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18 pages, 2665 KiB

Open AccessArticle

What We Learned about the Feasibility of Gene Electrotransfer for Vaccination on a Model of COVID-19 Vaccine

by Urska Kamensek, Maja Cemazar, Simona Kranjc Brezar, Tanja Jesenko, Spela Kos, Katarina Znidar, Bostjan Markelc, Ziva Modic, Tilen Komel, Tim Gorse, Eva Rebersek, Helena Jakopic and Gregor Sersa

Pharmaceutics 2023, 15(7), 1981; https://doi.org/10.3390/pharmaceutics15071981 - 19 Jul 2023

Viewed by 1764

Abstract

DNA vaccination is one of the emerging approaches for a wide range of applications, including prophylactic vaccination against infectious diseases and therapeutic vaccination against cancer. The aim of this study was to evaluate the feasibility of our previously optimized protocols for gene electrotransfer [...] Read more.

DNA vaccination is one of the emerging approaches for a wide range of applications, including prophylactic vaccination against infectious diseases and therapeutic vaccination against cancer. The aim of this study was to evaluate the feasibility of our previously optimized protocols for gene electrotransfer (GET)-mediated delivery of plasmid DNA into skin and muscle tissues on a model of COVID-19 vaccine. Plasmids encoding the SARS-CoV-2 proteins spike (S) and nucleocapsid (N) were used as the antigen source, and a plasmid encoding interleukin 12 (IL-12) was used as an adjuvant. Vaccination was performed in the skin or muscle tissue of C57BL/6J mice on days 0 and 14 (boost). Two weeks after the boost, blood, spleen, and transfected tissues were collected to determine the expression of S, N, IL-12, serum interferon-γ, the induction of antigen-specific IgG antibodies, and cytotoxic T-cells. In accordance with prior in vitro experiments that indicated problems with proper expression of the S protein, vaccination with S did not induce S-specific antibodies, whereas significant induction of N-specific antibodies was detected after vaccination with N. Intramuscular vaccination outperformed skin vaccination and resulted in significant induction of humoral and cell-mediated immunity. Moreover, both boost and adjuvant were found to be redundant for the induction of an immune response. Overall, the study confirmed the feasibility of the GET for DNA vaccination and provided valuable insights into this approach. Full article

(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications)

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23 pages, 4456 KiB

Open AccessArticle

Plasmid DNA Prime/Protein Boost Vaccination against Campylobacter jejuni in Broilers: Impact of Vaccine Candidates on Immune Responses and Gut Microbiota

by Noémie Gloanec, Muriel Guyard-Nicodème, Raphaël Brunetti, Ségolène Quesne, Alassane Keita, Marianne Chemaly and Daniel Dory

Pharmaceutics 2023, 15(5), 1397; https://doi.org/10.3390/pharmaceutics15051397 - 3 May 2023

Cited by 1 | Viewed by 1998

Abstract

Campylobacter infections, traced to poultry products, are major bacterial foodborne zoonoses, and vaccination is a potential solution to reduce these infections. In a previous experimental trial using a plasmid DNA prime/recombinant protein boost vaccine regimen, two vaccine candidates (YP437 and YP9817) induced a [...] Read more.

Campylobacter infections, traced to poultry products, are major bacterial foodborne zoonoses, and vaccination is a potential solution to reduce these infections. In a previous experimental trial using a plasmid DNA prime/recombinant protein boost vaccine regimen, two vaccine candidates (YP437 and YP9817) induced a partially protective immune response against Campylobacter in broilers, and an impact of the protein batch on vaccine efficacy was suspected. This new study was designed to evaluate different batches of the previously studied recombinant proteins (called YP437A, YP437P and YP9817P) and to enhance the immune responses and gut microbiota studies after a C. jejuni challenge. Throughout the 42-day trial in broilers, caecal Campylobacter load, specific antibodies in serum and bile, the relative expression of cytokines and β-defensins, and caecal microbiota were assessed. Despite there being no significant reduction in Campylobacter in the caecum of vaccinated groups, specific antibodies were detected in serum and bile, particularly for YP437A and YP9817P, whereas the production of cytokines and β-defensins was not significant. The immune responses differed according to the batch. A slight change in microbiota was demonstrated in response to vaccination against Campylobacter. The vaccine composition and/or regimen must be further optimised. Full article

(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications)

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16 pages, 4033 KiB

Open AccessArticle

Exploring the Fate of Antibody-Encoding pDNA after Intramuscular Electroporation in Mice

by Marie-Lynn Cuypers, Nick Geukens, Kevin Hollevoet, Paul Declerck and Maarten Dewilde

Pharmaceutics 2023, 15(4), 1160; https://doi.org/10.3390/pharmaceutics15041160 - 6 Apr 2023

Cited by 1 | Viewed by 1898

Abstract

DNA-based antibody therapy seeks to administer the encoding nucleotide sequence rather than the antibody protein. To further improve the in vivo monoclonal antibody (mAb) expression, a better understanding of what happens after the administration of the encoding plasmid DNA (pDNA) is required. This [...] Read more.

DNA-based antibody therapy seeks to administer the encoding nucleotide sequence rather than the antibody protein. To further improve the in vivo monoclonal antibody (mAb) expression, a better understanding of what happens after the administration of the encoding plasmid DNA (pDNA) is required. This study reports the quantitative evaluation and localization of the administered pDNA over time and its association with corresponding mRNA levels and systemic protein concentrations. pDNA encoding the murine anti-HER2 4D5 mAb was administered to BALB/c mice via intramuscular injection followed by electroporation. Muscle biopsies and blood samples were taken at different time points (up to 3 months). In muscle, pDNA levels decreased 90% between 24 h and one week post treatment (p < 0.0001). In contrast, mRNA levels remained stable over time. The 4D5 antibody plasma concentrations reached peak levels at week two followed by a slow decrease (50% after 12 weeks, p < 0.0001). Evaluation of pDNA localization revealed that extranuclear pDNA was cleared fast, whereas the nuclear fraction remained relatively stable. This is in line with the observed mRNA and protein levels over time and indicates that only a minor fraction of the administered pDNA is ultimately responsible for the observed systemic mAb levels. In conclusion, this study demonstrates that durable expression is dependent on the nuclear uptake of the pDNA. Therefore, efforts to increase the protein levels upon pDNA-based gene therapy should focus on strategies to increase both cellular entry and migration of the pDNA into the nucleus. The currently applied methodology can be used to guide the design and evaluation of novel plasmid-based vectors or alternative delivery methods in order to achieve a robust and prolonged protein expression. Full article

(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications)

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19 pages, 3846 KiB

Open AccessArticle

Gene Electrotransfer Efficiency in 2D and 3D Cancer Cell Models Using Different Electroporation Protocols: A Comparative Study

by Alexia de Caro, Elisabeth Bellard, Jelena Kolosnjaj-Tabi, Muriel Golzio and Marie-Pierre Rols

Pharmaceutics 2023, 15(3), 1004; https://doi.org/10.3390/pharmaceutics15031004 - 21 Mar 2023

Cited by 5 | Viewed by 2201

Abstract

Electroporation, a method relying on a pulsed electric field to induce transient cell membrane permeabilization, can be used as a non-viral method to transfer genes in vitro and in vivo. Such transfer holds great promise for cancer treatment, as it can induce or [...] Read more.

Electroporation, a method relying on a pulsed electric field to induce transient cell membrane permeabilization, can be used as a non-viral method to transfer genes in vitro and in vivo. Such transfer holds great promise for cancer treatment, as it can induce or replace missing or non-functioning genes. Yet, while efficient in vitro, gene-electrotherapy remains challenging in tumors. To assess the differences of gene electrotransfer in respect to applied pulses in multi-dimensional (2D, 3D) cellular organizations, we herein compared pulsed electric field protocols applicable to electrochemotherapy and gene electrotherapy and different “High Voltage–Low Voltage” pulses. Our results show that all protocols can result in efficient permeabilization of 2D- and 3D-grown cells. However, their efficiency for gene delivery varies. The gene-electrotherapy protocol is the most efficient in cell suspensions, with a transfection rate of about 50%. Conversely, despite homogenous permeabilization of the entire 3D structure, none of the tested protocols allowed gene delivery beyond the rims of multicellular spheroids. Taken together, our findings highlight the importance of electric field intensity and the occurrence of cell permeabilization, and underline the significance of pulses’ duration, impacting plasmids’ electrophoretic drag. The latter is sterically hindered in 3D structures and prevents the delivery of genes into spheroids’ core. Full article

(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications)

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18 pages, 3205 KiB

Open AccessArticle

Modification of the Tumor Microenvironment Enhances Anti-PD-1 Immunotherapy in Metastatic Melanoma

by Guilan Shi, Megan Scott, Cathryn G. Mangiamele and Richard Heller

Pharmaceutics 2022, 14(11), 2429; https://doi.org/10.3390/pharmaceutics14112429 - 10 Nov 2022

Cited by 3 | Viewed by 1961

Abstract

Resistance to checkpoint-blockade treatments is a challenge in the clinic. Both primary and acquired resistance have become major obstacles, greatly limiting the long-lasting effects and wide application of blockade therapy. Many patients with metastatic melanoma eventually require further therapy. The absence of T-cell [...] Read more.

Resistance to checkpoint-blockade treatments is a challenge in the clinic. Both primary and acquired resistance have become major obstacles, greatly limiting the long-lasting effects and wide application of blockade therapy. Many patients with metastatic melanoma eventually require further therapy. The absence of T-cell infiltration to the tumor site is a well-accepted contributor limiting immune checkpoint inhibitor efficacy. In this study, we combined intratumoral injection of plasmid IL-12 with electrotransfer and anti-PD-1 in metastatic B16F10 melanoma tumor model to increase tumor-infiltrating lymphocytes and improve therapeutic efficacy. We showed that effective anti-tumor responses required a subset of tumor-infiltrating CD8⁺ and CD4⁺ T cells. Additionally, the combination therapy induced higher MHC-I surface expression on tumor cells to hamper tumor cells escaping from immune recognition. Furthermore, we found that activating T cells by exposure to IL-12 resulted in tumors sensitized to anti-PD-1 treatment, suggesting a therapeutic strategy to improve responses to checkpoint blockade. Full article

(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications)

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12 pages, 2066 KiB

Open AccessArticle

Acute Effects of Intratumor DNA Electrotransfer

by Manya Bhandary, Amanda Sales Conniff, Kaitlyn Miranda and Loree C. Heller

Pharmaceutics 2022, 14(10), 2097; https://doi.org/10.3390/pharmaceutics14102097 - 30 Sep 2022

Cited by 5 | Viewed by 1845

Abstract

Intratumor therapeutic DNA electroporation or electrotransfer is in clinical trials in the United States and is under development in many other countries. Acute changes in endogenous gene expression in response to DNA or to pulse application may significantly modulate the therapeutic efficacy of [...] Read more.

Intratumor therapeutic DNA electroporation or electrotransfer is in clinical trials in the United States and is under development in many other countries. Acute changes in endogenous gene expression in response to DNA or to pulse application may significantly modulate the therapeutic efficacy of the expressed proteins. Oligonucleotide arrays were used in this study to quantify changes in mRNA expression in B16-F10 mouse melanoma tumors four hours after DNA electrotransfer. The data were subjected to the DAVID v6.8 web server for functional annotation to reveal regulated genes and genetic pathways. Gene ontology analysis revealed several molecular functions related to cytoskeletal remodeling and inflammatory signaling. In B16-F10 cells, F-actin remodeling was confirmed by phalloidin staining in cells that received pulse application alone or in the presence of DNA. Chemokine secretion was confirmed in cells receiving DNA electrotransfer. These results indicate that pulse application alone or in the presence of DNA may modulate the therapeutic efficacy of therapeutic DNA electrotransfer. Full article

(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications)

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21 pages, 3745 KiB

Open AccessArticle

Gene Electrotransfer into Mammalian Cells Using Commercial Cell Culture Inserts with Porous Substrate

by Tina Vindiš, Anja Blažič, Diaa Khayyat, Tjaša Potočnik, Shaurya Sachdev and Lea Rems

Pharmaceutics 2022, 14(9), 1959; https://doi.org/10.3390/pharmaceutics14091959 - 16 Sep 2022

Cited by 3 | Viewed by 2580

Abstract

Gene electrotransfer is one of the main non-viral methods for intracellular delivery of plasmid DNA, wherein pulsed electric fields are used to transiently permeabilize the cell membrane, allowing enhanced transmembrane transport. By localizing the electric field over small portions of the cell membrane [...] Read more.

Gene electrotransfer is one of the main non-viral methods for intracellular delivery of plasmid DNA, wherein pulsed electric fields are used to transiently permeabilize the cell membrane, allowing enhanced transmembrane transport. By localizing the electric field over small portions of the cell membrane using nanostructured substrates, it is possible to increase considerably the gene electrotransfer efficiency while preserving cell viability. In this study, we expand the frontier of localized electroporation by designing an electrotransfer approach based on commercially available cell culture inserts with polyethylene-terephthalate (PET) porous substrate. We first use multiscale numerical modeling to determine the pulse parameters, substrate pore size, and other factors that are expected to result in successful gene electrotransfer. Based on the numerical results, we design a simple device combining an insert with substrate containing pores with 0.4 µm or 1.0 µm diameter, a multiwell plate, and a pair of wire electrodes. We test the device in three mammalian cell lines and obtain transfection efficiencies similar to those achieved with conventional bulk electroporation, but at better cell viability and with low-voltage pulses that do not require the use of expensive electroporators. Our combined theoretical and experimental analysis calls for further systematic studies that will investigate the influence of substrate pore size and porosity on gene electrotransfer efficiency and cell viability. Full article

(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications)

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14 pages, 2888 KiB

Open AccessArticle

Design, Development, and Testing of a Device for Gene Electrotransfer to Skin Cells In Vivo

by Aleksandra Cvetkoska, Janja Dermol-Černe, Damijan Miklavčič, Simona Kranjc Brezar, Boštjan Markelc, Gregor Serša and Matej Reberšek

Pharmaceutics 2022, 14(9), 1826; https://doi.org/10.3390/pharmaceutics14091826 - 30 Aug 2022

Viewed by 1756

Abstract

Gene electrotransfer (GET) is considered one of the most efficient, safe, reproducible, and cost-effective methods of gene therapy, in which a gene is delivered to the cells in the form of a plasmid DNA vector by a method known as electroporation. To achieve [...] Read more.

Gene electrotransfer (GET) is considered one of the most efficient, safe, reproducible, and cost-effective methods of gene therapy, in which a gene is delivered to the cells in the form of a plasmid DNA vector by a method known as electroporation. To achieve successful electroporation, cells must be exposed to sufficiently high electric fields generated by short-duration, high-voltage electrical pulses that result in a temporary increase in plasma membrane permeability. The electrical pulses are generated by pulse generators (electroporators) and delivered to the cells via electrodes (applicators). However, there is a lack of standardized pulse delivery protocols as well as certified clinical pulse generators and applicators for gene delivery. In this paper, the development of a new pulse generator, applicator, and pulse delivery protocol for GET to skin cells is presented. A numerical model of electroporated skin developed and tested for two electrode configurations and two different pulse delivery protocols is also presented. An alternative pulse delivery protocol was proposed. The developed pulse generator, applicator, and the proposed pulse delivery protocol were then used in vivo for GET to skin cells in mice. The results showed high efficiency of the proposed pulse delivery protocol for the purpose of GET in mouse skin cells. Specifically, electroporation with the developed pulse generator, applicator, and proposed pulse delivery protocol resulted in higher gene expression in skin cells compared to the currently used pulse generator, applicator, and pulse delivery protocol. Full article

(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications)

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14 pages, 2888 KiB

Open AccessEditor’s ChoiceArticle

Efficient Delivery of DNA Using Lipid Nanoparticles

by Lishan Cui, Serena Renzi, Erica Quagliarini, Luca Digiacomo, Heinz Amenitsch, Laura Masuelli, Roberto Bei, Gianmarco Ferri, Francesco Cardarelli, Junbiao Wang, Augusto Amici, Daniela Pozzi, Cristina Marchini and Giulio Caracciolo

Pharmaceutics 2022, 14(8), 1698; https://doi.org/10.3390/pharmaceutics14081698 - 15 Aug 2022

Cited by 19 | Viewed by 6985

Abstract

DNA vaccination has been extensively studied as a promising strategy for tumor treatment. Despite the efforts, the therapeutic efficacy of DNA vaccines has been limited by their intrinsic poor cellular internalization. Electroporation, which is based on the application of a controlled electric field [...] Read more.

DNA vaccination has been extensively studied as a promising strategy for tumor treatment. Despite the efforts, the therapeutic efficacy of DNA vaccines has been limited by their intrinsic poor cellular internalization. Electroporation, which is based on the application of a controlled electric field to enhance DNA penetration into cells, has been the method of choice to produce acceptable levels of gene transfer in vivo. However, this method may cause cell damage or rupture, non-specific targeting, and even degradation of pDNA. Skin irritation, muscle contractions, pain, alterations in skin structure, and irreversible cell damage have been frequently reported. To overcome these limitations, in this work, we use a microfluidic platform to generate DNA-loaded lipid nanoparticles (LNPs) which are then characterized by a combination of dynamic light scattering (DLS), synchrotron small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM). Despite the clinical successes obtained by LNPs for mRNA and siRNA delivery, little is known about LNPs encapsulating bulkier DNA molecules, the clinical application of which remains challenging. For in vitro screening, LNPs were administered to human embryonic kidney 293 (HEK-293) and Chinese hamster ovary (CHO) cell lines and ranked for their transfection efficiency (TE) and cytotoxicity. The LNP formulation exhibiting the highest TE and the lowest cytotoxicity was then tested for the delivery of the DNA vaccine pVAX-hECTM targeting the human neoantigen HER2, an oncoprotein overexpressed in several cancer types. Using fluorescence-activated cell sorting (FACS), immunofluorescence assays and fluorescence confocal microscopy (FCS), we proved that pVAX-hECTM-loaded LNPs produce massive expression of the HER2 antigen on the cell membrane of HEK-293 cells. Our results provide new insights into the structure–activity relationship of DNA-loaded LNPs and pave the way for the access of this gene delivery technology to preclinical studies. Full article

(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications)

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18 pages, 7238 KiB

Open AccessArticle

Translating a Thin-Film Rehydration Method to Microfluidics for the Preparation of a SARS-CoV-2 DNA Vaccine: When Manufacturing Method Matters

by Allegra Peletta, Eakachai Prompetchara, Kittipan Tharakhet, Papatsara Kaewpang, Supranee Buranapraditkun, Nongnaphat Yostrerat, Suwimon Manopwisedjaroen, Arunee Thitithanyanont, Jonathan Avaro, Leonard Krupnik, Antonia Neels, Kiat Ruxrungtham, Chutitorn Ketloy and Gerrit Borchard

Pharmaceutics 2022, 14(7), 1427; https://doi.org/10.3390/pharmaceutics14071427 - 7 Jul 2022

Cited by 2 | Viewed by 2607

Abstract

Previous investigations conducted on a liposomal formulation for a SARS-CoV-2 DNA vaccine manufactured using the thin-film layer rehydration method showed promising immunogenicity results in mice. The adaptation of the liposomal formulation to a scalable and reproducible method of manufacture is necessary to continue [...] Read more.

Previous investigations conducted on a liposomal formulation for a SARS-CoV-2 DNA vaccine manufactured using the thin-film layer rehydration method showed promising immunogenicity results in mice. The adaptation of the liposomal formulation to a scalable and reproducible method of manufacture is necessary to continue the investigation of this vaccine candidate. Microfluidics manufacture shows high potential in method translation. The physicochemical characterization of the blank liposomes produced by thin-film layer rehydration or microfluidics were shown to be comparable. However, a difference in lipid nanostructure in the bilayer resulted in a significant difference in the hydration of the thin-film liposomes, ultimately altering their complexation behavior. A study on the complexation of liposomes with the DNA vaccine at various N/P ratios showed different sizes and Zeta-potential values between the two formulations. This difference in the complexation behavior resulted in distinct immunogenicity profiles in mice. The thin-film layer rehydration-manufactured liposomes induced a significantly higher response compared to the microfluidics-manufactured samples. The nanostructural analysis of the two samples revealed the critical importance of understanding the differences between the two formulations that resulted in the different immunogenicity in mice. Full article

(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications)

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16 pages, 2785 KiB

Open AccessArticle

Transfection by Electroporation of Cancer and Primary Cells Using Nanosecond and Microsecond Electric Fields

by Eivina Radzevičiūtė, Veronika Malyško-Ptašinskė, Jurij Novickij, Vitalij Novickij and Irutė Girkontaitė

Pharmaceutics 2022, 14(6), 1239; https://doi.org/10.3390/pharmaceutics14061239 - 11 Jun 2022

Cited by 10 | Viewed by 2796

Abstract

Gene transfer into primary immune cells as well as into cell lines is essential for scientific and therapeutical applications. One of the methods used for gene transfer is electroporation (EP). EP is a method where a pulsed electric field (PEF) causes a highly [...] Read more.

Gene transfer into primary immune cells as well as into cell lines is essential for scientific and therapeutical applications. One of the methods used for gene transfer is electroporation (EP). EP is a method where a pulsed electric field (PEF) causes a highly transient permeability of the targeted cell membrane. In this work, we present the electrotransfection of CHO-K1, 4T1 cell lines, and primary murine DCs with detectable protein-encoding plasmids in the sub-microsecond range. Microsecond (µs)- and nanosecond (ns)-range pulsed electric field transfection protocols were used. The efficiency of electrotransfection was evaluated using green fluorescent protein (GFP)-encoding plasmids (4.7 kbp; p-EGFP-N1) and plasmids expressing a firefly luciferase and red fluorescent protein (tdTomato) (8.5 kbp; pcDNA3.1(+)/Luc2 = tdT)). It was shown that the used nsPEFs protocol (7 kV/cm × 300 ns × 100, 1 MHz) ensured a better transfection efficiency than µsPEFs (1.2 kV/cm × 100 µs × 8, 1 Hz). Plasmid size and concentration had a strong impact on the cell transfection efficiency too. We also showed that there were no significant differences in transfection efficiency between immature and mature DCs. Finally, the nsPEF protocols were successfully applied for the stable transfection of the CHO-K1 cell line with the linearized pcDNA3.1(+)/Luc2 = tdT plasmid. The results of the study are applicable in gene therapy and DNA vaccination studies for the derivation of optimal electrotransfection conditions. Full article

(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications)

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13 pages, 1705 KiB

Open AccessArticle

Combination of DNA Vaccine and Immune Checkpoint Blockades Improves the Immune Response in an Orthotopic Unresectable Glioblastoma Model

by Mathilde Bausart, Kevin Vanvarenberg, Bernard Ucakar, Alessandra Lopes, Gaëlle Vandermeulen, Alessio Malfanti and Véronique Préat

Pharmaceutics 2022, 14(5), 1025; https://doi.org/10.3390/pharmaceutics14051025 - 10 May 2022

Cited by 12 | Viewed by 2662

Abstract

Combination immunotherapy has emerged as a promising strategy to increase the immune response in glioblastoma (GBM) and overcome the complex immunosuppression occurring in its microenvironment. In this study, we hypothesized that combining DNA vaccines—to stimulate a specific immune response—and dual immune checkpoint blockade [...] Read more.

Combination immunotherapy has emerged as a promising strategy to increase the immune response in glioblastoma (GBM) and overcome the complex immunosuppression occurring in its microenvironment. In this study, we hypothesized that combining DNA vaccines—to stimulate a specific immune response—and dual immune checkpoint blockade (ICB)—to decrease the immunosuppression exerted on T cells—will improve the immune response and the survival in an orthotopic unresectable GL261 model. We first highlighted the influence of the insertion position of a GBM epitope sequence in a plasmid DNA vaccine encoding a vesicular stomatitis virus glycoprotein (VSV-G) (here referred to as pTOP) in the generation of a specific and significant IFN-γ response against the GBM antigen TRP2 by inserting a CD8 epitope sequence in specific permissive sites. Then, we combined the pTOP vaccine with anti-PD-1 and anti-CTLA-4 ICBs. Immune cell analysis revealed an increase in effector T cell to Treg ratios in the spleens and an increase in infiltrated IFN-γ-secreting CD8 T cell frequency in the brains following combination therapy. Even if the survival was not significantly different between dual ICB and combination therapy, we offer a new immunotherapeutic perspective by improving the immune landscape in an orthotopic unresectable GBM model. Full article

(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications)

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Review

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22 pages, 1523 KiB

Open AccessReview

Exosomes as an Emerging Plasmid Delivery Vehicle for Gene Therapy

by Margaret Wallen, Farrukh Aqil, Wendy Spencer and Ramesh C. Gupta

Pharmaceutics 2023, 15(7), 1832; https://doi.org/10.3390/pharmaceutics15071832 - 27 Jun 2023

Cited by 4 | Viewed by 3449

Abstract

Despite its introduction more than three decades ago, gene therapy has fallen short of its expected potential for the treatment of a broad spectrum of diseases and continues to lack widespread clinical use. The fundamental limitation in clinical translatability of this therapeutic modality [...] Read more.

Despite its introduction more than three decades ago, gene therapy has fallen short of its expected potential for the treatment of a broad spectrum of diseases and continues to lack widespread clinical use. The fundamental limitation in clinical translatability of this therapeutic modality has always been an effective delivery system that circumvents degradation of the therapeutic nucleic acids, ensuring they reach the intended disease target. Plasmid DNA (pDNA) for the purpose of introducing exogenous genes presents an additional challenge due to its size and potential immunogenicity. Current pDNA methods include naked pDNA accompanied by electroporation or ultrasound, liposomes, other nanoparticles, and cell-penetrating peptides, to name a few. While the topic of numerous reviews, each of these methods has its own unique set of limitations, side effects, and efficacy concerns. In this review, we highlight emerging uses of exosomes for the delivery of pDNA for gene therapy. We specifically focus on bovine milk and colostrum-derived exosomes as a nano-delivery “platform”. Milk/colostrum represents an abundant, scalable, and cost-effective natural source of exosomes that can be loaded with nucleic acids for targeted delivery to a variety of tissue types in the body. These nanoparticles can be functionalized and loaded with pDNA for the exogenous expression of genes to target a wide variety of disease phenotypes, overcoming many of the limitations of current gene therapy delivery techniques. Full article

(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications)

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12 pages, 779 KiB

Open AccessReview

Application of DNA Replicons in Gene Therapy and Vaccine Development

by Kenneth Lundstrom

Pharmaceutics 2023, 15(3), 947; https://doi.org/10.3390/pharmaceutics15030947 - 15 Mar 2023

Cited by 2 | Viewed by 2796

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications)

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32 pages, 3623 KiB

Open AccessReview

Effect of Experimental Electrical and Biological Parameters on Gene Transfer by Electroporation: A Systematic Review and Meta-Analysis

by Tjaša Potočnik, Alenka Maček Lebar, Špela Kos, Matej Reberšek, Eva Pirc, Gregor Serša and Damijan Miklavčič

Pharmaceutics 2022, 14(12), 2700; https://doi.org/10.3390/pharmaceutics14122700 - 2 Dec 2022

Cited by 6 | Viewed by 2969

Abstract

The exact mechanisms of nucleic acid (NA) delivery with gene electrotransfer (GET) are still unknown, which represents a limitation for its broader use. Further, not knowing the effects that different experimental electrical and biological parameters have on GET additionally hinders GET optimization, resulting [...] Read more.

The exact mechanisms of nucleic acid (NA) delivery with gene electrotransfer (GET) are still unknown, which represents a limitation for its broader use. Further, not knowing the effects that different experimental electrical and biological parameters have on GET additionally hinders GET optimization, resulting in the majority of research being performed using a trial-and-error approach. To explore the current state of knowledge, we conducted a systematic literature review of GET papers in in vitro conditions and performed meta-analyses of the reported GET efficiency. For now, there is no universal GET strategy that would be appropriate for all experimental aims. Apart from the availability of the required electroporation device and electrodes, the choice of an optimal GET approach depends on parameters such as the electroporation medium; type and origin of cells; and the size, concentration, promoter, and type of the NA to be transfected. Equally important are appropriate controls and the measurement or evaluation of the output pulses to allow a fair and unbiased evaluation of the experimental results. Since many experimental electrical and biological parameters can affect GET, it is important that all used parameters are adequately reported to enable the comparison of results, as well as potentially faster and more efficient experiment planning and optimization. Full article

(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications)

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27 pages, 2576 KiB

Open AccessReview

Plasmid DNA for Therapeutic Applications in Cancer

by David Hernán Martínez-Puente, José Juan Pérez-Trujillo, Laura Mireya Zavala-Flores, Aracely García-García, Arnulfo Villanueva-Olivo, Humberto Rodríguez-Rocha, Jesús Valdés, Odila Saucedo-Cárdenas, Roberto Montes de Oca-Luna and María de Jesús Loera-Arias

Pharmaceutics 2022, 14(9), 1861; https://doi.org/10.3390/pharmaceutics14091861 - 3 Sep 2022

Cited by 24 | Viewed by 6483

Abstract

Recently, the interest in using nucleic acids for therapeutic applications has been increasing. DNA molecules can be manipulated to express a gene of interest for gene therapy applications or vaccine development. Plasmid DNA can be developed to treat different diseases, such as infections [...] Read more.

Recently, the interest in using nucleic acids for therapeutic applications has been increasing. DNA molecules can be manipulated to express a gene of interest for gene therapy applications or vaccine development. Plasmid DNA can be developed to treat different diseases, such as infections and cancer. In most cancers, the immune system is limited or suppressed, allowing cancer cells to grow. DNA vaccination has demonstrated its capacity to stimulate the immune system to fight against cancer cells. Furthermore, plasmids for cancer gene therapy can direct the expression of proteins with different functions, such as enzymes, toxins, and cytotoxic or proapoptotic proteins, to directly kill cancer cells. The progress and promising results reported in animal models in recent years have led to interesting clinical results. These DNA strategies are expected to be approved for cancer treatment in the near future. This review discusses the main strategies, challenges, and future perspectives of using plasmid DNA for cancer treatment. Full article

(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications)

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