By Michael Greenwood, M.Sc.Reviewed by Danielle Ellis, B.Sc.

Doggybone DNA ™ (dbDNA) is a technology developed by biotech company Touchlight, which is a form of linear plasmid with applications in genetic engineering and therapeutics.

Plasmids are small circular double-stranded DNA molecules naturally generated by bacterial cells, some eukaryotes, and archaea and separate from chromosomal DNA. Plasmids can self-replicate by using host enzymes, and each carries at least one gene, most of which are beneficial to the host.

The gene(s) carried by the plasmid may then be incorporated into the genome for expression. For example, a plasmid may encode a gene that produces a protein with antiphage functionality, allowing viral resistance to be incorporated into the bacterial chromosome. Plasmids are transferred between bacterial cells via pili; thus, beneficial genes carried by plasmids can be disseminated and incorporated into the chromosomal DNA of neighboring cells.

Owing to the natural function of plasmids, they can be utilized as vectors in genetic engineering, allowing specific genes to be inserted into the chromosomal DNA of bacterium and subsequently expressed. Large quantities of proteins are mass-produced by this method, and engineered strains of bacteria are cultured specifically for their generation. Alternatively, the DNA or RNA strand of the plasmid itself may be the therapeutic end product for applications in gene therapy, gene silencing, and the production of mRNA intended for vaccines.

How are Doggybone ™ DNA strands produced?

In dbDNA ™, protelomerase recognition sequences flank the gene of interest, and protelomerases are used in their production to form covalently closed hairpin ends, transforming the circular plasmid structure into a linear one. A closed linear structure improves plasmid stability by preventing exonuclease enzymes from interacting with the double-stranded DNA and also simplifies subsequent purification steps.

Once denatured, dbDNA ™ can then return to a circular form for use in further amplification steps, if necessary. Importantly, dbDNA ™ can be produced completely abiotically, without the use of bacterial cultures, using rolling circle amplification.

Rolling circle amplification uses a circular single-stranded DNA or RNA template onto which the complementary strand is ligated. As the complementary strand grows around the full circle of the template strand, the initial contact point is displaced, generating a continuous single strand of repeating DNA or RNA, which may then circularize to produce a second template construct and an intact double-stranded plasmid, propagating further generation.

Compared with plasmid manufacture using bacterial cultures, the process is comparatively cost-effective, being much quicker and requiring only simple benchtop equipment. dbDNA ™ production using enzymes is, therefore, rapidly scalable to meet demand. The technology is being explored both for the general production of vaccines and for rapid deployment against emerging novel viral variants.

How do Doggybone ™ DNA vaccines compare with other types of vaccine?

Recently, mRNA vaccines have become prominent thanks to their widespread and successful deployment in COVID-19 vaccines, with the constituent mRNA produced using bacterial vectors. mRNA produced from dbDNA ™ represents a more cost-effective and rapidly scalable method of vaccine manufacture. In the Summer of 2022, pharmaceutical manufacturer Pfizer purchased the right to utilize dbDNA in the production of COVID-19 mRNA vaccines.

COVID-19 dbDNA vaccines have been explored in animal models and show the induction of cross-variant neutralizing antibodies and protection from viral challenge. In a study by Mucker et al. (2022) hamsters were administered with either traditionally produced plasmids coding for the full length SARS-CoV-2 spike protein or the dbDNA equivalent, then challenged with wildtype SARS-CoV-2 or the one of several SARS-CoV-2 variants of concern.

Viral RNA and plaque titers were then quantified, demonstrating comparable protective effects from either vaccine that correlate with vaccine dose. The dbDNA ™ cassette may be further modified to enhance immunogenicity, and within this study, a stability-modified version was more effective in providing viral protection than the non-modified dbDNA ™ strand.

The minimal nature of the dbDNA ™ strand, containing only the functional gene of interest and the necessary end groups, may also be advantageous for patient safety and the minimization of off target effects. DNA plasmids produced using, for example, Escherichia coli fermentation, frequently contain superfluous bacterial genetic sequences that may be unnecessarily or detrimentally expressed. A lack of innate immune recognition towards dbDNA ™ lacking the same degree of immunogenicity as bacteria-derived plasmids may induce a lesser immune response, which may be desired for a vaccine. Finally, DNA and RNA-based vaccines typically employ lipid nanoparticle vectors, or those constructed from other materials, to aid in their delivery.

Given the novelty of dbDNA ™ as the basis for a vaccine, published work incorporating dbDNA ™ into comparable vector platforms is not yet available, though it will likely be a necessary hurdle before widespread clinical application. The great cost-effectiveness of dbDNA ™ manufacture as compared to traditional plasmids prepared in microorganisms, however, promotes their further exploration.

References

• Cunningham, D. S., Koepsel, R. R., Ataai, M. M., & Domach, M. M.. (2009). Factors affecting plasmid production in Escherichia coli from a resource allocation standpoint. Microbial Cell Factories, 8(1), 27. https://doi.org/10.1186/1475-2859-8-27
• Scott, V. L., Patel, A., Villarreal, D. O., Hensley, S. E., Ragwan, E., Yan, J., Sardesai, N. Y., Rothwell, P. J., Extance, J. P., Caproni, L. J., & Weiner, D. B.. (2015). Novel synthetic plasmid and Doggybone™ DNA vaccines induce neutralizing antibodies and provide protection from lethal influenza challenge in mice. Human Vaccines & Immunotherapeutics, 11(8), 1972–1982. https://doi.org/10.1080/21645515.2015.1022008
• Mucker, E. M., Brocato, R. L., Principe, L. M., Kim, R. K., Zeng, X., Smith, J. M., Kwilas, S. A., Kim, S., Horton, H., Caproni, L., & Hooper, J. W.. (2022). SARS-CoV-2 Doggybone DNA Vaccine Produces Cross-Variant Neutralizing Antibodies and Is Protective in a COVID-19 Animal Model. Vaccines, 10(7), 1104. https://doi.org/10.3390/vaccines10071104