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Advances in Non-viral Gene Transfer for Gene Therapy Applications
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Advances in Non-viral Gene Transfer for Gene Therapy Applications

A special issue of Genes (ISSN 2073-4425). This special issue belongs to the section "Technologies and Resources for Genetics".

Deadline for manuscript submissions: closed (15 November 2023) | Viewed by 13707

Special Issue Editors

Prof. Dr. Aglaia Athanassiadou

E-Mail Website
Guest Editor
Department of General Biology, Medical School, University of Patras, 26504 Patras, Greece
Interests: gene therapy; episomal vectors; autonomous replicon; haemoglobinopathies; animal models
Special Issues, Collections and Topics in MDPI journals
Prof. Dr. Axel Schambach

E-Mail Website
Guest Editor
1. Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany
2. Division of Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA
Interests: gene therapy; vector design; CARs; TRUCKs; hematology; stem cells; leukemia; iPSC; ovarian carcinoma
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

The field of gene therapy has experienced steady advances in recent years, particularly around viral gene transfer. At the same time, an array of nonviral vectors is currently developing, designed to address the drawbacks of viral vectors on issues such as safety, efficiency of industrial production, cost, etc.

The aim of this Special Issue is to provide a forum for scientists dedicated to research on the various kinds of non-viral vectors for gene therapy, spanning all diseases, acquired or inherited, to make their impact, no matter how big or small, and to allow new insights to be revealed and novel perspectives for gene therapy applications to be formulated.

Contributions can be reviews or articles of any size,  and reports are expected on all kinds of non-viral systems, such as episomal vectors including minicircles, nanoS/MAR, pFAR, or promoterless vectors and any other or newer similar systems, plasmids, transposons, etc. and their delivery, as well as on all kinds of disease or situations whether the vector is required to function transiently, e.g., in cancer, the production of CAR-T cells or iPS cells, or more long term, as is the case with the gene therapy of inherited diseases.

In the latter case, crucial aspects of episomal development are the identification of specific genetic and genomic features which, as part of a vector, enhance transfection efficiency, transgene expression or long-term retention of the free, circular, episomal vector within the eukaryotic nucleus. Therefore, reports are most welcome on the involvement of the compartmentalization of the nuclear matrix within the 3D nucleus and epigenetic factors deriving from the host cell on one hand, and sequences residing on the vector, on the other hand, namely origins of replication, insulators, S/MAR sequences, the backbone, etc., that may affect the establishment and maintenance of non-viral vectors in the host nucleus.

Our aim is for this Special Issue to promote scientific thinking on non-viral vectors’ structure and function that will benefit both the scientists submitting their articles and the readers.

Prof. Dr. Aglaia Athanassiadou
Prof. Dr. Axel Schambach
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Genes is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • gene therapy
  • non-viral vectors
  • non-viral vector delivery
  • plasmids
  • transposons
  • non-viral episomes
  • insulators
  • anchoring elements
  • S/MAR
  • minicircle
  • nanoS/MAR
  • pFAR
  • nuclear matrix
  • nuclear architecture
  • epigenetic factors
  • IR
  • β-globin Replicator

Published Papers (5 papers)

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Research

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17 pages, 3158 KiB  
Article
A Simple Nonviral Method to Generate Human Induced Pluripotent Stem Cells Using SMAR DNA Vectors
by Anna Hartley, Luisa Burger, Cornelia L. Wincek, Lieke Dons, Tracy Li, Annabel Grewenig, Toros Taşgın, Manuela Urban, Alicia Roig-Merino, Mehrnaz Ghazvini and Richard P. Harbottle
Genes 2024, 15(5), 575; https://doi.org/10.3390/genes15050575 - 30 Apr 2024
Viewed by 313
Abstract
Induced pluripotent stem cells (iPSCs) are a powerful tool for biomedical research, but their production presents challenges and safety concerns. Yamanaka and Takahashi revolutionised the field by demonstrating that somatic cells could be reprogrammed into pluripotent cells by overexpressing four key factors for [...] Read more.
Induced pluripotent stem cells (iPSCs) are a powerful tool for biomedical research, but their production presents challenges and safety concerns. Yamanaka and Takahashi revolutionised the field by demonstrating that somatic cells could be reprogrammed into pluripotent cells by overexpressing four key factors for a sufficient time. iPSCs are typically generated using viruses or virus-based methods, which have drawbacks such as vector persistence, risk of insertional mutagenesis, and oncogenesis. The application of less harmful nonviral vectors is limited as conventional plasmids cannot deliver the levels or duration of the factors necessary from a single transfection. Hence, plasmids that are most often used for reprogramming employ the potentially oncogenic Epstein–Barr nuclear antigen 1 (EBNA-1) system to ensure adequate levels and persistence of expression. In this study, we explored the use of nonviral SMAR DNA vectors to reprogram human fibroblasts into iPSCs. We show for the first time that iPSCs can be generated using nonviral plasmids without the use of EBNA-1 and that these DNA vectors can provide sufficient expression to induce pluripotency. We describe an optimised reprogramming protocol using these vectors that can produce high-quality iPSCs with comparable pluripotency and cellular function to those generated with viruses or EBNA-1 vectors. Full article
(This article belongs to the Special Issue Advances in Non-viral Gene Transfer for Gene Therapy Applications)
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18 pages, 3346 KiB  
Article
Non-Viral Episomal Vector Mediates Efficient Gene Transfer of the β-Globin Gene into K562 and Human Haematopoietic Progenitor Cells
by Vassileios M. Lazaris, Emmanouil Simantirakis, Eleana F. Stavrou, Meletios Verras, Argyro Sgourou, Maria K. Keramida, George Vassilopoulos and Aglaia Athanassiadou
Genes 2023, 14(9), 1774; https://doi.org/10.3390/genes14091774 - 08 Sep 2023
Viewed by 1284
Abstract
β-Thalassemia is a subgroup of inherited blood disorders associated with mild to severe anemia with few and limited conventional therapy options. Lately, lentiviral vector-based gene therapy has been successfully applied for disease treatment. However, the current development of non-viral episomal vectors (EV), non-integrating [...] Read more.
β-Thalassemia is a subgroup of inherited blood disorders associated with mild to severe anemia with few and limited conventional therapy options. Lately, lentiviral vector-based gene therapy has been successfully applied for disease treatment. However, the current development of non-viral episomal vectors (EV), non-integrating and non-coding for viral proteins, may be helpful in generating valid alternatives to viral vectors. We constructed a non-viral, episomal vector pEPβ-globin for the physiological β-globin gene based on two human chromosomal elements: the scaffold or matrix attachment region (S/MAR), allowing for long nuclear retention and non-integration and the β-globin replication initiation region (IR), allowing for enhancement of replication and establishment. After nucleofections into K562 cells with a transfection efficiency of 24.62 ± 7.7%, the vector induces stable transfection and is detected in long-term cultures as a non-integrating, circular episome expressing the β-globin gene efficiently. Transfections into CD34+ cells demonstrate an average efficiency of 15.57 ± 11.64%. In the colony-forming cell assay, fluorescent colonies are 92.21%, which is comparable to those transfected with vector pEP-IR at 92.68%. Additionally, fluorescent colonies produce β-globin mRNA at a physiologically 3-fold higher level than the corresponding non-transfected cells. Vector pEPβ-globin provides the basis for the development of therapeutic EV for gene therapy of β-thalassemias. Full article
(This article belongs to the Special Issue Advances in Non-viral Gene Transfer for Gene Therapy Applications)
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Review

Jump to: Research

30 pages, 2154 KiB  
Review
Antibiotic-Free Gene Vectors: A 25-Year Journey to Clinical Trials
by Corinne Marie and Daniel Scherman
Genes 2024, 15(3), 261; https://doi.org/10.3390/genes15030261 - 20 Feb 2024
Viewed by 1133
Abstract
Until very recently, the major use, for gene therapy, specifically of linear or circular DNA, such as plasmids, was as ancillary products for viral vectors’ production or as a genetic template for mRNA production. Thanks to targeted and more efficient physical or chemical [...] Read more.
Until very recently, the major use, for gene therapy, specifically of linear or circular DNA, such as plasmids, was as ancillary products for viral vectors’ production or as a genetic template for mRNA production. Thanks to targeted and more efficient physical or chemical delivery techniques and to the refinement of their structure, non-viral plasmid DNA are now under intensive consideration as pharmaceutical drugs. Plasmids traditionally carry an antibiotic resistance gene for providing the selection pressure necessary for maintenance in a bacterial host. Nearly a dozen different antibiotic-free gene vectors have now been developed and are currently assessed in preclinical assays and phase I/II clinical trials. Their reduced size leads to increased transfection efficiency and prolonged transgene expression. In addition, associating non-viral gene vectors and DNA transposons, which mediate transgene integration into the host genome, circumvents plasmid dilution in dividing eukaryotic cells which generate a loss of the therapeutic gene. Combining these novel molecular tools allowed a significantly higher yield of genetically engineered T and Natural Killer cells for adoptive immunotherapies due to a reduced cytotoxicity and increased transposition rate. This review describes the main progresses accomplished for safer, more efficient and cost-effective gene and cell therapies using non-viral approaches and antibiotic-free gene vectors. Full article
(This article belongs to the Special Issue Advances in Non-viral Gene Transfer for Gene Therapy Applications)
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22 pages, 1110 KiB  
Review
Episomes and Transposases—Utilities to Maintain Transgene Expression from Nonviral Vectors
by Florian Kreppel and Claudia Hagedorn
Genes 2022, 13(10), 1872; https://doi.org/10.3390/genes13101872 - 16 Oct 2022
Viewed by 2799
Abstract
The efficient delivery and stable transgene expression are critical for applications in gene therapy. While carefully selected and engineered viral vectors allowed for remarkable clinical successes, they still bear significant safety risks. Thus, nonviral vectors are a sound alternative and avoid genotoxicity and [...] Read more.
The efficient delivery and stable transgene expression are critical for applications in gene therapy. While carefully selected and engineered viral vectors allowed for remarkable clinical successes, they still bear significant safety risks. Thus, nonviral vectors are a sound alternative and avoid genotoxicity and adverse immunological reactions. Nonviral vector systems have been extensively studied and refined during the last decades. Emerging knowledge of the epigenetic regulation of replication and spatial chromatin organisation, as well as new technologies, such as Crispr/Cas, were employed to enhance the performance of different nonviral vector systems. Thus, nonviral vectors are in focus and hold some promising perspectives for future applications in gene therapy. This review addresses three prominent nonviral vector systems: the Sleeping Beauty transposase, S/MAR-based episomes, and viral plasmid replicon-based EBV vectors. Exemplarily, we review different utilities, modifications, and new concepts that were pursued to overcome limitations regarding stable transgene expression and mitotic stability. New insights into the nuclear localisation of nonviral vector molecules and the potential consequences thereof are highlighted. Finally, we discuss the remaining limitations and provide an outlook on possible future developments in nonviral vector technology. Full article
(This article belongs to the Special Issue Advances in Non-viral Gene Transfer for Gene Therapy Applications)
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27 pages, 3571 KiB  
Review
Appraisal for the Potential of Viral and Nonviral Vectors in Gene Therapy: A Review
by Muhammad Hammad Butt, Muhammad Zaman, Abrar Ahmad, Rahima Khan, Tauqeer Hussain Mallhi, Mohammad Mehedi Hasan, Yusra Habib Khan, Sara Hafeez, Ehab El Sayed Massoud, Md. Habibur Rahman and Simona Cavalu
Genes 2022, 13(8), 1370; https://doi.org/10.3390/genes13081370 - 30 Jul 2022
Cited by 37 | Viewed by 7598
Abstract
Over the past few decades, gene therapy has gained immense importance in medical research as a promising treatment strategy for diseases such as cancer, AIDS, Alzheimer’s disease, and many genetic disorders. When a gene needs to be delivered to a target cell inside [...] Read more.
Over the past few decades, gene therapy has gained immense importance in medical research as a promising treatment strategy for diseases such as cancer, AIDS, Alzheimer’s disease, and many genetic disorders. When a gene needs to be delivered to a target cell inside the human body, it has to pass a large number of barriers through the extracellular and intracellular environment. This is why the delivery of naked genes and nucleic acids is highly unfavorable, and gene delivery requires suitable vectors that can carry the gene cargo to the target site and protect it from biological degradation. To date, medical research has come up with two types of gene delivery vectors, which are viral and nonviral vectors. The ability of viruses to protect transgenes from biological degradation and their capability to efficiently cross cellular barriers have allowed gene therapy research to develop new approaches utilizing viruses and their different genomes as vectors for gene delivery. Although viral vectors are very efficient, science has also come up with numerous nonviral systems based on cationic lipids, cationic polymers, and inorganic particles that provide sustainable gene expression without triggering unwanted inflammatory and immune reactions, and that are considered nontoxic. In this review, we discuss in detail the latest data available on all viral and nonviral vectors used in gene delivery. The mechanisms of viral and nonviral vector-based gene delivery are presented, and the advantages and disadvantages of all types of vectors are also given. Full article
(This article belongs to the Special Issue Advances in Non-viral Gene Transfer for Gene Therapy Applications)
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