Since the first demonstration of in vivo gene expression from an injected RNA molecule almost two decades ago, the field of RNA-based therapeutics is now taking significant strides, with many cancer and infectious disease targets entering clinical trials. Critical to this success has been advances in the knowledge and application of delivery formulations. Currently, various lipid nanoparticle (LNP) platforms are at the forefront, but the encapsulation approach underpinning LNP formulations offsets the synthetic and rapid-response nature of RNA vaccines. Second, limited stability of LNP formulated RNA precludes stockpiling for pandemic readiness. Here, we show the development of a two-vialed approach wherein the delivery formulation, a highly stable nanostructured lipid carrier (NLC), can be manufactured and stockpiled separate from the target RNA, which is admixed prior to administration. Furthermore, specific physicochemical modifications to the NLC modulate immune responses, either enhancing or diminishing neutralizing antibody responses. We have combined this approach with a replicating viral RNA (rvRNA) encoding Zika virus (ZIKV) antigens and demonstrated a single dose as low as 10 ng can completely protect mice against a lethal ZIKV challenge, representing what might be the most potent approach to date of any Zika vaccine.

siRNAs confer sequence specific and robust silencing of mRNA. By virtue of these properties, siRNAs have become therapeutic candidates for disease intervention. However, their use as therapeutic agents can be hampered by unintended off-target effects by either or both strands of the siRNA duplex. We report here that unlocked nucleobase analogs (UNAs) confer desirable properties to siRNAs. Addition of a single UNA at the 5′-terminus of the passenger strand blocks participation of the passenger strand in RISC-mediated target down-regulation with a concomitant increase in guide strand activity. Placement of a UNA in the seed region of the guide strand prevents miRNA-like off-target silencing without compromising siRNA activity. Most significantly, combined substitution of UNA at the 3′-termini of both strands, the addition of a UNA at the 5′-terminus of the passenger strand, and a single UNA in the seed region of the guide strand, reduced the global off-target events by more than 10-fold compared to unmodified siRNA. The reduction in off-target events was specific to UNA placement in the siRNA, with no apparent new off-target events. Taken together, these results indicate that when strategically placed, UNA substitutions have important implications for the design of safe and effective siRNA-based therapeutics.

Monoclonal antibody (mAb) therapeutics are an effective modality for the treatment of infectious, autoimmune, and cancer-related diseases. However, the discovery, development, and manufacturing processes are complex, resource-consuming activities that preclude the rapid deployment of mAbs in outbreaks of emerging infectious diseases. Given recent advances in nucleic acid delivery technology, it is now possible to deliver exogenous mRNA encoding mAbs for in situ expression following intravenous (i.v.) infusion of lipid nanoparticle-encapsulated mRNA. However, the requirement for i.v. administration limits the application to settings where infusion is an option, increasing the cost of treatment. As an alternative strategy, and to enable intramuscular (IM) administration of mRNA-encoded mAbs, we describe a nanostructured lipid carrier for delivery of an alphavirus replicon encoding a previously described highly neutralizing human mAb, ZIKV-117. Using a lethal Zika virus challenge model in mice, our studies show robust protection following alphavirus-driven expression of ZIKV-117 mRNA when given by IM administration as pre-exposure prophylaxis or post-exposure therapy.

Since 1997, highly pathogenic avian influenza viruses of the H5N1 subtype have been transmitted from avian hosts to humans. The severity of H5N1 infection in humans, as well as the sporadic nature of H5N1 outbreaks, both geographically and temporally, make generation of an effective vaccine a global public health priority. An effective H5N1 vaccine must ultimately provide protection against viruses from diverse clades. Toll-like receptor (TLR) agonist adjuvant formulations have a demonstrated ability to broaden H5N1 vaccine responses in pre-clinical models. However, many of these agonist molecules have proven difficult to develop clinically. Here, we describe comprehensive adjuvant formulation development of the imidazoquinoline TLR-7/8 agonist 3M-052, in combination with H5N1 hemagglutinin (HA) based antigens. We find that 3M-052 in multiple formulations protects both mice and ferrets from lethal H5N1 homologous virus challenge. Furthermore, we conclusively demonstrate the ability of 3M-052 adjuvant formulations to broaden responses to H5N1 HA based antigens, and show that this broadening is functional using a heterologous lethal virus challenge in ferrets. Given the extensive clinical use of imidazoquinoline TLR agonists for other indications, these studies identify multiple adjuvant formulations which may be rapidly advanced into clinical trials in an H5N1 vaccine.

The emergence and re-emergence of highly virulent viral pathogens with the potential to cause a pandemic creates an urgent need for the accelerated discovery of antiviral therapeutics. Antiviral human monoclonal antibodies (mAbs) are promising candidates for the prevention and treatment of severe viral diseases, but their long development timeframes limit their rapid deployment and use. Here, we report the development of an integrated sequence of technologies, including single-cell mRNA-sequence analysis, bioinformatics, synthetic biology and high-throughput functional analysis, that enables the rapid discovery of highly potent antiviral human mAbs, the activity of which we validated in vivo. In a 78-d study modelling the deployment of a rapid response to an outbreak, we isolated more than 100 human mAbs that are specific to Zika virus, assessed their function, identified that 29 of these mAbs have broadly neutralizing activity, and verified the therapeutic potency of the lead candidates in mice and non-human primate models of infection through the delivery of an antibody-encoding mRNA formulation and of the respective IgG antibody. The pipeline provides a roadmap for rapid antibody-discovery programmes against viral pathogens of global concern.