The application of induced pluripotent stem cells (iPSCs) in advanced therapies is increasing at pace, but concerns remain over their clinical safety profile. We report the first-ever application of doggybone DNA (dbDNA) vectors to generate human iPSCs. dbDNA vectors are closed-capped linear double-stranded DNA gene expression cassettes that contain no bacterial DNA and are amplified by a chemically defined, current good manufacturing practice (cGMP)-compliant methodology. We achieved comparable iPSC reprogramming efficiencies using transiently expressing dbDNA vectors with the same iPSC reprogramming coding sequences as the state-of-the-art OriP/EBNA1 episomal vectors but, crucially, in the absence of p53 shRNA repression. Moreover, persistent expression of EBNA1 from bacterially derived episomes resulted in stimulation of the interferon response, elevated DNA damage, and increased spontaneous differentiation. These cellular activities were diminished or absent in dbDNA-iPSCs, resulting in lines with a greater stability and safety potential for cell therapy.

CD19-specific chimeric antigen receptor (CAR19) T cells, generated using viral vectors, are an efficacious but costly treatment for B cell malignancies. The nonviral piggyBac transposon system provides a simple and inexpensive alternative for CAR19 T cell production. Until now, piggyBac has been plasmid based, facilitating economical vector amplification in bacteria. However, amplified plasmids have several undesirable qualities for clinical translation, including bacterial genetic elements, antibiotic-resistance genes, and the requirement for purification to remove endotoxin. Doggybones (dbDNA) are linear, covalently closed, minimal DNA vectors that can be inexpensively produced enzymatically in vitro at large scale. Importantly, they lack the undesirable features of plasmids. We used dbDNA incorporating piggyBac to generate CAR19 T cells. Initially, expression of functional transposase was evident, but stable CAR expression did not occur. After excluding other causes, additional random DNA flanking the transposon within the dbDNA was introduced, promoting stable CAR expression comparable to that of using plasmid components. Our findings demonstrate that dbDNA incorporating piggyBac can be used to generate CAR T cells and indicate that there is a requirement for DNA flanking the piggyBac transposon to enable effective transposition. dbDNA may further reduce the cost and improve the safety of CAR T cell production with transposon systems.

The manufacture of large quantities of high-quality DNA is a major bottleneck in the production of viral vectors for gene therapy. Touchlight Genetics has developed a proprietary abiological technology that addresses the major issues in commercial DNA supply. The technology uses 'rolling-circle' amplification to produce large quantities of concatameric DNA that is then processed to create closed linear double-stranded DNA by enzymatic digestion. This novel form of DNA, Doggybone™ DNA (dbDNA™), is structurally distinct from plasmid DNA. Here we compare lentiviral vectors production from dbDNA™ and plasmid DNA. Lentiviral vectors were administered to neonatal mice via intracerebroventricular injection. Luciferase expression was quantified in conscious mice continually by whole-body bioluminescent imaging. We observed long-term luciferase expression using dbDNA™-derived vectors, which was comparable to plasmid-derived lentivirus vectors. Here we have demonstrated that functional lentiviral vectors can be produced using the novel dbDNA™ configuration for delivery in vitro and in vivo. Importantly, this could enable lentiviral vector packaging of complex DNA sequences that have previously been incompatible with bacterial propagation systems, as dbDNA™ technology could circumvent such restrictions through its phi29-based rolling-circle amplification.

Vaccination with DNA that encodes cancer antigens is a simple and convenient way to raise immunity against cancer and has already shown promise in the clinical setting. Conventional plasmid DNA is commonly used which together with the encoded antigen also includes bacterial immunostimulatory CpG motifs to target the DNA sensor Toll-like receptor 9. Recently DNA vaccines using doggybone DNA (dbDNA™), have been developed without the use of bacteria. The cell-free process relies on the use of Phi29 DNA polymerase to amplify the template followed by protelomerase TelN to complete individual closed linear DNA. The resulting DNA contains the required antigenic sequence, a promoter and a poly A tail but lacks bacterial sequences such as an antibiotic resistance gene, prompting the question of immunogenicity. Here we compared the ability of doggybone DNA vaccine with plasmid DNA vaccine to induce adaptive immunity using clinically relevant oncotargets E6 and E7 from HPV. We demonstrate that despite the inability to trigger TLR9, doggybone DNA was able to induce similar levels of cellular and humoral immunity as plasmid DNA, with suppression of established TC-1 tumours.

The cover picture shows a novel bicyclic peptide inhibitor of the serine protease plasma kallikrein identified by phage display. By modulating the loop size of the peptide macrocycles, inhibitors with sub-nanomolar potency and an optimal specificity profile could be generated. For more details, see the Communication by Greg Winter, John Tite, Christian Heinis et al. on p. 1173 ff.

Immunotherapies that induce durable immune control of chronic HIV infection may eliminate the need for life-long dependence on drugs. We investigated a DNA vaccine formulated with a novel genetic adjuvant that stimulates immune responses in the blood and gut for the ability to improve therapy in rhesus macaques chronically infected with SIV. Using the SIV-macaque model for AIDS, we show that epidermal co-delivery of plasmids expressing SIV Gag, RT, Nef and Env, and the mucosal adjuvant, heat-labile E. coli enterotoxin (LT), during antiretroviral therapy (ART) induced a substantial 2-4-log fold reduction in mean virus burden in both the gut and blood when compared to unvaccinated controls and provided durable protection from viral rebound and disease progression after the drug was discontinued. This effect was associated with significant increases in IFN-γ T cell responses in both the blood and gut and SIV-specific CD8+ T cells with dual TNF-α and cytolytic effector functions in the blood. Importantly, a broader specificity in the T cell response seen in the gut, but not the blood, significantly correlated with a reduction in virus production in mucosal tissues and a lower virus burden in plasma. We conclude that immunizing with vaccines that induce immune responses in mucosal gut tissue could reduce residual viral reservoirs during drug therapy and improve long-term treatment of HIV infection in humans.

Picky push-bikes! Bicyclic peptides with low to sub-nanomolar inhibitory activities towards the serine protease plasma kallikrein were developed. By modulating the size of the macrocyclic rings, inhibitors with the desired specificity profile could be generated.

The development of a successful cancer vaccine requires the ability to break immunological tolerance to self-Ags expressed on tumor cells. The transgenic rat insulin promoter (RIP) OVALOW mouse model has been reported to be hyporesponsive for both OVA-specific CD4 and CD8 T cell responses. The experiments described in the current study show that this hyporesponsiveness can be overcome by inclusion of GM-CSF and the TLR7 agonist imiquimod as adjuvants in a DNA immunization regimen with OVA-encoding plasmids. High frequencies of OVA-specific CD8 and CD4 T cells, including a response to a CD4 T cell epitope seen only in the RIP OVALOW mice, were generated by this regimen. These responses were associated with the development of autoimmunity and increased protection to tumor challenge in the RIP OVALOW mice. Heterologous CD4 T cell help has been shown to improve functional CD8 T cell responses, and we confirmed that inclusion of the CD4 T cell epitope pan HLA-DR–binding epitope improved CD8 T cell responses compared with self-Ag alone. Addition of GM-CSF and imiquimod, however, resulted in dominance of the pan HLA-DR–binding epitope-specific response over the OVA-specific CD4 T cell responses, decreased OVA-specific CD8 T cell numbers and function in tolerant RIP OVALOW mice, and failure to induce diabetes. The results of this study suggest that the use of heterologous help needs to be evaluated carefully in the context of specific immunization regimes and that a preferable approach may be adjuvantization of DNA vaccines.

Two discrete mechanisms of T-B cell collaboration appear to exist. In cognate recognition, B cell triggering results from a direct recognition of antigen and MHC determinants at the B cell surface. Alternatively, B cells can be triggered by transstimulation, in which the Th cell is activated by an antigen-presenting cell to produce soluble factors which in turn trigger the B cell. This report addresses the question of whether antigen recognition at the B cell surface in association with Ia determinants delivers a signal to the B cell, which is qualitatively different from the signals delivered by the soluble mediators released by the activated Th cell. Previous reports from a number of laboratories suggest that cognate recognition is obligatory for the triggering of small resting B cells and B cells of the Lyb-5- phenotype, whereas enlarged B cell blasts and the Lyb-5+ subset can be triggered solely by soluble mediators. Contrary to these findings, the experiments described here indicate that B cells isolated in different states of activation from normal spleens on the basis of their buoyant density in Percoll density gradients, or unfractionated B cells from mice differing genetically due to the xid defect [Lyb-5- B cells from (CBA/N X BALB/c)F1 male mice], do not discriminate between the two modes of Th cell function. In both stimulation modes, the high density B cells, and the B cells from xid mice made very poor immunoglobulin secretory responses measured in terms of reverse plaque formation on protein A-coupled erythrocytes. When the responses of different density fractions of B cells were compared under conditions where stimulation occurred either directly or indirectly via transstimulation, the following hierarchy of responsiveness in both the proliferative and plaque-forming cell (PFC) responses was observed in the density fractions 60% greater than 65% greater than 70% greater than 75%. The hierarchy was the same in both modes of interaction and the deficiency of the high density, small B cells was far more marked in the PFC assay than in the proliferative assay. We conclude that the initial proliferative response of the resting B cell can be triggered comparably in vitro under conditions of direct or transstimulation. Thus, recognition of B cell surface Ia by Th cells is not obligatory for B cell activation and does not transfer an essential transmembrane signal to the B cell.