DNA-based therapies (vaccines/ gene therapies) utilise plasmid vectors for the delivery of
potent biologics, however, their promising application remains restricted by uncontrolled
host immunity. This study presents novel technologies for the quantification,
characterisation, and control of these DNA-driven immune responses, facilitating the
development of safer and more efficacious therapeutics.
Firstly, novel immune-profiling tool ‘Nanopanel’ was developed, capable of characterising
plasmid DNA (pDNA) immune effects across DNA-specific response pathways in murine
dendritic cells (DC). An experimental pipeline was designed, validated, and utilised,
incorporating an automated analysis app to facilitate medium-throughput screening of
vector immunity. Application of this technology will allow highly sensitive quantification
of DNA-therapy vector immunogenicity, informing sequence-specific design and/or selection to improve their safety and efficacy.
Secondly, double-stranded DNA (dsDNA) motifs able to suppress immune genes upregulated by pDNA immunity were identified and characterised. A comprehensive library of homotypic and heterotypic constructs were designed and screened, testing 20 DNA motifs in multiple design conditions. Multiple linear regression (MLR) modelling identified six statistically significant sequences capable of gene-level suppression, and seven critical design rules. These findings provide novel insight into pDNA integrated sequence effects, informing the design of immunomodulatory vectors.
Finally, novel application of sequence-specific oligodeoxynucleotide (ODN)’s as immune
suppressors of pDNA-driven responses was investigated. dsDNA screening informed the
selection of six motifs which, as ODNs, exhibited potent modulatory activity across
inflammatory and antiviral pathways. This established a toolbox of ODNs for the control of
pDNA immunogenicity. Further optimisation identified M16, a microsatellite-derived ODN
capable of counteracting vector cytosine-phosphate-guanine (CpG) stimulation, suppressing TNFa production up to ~70%. M16 expands the design space for vector CpG incorporation, enabling the use of more effective functional components (promoters/ intragenic regions) in gene therapy plasmids, without compromising safety.
