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Pharmaceutics
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Plasmid DNA for Gene Therapy and DNA Vaccine Applications, 2nd...
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Plasmid DNA for Gene Therapy and DNA Vaccine Applications, 2nd Edition

A special issue of Pharmaceutics (ISSN 1999-4923). This special issue belongs to the section "Gene and Cell Therapy".

Deadline for manuscript submissions: 10 August 2024 | Viewed by 4112

Special Issue Editor

Dr. Urska Kamensek

E-Mail Website
Guest Editor
Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska Cesta 2, SI-1000 Ljubljana, Slovenia
Interests: plasmid design; nonviral gene therapy; cancer immunotherapy; electroporation
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

In recent years, there has been an increased interest in plasmid DNA as a vector for gene therapies and DNA vaccines against infectious, acquired, and genetic diseases, including cancer and COVID-19. Additionally, plasmids are used for the production of viral particles intended for viral gene and oncolytic therapy. With the recent event of artificial gene synthesis methods, the assembly of new plasmids has become easier than ever. Plasmids can be designed to encode for various therapeutic targets, vaccine antigens, therapeutic antibodies, shRNA molecules, elements of CRSPR/Cas9 systems, viral vector elements, etc. Additionally, expression cassettes can now be easily tweaked and codon-optimized for higher and more targeted expression, and bacterial backbones minimized for more efficient and safer plasmid production. However, there are still some challenges that need to be overcome to achieve the widespread application of plasmid DNA therapeutics. First of all, as plasmids do not enter cells actively, their therapeutic success depends heavily on physical and chemical delivery methods, which are constantly being developed and optimized. Secondly, the exact mechanisms leading from a plasmid’s cell entrance to its expression in the nucleus remain elusive. Thirdly, although considered less immunogenic than viral vectors, plasmid DNA still activates a cell’s defense mechanisms against foreign DNA. While this self-adjuvanting effect of plasmid DNA can be advantageous for applications in DNA vaccination and cancer gene therapy, it can uncontrollably influence transfection efficiency and safety. Finally, there are still some challenges in preparing large quantities of clinical-grade plasmid DNA.

This Special Issue welcomes research papers dealing with all aspects of gene therapy and DNA vaccine applications of plasmid DNA: from basic plasmid design studies, mechanistic studies of plasmid fate inside cells, and the testing of new therapeutic approaches or new plasmid delivery systems to large-scale manufacturing and regulatory compliances of plasmid DNA therapeutics as well as their clinical applications. Review articles on the abovementioned topics are also welcomed.

Dr. Urska Kamensek
Guest Editor

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. Pharmaceutics 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 2900 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

  • plasmid DNA
  • plasmid vector
  • non-viral gene delivery
  • transfection
  • gene electrotransfer
  • plasmid DNA vaccine
  • plasmid gene therapy
  • plasmid production
  • plasmid regulatory compliance

Related Special Issue

Published Papers (5 papers)

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Research

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18 pages, 4678 KiB  
Article
Enhancing In Vivo Electroporation Efficiency through Hyaluronidase: Insights into Plasmid Distribution and Optimization Strategies
by Debnath Maji, Verónica Miguela, Andrew D. Cameron, Delcora A. Campbell, Linda Sasset, Xin Yao, Andy T. Thompson, Carleigh Sussman, David Yang, Robert Miller, Marek M. Drozdz and Rachel A. Liberatore
Pharmaceutics 2024, 16(4), 547; https://doi.org/10.3390/pharmaceutics16040547 - 17 Apr 2024
Viewed by 596
Abstract
Electroporation (EP) stands out as a promising non-viral plasmid delivery strategy, although achieving optimal transfection efficiency in vivo remains a challenge. A noteworthy advancement in the field of in vivo EP is the application of hyaluronidase, an enzyme with the capacity to degrade [...] Read more.
Electroporation (EP) stands out as a promising non-viral plasmid delivery strategy, although achieving optimal transfection efficiency in vivo remains a challenge. A noteworthy advancement in the field of in vivo EP is the application of hyaluronidase, an enzyme with the capacity to degrade hyaluronic acid in the extracellular matrix, which thereby enhances DNA transfer efficiency by 2- to 3-fold. This paper focuses on elucidating the mechanism of hyaluronidase’s impact on transfection efficiency. We demonstrate that hyaluronidase promotes a more uniform distribution of plasmid DNA (pDNA) within skeletal muscle. Additionally, our study investigates the effect of the timing of hyaluronidase pretreatment on EP efficiency by including time intervals of 0, 5, and 30 min between hyaluronidase treatment and the application of pulses. Serum levels of the pDNA-encoded transgene reveal a minimal influence of the hyaluronidase pretreatment time on the final serum protein levels following delivery in both mice and rabbit models. Leveraging bioimpedance measurements, we capture morphological changes in muscle induced by hyaluronidase treatment, which result in a varied pDNA distribution. Subsequently, these findings are employed to optimize EP electrical parameters following hyaluronidase treatment in animal models. This paper offers novel insights into the potential of hyaluronidase in enhancing the effectiveness of in vivo EP, as well as guides optimized electroporation strategies following hyaluronidase use. Full article
(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications, 2nd Edition)
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18 pages, 2771 KiB  
Article
Transcription Factor Binding Site in Promoter Determines the Pattern of Plasmid-Based Transgene Expression In Vivo
by Chunbo Zhang and Dexi Liu
Pharmaceutics 2024, 16(4), 544; https://doi.org/10.3390/pharmaceutics16040544 - 15 Apr 2024
Viewed by 682
Abstract
Understanding the regulation of transgene expression is critical for the success of plasmid-based gene therapy and vaccine development. In this study, we used two sets of plasmid vectors containing secreted embryonic alkaline phosphatase or the mouse IL-10 gene as a reporter and investigated [...] Read more.
Understanding the regulation of transgene expression is critical for the success of plasmid-based gene therapy and vaccine development. In this study, we used two sets of plasmid vectors containing secreted embryonic alkaline phosphatase or the mouse IL-10 gene as a reporter and investigated the role of promoter elements in regulating transgene expression in vivo. We demonstrated in mice that hydrodynamic transfer of plasmids with the CMV promoter resulted in a high level of reporter gene expression that declined rapidly over time. In contrast, when plasmids with albumin promoters were used, a lower but sustained gene expression pattern was observed. We also found that plasmids containing a shorter CMV promoter sequence with fewer transcription factor binding sites showed a decrease in the peak level of gene expression without changing the overall pattern of reporter gene expression. The replacement of regulatory elements in the CMV promoter with a single regulatory element of the albumin promoter changed the pattern of transient gene expression seen in the CMV promoter to a pattern of sustained gene expression identical to that of a full albumin promoter. ChIP analyses demonstrated an elevated binding of acetylated histones and TATA box-binding protein to the promoter carrying regulatory elements of the albumin promoter. These results suggest that the strength of a promoter is determined by the number of appropriate transcription factor binding sites, while gene expression persistence is determined by the presence of regulatory elements capable of recruiting epigenetic modifying complexes that make the promoter accessible for transcription. This study provides important insights into the mechanisms underlying gene expression regulation in vivo, which can be used to improve plasmid-based gene therapy and vaccine development. Full article
(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications, 2nd Edition)
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12 pages, 3216 KiB  
Article
Sucrose Treatment Enhances the Electrotransfer of DNA by Activating Phospholipase A2
by Chunxi Wang, Chun-Chi Chang, Jen-Tsan Chi and Fan Yuan
Pharmaceutics 2024, 16(4), 475; https://doi.org/10.3390/pharmaceutics16040475 - 29 Mar 2024
Viewed by 611
Abstract
Our previous study discovered that sucrose and other non-reducing sugars (e.g., trehalose and raffinose) could be used to improve the electrotransfer (ET) of molecular cargo, including DNA, mRNA, and ribonucleoprotein in various cell lines and primary human cells in vitro and in vivo. [...] Read more.
Our previous study discovered that sucrose and other non-reducing sugars (e.g., trehalose and raffinose) could be used to improve the electrotransfer (ET) of molecular cargo, including DNA, mRNA, and ribonucleoprotein in various cell lines and primary human cells in vitro and in vivo. To understand the molecular mechanisms of this improvement, we used RNA sequencing technology to analyze changes in the cell transcriptome after sucrose treatment. The results from our analysis demonstrated that the sucrose treatment upregulated phospholipase A2 and V-ATPase gene families, which could potentially influence the acidity of intracellular vesicles through augmenting vesicle fusion and the influx of proton, respectively. To determine how this upregulation affects ET efficiency, we treated cells with pharmaceutical inhibitors of phospholipase A2 and V-ATPase. The data demonstrated that the treatment with the phospholipase A2 inhibitor could reverse the ET improvement elicited by the sucrose treatment. The V-ATPase inhibitor treatment either had little influence or further enhanced the effect of the sucrose treatment on the ET efficiency. These observations provide a molecular explanation for our previous findings, demonstrating that the sucrose treatment primarily enhanced the ET efficiency by promoting vesicle trafficking and fusion through the activation of phospholipase A2. Full article
(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications, 2nd Edition)
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15 pages, 3104 KiB  
Article
Pulsed Electric Fields Induce STING Palmitoylation and Polymerization Independently of Plasmid DNA Electrotransfer
by Amanda Sales Conniff, Julie Singh, Richard Heller and Loree C. Heller
Pharmaceutics 2024, 16(3), 363; https://doi.org/10.3390/pharmaceutics16030363 - 5 Mar 2024
Viewed by 911
Abstract
Gene therapy approaches may target skeletal muscle due to its high protein-expressing nature and vascularization. Intramuscular plasmid DNA (pDNA) delivery via pulsed electric fields (PEFs) can be termed electroporation or electrotransfer. Nonviral delivery of plasmids to cells and tissues activates DNA-sensing pathways. The [...] Read more.
Gene therapy approaches may target skeletal muscle due to its high protein-expressing nature and vascularization. Intramuscular plasmid DNA (pDNA) delivery via pulsed electric fields (PEFs) can be termed electroporation or electrotransfer. Nonviral delivery of plasmids to cells and tissues activates DNA-sensing pathways. The central signaling complex in cytosolic DNA sensing is the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING). The effects of pDNA electrotransfer on the signaling of STING, a key adapter protein, remain incompletely characterized. STING undergoes several post-translational modifications which modulate its function, including palmitoylation. This study demonstrated that in mouse skeletal muscle, STING was constitutively palmitoylated at two sites, while an additional site was modified following electroporation independent of the presence of pDNA. This third palmitoylation site correlated with STING polymerization but not with STING activation. Expression of several palmitoyl acyltransferases, including zinc finger and DHHC motif containing 1 (zDHHC1), coincided with STING activation. Expression of several depalmitoylases, including palmitoyl protein thioesterase 2 (PPT2), was diminished in all PEF application groups. Therefore, STING may not be regulated by active modification by palmitate after electroporation but inversely by the downregulation of palmitate removal. These findings unveil intricate molecular changes induced by PEF application. Full article
(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications, 2nd Edition)
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Review

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17 pages, 872 KiB  
Review
Delivery of DNA-Based Therapeutics for Treatment of Chronic Diseases
by Carleigh Sussman, Rachel A. Liberatore and Marek M. Drozdz
Pharmaceutics 2024, 16(4), 535; https://doi.org/10.3390/pharmaceutics16040535 - 13 Apr 2024
Viewed by 665
Abstract
Gene therapy and its role in the medical field have evolved drastically in recent decades. Studies aim to define DNA-based medicine as well as encourage innovation and the further development of novel approaches. Gene therapy has been established as an alternative approach to [...] Read more.
Gene therapy and its role in the medical field have evolved drastically in recent decades. Studies aim to define DNA-based medicine as well as encourage innovation and the further development of novel approaches. Gene therapy has been established as an alternative approach to treat a variety of diseases. Its range of mechanistic applicability is wide; gene therapy has the capacity to address the symptoms of disease, the body’s ability to fight disease, and in some cases has the ability to cure disease, making it a more attractive intervention than some traditional approaches to treatment (i.e., medicine and surgery). Such versatility also suggests gene therapy has the potential to address a greater number of indications than conventional treatments. Many DNA-based therapies have shown promise in clinical trials, and several have been approved for use in humans. Whereas current treatment regimens for chronic disease often require frequent dosing, DNA-based therapies can produce robust and durable expression of therapeutic genes with fewer treatments. This benefit encourages the application of DNA-based gene therapy to manage chronic diseases, an area where improving efficiency of current treatments is urgent. Here, we provide an overview of two DNA-based gene therapies as well as their delivery methods: adeno associated virus (AAV)-based gene therapy and plasmid DNA (pDNA)-based gene therapy. We will focus on how these therapies have already been utilized to improve treatment of chronic disease, as well as how current literature supports the expansion of these therapies to treat additional chronic indications in the future. Full article
(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications, 2nd Edition)
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