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1
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Cho SW, Shin SC, Nam Y, Ahn HJ. A T7 autogene-mediated DNA vaccine platform for SARS-CoV-2: Overcoming DNA vaccine limitations with enhanced spike mRNA production. J Control Release 2025; 383:113776. [PMID: 40287096 DOI: 10.1016/j.jconrel.2025.113776] [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: 12/11/2024] [Revised: 04/22/2025] [Accepted: 04/23/2025] [Indexed: 04/29/2025]
Abstract
The COVID-19 pandemic has underscored the urgent need for innovative vaccine platforms that combine potent immunogenicity, thermal stability, and global accessibility. While mRNA vaccines have proven highly efficacious, their fragility and reliance on ultracold storage complicates distribution in resource-constrained settings. DNA vaccines, by contrast, are more stable and easier to distribute but suffer from low immunogenicity due to inefficient nuclear translocation. Here, we introduce the mRNA Factory (mRF) vaccine platform, a DNA-based system that enables direct cytoplasmic transcription of spike mRNA via self-amplifying T7 RNA polymerase. The Rpol/spk mRF vaccine elicited markedly enhanced immunogenicity compared to conventional plasmid DNA vaccines, as evidenced by elevated spike protein expression, robust T cell activation, Th1-biased cytokine profiles, and higher titers of SARS-CoV-2-specific IgG. Compared with an IVT spike mRNA vaccine, the mRF vaccine elicited comparable levels of overall immune responses, including T effector cell activation, IFNγ+CD8+ cytotoxic T cell activation, Th1 polarization, and specific IgG production. These results highlight the mRF platform's potential to overcome key limitations of conventional DNA vaccines while addressing distribution challenges associated with mRNA vaccines. Our findings support the mRF platform as a promising strategy for global vaccine deployment and future pandemic preparedness.
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Affiliation(s)
- Sang Won Cho
- Medicinal Materials Research Center, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea
| | - Sang Chul Shin
- Technological Convergence Center, Research Resources Division, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea
| | - Yunju Nam
- Medicinal Materials Research Center, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea
| | - Hyung Jun Ahn
- Medicinal Materials Research Center, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea.
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2
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Casper J, Schenk SH, Parhizkar E, Detampel P, Dehshahri A, Huwyler J. Polyethylenimine (PEI) in gene therapy: Current status and clinical applications. J Control Release 2023; 362:667-691. [PMID: 37666302 DOI: 10.1016/j.jconrel.2023.09.001] [Citation(s) in RCA: 86] [Impact Index Per Article: 43.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2023] [Revised: 08/24/2023] [Accepted: 09/01/2023] [Indexed: 09/06/2023]
Abstract
Polyethlyenimine (PEI) was introduced 1995 as a cationic polymer for nucleic acid delivery. PEI and its derivatives are extensively used in basic research and as reference formulations in the field of polymer-based gene delivery. Despite its widespread use, the number of clinical applications to date is limited. Thus, this review aims to consolidate the past applications of PEI in DNA delivery, elucidate the obstacles that hinder its transition to clinical use, and highlight potential prospects for novel iterations of PEI derivatives. The present review article is divided into three sections. The first section examines the mechanism of action employed by PEI, examining fundamental aspects of cellular delivery including uptake mechanisms, release from endosomes, and transport into the cell nucleus, along with potential strategies for enhancing these delivery phases. Moreover, an in-depth analysis is conducted concerning the mechanism underlying cellular toxicity, accompanied with approaches to overcome this major challenge. The second part is devoted to the in vivo performance of PEI and its application in various therapeutic indications. While systemic administration has proven to be challenging, alternative localized delivery routes hold promise, such as treatment of solid tumors, application as a vaccine, or serving as a therapeutic agent for pulmonary delivery. In the last section, the outcome of completed and ongoing clinical trials is summarized. Finally, an expert opinion is provided on the potential of PEI and its future applications. PEI-based formulations for nucleic acid delivery have a promising potential, it will be an important task for the years to come to introduce innovations that address PEI-associated shortcomings by introducing well-designed PEI formulations in combination with an appropriate route of administration.
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Affiliation(s)
- Jens Casper
- Division of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland
| | - Susanne H Schenk
- Division of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland
| | - Elahehnaz Parhizkar
- Department of Pharmaceutics, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Pascal Detampel
- Division of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland
| | - Ali Dehshahri
- Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.
| | - Jörg Huwyler
- Division of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
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3
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Lundstrom K. Application of DNA Replicons in Gene Therapy and Vaccine Development. Pharmaceutics 2023; 15:pharmaceutics15030947. [PMID: 36986808 PMCID: PMC10054396 DOI: 10.3390/pharmaceutics15030947] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2023] [Revised: 03/04/2023] [Accepted: 03/13/2023] [Indexed: 03/17/2023] Open
Abstract
DNA-based gene therapy and vaccine development has received plenty of attention lately. DNA replicons based on self-replicating RNA viruses such as alphaviruses and flaviviruses have been of particular interest due to the amplification of RNA transcripts leading to enhanced transgene expression in transfected host cells. Moreover, significantly reduced doses of DNA replicons compared to conventional DNA plasmids can elicit equivalent immune responses. DNA replicons have been evaluated in preclinical animal models for cancer immunotherapy and for vaccines against infectious diseases and various cancers. Strong immune responses and tumor regression have been obtained in rodent tumor models. Immunization with DNA replicons has provided robust immune responses and protection against challenges with pathogens and tumor cells. DNA replicon-based COVID-19 vaccines have shown positive results in preclinical animal models.
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4
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Vlach M, Coppens-Exandier H, Jamin A, Berchel M, Scaviner J, Chesné C, Montier T, Jaffrès PA, Corlu A, Loyer P. Liposome-Mediated Gene Transfer in Differentiated HepaRG™ Cells: Expression of Liver Specific Functions and Application to the Cytochrome P450 2D6 Expression. Cells 2022; 11:cells11233904. [PMID: 36497165 PMCID: PMC9737581 DOI: 10.3390/cells11233904] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2022] [Revised: 11/19/2022] [Accepted: 11/29/2022] [Indexed: 12/12/2022] Open
Abstract
The goal of this study was to establish a procedure for gene delivery mediated by cationic liposomes in quiescent differentiated HepaRG™ human hepatoma cells. We first identified several cationic lipids promoting efficient gene transfer with low toxicity in actively dividing HepG2, HuH7, BC2 and progenitor HepaRG™ human hepatoma cells. The lipophosphoramidate Syn1-based nanovector, which allowed the highest transfection efficiencies of progenitor HepaRG™ cells, was next used to transfect differentiated HepaRG™ cells. Lipofection of these cells using Syn1-based liposome was poorly efficient most likely because the differentiated HepaRG™ cells are highly quiescent. Thus, we engineered the differentiated HepaRG™ Mitogenic medium supplement (ADD1001) that triggered robust proliferation of differentiated cells. Importantly, we characterized the phenotypical changes occurring during proliferation of differentiated HepaRG™ cells and demonstrated that mitogenic stimulation induced a partial and transient decrease in the expression levels of some liver specific functions followed by a fast recovery of the full differentiation status upon removal of the mitogens. Taking advantage of the proliferation of HepaRG™ cells, we defined lipofection conditions using Syn1-based liposomes allowing transient expression of the cytochrome P450 2D6, a phase I enzyme poorly expressed in HepaRG cells, which opens new means for drug metabolism studies in HepaRG™ cells.
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Affiliation(s)
- Manuel Vlach
- Institut NUMECAN (Nutrition Metabolisms and Cancer), F-35000 Rennes, France
- Institut AGRO Rennes-Angers, F-35042 Rennes, France
| | - Hugo Coppens-Exandier
- Institut NUMECAN (Nutrition Metabolisms and Cancer), F-35000 Rennes, France
- Biopredic International, F-35760 Saint Grégoire, France
| | - Agnès Jamin
- Biopredic International, F-35760 Saint Grégoire, France
| | - Mathieu Berchel
- Univ. Brest, CNRS, CEMCA, UMR 6521, F-29238 Brest, France
- Plateforme BiogenOuest SynNanoVect, F-44035 Nantes, France
| | - Julien Scaviner
- Institut NUMECAN (Nutrition Metabolisms and Cancer), F-35000 Rennes, France
- Biopredic International, F-35760 Saint Grégoire, France
| | | | - Tristan Montier
- Plateforme BiogenOuest SynNanoVect, F-44035 Nantes, France
- Univ. Brest, INSERM, EFS, UMR 1078, GGB-GTCA, F-29200 Brest, France
| | - Paul-Alain Jaffrès
- Univ. Brest, CNRS, CEMCA, UMR 6521, F-29238 Brest, France
- Plateforme BiogenOuest SynNanoVect, F-44035 Nantes, France
| | - Anne Corlu
- Institut NUMECAN (Nutrition Metabolisms and Cancer), F-35000 Rennes, France
- Correspondence: (A.C.); (P.L.); Tel.: +33-(02)-23233873 (P.L.)
| | - Pascal Loyer
- Institut NUMECAN (Nutrition Metabolisms and Cancer), F-35000 Rennes, France
- Plateforme BiogenOuest SynNanoVect, F-44035 Nantes, France
- Correspondence: (A.C.); (P.L.); Tel.: +33-(02)-23233873 (P.L.)
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5
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Heinemann D, Zabic M, Terakawa M, Boch J. Laser-based molecular delivery and its applications in plant science. PLANT METHODS 2022; 18:82. [PMID: 35690858 PMCID: PMC9188231 DOI: 10.1186/s13007-022-00908-9] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/11/2021] [Accepted: 05/12/2022] [Indexed: 05/14/2023]
Abstract
Lasers enable modification of living and non-living matter with submicron precision in a contact-free manner which has raised the interest of researchers for decades. Accordingly, laser technologies have drawn interest across disciplines. They have been established as a valuable tool to permeabilize cellular membranes for molecular delivery in a process termed photoinjection. Laser-based molecular delivery was first reported in 1984, when normal kidney cells were successfully transfected with a frequency-multiplied Nd:YAG laser. Due to the rapid development of optical technologies, far more sophisticated laser platforms have become available. In particular, near infrared femtosecond (NIR fs) laser sources enable an increasing progress of laser-based molecular delivery procedures and opened up multiple variations and applications of this technique.This review is intended to provide a plant science audience with the physical principles as well as the application potentials of laser-based molecular delivery. The historical origins and technical development of laser-based molecular delivery are summarized and the principle physical processes involved in these approaches and their implications for practical use are introduced. Successful cases of laser-based molecular delivery in plant science will be reviewed in detail, and the specific hurdles that plant materials pose will be discussed. Finally, we will give an outlook on current limitations and possible future applications of laser-based molecular delivery in the field of plant science.
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Affiliation(s)
- Dag Heinemann
- Hannover Centre for Optical Technologies, Leibniz University Hannover, Nienburger Str. 17, 30167, Hannover, Germany.
- Institute of Horticultural Production Systems, Leibniz University Hannover, Herrenhäuser Str. 2, 30419, Hannover, Germany.
- Cluster of Excellence PhoenixD, Leibniz University Hannover, Welfengarten 1, 30167, Hannover, Germany.
| | - Miroslav Zabic
- Hannover Centre for Optical Technologies, Leibniz University Hannover, Nienburger Str. 17, 30167, Hannover, Germany
- Institute of Horticultural Production Systems, Leibniz University Hannover, Herrenhäuser Str. 2, 30419, Hannover, Germany
| | - Mitsuhiro Terakawa
- Department of Electronics and Electrical Engineering, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Japan
| | - Jens Boch
- Institute of Plant Genetics, Leibniz University Hannover, Herrenhäuser Str. 2, 30419, Hannover, Germany
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6
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Mitdank H, Sama S, Tröger M, Testa MF, Ferrarese M, Balestra D, Pinotti M, Weng A. An advanced method for the small-scale production of high-quality minicircle DNA. Int J Pharm 2021; 605:120830. [PMID: 34214654 DOI: 10.1016/j.ijpharm.2021.120830] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2021] [Revised: 05/04/2021] [Accepted: 06/22/2021] [Indexed: 11/24/2022]
Abstract
Minicircle DNA is a promising tool in the field of gene therapy, whose products are increasingly gaining market access. Greater transfection efficiency and longer expression time as well as lower immunogenicity contrast with cost-intensive production, which also stands in the way of a broader use of the advantages of this technology in research. Starting from a commercial minicircle production kit a simple protocol for the cost-effective small-scale production of high-quality minicircle DNA to be used at a research scale has been developed by combining and improving procedures of various publications. An optimized size-exclusion chromatography method led to almost pure minicircle DNA with a superior proportion of the desired supercoiled plasmid conformation. The pharmaceutical potential of the produced minicircle DNA was investigated in vitro by real-time impedance assays in a tumor cell model in case of coded suicide genes as well as by ELISA of the translation product in case of coded human coagulation factor IX.
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Affiliation(s)
- Hardy Mitdank
- Institute of Pharmacy, Free University of Berlin, Königin-Luise-Str. 2+4, 14195 Berlin, Germany.
| | - Simko Sama
- Institute of Pharmacy, Free University of Berlin, Königin-Luise-Str. 2+4, 14195 Berlin, Germany
| | - Meike Tröger
- Institute of Pharmacy, Free University of Berlin, Königin-Luise-Str. 2+4, 14195 Berlin, Germany
| | - Maria Francesca Testa
- Department of Life Sciences and Biotechnologies, University of Ferrara, Via Luigi Borsari 64, 44121 Ferrara, Italy
| | - Mattia Ferrarese
- Department of Life Sciences and Biotechnologies, University of Ferrara, Via Luigi Borsari 64, 44121 Ferrara, Italy
| | - Dario Balestra
- Department of Life Sciences and Biotechnologies, University of Ferrara, Via Luigi Borsari 64, 44121 Ferrara, Italy
| | - Mirko Pinotti
- Department of Life Sciences and Biotechnologies, University of Ferrara, Via Luigi Borsari 64, 44121 Ferrara, Italy
| | - Alexander Weng
- Institute of Pharmacy, Free University of Berlin, Königin-Luise-Str. 2+4, 14195 Berlin, Germany.
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7
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Lundstrom K. Impact of a Plasmid DNA-Based Alphavirus Vaccine on Immunization Efficiency. Methods Mol Biol 2021; 2197:33-47. [PMID: 32827131 DOI: 10.1007/978-1-0716-0872-2_3] [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: 06/11/2023]
Abstract
Alphavirus vectors have been engineered for high-level gene expression relying originally on replication-deficient recombinant particles, more recently designed for plasmid DNA-based administration. As alphavirus-based DNA vectors encode the alphavirus RNA replicon genes, enhanced transgene expression in comparison to conventional DNA plasmids is achieved. Immunization studies with alphavirus-based DNA plasmids have elicited specific antibody production, have generated tumor regression and protection against challenges with infectious agents and tumor cells in various animal models. A limited number of clinical trials have been conducted with alphavirus DNA vectors. Compared to conventional plasmid DNA-based immunization, alphavirus DNA vectors required 1000-fold less DNA to elicit similar immune responses in rodents.
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8
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Hager S, Fittler FJ, Wagner E, Bros M. Nucleic Acid-Based Approaches for Tumor Therapy. Cells 2020; 9:E2061. [PMID: 32917034 PMCID: PMC7564019 DOI: 10.3390/cells9092061] [Citation(s) in RCA: 43] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2020] [Revised: 09/06/2020] [Accepted: 09/07/2020] [Indexed: 12/24/2022] Open
Abstract
Within the last decade, the introduction of checkpoint inhibitors proposed to boost the patients' anti-tumor immune response has proven the efficacy of immunotherapeutic approaches for tumor therapy. Furthermore, especially in the context of the development of biocompatible, cell type targeting nano-carriers, nucleic acid-based drugs aimed to initiate and to enhance anti-tumor responses have come of age. This review intends to provide a comprehensive overview of the current state of the therapeutic use of nucleic acids for cancer treatment on various levels, comprising (i) mRNA and DNA-based vaccines to be expressed by antigen presenting cells evoking sustained anti-tumor T cell responses, (ii) molecular adjuvants, (iii) strategies to inhibit/reprogram tumor-induced regulatory immune cells e.g., by RNA interference (RNAi), (iv) genetically tailored T cells and natural killer cells to directly recognize tumor antigens, and (v) killing of tumor cells, and reprograming of constituents of the tumor microenvironment by gene transfer and RNAi. Aside from further improvements of individual nucleic acid-based drugs, the major perspective for successful cancer therapy will be combination treatments employing conventional regimens as well as immunotherapeutics like checkpoint inhibitors and nucleic acid-based drugs, each acting on several levels to adequately counter-act tumor immune evasion.
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Affiliation(s)
- Simone Hager
- Department of Chemistry and Pharmacy, Ludwig-Maximilians-University (LMU), 81377 Munich, Germany;
| | | | - Ernst Wagner
- Department of Chemistry and Pharmacy, Ludwig-Maximilians-University (LMU), 81377 Munich, Germany;
| | - Matthias Bros
- Department of Dermatology, University Medical Center, 55131 Mainz, Germany;
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9
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Affiliation(s)
- Zhiyu Yang
- Key Laboratory of Polymer Ecomaterials Chinese Academy of Sciences Changchun Institute of Applied Chemistry Changchun China
- University of Science and Technology of China Hefei China
| | - Zhaopei Guo
- Key Laboratory of Polymer Ecomaterials Chinese Academy of Sciences Changchun Institute of Applied Chemistry Changchun China
- Jilin Biomedical Polymers Engineering Laboratory Changchun China
| | - Huayu Tian
- Key Laboratory of Polymer Ecomaterials Chinese Academy of Sciences Changchun Institute of Applied Chemistry Changchun China
- University of Science and Technology of China Hefei China
- Jilin Biomedical Polymers Engineering Laboratory Changchun China
| | - Xuesi Chen
- Key Laboratory of Polymer Ecomaterials Chinese Academy of Sciences Changchun Institute of Applied Chemistry Changchun China
- University of Science and Technology of China Hefei China
- Jilin Biomedical Polymers Engineering Laboratory Changchun China
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10
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Kiełbik A, Szlasa W, Saczko J, Kulbacka J. Electroporation-Based Treatments in Urology. Cancers (Basel) 2020; 12:E2208. [PMID: 32784598 PMCID: PMC7465806 DOI: 10.3390/cancers12082208] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2020] [Revised: 08/03/2020] [Accepted: 08/05/2020] [Indexed: 02/06/2023] Open
Abstract
The observation that an application of a pulsed electric field (PEF) resulted in an increased permeability of the cell membrane has led to the discovery of the phenomenon called electroporation (EP). Depending on the parameters of the electric current and cell features, electroporation can be either reversible or irreversible. The irreversible electroporation (IRE) found its use in urology as a non-thermal ablative method of prostate and renal cancer. As its mechanism is based on the permeabilization of cell membrane phospholipids, IRE (as well as other treatments based on EP) provides selectivity sparing extracellular proteins and matrix. Reversible EP enables the transfer of genes, drugs, and small exogenous proteins. In clinical practice, reversible EP can locally increase the uptake of cytotoxic drugs such as cisplatin and bleomycin. This approach is known as electrochemotherapy (ECT). Few in vivo and in vitro trials of ECT have been performed on urological cancers. EP provides the possibility of transmission of genes across the cell membrane. As the protocols of gene electrotransfer (GET) over the last few years have improved, EP has become a well-known technique for non-viral cell transfection. GET involves DNA transfection directly to the cancer or the host skin and muscle tissue. Among urological cancers, the GET of several plasmids encoding prostate cancer antigens has been investigated in clinical trials. This review brings into discussion the underlying mechanism of EP and an overview of the latest progress and development perspectives of EP-based treatments in urology.
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Affiliation(s)
- Aleksander Kiełbik
- Faculty of Medicine, Wroclaw Medical University, 50-367 Wroclaw, Poland; (A.K.); (W.S.)
| | - Wojciech Szlasa
- Faculty of Medicine, Wroclaw Medical University, 50-367 Wroclaw, Poland; (A.K.); (W.S.)
| | - Jolanta Saczko
- Department of Molecular and Cellular Biology, Wroclaw Medical University, 50-556 Wroclaw, Poland;
| | - Julita Kulbacka
- Department of Molecular and Cellular Biology, Wroclaw Medical University, 50-556 Wroclaw, Poland;
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11
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Vaughan HJ, Green JJ, Tzeng SY. Cancer-Targeting Nanoparticles for Combinatorial Nucleic Acid Delivery. ADVANCED MATERIALS (DEERFIELD BEACH, FLA.) 2020; 32:e1901081. [PMID: 31222852 PMCID: PMC6923623 DOI: 10.1002/adma.201901081] [Citation(s) in RCA: 151] [Impact Index Per Article: 30.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/15/2019] [Revised: 04/13/2019] [Indexed: 05/03/2023]
Abstract
Nucleic acids are a promising type of therapeutic for the treatment of a wide range of conditions, including cancer, but they also pose many delivery challenges. For efficient and safe delivery to cancer cells, nucleic acids must generally be packaged into a vehicle, such as a nanoparticle, that will allow them to be taken up by the target cells and then released in the appropriate cellular compartment to function. As with other types of therapeutics, delivery vehicles for nucleic acids must also be designed to avoid unwanted side effects; thus, the ability of such carriers to target their cargo to cancer cells is crucial. Classes of nucleic acids, hurdles that must be overcome for effective intracellular delivery, types of nonviral nanomaterials used as delivery vehicles, and the different strategies that can be employed to target nucleic acid delivery specifically to tumor cells are discussed. Additonally, nanoparticle designs that facilitate multiplexed delivery of combinations of nucleic acids are reviewed.
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Affiliation(s)
- Hannah J Vaughan
- Department of Biomedical Engineering, Translational Tissue Engineering Center and Institute for NanoBioTechnology, Johns Hopkins University School of Medicine, 400 North Broadway, Smith Building 5001, Baltimore, MD, 21231, USA
| | - Jordan J Green
- Department of Biomedical Engineering, Translational Tissue Engineering Center and Institute for NanoBioTechnology, Johns Hopkins University School of Medicine, 400 North Broadway, Smith Building 5001, Baltimore, MD, 21231, USA
| | - Stephany Y Tzeng
- Department of Biomedical Engineering, Translational Tissue Engineering Center and Institute for NanoBioTechnology, Johns Hopkins University School of Medicine, 400 North Broadway, Smith Building 5001, Baltimore, MD, 21231, USA
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12
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Jia N, Zhang X, Li W, Chen D, Hu H. Phospholipid-Coated Guanosine Diphosphate Auxiliary CaP Active Nanoparticles Can Systematically Improve the Efficiency of Gene Therapy for Cancer Disease. ACS Biomater Sci Eng 2020; 6:2107-2116. [PMID: 33455334 DOI: 10.1021/acsbiomaterials.0c00316] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Endogenous active substance guanosine diphosphate (GDP) is involved in the physiological process of DNA transfection and expression in the cytoplasm by binding to Ran proteins. To substantially improve the gene delivery efficiency of nanoparticles, phospholipid-coated Ca(P-GDP)/pDNA/NLS hybrid nanoparticles were prepared using GDP as a common biophosphorus source based on the biological process of exogenous gene expression in the cells. This nanoparticle has a relative uniform particle size distribution and in vitro stability. The addition of GDP in nanoparticles significantly enhanced the gene expression efficiency with good biocompatibility. Moreover, an in vivo study further verified that hybrid nanoparticles were more effective in increasing the p53 gene expression, thus significantly inhibiting the tumor growth in the heterotopic tumor model of nude mice. These results demonstrated that phospholipid-coated Ca(P-GDP) nanoparticles were a potential nonviral gene vector to promote gene expression. The experimental results confirmed the feasibility of designing a delivery system based on active substances and provided a new solution to improve the transfection efficiency of gene drugs.
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Affiliation(s)
- Nan Jia
- Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang 110016, PR China
| | - Xirui Zhang
- Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang 110016, PR China
| | - Wenpan Li
- Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang 110016, PR China
| | - Dawei Chen
- Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang 110016, PR China
| | - Haiyang Hu
- Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang 110016, PR China
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13
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Coutinho De Oliveira B, Duthie MS, Alves Pereira VR. Vaccines for leishmaniasis and the implications of their development for American tegumentary leishmaniasis. Hum Vaccin Immunother 2019; 16:919-930. [PMID: 31634036 DOI: 10.1080/21645515.2019.1678998] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
The leishmaniases are a collection of vector-borne parasitic diseases caused by a number of different Leishmania species that are distributed worldwide. Clinical and laboratory research have together revealed several important immune components that control Leishmania infection and indicate the potential of immunization to prevent leishmaniasis. In this review we introduce previous and ongoing experimental research efforts to develop vaccines against Leishmania species. First, second and third generation vaccine strategies that have been proposed to counter cutaneous and visceral leishmaniasis (CL and VL, respectively) are summarized. One of the major bottlenecks in development is the transition from results in animal model studies to humans, and we highlight that although American tegumentary leishmaniasis (ATL; New World CL) can progress to destructive and disfiguring mucosal lesions, most research has been conducted using mouse models and Old World Leishmania species. We conclude that assessment of vaccine candidates in ATL settings therefore appears merited.
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Affiliation(s)
- Beatriz Coutinho De Oliveira
- Pós-Graduação em Inovação Terapêutica, Universidade Federal de Pernambuco (UFPE), Recife, Brazil.,Departamento de Imunologia, Instituto Aggeu Magalhães, Recife, Brazil
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14
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Lundstrom K. Plasmid DNA-based Alphavirus Vaccines. Vaccines (Basel) 2019; 7:vaccines7010029. [PMID: 30857255 PMCID: PMC6466081 DOI: 10.3390/vaccines7010029] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2019] [Revised: 03/01/2019] [Accepted: 03/04/2019] [Indexed: 12/28/2022] Open
Abstract
Alphaviruses have been engineered as vectors for high-level transgene expression. Originally, alphavirus-based vectors were applied as recombinant replication-deficient particles, subjected to expression studies in mammalian and non-mammalian cell lines, primary cell cultures, and in vivo. However, vector engineering has expanded the application range to plasmid DNA-based delivery and expression. Immunization studies with DNA-based alphavirus vectors have demonstrated tumor regression and protection against challenges with infectious agents and tumor cells in animal tumor models. The presence of the RNA replicon genes responsible for extensive RNA replication in the RNA/DNA layered alphavirus vectors provides superior transgene expression in comparison to conventional plasmid DNA-based expression. Immunization with alphavirus DNA vectors revealed that 1000-fold less DNA was required to elicit similar immune responses compared to conventional plasmid DNA. In addition to DNA-based delivery, immunization with recombinant alphavirus particles and RNA replicons has demonstrated efficacy in providing protection against lethal challenges by infectious agents and tumor cells.
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15
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Hobernik D, Bros M. DNA Vaccines-How Far From Clinical Use? Int J Mol Sci 2018; 19:ijms19113605. [PMID: 30445702 PMCID: PMC6274812 DOI: 10.3390/ijms19113605] [Citation(s) in RCA: 323] [Impact Index Per Article: 46.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2018] [Revised: 11/07/2018] [Accepted: 11/09/2018] [Indexed: 12/12/2022] Open
Abstract
Two decades ago successful transfection of antigen presenting cells (APC) in vivo was demonstrated which resulted in the induction of primary adaptive immune responses. Due to the good biocompatibility of plasmid DNA, their cost-efficient production and long shelf life, many researchers aimed to develop DNA vaccine-based immunotherapeutic strategies for treatment of infections and cancer, but also autoimmune diseases and allergies. This review aims to summarize our current knowledge on the course of action of DNA vaccines, and which factors are responsible for the poor immunogenicity in human so far. Important optimization steps that improve DNA transfection efficiency comprise the introduction of DNA-complexing nano-carriers aimed to prevent extracellular DNA degradation, enabling APC targeting, and enhanced endo/lysosomal escape of DNA. Attachment of virus-derived nuclear localization sequences facilitates nuclear entry of DNA. Improvements in DNA vaccine design include the use of APC-specific promotors for transcriptional targeting, the arrangement of multiple antigen sequences, the co-delivery of molecular adjuvants to prevent tolerance induction, and strategies to circumvent potential inhibitory effects of the vector backbone. Successful clinical use of DNA vaccines may require combined employment of all of these parameters, and combination treatment with additional drugs.
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Affiliation(s)
- Dominika Hobernik
- Department of Dermatology, University Medical Center, 55131 Mainz, Germany.
| | - Matthias Bros
- Department of Dermatology, University Medical Center, 55131 Mainz, Germany.
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16
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A Nanostructured Lipid Carrier for Delivery of a Replicating Viral RNA Provides Single, Low-Dose Protection against Zika. Mol Ther 2018; 26:2507-2522. [PMID: 30078765 DOI: 10.1016/j.ymthe.2018.07.010] [Citation(s) in RCA: 109] [Impact Index Per Article: 15.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2018] [Revised: 07/02/2018] [Accepted: 07/10/2018] [Indexed: 12/26/2022] Open
Abstract
Since the first demonstration of in vivo gene expression from an injected RNA molecule almost two decades ago,1 the field of RNA-based therapeutics is now taking significant strides, with many cancer and infectious disease targets entering clinical trials.2 Critical to this success has been advances in the knowledge and application of delivery formulations. Currently, various lipid nanoparticle (LNP) platforms are at the forefront,3 but the encapsulation approach underpinning LNP formulations offsets the synthetic and rapid-response nature of RNA vaccines.4 Second, limited stability of LNP formulated RNA precludes stockpiling for pandemic readiness.5 Here, we show the development of a two-vialed approach wherein the delivery formulation, a highly stable nanostructured lipid carrier (NLC), can be manufactured and stockpiled separate from the target RNA, which is admixed prior to administration. Furthermore, specific physicochemical modifications to the NLC modulate immune responses, either enhancing or diminishing neutralizing antibody responses. We have combined this approach with a replicating viral RNA (rvRNA) encoding Zika virus (ZIKV) antigens and demonstrated a single dose as low as 10 ng can completely protect mice against a lethal ZIKV challenge, representing what might be the most potent approach to date of any Zika vaccine.
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17
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Li L, Hu S, Chen X. Non-viral delivery systems for CRISPR/Cas9-based genome editing: Challenges and opportunities. Biomaterials 2018; 171:207-218. [PMID: 29704747 DOI: 10.1016/j.biomaterials.2018.04.031] [Citation(s) in RCA: 262] [Impact Index Per Article: 37.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2018] [Revised: 04/13/2018] [Accepted: 04/14/2018] [Indexed: 02/06/2023]
Abstract
In recent years, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) genome editing systems have become one of the most robust platforms in basic biomedical research and therapeutic applications. To date, efficient in vivo delivery of the CRISPR/Cas9 system to the targeted cells remains a challenge. Although viral vectors have been widely used in the delivery of the CRISPR/Cas9 system in vitro and in vivo, their fundamental shortcomings, such as the risk of carcinogenesis, limited insertion size, immune responses and difficulty in large-scale production, severely limit their further applications. Alternative non-viral delivery systems for CRISPR/Cas9 are urgently needed. With the rapid development of non-viral vectors, lipid- or polymer-based nanocarriers have shown great potential for CRISPR/Cas9 delivery. In this review, we analyze the pros and cons of delivering CRISPR/Cas9 systems in the form of plasmid, mRNA, or protein and then discuss the limitations and challenges of CRISPR/Cas9-based genome editing. Furthermore, current non-viral vectors that have been applied for CRISPR/Cas9 delivery in vitro and in vivo are outlined in details. Finally, critical obstacles for non-viral delivery of CRISPR/Cas9 system are highlighted and promising strategies to overcome these barriers are proposed.
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Affiliation(s)
- Ling Li
- Department of PET Center, Xiangya Hospital, Central South University, Changsha, 410008, China; Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, MD 20892, USA
| | - Shuo Hu
- Department of PET Center, Xiangya Hospital, Central South University, Changsha, 410008, China.
| | - Xiaoyuan Chen
- Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, MD 20892, USA.
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18
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Huang Y, Chen Q, Lu H, An J, Zhu H, Yan X, Li W, Gao H. Near-infrared AIEgen-functionalized and diselenide-linked oligo-ethylenimine with self-sufficing ROS to exert spatiotemporal responsibility for promoted gene delivery. J Mater Chem B 2018; 6:6660-6666. [DOI: 10.1039/c8tb02207k] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
We attempted to synthesize an oligo-ethyleneimine (OEI)-crosslinked polycation, characterized with self-sufficing ROS by virtue of a functional AIE component and an ROS-labile diselenide linkage.
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Affiliation(s)
- Yongkang Huang
- School of Chemistry and Chemical Engineering
- Tianjin Key Laboratory of Organic Solar Cells and Photochemical Conversion
- Tianjin University of Technology
- Tianjin 300384
- P. R. China
| | - Qixian Chen
- School of Life Science and Biotechnology
- Dalian University of Technology
- Dalian 116024
- P. R. China
| | - Hongguang Lu
- School of Chemistry and Chemical Engineering
- Tianjin Key Laboratory of Organic Solar Cells and Photochemical Conversion
- Tianjin University of Technology
- Tianjin 300384
- P. R. China
| | - Jinxia An
- School of Chemistry and Chemical Engineering
- Tianjin Key Laboratory of Organic Solar Cells and Photochemical Conversion
- Tianjin University of Technology
- Tianjin 300384
- P. R. China
| | - Huajie Zhu
- School of Chemistry and Chemical Engineering
- Tianjin Key Laboratory of Organic Solar Cells and Photochemical Conversion
- Tianjin University of Technology
- Tianjin 300384
- P. R. China
| | - Xiangjie Yan
- School of Chemistry and Chemical Engineering
- Tianjin Key Laboratory of Organic Solar Cells and Photochemical Conversion
- Tianjin University of Technology
- Tianjin 300384
- P. R. China
| | - Wei Li
- School of Chemistry and Chemical Engineering
- Tianjin Key Laboratory of Organic Solar Cells and Photochemical Conversion
- Tianjin University of Technology
- Tianjin 300384
- P. R. China
| | - Hui Gao
- School of Chemistry and Chemical Engineering
- Tianjin Key Laboratory of Organic Solar Cells and Photochemical Conversion
- Tianjin University of Technology
- Tianjin 300384
- P. R. China
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19
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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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20
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Demazeau M, Quesnot N, Ripoche N, Rauch C, Jeftić J, Morel F, Gauffre F, Benvegnu T, Loyer P. Efficient transfection of Xenobiotic Responsive Element-biosensor plasmid using diether lipid and phosphatidylcholine liposomes in differentiated HepaRG cells. Int J Pharm 2017; 524:268-278. [PMID: 28365389 DOI: 10.1016/j.ijpharm.2017.03.080] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2017] [Revised: 03/27/2017] [Accepted: 03/28/2017] [Indexed: 10/19/2022]
Abstract
In this study, we evaluated cationic liposomes prepared from diether-NH2 and egg phosphatidylcholine (EPC) for in vitro gene delivery. The impact of the lipid composition, i.e. the EPC and Diether-NH2 molar ratio, on in vitro transfection efficiency and cytotoxicity was investigated using the human HEK293T and hepatoma HepaRG cells known to be permissive and poorly permissive cells for liposome-mediated gene transfer, respectively. Here, we report that EPC/Diether-NH2-based liposomes enabled a very efficient transfection with low cytotoxicity compared to commercial transfection reagents in both HEK293T and proliferating progenitor HepaRG cells. Taking advantage of these non-toxic EPC/Diether-NH2-based liposomes, we developed a method to efficiently transfect differentiated hepatocyte-like HepaRG cells and a biosensor plasmid containing a Xenobiotic Responsive Element and a minimal promoter driving the transcription of the luciferase reporter gene. We demonstrated that the luciferase activity was induced by a canonical inducer of cytochrome P450 genes, the benzo[a]pyrene, and two environmental contaminants, the fluoranthene, a polycyclic aromatic hydrocarbon, and the endosulfan, an organochlorine insecticide, known to induce toxicity and genotoxicity in differentiated HepaRG cells. In conclusion, we established a new efficient lipofection-mediated gene transfer in hepatocyte-like HepaRG cells opening new perspectives in drug evaluation relying on xenobiotic inducible biosensor plasmids.
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Affiliation(s)
- Maxime Demazeau
- Ecole Nationale Supérieure de Chimie de Rennes, Institut des Sciences Chimiques de Rennes, UMR 6226 CNRS, Plateforme SynNanoVect, Biogenouest, 11 allée de Beaulieu, CS 50837, 35708 Rennes Cedex 7, France
| | - Nicolas Quesnot
- INSERM, INRA, Univ Rennes 1, Univ Bretagne Loire, Nutrition Metabolisms and Cancer (NuMeCan), Plateforme SynNanoVect, Biogenouest, Rennes, France
| | - Nicolas Ripoche
- Ecole Nationale Supérieure de Chimie de Rennes, Institut des Sciences Chimiques de Rennes, UMR 6226 CNRS, Plateforme SynNanoVect, Biogenouest, 11 allée de Beaulieu, CS 50837, 35708 Rennes Cedex 7, France
| | - Claudine Rauch
- INSERM, INRA, Univ Rennes 1, Univ Bretagne Loire, Nutrition Metabolisms and Cancer (NuMeCan), Plateforme SynNanoVect, Biogenouest, Rennes, France
| | - Jelena Jeftić
- Ecole Nationale Supérieure de Chimie de Rennes, Institut des Sciences Chimiques de Rennes, UMR 6226 CNRS, Plateforme SynNanoVect, Biogenouest, 11 allée de Beaulieu, CS 50837, 35708 Rennes Cedex 7, France
| | - Fabrice Morel
- INSERM, INRA, Univ Rennes 1, Univ Bretagne Loire, Nutrition Metabolisms and Cancer (NuMeCan), Plateforme SynNanoVect, Biogenouest, Rennes, France
| | - Fabienne Gauffre
- Institut des Sciences Chimiques de Rennes, UMR 6226 CNRS, Université de Rennes 1, Campus de Beaulieu, 263 Avenue du Général Leclerc, F-35042 Rennes Cedex, France.
| | - Thierry Benvegnu
- Ecole Nationale Supérieure de Chimie de Rennes, Institut des Sciences Chimiques de Rennes, UMR 6226 CNRS, Plateforme SynNanoVect, Biogenouest, 11 allée de Beaulieu, CS 50837, 35708 Rennes Cedex 7, France.
| | - Pascal Loyer
- INSERM, INRA, Univ Rennes 1, Univ Bretagne Loire, Nutrition Metabolisms and Cancer (NuMeCan), Plateforme SynNanoVect, Biogenouest, Rennes, France.
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21
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Upadhya A, Sangave PC. Hydrophobic and electrostatic interactions between cell penetrating peptides and plasmid DNA are important for stable non-covalent complexation and intracellular delivery. J Pept Sci 2016; 22:647-659. [DOI: 10.1002/psc.2927] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2016] [Revised: 06/27/2016] [Accepted: 08/24/2016] [Indexed: 12/13/2022]
Affiliation(s)
- Archana Upadhya
- Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management; SVKM's NMIMS University; V.L. Mehta Road, Vile Parle (West) Mumbai 400056 Maharashtra India
| | - Preeti C. Sangave
- Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management; SVKM's NMIMS University; V.L. Mehta Road, Vile Parle (West) Mumbai 400056 Maharashtra India
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22
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Vaidyanathan S, Orr BG, Banaszak Holl MM. Role of Cell Membrane-Vector Interactions in Successful Gene Delivery. Acc Chem Res 2016; 49:1486-93. [PMID: 27459207 DOI: 10.1021/acs.accounts.6b00200] [Citation(s) in RCA: 63] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
Cationic polymers have been investigated as nonviral vectors for gene delivery due to their favorable safety profile when compared to viral vectors. However, nonviral vectors are limited by poor efficacy in inducing gene expression. The physicochemical properties of cationic polymers enabling successful gene expression have been investigated in order to improve expression efficiency and safety. Studies over the past several years have focused on five possible rate-limiting processes to explain the differences in gene expression: (1) endosomal release, (2) transport within specific intracellular pathways, (3) protection of DNA from nucleases, (4) transport into the nucleus, and (5) DNA release from vectors. However, determining the relative importance of these processes and the vector properties necessary for optimization remain a challenge to the field. In this Account, we describe over a decade of studies focused on understanding the interaction of cationic polymer and cationic polymer/oligonucleotide (polyplex) interactions with model lipid membranes, cell membranes, and cells in culture. In particular, we have been interested in how the interaction between cationic polymers and the membrane influences the intracellular transport of intact DNA to the nucleus. Recent advances in microfluidic patch clamp techniques enabled us to quantify polyplex cell membrane interactions at the cellular level with precise control over material concentrations and exposure times. In attempting to relate these findings to subsequent intracellular transport of DNA and expression of protein, we needed to develop an approach that could distinguish DNA that was intact and potentially functional for gene expression from the much larger pool of degraded, nonfunctional DNA within the cell. We addressed this need by developing a FRET oligonucleotide molecular beacon (OMB) to monitor intact DNA transport. The research highlighted in this Account builds to the conclusion that polyplex transported DNA is released from endosomes by free cationic polymer intercalated into the endosomal membrane. This cationic polymer initially interacts with the cell plasma membrane and appears to reach the endosome by lipid cycling mechanisms. The fraction of cells displaying release of intact DNA from endosomes quantitatively predicts the fraction of cells displaying gene expression for both linear poly(ethylenimine) (L-PEI; an effective vector) and generation five poly(amidoamine) dendrimer (G5 PAMAM; an ineffective vector). Moreover, intact OMB delivered with G5 PAMAM, which normally is confined to endosomes, was released by the subsequent addition of L-PEI with a corresponding 10-fold increase in transgene expression. These observations are consistent with experiments demonstrating that cationic polymer/membrane partition coefficients, not polyplex/membrane partition coefficients, predict successful gene expression. Interestingly, a similar partitioning of cationic polymers into the mitochondrial membranes has been proposed to explain the cytotoxicity of these materials. Thus, the proposed model indicates the same physicochemical property (partitioning into lipid bilayers) is linked to release from endosomes, giving protein expression, and to cytotoxicity.
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Affiliation(s)
- Sriram Vaidyanathan
- Departments of Biomedical Engineering, ‡Chemistry, and §Physics, ∥Program in Applied Physics and ⊥Macromolecular
Science and Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States
| | - Bradford G. Orr
- Departments of Biomedical Engineering, ‡Chemistry, and §Physics, ∥Program in Applied Physics and ⊥Macromolecular
Science and Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States
| | - Mark M. Banaszak Holl
- Departments of Biomedical Engineering, ‡Chemistry, and §Physics, ∥Program in Applied Physics and ⊥Macromolecular
Science and Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States
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23
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Schott JW, Morgan M, Galla M, Schambach A. Viral and Synthetic RNA Vector Technologies and Applications. Mol Ther 2016; 24:1513-27. [PMID: 27377044 DOI: 10.1038/mt.2016.143] [Citation(s) in RCA: 56] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2015] [Accepted: 06/30/2016] [Indexed: 12/21/2022] Open
Abstract
Use of RNA is an increasingly popular method to transiently deliver genetic information for cell manipulation in basic research and clinical therapy. In these settings, viral and nonviral RNA platforms are employed for delivery of small interfering RNA and protein-coding mRNA. Technological advances allowing RNA modification for increased stability, improved translation and reduced immunogenicity have led to increased use of nonviral synthetic RNA, which is delivered in naked form or upon formulation. Alternatively, highly efficient viral entry pathways are exploited to transfer genes of interest as RNA incorporated into viral particles. Current viral RNA transfer technologies are derived from Retroviruses, nonsegmented negative-strand RNA viruses or positive-stranded Alpha- and Flaviviruses. In retroviral particles, the genes of interest can either be incorporated directly into the viral RNA genome or as nonviral RNA. Nonsegmented negative-strand virus-, Alpha- and Flavivirus-derived vectors support prolonged expression windows through replication of viral RNA encoding genes of interest. Mixed technologies combining viral and nonviral components are also available. RNA transfer is ideal for all settings that do not require permanent transgene expression and excludes potentially detrimental DNA integration into the target cell genome. Thus, RNA-based technologies are successfully applied for reprogramming, transdifferentiation, gene editing, vaccination, tumor therapy, and gene therapy.
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Affiliation(s)
- Juliane W Schott
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany
| | - Michael Morgan
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany
| | - Melanie Galla
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany
| | - Axel Schambach
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany.,Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA
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24
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Liu Y, Yan J, Santangelo PJ, Prausnitz MR. DNA uptake, intracellular trafficking and gene transfection after ultrasound exposure. J Control Release 2016; 234:1-9. [PMID: 27165808 DOI: 10.1016/j.jconrel.2016.05.013] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2016] [Revised: 04/26/2016] [Accepted: 05/06/2016] [Indexed: 11/17/2022]
Abstract
Ultrasound has been studied as a promising tool for intracellular gene delivery. In this work, we studied gene transfection of a human prostate cancer cell line exposed to megahertz pulsed ultrasound in the presence of contrast agent and assessed the efficiency of fluorescently labelled DNA delivery into cell nuclei, which is necessary for gene transfection. At the sonication conditions studied, ~30% of cells showed DNA uptake 30min after sonication, but that fraction decreased over time to ~10% of cells after 24h. Most cells containing DNA had DNA in their nuclei, but the amount varied significantly. Transfection efficiency peaked at ~10% at 8h post sonication. Among those cells containing DNA, ~30% of DNA was localized in the cell nuclei, ~30% was in autophagosomes/autophagolysosomes and the remainder was "free" in the cytoplasm 30min after sonication. At later times up to 24h, ~30% of DNA continued to be found in the nuclei and most or all of the rest of the DNA was in autophagosomes/autophagolysosomes. These results demonstrate that ultrasound can deliver DNA into cell nuclei shortly after sonication and that the rest of the DNA can be cleared by autophagosomes/autophagolysosomes.
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Affiliation(s)
- Ying Liu
- School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332-0100, USA
| | - Jing Yan
- School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332-0100, USA
| | - Philip J Santangelo
- Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, Georgia Institute of Technology, Atlanta, GA 30332, USA
| | - Mark R Prausnitz
- School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332-0100, USA; Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, Georgia Institute of Technology, Atlanta, GA 30332, USA.
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25
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Vaidyanathan S, Chen J, Orr BG, Banaszak Holl MM. Cationic Polymer Intercalation into the Lipid Membrane Enables Intact Polyplex DNA Escape from Endosomes for Gene Delivery. Mol Pharm 2016; 13:1967-78. [PMID: 27111496 DOI: 10.1021/acs.molpharmaceut.6b00139] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Developing improved cationic polymer-DNA polyplexes for gene delivery requires improved understanding of DNA transport from endosomes into the nucleus. Using a FRET-capable oligonucleotide molecular beacon (OMB), we monitored the transport of intact DNA to cell organelles. We observed that for effective (jetPEI) and ineffective (G5 PAMAM) vectors, the fraction of cells displaying intact OMB in the cytosol (jetPEI ≫ G5 PAMAM) quantitatively predicted the fraction expressing transgene (jetPEI ≫ G5 PAMAM). Intact OMB delivered with PAMAM and confined to endosomes could be released to the cytosol by the subsequent addition of L-PEI, with a corresponding 10-fold increase in transgene expression. These results suggest that future vector development should optimize vectors for intercalation into, and destabilization of, the endosomal membrane. Finally, the study highlights a two-step strategy in which the pDNA is loaded in cells using one vector and endosomal release is mediated by a second agent.
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Affiliation(s)
- Sriram Vaidyanathan
- Departments of †Biomedical Engineering and ‡Chemistry and Programs in §Applied Physics and ⊥Macromolecular Science and Engineering, University of Michigan , Ann Arbor, Michigan 48109, United States
| | - Junjie Chen
- Departments of †Biomedical Engineering and ‡Chemistry and Programs in §Applied Physics and ⊥Macromolecular Science and Engineering, University of Michigan , Ann Arbor, Michigan 48109, United States
| | - Bradford G Orr
- Departments of †Biomedical Engineering and ‡Chemistry and Programs in §Applied Physics and ⊥Macromolecular Science and Engineering, University of Michigan , Ann Arbor, Michigan 48109, United States
| | - Mark M Banaszak Holl
- Departments of †Biomedical Engineering and ‡Chemistry and Programs in §Applied Physics and ⊥Macromolecular Science and Engineering, University of Michigan , Ann Arbor, Michigan 48109, United States
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26
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Fiume G, Di Rienzo C, Marchetti L, Pozzi D, Caracciolo G, Cardarelli F. Single-cell real-time imaging of transgene expression upon lipofection. Biochem Biophys Res Commun 2016; 474:8-14. [DOI: 10.1016/j.bbrc.2016.03.088] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2016] [Accepted: 03/18/2016] [Indexed: 10/22/2022]
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27
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Peng Q, Zhu J, Yu Y, Hoffman L, Yang X. Hyperbranched lysine−arginine copolymer for gene delivery. JOURNAL OF BIOMATERIALS SCIENCE-POLYMER EDITION 2015; 26:1163-77. [DOI: 10.1080/09205063.2015.1080482] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
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28
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Abstract
Many viruses deliver their genomes into the host cell’s nucleus before they replicate. While onco-retroviruses and papillomaviruses tether their genomes to host chromatin upon mitotic breakdown of the nuclear envelope, lentiviruses, such as human immunodeficiency virus, adenoviruses, herpesviruses, parvoviruses, influenza viruses, hepatitis B virus, polyomaviruses, and baculoviruses deliver their genomes into the nucleus of post-mitotic cells. This poses the significant challenge of slipping a DNA or RNA genome past the nuclear pore complex (NPC) embedded in the nuclear envelope. Quantitative fluorescence imaging is shedding new light on this process, with recent data implicating misdelivery of viral genomes at nuclear pores as a bottleneck to virus replication. Here, we infer NPC functions for nuclear import of viral genomes from cell biology experiments and explore potential causes of misdelivery, including improper virus docking at NPCs, incomplete translocation, virus-induced stress and innate immunity reactions. We conclude by discussing consequences of viral genome misdelivery for viruses and host cells, and lay out future questions to enhance our understanding of this phenomenon. Further studies into viral genome misdelivery may reveal unexpected aspects about NPC structure and function, as well as aid in developing strategies for controlling viral infections to improve human health.
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29
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Maizels Y, Gerlitz G. Shaping of interphase chromosomes by the microtubule network. FEBS J 2015; 282:3500-24. [PMID: 26040675 DOI: 10.1111/febs.13334] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2015] [Revised: 05/11/2015] [Accepted: 06/01/2015] [Indexed: 12/31/2022]
Abstract
It is well established that microtubule dynamics play a major role in chromosome condensation and localization during mitosis. During interphase, however, it is assumed that the metazoan nuclear envelope presents a physical barrier, which inhibits interaction between the microtubules located in the cytoplasm and the chromatin fibers located in the nucleus. In recent years, it has become apparent that microtubule dynamics alter chromatin structure and function during interphase as well. Microtubule motor proteins transport several transcription factors and exogenous DNA (such as plasmid DNA) from the cytoplasm to the nucleus. Various soluble microtubule components are able to translocate into the nucleus, where they bind various chromatin elements leading to transcriptional alterations. In addition, microtubules may apply force on the nuclear envelope, which is transmitted into the nucleus, leading to changes in chromatin structure. Thus, microtubule dynamics during interphase may affect chromatin spatial organization, as well as transcription, replication and repair.
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Affiliation(s)
- Yael Maizels
- Department of Molecular Biology, Faculty of Natural Sciences, Ariel University, Israel
| | - Gabi Gerlitz
- Department of Molecular Biology, Faculty of Natural Sciences, Ariel University, Israel
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30
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Magalhães S, Duarte S, Monteiro GA, Fernandes F. Quantitative evaluation of DNA dissociation from liposome carriers and DNA escape from endosomes during lipid-mediated gene delivery. Hum Gene Ther Methods 2015; 25:303-13. [PMID: 25285806 DOI: 10.1089/hgtb.2014.080] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Nonviral vectors are highly attractive for gene therapy from a clinical point of view, and cationic lipid nanoparticles in particular have generated considerable interest. However, despite considerable recent advances, problems associated with low transfection efficiencies remain to be resolved to fully meet the potential of these vectors. The trafficking of plasmid DNA (pDNA) from the extracellular space up to the nucleus is prevented by several barriers, including liposome/pDNA dissociation within the endosome and pDNA escape into the cytosol. The aim of this work was to develop and optimize a tool that could offer simultaneous quantitative information both on the intracellular dissociation of oligonucleotides from lipid nanoparticles, and on the DNA escape from endocytic compartments. The ability to follow in real time both of these processes simultaneously (in a quantitative manner) is expected to be of high value in the rationalization and conception of new lipid nanoparticle vectors for gene delivery for therapeutic purposes. To this effect, a combination of Förster resonance energy transfer (FRET) and colocalization microscopy was employed. We show that it is possible to distinguish between liposome/pDNA dissociation and depletion of DNA within endosomes, providing resolution for the detection of intermediate species between endocytic particles with intact lipoplexes and endosomes devoid of DNA because of DNA escape or degradation. We demonstrate that after endocytosis, exceptionally few endocytic particles are found to exhibit simultaneously DNA/lipid colocalization and low FRET (DNA/lipid dissociation). These results clearly point to an extremely short-lived state for free plasmid within endosomes, which either escapes at once to the cytosol or is degraded within the endocytic compartment (because of exposure of DNA). It is possible that this limitation greatly contributes to reduction in probability of successful gene delivery through cationic lipid particles.
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Affiliation(s)
- Salomé Magalhães
- 1 Institute of Biotechnology and Bioengineering, Centre for Chemical and Biological Engineering, Instituto Superior Técnico, Universidade de Lisboa , 1049-001 Lisbon, Portugal
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Zeghimi A, Novell A, Thépault RA, Vourc'h P, Bouakaz A, Escoffre JM. Serum influence on in-vitro gene delivery using microbubble-assisted ultrasound. J Drug Target 2014; 22:748-60. [PMID: 24878379 DOI: 10.3109/1061186x.2014.921922] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
BACKGROUND Plasmid DNA (pDNA) is attractive molecule for gene therapy. pDNA-targeted delivery by efficient and safe methods is required to enhance its intra-tissue bioavailability. Among non-viral methods, sonoporation has become a promising method for in-vitro and in-vivo pDNA delivery. The efficiency of non-viral delivery methods of pDNA is generally limited by the presence of serum. PURPOSE The aim of this study was to evaluate the influence of serum on in-vitro pDNA delivery using microbubble-assisted ultrasound. METHODS The effects of a range of serum concentrations (0-50%) on efficiency of in-vitro pDNA delivery by sonoporation were determined on human glioblastoma cells. Furthermore, the influence of the serum on cell viability, membrane permeabilization, microbubble destruction, and pDNA topology were also assessed. RESULTS In-vitro results showed that a low serum concentration (i.e. ≤1%) induced a significant increase in transfection level through an increase in cell viability. However, a high serum concentration (i.e. ≥5%) resulted in a significant decrease in cell transfection, which was not associated with a decrease in membrane permeabilization or loss in cell viability. This decrease in transfection level was in fact positively correlated to changes in pDNA topology. CONCLUSION Serum influences the efficiency of in-vitro pDNA delivery by sonoporation through change in pDNA topology.
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Affiliation(s)
- Aya Zeghimi
- UMR Inserm U930, Université François-Rabelais de Tours , Tours , France
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Mladenova V, Mladenov E, Russev G. Organization of Plasmid DNA into Nucleosome-Like Structures after Transfection in Eukaryotic Cells. BIOTECHNOL BIOTEC EQ 2014. [DOI: 10.1080/13102818.2009.10817609] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022] Open
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Tschiche A, Malhotra S, Haag R. Nonviral gene delivery with dendritic self-assembling architectures. Nanomedicine (Lond) 2014; 9:667-93. [DOI: 10.2217/nnm.14.32] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
In this review, we outline the concept and applicability of self-assembling dendrimers for gene-delivery applications. Low-molecular-weight, well-defined cationic dendritic arrays which have been modified with hydrophobic domains can form self-organized multivalent systems that have significant advantages over nonassembling, high-molecular-weight/polymeric gene vectors. Particular structural variations have been highlighted with respect to the individual components of the displayed dendritic amphiphiles, namely, the employed amine termini, the hydrophobic segment, the size of the dendritic array, and the integration of special features such as targeting ability and cleavability/degradability, which can all have a crucial effect on gene-transfection efficiencies. Accordingly, the scientific efforts to create new synthetic gene-delivery vectors to act as promising in vivo transfection agents in the future will be presented and discussed here.
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Affiliation(s)
- Ariane Tschiche
- Institute of Chemistry & Biochemistry, Freie Universität Berlin, Takustrasse 3, 14195 Berlin, Germany
| | - Shashwat Malhotra
- Institute of Chemistry & Biochemistry, Freie Universität Berlin, Takustrasse 3, 14195 Berlin, Germany
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Abstract
Evolutionary selection for optimal genome preservation, replication, and expression should yield similar chromosome organizations in any type of cells. And yet, the chromosome organization is surprisingly different between eukaryotes and prokaryotes. The nuclear versus cytoplasmic accommodation of genetic material accounts for the distinct eukaryotic and prokaryotic modes of genome evolution, but it falls short of explaining the differences in the chromosome organization. I propose that the two distinct ways to organize chromosomes are driven by the differences between the global-consecutive chromosome cycle of eukaryotes and the local-concurrent chromosome cycle of prokaryotes. Specifically, progressive chromosome segregation in prokaryotes demands a single duplicon per chromosome, while other "precarious" features of the prokaryotic chromosomes can be viewed as compensations for this severe restriction.
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Weiss R, Scheiblhofer S, Roesler E, Weinberger E, Thalhamer J. mRNA vaccination as a safe approach for specific protection from type I allergy. Expert Rev Vaccines 2014; 11:55-67. [DOI: 10.1586/erv.11.168] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
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Silva CL, Bonato VLD, dos Santos-Júnior RR, Zárate-Bladés CR, Sartori A. Recent advances in DNA vaccines for autoimmune diseases. Expert Rev Vaccines 2014; 8:239-52. [DOI: 10.1586/14760584.8.2.239] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
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Abstract
Polyethylenimines (PEIs) have proven to be highly efficient and versatile agents for nucleic acid delivery in vitro and in vivo. Despite the low biodegradability of these polymers, they have been used in several clinical trials and the results suggest that the nucleic acid/PEI complexes have a good safety profile. The high transfection efficiency of PEIs probably relies on the fact that these polymers possess a stock of amines that can undergo protonation during the acidification of endosomes. This buffering capacity likely enhances endosomal escape of the polyplexes through the "proton sponge" effect. PEIs have also attracted great interest because the presence of many amino groups allow for easy chemical modifications or conjugation of targeting moieties and hydrophilic polymers. In the present chapter, we summarize and discuss the mechanism of PEI-mediated transfection, as well as the recent developments in PEI-mediated DNA, antisense oligonucleotide, and siRNA delivery.
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Affiliation(s)
- Patrick Neuberg
- Laboratoire "Vecteurs: Synthèse et Applications Thérapeutiques", UMR7199 CNRS-Université de Strasbourg, Faculté de Pharmacie, Illkirch, France
| | - Antoine Kichler
- Laboratoire "Vecteurs: Synthèse et Applications Thérapeutiques", UMR7199 CNRS-Université de Strasbourg, Faculté de Pharmacie, Illkirch, France
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Ramamoorthi K, Curtis D, Asuri P. Advances in homology directed genetic engineering of human pluripotent and adult stem cells. World J Stem Cells 2013; 5:98-105. [PMID: 24179598 PMCID: PMC3812527 DOI: 10.4252/wjsc.v5.i4.98] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/26/2013] [Revised: 09/06/2013] [Accepted: 09/17/2013] [Indexed: 02/06/2023] Open
Abstract
The ability to introduce precise genomic modifications in human cells has profound implications for both basic and applied research in stem cells, ranging from identification of genes regulating stem cell self-renewal and multilineage differentiation to therapeutic gene correction and creation of in vitro models of human diseases. However, the overall efficiency of this process is challenged by several factors including inefficient gene delivery into stem cells and low rates of homology directed site-specific targeting. Recent studies report the development of novel techniques to improve gene targeting efficiencies in human stem cells; these methods include molecular engineering of viral vectors to efficiently deliver episomal genetic sequences that can participate in homology directed targeting, as well as the design of synthetic proteins that can introduce double-stranded breaks in DNA to initiate such recombination events. This review focuses on the potential of these new technologies to precisely alter the human stem cell genome and also highlights the possibilities offered by the combination of these complementary strategies.
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Symens N, Rejman J, Lucas B, Demeester J, De Smedt SC, Remaut K. Noncoding DNA in lipofection of HeLa cells-a few insights. Mol Pharm 2013; 10:1070-9. [PMID: 23421924 DOI: 10.1021/mp300569j] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
In cationic carrier-mediated gene delivery, the disproportional relationship between the quantity of delivered DNA and the amount of encoded protein produced is a well-known phenomenon. The numerous intracellular barriers which need to be overcome by pDNA to reach the nucleoplasm play a major role in it. In contrast to what one would expect, a partial replacement of coding pDNA by noncoding DNA does not lead to a decrease in transfection efficiency. The mechanism underlying this observation is still unclear. Therefore, we investigated which constituents of the transfection process might contribute to this phenomenon. Our data reveal that the topology of the noncoding plasmid DNA plays a major role. Noncoding pDNA can be used only in a supercoiled form to replace coding pDNA in Lipofectamine lipoplexes, without a loss in transfection levels. When noncoding pDNA is linearized or partly digested, it diminishes the transfection potential of coding pDNA, as does noncoding salmon DNA. The difference in transfection efficiencies could not be attributed to diverse physicochemical characteristics of the Lipofectamine lipoplexes containing different types of noncoding DNA or to the extent of their internalization. At the level of endosomal release, however, nucleic acid release from the endosomal compartment proceeds faster when lipoplexes contain noncoding salmon DNA. Since the half-life of pDNA in the cytosol hardly exceeds 90 min, it is conceivable that prolonged release of coding pDNA from complexes carrying supercoiled noncoding pDNA may explain its positive effect on transfection, while this depot effect does not exist when noncoding salmon DNA is used.
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Affiliation(s)
- Nathalie Symens
- Laboratory of General Biochemistry and Physical Pharmacy, Ghent University , Harelbekestraat 72, Ghent, B-9000, Belgium
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Fichter KM, Ingle NP, McLendon PM, Reineke TM. Polymeric nucleic acid vehicles exploit active interorganelle trafficking mechanisms. ACS NANO 2013; 7:347-64. [PMID: 23234474 PMCID: PMC3586558 DOI: 10.1021/nn304218q] [Citation(s) in RCA: 60] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/21/2023]
Abstract
Materials that self-assemble with nucleic acids into nanocomplexes (e.g. polyplexes) are widely used in many fundamental biological and biomedical experiments. However, understanding the intracellular transport mechanisms of these vehicles remains a major hurdle in their effective usage. Here, we investigate two polycation models, Glycofect (which slowly degrades via hydrolysis) and linear polyethyleneimine (PEI) (which does not rapidly hydrolyze), to determine the impact of polymeric structure on intracellular trafficking. Cells transfected using Glycofect underwent increasing transgene expression over the course of 40 h and remained benign over the course of 7 days. Transgene expression in cells transfected with PEI peaked at 16 h post-transfection and resulted in less than 10% survival after 7 days. While saccharide-containing Glycofect has a higher buffering capacity than PEI, polyplexes created with Glycofect demonstrate more sustained endosomal release, possibly suggesting an additional or alternative delivery mechanism to the classical "proton sponge mechanism". PEI appeared to promote release of DNA from acidic organelles more than Glycofect. Immunofluorescence images indicate that both Glycofect and linear PEI traffic oligodeoxynucleotides to the Golgi and endoplasmic reticulum, which may be a route towards nuclear delivery. However, Glycofect polyplexes demonstrated higher co-localization with the ER than PEI polyplexes, and co-localization experiments indicate the retrograde transport of polyplexes via COP I vesicles from the Golgi to the ER. We conclude that slow release and unique trafficking behaviors of Glycofect polyplexes may be due to the presence of saccharide units and the degradable nature of the polymer, allowing more efficacious and benign delivery.
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Affiliation(s)
- Katye M. Fichter
- Department Chemistry, Missouri State University, Springfield, MO
| | - Nilesh. P. Ingle
- Department of Chemistry, University of Minnesota-Twin Cities, Minneapolis, MN
| | - Patrick M. McLendon
- Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH
| | - Theresa M. Reineke
- Department of Chemistry, University of Minnesota-Twin Cities, Minneapolis, MN
- Corresponding Author. Correspondence should be addressed to Professor Theresa M. Reineke, Department of Chemistry, University of Minnesota-Twin Cities, Minneapolis, MN. Phone: 612-624-8042.
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Functional eukaryotic nuclear localization signals are widespread in terminal proteins of bacteriophages. Proc Natl Acad Sci U S A 2012; 109:18482-7. [PMID: 23091024 DOI: 10.1073/pnas.1216635109] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
A number of prokaryotic proteins have been shown to contain nuclear localization signals (NLSs), although its biological role remains sometimes unclear. Terminal proteins (TPs) of bacteriophages prime DNA replication and become covalently linked to the genome ends. We predicted NLSs within the TPs of bacteriophages from diverse families and hosts and, indeed, the TPs of Φ29, Nf, PRD1, Bam35, and Cp-1, out of seven TPs tested, were found to localize to the nucleus when expressed in mammalian cells. Detailed analysis of Φ29 TP led us to identify a bona fide NLS within residues 1-37. Importantly, gene delivery into the eukaryotic nucleus is enhanced by the presence of Φ29 TP attached to the 5' DNA ends. These findings show a common feature of TPs from diverse bacteriophages targeting the eukaryotic nucleus and suggest a possible common function by facilitating the horizontal transfer of genes between prokaryotes and eukaryotes.
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Ilina P, Hyvonen Z, Saura M, Sandvig K, Yliperttula M, Ruponen M. Genetic blockage of endocytic pathways reveals differences in the intracellular processing of non-viral gene delivery systems. J Control Release 2012; 163:385-95. [PMID: 23041276 DOI: 10.1016/j.jconrel.2012.09.016] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2012] [Revised: 09/03/2012] [Accepted: 09/24/2012] [Indexed: 12/21/2022]
Abstract
Detailed understanding of the uptake mechanisms and intracellular processing of nonviral gene delivery systems will allow design of more effective carriers. This work gets insight into the intracellular kinetics of pDNA delivered by polyethyleneimine (PEI), cationic lipid DOTAP and calcium phosphate (CaP) precipitates. Amount of cell- and nuclear-associated pDNA was quantified by qRT-PCR at multiple time points after transfection. Moreover, the impact of specific endocytic pathways on the cell entry and intracellular kinetics of pDNA was studied by inhibition (blockage) of either clathrin- or dynamin-mediated endocytosis by using both genetically manipulated cell lines and chemical inhibitors of endocytosis. Quantitative analysis of defined kinetic parameters revealed that neither cellular nor nuclear uptake of pDNA correlated with transgene expression, emphasizing the importance of the post-nuclear processes in overall transfection efficacy. Changes in transgene expression observed upon blockage of endocytosis was carrier dependent and correlated relatively well with the changes at the cellular and nuclear uptake levels but not with the amount of cell-associated pDNA. Due to low specificity of chemical inhibitors and activation of alternative endocytosis pathways after genetic blockage of endocytosis neither of these methods is optimal for studying the role of endocytosis. Therefore, one should be careful when interpreting the obtained results from such studies and not to trust the data obtained only from one method.
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Affiliation(s)
- Polina Ilina
- Division of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, P.O. Box 56, FI-00014, University of Helsinki, Finland.
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Badding MA, Dean DA. Highly acetylated tubulin permits enhanced interactions with and trafficking of plasmids along microtubules. Gene Ther 2012; 20:616-24. [PMID: 23013836 PMCID: PMC3587030 DOI: 10.1038/gt.2012.77] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Microtubule-based transport is required for plasmid translocation to the nucleus during transfections, and having stable structures could enhance this movement. In previous studies in which the cytoskeleton was disrupted, we found that populations of microtubules remain that are stable and highly acetylated. By increasing the levels of acetylated tubulin through inhibition of the tubulin deacetylase HDAC6, we observe more rapid plasmid nuclear localization of transfected plasmids and greater levels of gene transfer. In this study, we sought to understand plasmid movement in cells with enhanced tubulin acetylation. Using variations of a microtubule spin down assay, we found that plasmids bound to hyper-acetylated microtubules to a greater degree than they did to unmodified microtubules. To determine if microtubule acetylation also affects cytoplasmic trafficking, plasmid movement was evaluated in real time by particle tracking in cells with varying levels of acetylated microtubules. We found that plasmids display greater net rates of movement, spend more time in productive motion and display longer runs of continuous motion in cells with highly acetylated microtubules compared to those with fewer modifications. These results all suggest that plasmid movement is enhanced along highly acetylated microtubules, reducing the time spent in the cytoplasm prior to nuclear import. Taken together, these findings provide a foundation for determining how modulation of microtubule acetylation can be used as a means to increase intracellular trafficking of plasmids and enhance gene therapy.
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Affiliation(s)
- M A Badding
- Department of Pediatrics, Division of Neonatology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA
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Satkauskas S, Ruzgys P, Venslauskas MS. Towards the mechanisms for efficient gene transfer into cells and tissues by means of cell electroporation. Expert Opin Biol Ther 2012; 12:275-86. [PMID: 22339479 DOI: 10.1517/14712598.2012.654775] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
INTRODUCTION Intracellular gene electrotransfer by means of electroporation has been on the increase during the past decade. Significant progress has been achieved both in characterizing mechanisms of gene electrotransfer and in optimizing the protocol in many preclinical trials. Recently this has led to initiation of clinical trials of gene electrotransfer to treat metastatic melanomas. Further progress with the method in various clinical trials requires better understanding of mechanisms of gene electrotransfer. AREAS COVERED A summary of recent progress in understanding mechanisms of gene electrotransfer, imparting general knowledge of cell electroporation and intracellular molecule electrotransfer. EXPERT OPINION Gene electrotransfer into cells and tissues is a complex process involving multiple steps that lead to plasmid DNA passage from the extracellular region to the cell nucleus crossing the barriers of the plasma membrane, cytoplasm and nucleus membrane. Electrical parameters of pulses used for gene electrotransfer affect the initial steps of DNA translocation through the plasma membrane and play a crucial role in determining the transfection efficiency. When considering gene electrotransfer into tissues it becomes clear that other nonelectrical conditions are also of primary importance.
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Affiliation(s)
- Saulius Satkauskas
- Vytautas Magnus University, Biology Department, Biophysical Research Group, Vileikos 8, Kaunas LT-44404, Lithuania.
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Symens N, Walczak R, Demeester J, Mattaj I, De Smedt SC, Remaut K. Nuclear inclusion of nontargeted and chromatin-targeted polystyrene beads and plasmid DNA containing nanoparticles. Mol Pharm 2011; 8:1757-66. [PMID: 21859089 DOI: 10.1021/mp200120v] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The nuclear membrane is one of the major cellular barriers in the delivery of plasmid DNA (pDNA). Cell division has a positive influence on the expression efficiency since, at the end of mitosis, pDNA or pDNA containing complexes near the chromatin are probably included by a random process in the nuclei of the daughter cells. However, very little is known about the nuclear inclusion of nanoparticles during cell division. Using the Xenopus nuclear envelope reassembly (XNER) assay, we found that the nuclear enclosure of nanoparticles was dependent on size (with 100 and 200 nm particles being better included than the 500 nm ones) and charge (with positively charged particles being better included than negatively charged or polyethyleneglycolated (PEGylated) ones) of the beads. Also, coupling chromatin-targeting peptides to the polystyrene beads or pDNA complexes improved their inclusion by 2- to 3-fold. Upon microinjection in living HeLa cells, however, nanoparticles were never observed in the nuclei of cells postdivision but accumulated in a specific perinuclear region, which was identified as the lysosomal compartment. This indicates that nanoparticles can end up in the lysosomes even when they were not delivered through endocytosis. To elucidate if the chromatin binding peptides also have potential in living cells, this additional barrier first has to be tackled, since it prevents free particles from being present near the chromatin at the moment of cell division.
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Affiliation(s)
- Nathalie Symens
- Laboratory of General Biochemistry and Physical Pharmacy, Ghent Research Group on Nanomedicines, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium
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Kafil V, Omidi Y. Cytotoxic impacts of linear and branched polyethylenimine nanostructures in a431 cells. BIOIMPACTS : BI 2011; 1:23-30. [PMID: 23678404 DOI: 10.5681/bi.2011.004] [Citation(s) in RCA: 90] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Received: 12/11/2010] [Revised: 12/20/2010] [Accepted: 12/28/2010] [Indexed: 02/02/2023]
Abstract
INTRODUCTION Polyethylenimine (PEI), as a nonviral cationic polymer, has been widely used as gene delivery nanosystem. Although a number of investigations have highlighted its toxic impacts on target cells through induction of apoptosis/necrosis, still it is essential to look at its structural impacts on target cells. METHODS In this current study, cytogenomic impacts of 25 kD linear and branched PEI (LPEI and BPEI, respectively) in A431 cells are reported to address possible mechanism for induction of apoptosis. At 40-50% confluency, A431 cells were exposed to PEI at a recommended concentration for 4 hr. After 24 hr, to detect apoptosis and DNA damage, the treated cells were subjected to MTT assay, FITC-labeled annexin V flow cytometry and comet assay. RESULTS Flow cytometry assessments revealed that the BPEI can result in greater internalization than the linear PEI, which also induced greater cytotoxicity. Annexin V assay confirmed early and late apoptosis by BPEI, imposing somewhat DNA damage detected by comet assay. Western blot analysis resulted in induction of Akt-kinase which is possibly one of biomolecules affected by PEI. CONCLUSION These results highlight that, despite induction of Akt-kinase, the BPEI can elicit apoptosis in target cells.
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Affiliation(s)
- Vala Kafil
- Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
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Functional Polymer Conjugates for Medicinal Nucleic Acid Delivery. POLYMERS IN NANOMEDICINE 2011. [DOI: 10.1007/12_2011_148] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
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Synergistic effects between natural histone mixtures and polyethylenimine in non-viral gene delivery in vitro. Int J Pharm 2010; 400:86-95. [PMID: 20816738 DOI: 10.1016/j.ijpharm.2010.08.036] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2010] [Revised: 08/18/2010] [Accepted: 08/25/2010] [Indexed: 10/19/2022]
Abstract
Nanoparticles made of plasmid DNA (pDNA) and cationic polymers are promising strategies for non-viral gene delivery. However, many cationic polymers are toxic to cells when used in higher concentrations. Positively charged proteins, such as histones, are biodegradable and a good alternative, especially for potential in vivo applications. It has previously been shown that histones are able to complex DNA and mediate transfection of cells. To investigate possible synergistic effects between the different histone types and to avoid the use of recombinant proteins, we analysed whether natural histone mixtures would be functional as gene carriers. Core and linker histones from calf thymus and from chicken erythrocytes were used to transfect different cell lines. The protein mixtures efficiently complexed the pDNA, and the resulting particles entered the cells. However, only marginal expression of the gene encoded by the pDNA was observed. Transfection rates increased drastically when minimal amounts of the basic polymer polyethylenimine (PEI) were added to the particles. Neither PEI nor histones alone mediated any transfection under the conditions where a combination of both worked efficiently, and the combined particles were well tolerated by the cells. These results demonstrate that histone mixtures from natural sources in combination with minimal amounts of PEI can be used as gene carriers. This might have consequences for the development of novel gene delivery strategies, such as DNA vaccines, with minimal side-effects.
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