Lately, the FDA paused the planned Phase II/III trial of the vaccine candidate INO-4800 due to questions about the design and use of the INOVIO delivery machine CELLECTRA? 2000, which is used to deliver the vaccine directly into the skin [127]

Lately, the FDA paused the planned Phase II/III trial of the vaccine candidate INO-4800 due to questions about the design and use of the INOVIO delivery machine CELLECTRA? 2000, which is used to deliver the vaccine directly into the skin [127]. of DNA in vaccine development. DNA vaccines are DNA vehicles such as bacterial Mouse monoclonal to beta Actin.beta Actin is one of six different actin isoforms that have been identified. The actin molecules found in cells of various species and tissues tend to be very similar in their immunological and physical properties. Therefore, Antibodies againstbeta Actin are useful as loading controls for Western Blotting. However it should be noted that levels ofbeta Actin may not be stable in certain cells. For example, expression ofbeta Actin in adipose tissue is very low and therefore it should not be used as loading control for these tissues plasmids, minicircle DNA or linear, covalently-closed minimalistic expression constructs including minimalistic, immunologically defined gene expression (MIDGE) DNA and Doggybone? DNA that contain at least one eukaryotic expression cassette encoding for the antigen of interest. The expression cassettes usually consist of a eukaryotic promoter/enhancer, the antigen gene and a poly(A) signal sequence, which are essential for the expression of the antigen in eukaryotic cells (e.g., muscular cells) [5C7]. DNA vaccines have shown compelling safety and immunogenicity in preclinical studies [8]. Several DNA vaccines are currently licensed for veterinary use in large animals such as horses as well as small animals such as chickens [9,10]. The results from clinical trials of DNA vaccines for West Nile virus (WNV) [11,12], Ebola and Marburg viruses [13, 14] and SARS-CoV-2 [15C19] have shown that antibodies are generated in humans a few weeks after immunization. However, there are also many cases of poor immunogenicity in clinical trials. Target antigen and optimization of construct, formulation and delivery methods appear to be key elements in the immunogenicity of DNA vaccines [8]. This review will discuss the design, production, delivery and administration of DNA vaccines, factors that may improve the immunogenicity of DNA vaccines, and summarize the recent preclinical and clinical trials of DNA vaccines for COVID-19. Design of DNA vaccines The plasmid DNA vaccines are comprised of a bacterial origin of replication and AMG-3969 at least one antibiotic resistance gene as a selectable marker. It was shown that bacterial backbone can reduce gene expression in mammalian cells [20]. The formation of heterochromatin in bacterial sequences spreading into the expression cassette may be one of the reasons for the silencing of transgene expression [21]. In addition, it was found that increasing the A/T sequence composition in plasmid antibiotic resistance genes can increase the stable transcription of backbone genes as well as adjacent expression cassettes in mice [22]. Changes in sequence composition and deletion of bacterial backbone sequences in DNA vaccines may increase antigen expression. Minicircle DNA, MIDGE and Doggybone? are DNA constructs composed of the gene expression cassette(s) without the bacterial backbone of plasmids [5C7]. Sometimes, we need to express multiple genes in a single DNA vaccine, for example, designing multi-antigen DNA vaccines or expressing a genetic adjuvant combined with the antigen. In this regard, three strategies were used: first, we can use different expression cassettes for each AMG-3969 gene with individual promoters for independent expression of multiple transgenes; second, we can use bi-cistronic or multi-cistronic vectors with a single promoter for the expression of multiple genes which are separated by internal ribosome entry site (IRES) elements for independent translation of multiple genes; third, we can use a virus-derived T2A sequence instead of IRES between genes, where, after translation, the corresponding peptide sequence is recognized and cleaved by an endogenous protease [23C27]. The genetic material of a DNA vaccine must first enter the nucleus for subsequent transcription of the encoding genes of antigens or genetic adjuvants. Then, the transcribed mRNA(s) are exported from the nucleus into the cytoplasm for translation. The efficient DNA transfer to the cell nucleus is an important barrier for the expression of transgenes in DNA vaccines, especially for mitotically inactive cells such as antigen-presenting cells (APCs) [27]. Certain DNA sequences such as the simian virus (SV) 40 enhancer have a nuclear localization signal (NLS), and binding of specific transcription factors to this NLS signal in the cytoplasm leads to active nuclear transport of DNA [28]. AMG-3969 In addition, insertion of some tissue-specific transcription factor-binding sequences in DNA plasmids may lead to tissue-specific nuclear import of plasmid DNA [29]. Alternatively, AMG-3969 some DNA binding proteins such as NFB (p50) and engineered NLS-tetracycline repressor can be used to form proteinCDNA complexes before AMG-3969 administration, improving the nuclear localization of DNA in cells [30C32]. In addition, covalent or noncovalent conjugation of a virus-derived NLS peptide to either natural.