Novel enzymatic DNA produced from a text file achieves comparable immune responses as plasmid vaccine

Computational code used for sequence analysis is hosted on GitHub (see https://github.com/timplab/pacbio_eds_rca).

1. Ashley, J., Potts, I. M. & Olorunniji, F. J. Applications of terminal deoxynucleotidyl transferase enzyme in biotechnology. ChemBioChem 24, e202200510 (2023).

   Google Scholar 

2. Challener, C. A. Synthetic DNA as an alternative to plasmids. BioPharm. Int. 37, 14–17 (2024).

   Google Scholar 

3. Ohlson, J. Plasmid manufacture is the bottleneck of the genetic medicine revolution. Drug Discov. Today 25, 1891–1893 (2020).

   Google Scholar 

4. Conforti, A. et al. Linear DNA amplicons as a novel cancer vaccine strategy. J. Exp. Clin. Cancer Res. 41, 195 (2022).

   Google Scholar 

5. Vilalta, A. et al. Vaccination with polymerase chain reaction-generated linear expression cassettes protects mice against lethal influenza A challenge. Hum. Gene Ther. 18, 763–771 (2007).

   Google Scholar 

6. Shen, X. et al. Influenza A vaccines using linear expression cassettes delivered via electroporation afford full protection against challenge in a mouse model. Vaccine 30, 6946–6954 (2012).

   Google Scholar 

7. Moreno, S. et al. DNA immunisation with minimalistic expression constructs. Vaccine 22, 1709–1716 (2004).

   Google Scholar 

8. Hughes, R. A. & Ellington, A. D. Synthetic DNA synthesis and assembly: putting the synthetic in synthetic biology. Cold Spring Harb. Perspect. Biol. 9 https://doi.org/10.1101/cshperspect.a023812 (2017).

9. de Mey, W. et al. A synthetic DNA template for fast manufacturing of versatile single epitope mRNA. Mol. Ther. Nucleic Acids 29, 943–954 (2022).

   Google Scholar 

10. Hekele, A. et al. Rapidly produced SAM® vaccine against H7N9 influenza is immunogenic in mice. Emerg. Microbes Infect. 2, 1–7 (2013).

    Google Scholar 

11. Dormitzer, P. R. et al. Synthetic generation of influenza vaccine viruses for rapid response to pandemics. Sci. Transl. Med. 5, 185ra168–185ra168 (2013).

    Google Scholar 

12. Simmons, B. L., McDonald, N. D. & Robinett, N. G. Assessment of enzymatically synthesized DNA for gene assembly. Front. Bioeng. Biotechnol. 11 https://doi.org/10.3389/fbioe.2023.1208784 (2023).

13. Hutchison, C. A., Smith, H. O., Pfannkoch, C. & Venter, J. C. Cell-free cloning using φ29 DNA polymerase. Proc. Natl. Acad. Sci. USA 102, 17332–17336 (2005).

    Google Scholar 

14. Dean, F. B., Nelson, J. R., Giesler, T. L. & Lasken, R. S. Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11, 1095–1099 (2001).

    Google Scholar 

15. Kendirgi, F. et al. Novel linear DNA vaccines induce protective immune responses against lethal infection with influenza virus type A/H5N1. Hum. Vaccines 4, 410–419 (2008).

    Google Scholar 

16. de Graaf, J. F. et al. Neoantigen-specific T cell help outperforms non-specific help in multi-antigen DNA vaccination against cancer. Mol. Ther. Oncol. 32, 200835 (2024).

    Google Scholar 

17. Scott, V. L. et al. Novel synthetic plasmid and Doggybone DNA vaccines induce neutralizing antibodies and provide protection from lethal influenza challenge in mice. Hum. Vaccine Immunother. 11, 1972–1982 (2015).

    Google Scholar 

18. Walters, A. A. et al. Comparative analysis of enzymatically produced novel linear DNA constructs with plasmids for use as DNA vaccines. Gene Ther. 21, 645–652 (2014).

    Google Scholar 

19. Masaki, Y., Onishi, Y. & Seio, K. Quantification of synthetic errors during chemical synthesis of DNA and its suppression by non-canonical nucleosides. Sci. Rep. 12, 12095 (2022).

    Google Scholar 

20. Filges, S., Mouhanna, P. & Ståhlberg, A. Digital quantification of chemical oligonucleotide synthesis errors. Clin. Chem. 67, 1384–1394 (2021).

    Google Scholar 

21. Ma, S., Saaem, I. & Tian, J. Error correction in gene synthesis technology. Trends Biotechnol. 30, 147–154 (2012).

    Google Scholar 

22. Potapov, V. et al. A single-molecule sequencing assay for the comprehensive profiling of T4 DNA ligase fidelity and bias during DNA end-joining. Nucleic Acids Res. 46, e79 (2018).

    Google Scholar 

23. Potapov, V. et al. Comprehensive profiling of four base overhang ligation fidelity by T4 DNA ligase and application to DNA assembly. ACS Synth. Biol. 7, 2665–2674 (2018).

    Google Scholar 

24. Pertmer, T. M. et al. Gene gun-based nucleic acid immunization: elicitation of humoral and cytotoxic T lymphocyte responses following epidermal delivery of nanogram quantities of DNA. Vaccine 13, 1427–1430 (1995).

    Google Scholar 

25. Wang, S. et al. The relative immunogenicity of DNA vaccines delivered by the intramuscular needle injection, electroporation and gene gun methods. Vaccine 26, 2100–2110 (2008).

    Google Scholar 

26. Fynan, E. F. et al. DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations. Proc. Natl. Acad. Sci. USA 90, 11478–11482 (1993).

    Google Scholar 

27. Nelson, J. R. et al. TempliPhi, phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing. Biotechniques 32, 44–47 (2002).

28. Bradley, C. C. et al. RNA polymerase inaccuracy underlies SARS-CoV-2 variants and vaccine heterogeneity. Res. Sq. https://doi.org/10.21203/rs.3.rs-1690086/v1 (2022).

    Google Scholar 

29. Chen, T. H., Potapov, V., Dai, N., Ong, J. L. & Roy, B. N(1)-methyl-pseudouridine is incorporated with higher fidelity than pseudouridine in synthetic RNAs. Sci. Rep. 12, 13017 (2022).

    Google Scholar 

30. Wong, H. E., Huang, C. J. & Zhang, Z. Amino acid misincorporation in recombinant proteins. Biotechnol. Adv. 36, 168–181 (2018).

    Google Scholar 

31. Harris, R. P. & Kilby, P. M. Amino acid misincorporation in recombinant biopharmaceutical products. Curr. Opin. Biotechnol. 30, 45–50 (2014).

    Google Scholar 

32. Amante, D. H. et al. Skin transfection patterns and expression kinetics of electroporation-enhanced plasmid delivery using the CELLECTRA-3P, a portable next-generation dermal electroporation device. Hum. Gene Ther. Methods 26, 134–146 (2015).

    Google Scholar 

33. Morrow, M. P. et al. DNA immunotherapy for recurrent respiratory papillomatosis (RRP): phase 1/2 study assessing efficacy, safety, and immunogenicity of INO-3107. Nat. Commun. 16, 1518 (2025).

    Google Scholar 

34. Marano, J. M., Chuong, C. & Weger-Lucarelli, J. Rolling circle amplification: a high fidelity and efficient alternative to plasmid preparation for the rescue of infectious clones. Virology 551, 58–63 (2020).

    Google Scholar 

35. Mizuta, R., Mizuta, M. & Kitamura, D. Atomic force microscopy analysis of rolling circle amplification of plasmid DNA. Arch. Histol. Cytol. 66, 175–181 (2003).

    Google Scholar 

36. Sato, M., Ohtsuka, M. & Ohmi, Y. Repeated GenomiPhi, phi29 DNA polymerase-based rolling circle amplification, is useful for generation of large amounts of plasmid DNA. Nucleic Acids Symp. Ser. 48, 147–148 (2004).

37. Loghin, E. et al. Transient Transfection of Rolling-Circle Amplified DNA in Biomanufacturing-Relevant Mammalian Cell Lines: A Comparison of Transfection Conditions for Optimal Protein Expression. Biotechnol. Appl. Biochem. https://doi.org/10.1002/bab.70100 (2025).

38. Cox, R. J. Correlates of protection to influenza virus, where do we go from here? Hum. Vaccine Immunother. 9, 405–408 (2013).

    Google Scholar 

39. Yarchoan, M. et al. Personalized neoantigen vaccine and pembrolizumab in advanced hepatocellular carcinoma: a phase 1/2 trial. Nat. Med. 30, 1044–1053 (2024).

    Google Scholar 

40. Duperret, E. K. et al. A synthetic DNA, multi-neoantigen vaccine drives predominately MHC Class I CD8(+) T-cell responses, impacting tumor challenge. Cancer Immunol. Res. 7, 174–182 (2019).

    Google Scholar 

41. Khobragade, A. et al. Efficacy, safety, and immunogenicity of the DNA SARS-CoV-2 vaccine (ZyCoV-D): the interim efficacy results of a phase 3, randomised, double-blind, placebo-controlled study in India. Lancet 399, 1313–1321 (2022).

    Google Scholar 

42. Tebas, P. et al. Safety and immunogenicity of INO-4800 DNA vaccine against SARS-CoV-2: a preliminary report of an open-label, phase 1 clinical trial. EClinicalMedicine 31, 100689 (2021).

    Google Scholar 

43. De Rosa, S. C. et al. Robust antibody and cellular responses induced by DNA-only vaccination for HIV. JCI Insight 5 https://doi.org/10.1172/jci.insight.137079 (2020).

44. Zanta-Boussif, M. A. et al. Validation of a mutated PRE sequence allowing high and sustained transgene expression while abrogating WHV-X protein synthesis: application to the gene therapy of WAS. Gene Ther. 16, 605–619 (2009).

    Google Scholar 

45. Garg, S., Oran, A. E., Hon, H. & Jacob, J. The hybrid cytomegalovirus enhancer/chicken beta-actin promoter along with woodchuck hepatitis virus posttranscriptional regulatory element enhances the protective efficacy of DNA vaccines. J. Immunol. 173, 550–558 (2004).

    Google Scholar 

46. Loudon, P. T. et al. GM-CSF increases mucosal and systemic immunogenicity of an H1N1 influenza DNA vaccine administered into the epidermis of non-human primates. PLoS ONE 5, e11021 (2010).

    Google Scholar 

47. How does CCS work. CCS Docs. https://ccs.how/how-does-ccs-work.

48. Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Google Scholar 

49. Li, H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34, 3094–3100 (2018).

    Google Scholar 

50. Ajay Khanna, D. E. L. et al. Bam-readcount – rapid generation of basepair-resolution sequence metrics. J. Open Source Softw. 7, 3722 (2022).

    Google Scholar 

51. Chen, D., Maa, Y. F. & Haynes, J. R. Needle-free epidermal powder immunization. Expert Rev. Vaccines 1, 265–276 (2002).

    Google Scholar 

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Authors and Affiliations

1. Department of Microbiology, University of Washington, Seattle, WA, USA

   James Fuller & Deborah H. Fuller

2. GE HealthCare Technology & Innovation Center, One Research Circle, Niskayuna, NY, USA

   Erik Kvam, Weston Griffin & John Nelson

3. DNA Script Inc., Le Kremlin-Bicêtre, France

   Sandrine Creton, Rebecca Ryan & Xavier Godron

4. Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA

   Courtney Hall & Winston Timp

5. Vaccine and Immunotherapy Center, The Wistar Institute, Philadelphia, PA, USA

   Nicholas J. Tursi, Kerry Blatney & David B. Weiner

6. Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

   Nicholas J. Tursi

Contributions

S.C. and R.R. lead enzymatic oligo synthesis and gene assembly. E.K., J.N., and W.G. lead DNA propagation, including rolling circle amplification and DNA purification. C.H. and W.T. conducted sequence analysis of the generated DNA. J.F., N.J.T., and K.B. performed DNA immunization and analyzed mouse results. X.G., D.B.W., J.N., W.G., E.K., and D.H.F. assisted in supervision and manuscript editing. E.K., N.J.T., S.C., and C.H. prepared the initial draft manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Erik Kvam or Deborah H. Fuller.

Competing interests

The authors declare the following, which may be considered as potential competing interests: S.C., R.R., and X.G. are employees of DNA Script and have filed patent applications pertaining to aspects of this work. E.K., J.N., and W.G. are employees of GE HealthCare and have filed patent applications pertaining to aspects of this work. D.B.W. is a consultant for and a member of the Scientific Advisory Board for INOVIO Pharmaceuticals. D.H.F. is a consultant for and co-founder of Orlance Inc. Neither Orlance nor INOVIO were involved in the study design, data collection, data analysis, or manuscript preparation. D.B.W. participates in industry collaborations; has received speaking honoraria; and has received fees for consulting, including serving on scientific review committees. Remunerations received by D.B.W. include direct payments and equity/options. D.B.W. also discloses the following associations with commercial partners: Geneos (consultant/advisory board), AstraZeneca (advisory board, speaker), and Pfizer (advisory board). All other authors declare that they have no competing interests related to this work.

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