Comparative analysis of enzymatically produced novel linear DNA constructs with plasmids for use as DNA vaccines

Abstract

The use of DNA to deliver vaccine antigens offers many advantages, including ease of manufacture and cost. However, most DNA vaccines are plasmids and must be grown in bacterial culture, necessitating elements which are either unnecessary for effective gene delivery (e.g. bacterial origins of replication) or undesirable (e.g. antibiotic resistance genes). Removing these elements may improve the safety profile of DNA for the delivery of vaccines. Here we describe a novel, double-stranded, linear DNA construct produced by an enzymatic process that solely encodes an antigen expression cassette, comprising antigen, promoter, polyA tail and telomeric ends. We compared these constructs (called ‘Doggybones’ because of their shape) with conventional plasmid DNA. Using luciferase-expressing constructs, we demonstrated that expression levels were equivalent between Doggybones and plasmids both in vitro and in vivo. When mice were immunized with DNA constructs expressing the HIV envelope protein gp140, equivalent humoral and cellular responses were induced. Immunizations with either construct type expressing haemagluttinin were protective against H1N1 influenza challenge. This is the first example of an effective DNA vaccine which can be produced on a large scale by enzymatic processes.

CAR T Cell Generation by piggyBac Transposition from Linear Doggybone DNA Vectors Requires Transposon DNA-Flanking Regions

“…Although not statistically significant, there was also a trend toward a lower proportion of T cells expressing CAR in CAR19 T cell cultures generated using only dbDNA. These findings are consistent with previous reports of a trend toward reduced expression from dbDNA compared to an equimolar amount of plasmid 30 and that piggyBac transposition occurs more efficiently from circular than linear donor vectors. 45 Transfection of an equal mass of dbDNA to plasmid leads to equivalent levels of expression, 30,31 and this approach might also improve CAR expression in this setting.…”

“…Doggybones (dbDNA) are minimal DNA vectors that can be produced enzymatically in vitro at scale. [30][31][32] As production does not involve bacteria, the issues associated with plasmids are avoided. No origin of replication sequences or antibiotic-resistance genes is required, and the risk of recombination events within bacteria is eliminated.…”

Resolving systematic errors in widely-used enhancer activity assays in human cells enables genome-wide functional enhancer characterization

“…Alternatively, one might create plasmids that do not contain the ORI and thus only a single corepromoter that one should be able to choose freely. A family of vectors designed to remove prokaryotic parts of the plasmid after amplification in bacteria (minicircles [10][11][12] or in vitro synthesis (doggybones 13 ) could be such alternatives, as might be different lower copy ORIs. Alternatively, one could screen linear DNA fragments that lack the ORI (e.g.…”

Novel approaches for the design, delivery and administration of vaccine technologies

“…For infectious pathogens, two major types of RNA vaccine have been utilized: nonreplicating and self-amplifying. The recent development of scalable, cell-free, enzyme-driven DNA production technologies strengthens the case for the translatability of DNA vaccines [51]. Self-amplifying RNA systems can be based on sequences and principles borrowed from single-positive strand RNA viruses, such as alphaviruses (Alphavax).…”

“…Should the delivery barrier faced by DNA be overcome, a resurgence in the interest in its use may follow. The recent development of scalable, cell-free, enzyme-driven DNA production technologies strengthens the case for the translatability of DNA vaccines [51].…”