene delivery and genome editing with modern techniques for insertion of the neurotrophic factors into clinically relevant cells for a more sustained pharmaceutical effect. It has been suggested that the long-term expression of neurotrophic factors is the ultimate approach to prevent and/or treat neurodegenerative disorders such as glaucoma in patients who do not respond to available treatments or are at the progressive stage of the disease. Recent preclinical research suggests that novel neuroprotective gene and cell therapeutics could be promising approaches for both non-invasive neuroprotection and regenerative functions in the eye. Several progenitor and retinal cell types have been investigated as potential candidates for glaucoma neurotrophin therapy either as targets for gene therapy, options for cell replacement therapy, or as vehicles for gene delivery. Therefore, in parallel with deeper understanding of the specific protective effects of different neurotrophic factors and the potential therapeutic cell candidates for glaucoma neuroprotection, the development of non-invasive and highly specific gene delivery methods with safe and effective technologies to modify cell candidates for life-long neuroprotection in the eye is essential before investing in this field.
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Affiliation(s)
| | - Marianna Foldvari
- School of Pharmacy and Waterloo Institute of Nanotechnology, University of WaterlooWaterloo, ON, Canada
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Nafissi N, Foldvari M. Neuroprotective therapies in glaucoma: I. Neurotrophic factor delivery. WILEY INTERDISCIPLINARY REVIEWS-NANOMEDICINE AND NANOBIOTECHNOLOGY 2015; 8:240-54. [PMID: 26306832 DOI: 10.1002/wnan.1361] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/01/2015] [Revised: 06/15/2015] [Accepted: 07/04/2015] [Indexed: 12/11/2022]
Abstract
Glaucoma is a neurodegenerative eye disease that causes permanent blindness at the progressive stage and the number of people affected worldwide is expected to reach over 79 million by 2020. Currently, glaucoma management relies on pharmacological and invasive surgical treatments mainly by reducing the intraocular pressure (IOP), which is the most important risk factor for the progression of the visual field loss. Recent research suggests that neuroprotective or neuroregenerative approaches are necessary to prevent retinal ganglion cells (RGCs) loss and visual impairment over time. Neuroprotection is a new therapeutic strategy that attempts to keep RGCs alive and functional. New gene and cell therapeutics encoding neurotrophic factors (NTFs) are emerging for both neuroprotection and regenerative treatments for retinal diseases. This article briefly reviews the role of NTFs in glaucoma and the potential delivery systems.
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Affiliation(s)
- Nafiseh Nafissi
- School of Pharmacy and Waterloo Institute of Nanotechnology, University of Waterloo, Waterloo, Ontario, Canada
| | - Marianna Foldvari
- School of Pharmacy and Waterloo Institute of Nanotechnology, University of Waterloo, Waterloo, Ontario, Canada
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45
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Schomberg D, Miranpuri G, Duellman T, Crowell A, Vemuganti R, Resnick D. Spinal cord injury induced neuropathic pain: Molecular targets and therapeutic approaches. Metab Brain Dis 2015; 30:645-58. [PMID: 25588751 DOI: 10.1007/s11011-014-9642-0] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/21/2014] [Accepted: 12/05/2014] [Indexed: 10/24/2022]
Abstract
Neuropathic pain, especially that resulting from spinal cord injury, is a tremendous clinical challenge. A myriad of biological changes have been implicated in producing these pain states including cellular interactions, extracellular proteins, ion channel expression, and epigenetic influences. Physiological consequences of these changes are varied and include functional deficits and pain responses. Developing therapies that effectively address the cause of these symptoms require a deeper knowledge of alterations in the molecular pathways. Matrix metalloproteinases and tissue inhibitors of metalloproteinases are two promising therapeutic targets. Matrix metalloproteinases interact with and influence many of the studied pain pathways. Gene expression of ion channels and inflammatory mediators clearly contributes to neuropathic pain. Localized and time dependent targeting of these proteins could alleviate and even prevent neuropathic pain from developing. Current therapeutic options for neuropathic pain are limited primarily to analgesics targeting the opioid pathway. Therapies directed at molecular targets are highly desirable and in early stages of development. These include transplantation of exogenously engineered cell populations and targeted gene manipulation. This review describes specific molecular targets amenable to therapeutic intervention using currently available delivery systems.
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Affiliation(s)
- Dominic Schomberg
- Department of Neurological Surgery, University of Wisconsin School of Medicine and Public Health, 600 Highland Ave, Madison, WI, 53792, USA
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Sanei Ata-Abadi N, Dormiani K, Khazaie Y, Ghaedi K, Forouzanfar M, Lachinani L, Rezaei N, Kiani-Esfahani A, Nasr-Esfahani MH. Construction of a new minicircle DNA carrying an enhanced green florescent protein reporter gene for efficient expression into mammalian cell lines. Mol Biol Rep 2015; 42:1175-85. [DOI: 10.1007/s11033-015-3864-z] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2014] [Accepted: 02/16/2015] [Indexed: 01/15/2023]
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Majidi S, Zeinali Sehrig F, Samiei M, Milani M, Abbasi E, Dadashzadeh K, Akbarzadeh A. Magnetic nanoparticles: Applications in gene delivery and gene therapy. ARTIFICIAL CELLS NANOMEDICINE AND BIOTECHNOLOGY 2015; 44:1186-93. [DOI: 10.3109/21691401.2015.1014093] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
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Xu Z, Ye J, Zhang A, Xie L, Shen Q, Xue J, Chen J. Gene Therapy for Hemophilia B With Liver-specific Element Mediated by Rep-RBE Site-specific Integration System. J Cardiovasc Pharmacol 2015; 65:153-9. [DOI: 10.1097/fjc.0000000000000172] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
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49
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Hou XH, Guo XY, Chen Y, He CY, Chen ZY. Increasing the minicircle DNA purity using an enhanced triplex DNA technology to eliminate DNA contaminants. Mol Ther Methods Clin Dev 2015; 1:14062. [PMID: 26052527 PMCID: PMC4449018 DOI: 10.1038/mtm.2014.62] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2014] [Accepted: 11/24/2014] [Indexed: 12/01/2022]
Abstract
DNA vectors for human gene therapy have to meet the efficacy and safety requirements. Minicircles (MCs), a class of optimized DNA vectors free of plasmid backbone (PB) DNAs, have emerged as promising candidates because of their superior transgene expression profiles. However, the existence of impure DNAs, including the unrecombined MC producing plasmid (PP) and PB circle, in the MC products made using the current technologies exceed the safety limit. Here, we report the development of an enhanced triplex DNA (TriD) technology to eliminate almost all the impure DNAs from the MC products. To do this, a pair of optimized TriD forming sequences was placed to flank the kanamycin resistance gene in the PP. The MC products were incubated with a biotinylated TriD forming DNA oligonucleotide (olig), and the resulted TriDs were removed by binding to streptovidin-coated magnetic beads. Consequently, the residual impure DNAs were 0.03% or less in the final MC products. The reproducibility of this technique was confirmed with MCs of various transgene expression cassettes, sizes, and quantities, suggesting its great potential in making high quality MC for human gene therapy.
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Affiliation(s)
- Xiaohu H Hou
- The Laboratory for Gene and Cell Therapy, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China
| | - Xiaoyan Y Guo
- The Laboratory for Gene and Cell Therapy, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China
- Department of Infectious Diseases, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China
| | - Yusheng Chen
- The Laboratory for Gene and Cell Therapy, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China
| | - Cheng-Yi He
- The Laboratory for Gene and Cell Therapy, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China
| | - Zhi-Ying Chen
- The Laboratory for Gene and Cell Therapy, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China
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Mikkelsen JG. Nonviral Gene Therapy—The Challenge of Mobilizing DNA. SOMATIC GENOME MANIPULATION 2015:69-104. [DOI: 10.1007/978-1-4939-2389-2_4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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