Ivan Vuong

Versatile technologies that can deliver both RNA and protein payloads could streamline development, simplify manufacturing and expand the capabilities of combination therapies. Here we demonstrate an efficient approach to forming ca. 100 nm polymer vesicles (polymersomes) capable of rapid self-assembly without organic solvents, avoiding the need for post-encapsulation purification. Block copolymers are designed with a lower critical solution temperature that renders them soluble in aqueous medium under standard refrigeration, but they spontaneously assemble at room temperature into large batches of nanoparticles with predictable size and morphology. The nanomaterials are designed with charged and biofunctional moieties to drive payload affinity and in vivo targeting, while both siRNA and proteins can be encapsulated during warming at >75% loading efficiencies. Formulations can be stored in a dry state for greater hydrolytic stability under standard refrigeration and can be diluted directly from the vial, bypassing the need for purification required for high scalability. We use our system for in vivo delivery in protein subunit vaccination, immune tolerance induction and siRNA interference therapy in cancer.

Current asthma treatments manage disease symptoms but fail to address the underlying cause of allergic disease. Allergen immunotherapy holds the promise for durable disease control by establishing allergen-specific tolerance through repeated introduction of native allergens; however, its efficacy can be limited by long interventions, reactions upon administration, and poor patient compliance. Here, we developed a rapid, safe, and effective liver-targeted allergen immunotherapy (LIT) to provide long-term disease control. We developed LIT tolerogens from native respiratory allergens, which induced antigen-specific regulatory T (T _reg ) cells in vivo with only two interventions. Synthetic mannosylation of native allergens prevented antibody-mediated recognition and subsequent life-threatening anaphylaxis upon administration. Protein engineering prevented sensitization events that occurred because of the proteolytic activity of native respiratory allergens, which limit the effectiveness of allergen immunotherapy. In preclinical models of allergic asthma, LIT ameliorated clinical, pathological, and serological features, and protection was dependent on antigen-specific T _reg cells. In sensitized mice, LIT provided a year-long control of allergic asthma symptoms in the absence of additional intervention. Last, LIT induced antigen-specific T _reg cells against Der p 1, a major protein in the clinically relevant house dust mite (HDM) respiratory allergen. In mice with established HDM allergy, LIT was well tolerated and provided allergic symptom relief. Together, our data provide a proof of concept that LIT with synthetically mannosylated tolerogens provides a rapid, safe, and effective approach to allergen immunotherapy and holds promise for durable control of allergic asthma.

Hydrogel materials have emerged as versatile platforms for various biomedical applications. Notably, the engineered nanofiber-hydrogel composite (NHC) has proven effective in mimicking the soft tissue extracellular matrix, facilitating substantial recruitment of host immune cells and the formation of a local immunostimulatory microenvironment. Leveraging this feature, here we report an mRNA lipid nanoparticle (LNP)-incorporated NHC microgel matrix, termed LiNx, by incorporating LNPs loaded with mRNA encoding tumour antigens. Harnessing the potent transfection efficiency of LNPs in antigen-presenting cells (APCs), LiNx demonstrates remarkable immune cell recruitment, antigen expression and presentation, and cellular interaction. These attributes collectively create an immunostimulating milieu and yield a potent immune response achievable with a single dose, comparable to the conventional three-dose LNP immunization regimen. Further investigations reveal that the LiNx not only generates heightened Th1 and Th2 responses but also elicits a distinctive Type 17 T helper cell-mediated response pivotal for bolstering antitumour efficacy. Our findings elucidate the mechanism underlying LiNx’s role in potentiating antigen-specific immune responses, presenting a new strategy for cancer immunotherapy.

Although great advances in siRNA and mRNA delivery have been made (e.g. with lipid nanoparticles), there remains a pressing need for a versatile platform that can deliver both RNA and protein payloads with convenient processing and storage. Here, we demonstrate a highly efficient approach to forming ca. 100 nm vesicular polymer nanoparticles (polymersomes) that requires no organic solvents by utilizing thermoresponsive block copolymers capable of self-assembling in aqueous medium as the solution is raised to room temperature. This is achieved using block copolymers with a domain that has a lower critical solution temperature (LCST) such that they are soluble in aqueous medium under standard refrigeration (4-7°C) temperature but assemble upon warming to room temperature, resulting in large batches of nanoparticles with predictable size and morphology as dictated by polymer structure. The nanomaterials are designed with charged and biofunctional moieties to drive payload affinity as well as in-vivo targeting, respectively. Both siRNA and protein payloads can be incorporated during warming at higher than 75% loading efficiencies. Payload-polymer suspensions can also be lyophilized into a dry state, allowing for greater hydrolytic stability under mild (4-7°C) refrigeration conditions. This greatly reduces processing and storage requirements, as the powder can simply be reconstituted in cold aqueous medium, then used as described. Here, we selected biomedical applications that did not require removal of unencapsulated payload, bypassing further purification processes and demonstrating a highly scalable approach for formulating nanoparticle-based treatments. Finally, we demonstrate that our system is capable of in-vivo delivery in the contexts of protein subunit vaccination, prophylactic immune tolerance induction, and siRNA interference therapy in cancer. We believe this polymersome platform addresses several challenges to broad translation of nanoparticle delivery of biologics to the clinic, such as scalability, loading efficiency, quality control, and dry storage.

Lipid nanoparticles (LNPs) can be designed to potentiate cancer immunotherapy by promoting their uptake by antigen-presenting cells, stimulating the maturation of these cells and modulating the activity of adjuvants. Here we report an LNP-screening method for the optimization of the type of helper lipid and of lipid-component ratios to enhance the delivery of tumour-antigen-encoding mRNA to dendritic cells and their immune-activation profile towards enhanced antitumour activity. The method involves screening for LNPs that enhance the maturation of bone-marrow-derived dendritic cells and antigen presentation in vitro, followed by assessing immune activation and tumour-growth suppression in a mouse model of melanoma after subcutaneous or intramuscular delivery of the LNPs. We found that the most potent antitumour activity, especially when combined with immune checkpoint inhibitors, resulted from a coordinated attack by T cells and NK cells, triggered by LNPs that elicited strong immune activity in both type-1 and type-2 T helper cells. Our findings highlight the importance of optimizing the LNP composition of mRNA-based cancer vaccines to tailor antigen-specific immune-activation profiles.

A pan-caspase inhibitor, Q-VD-OPH, has therapeutic efficacy against preclinical methicillin-resistant Staphylococcus aureus skin infection.