High-Throughput Automation of Endosomolytic Polymers for mRNA Delivery.

In recent years, there has been an increasing interest in designing delivery systems to enhance the efficacy of RNA-based therapeutics. Here, we have synthesized copolymers comprised of dimethylaminoethyl methacrylate (DMAEMA) or diethylaminoethyl methacrylate (DEAEMA) copolymerized with alkyl methacrylate monomers ranging from 2 to 12 carbons, and developed a high throughput workflow for rapid investigation of their applicability for mRNA delivery. The structure activity relationship revealed that the mRNA encapsulation efficiency is improved by increasing the cationic density and use of shorter alkyl side chains (2-6 carbons). Minimal cytotoxicity was observed when using DEAEMA-co-BMA (EB) polyplexes up to 18 h after dosing, independent of a poly(ethylene glycol) (PEG) first block. The lowest molecular weight polymer (EB10,250) performed best, exhibiting greater transfection than polyethyenimine (PEI) based upon the number of cells transfected and mean intensity. Conventional investigations into the performance of polymeric materials for mRNA delivery is quite tedious, consequently limiting the number of materials and formulation conditions that can be studied. The high throughput approach presented here can accelerate the screening of polymeric systems and paves the way for expanding this generalizable approach to assess various materials for mRNA delivery.

Structural Optimization of Polymeric Carriers to Enhance the Immunostimulatory Activity of Molecularly Defined RIG-I Agonists.

RNA ligands of retinoic acid-inducible gene I (RIG-I) hold significant promise as antiviral agents, vaccine adjuvants, and cancer immunotherapeutics, but their efficacy is hindered by inefficient intracellular delivery to the cytosol where RIG-I is localized. Here, we address this challenge through the synthesis and evaluation of a library of polymeric carriers rationally designed to promote the endosomal escape of 5'-triphosphate RNA (3pRNA) RIG-I agonists. We synthesized a series of PEG-block-(DMAEMA-co-A n MA) polymers, where A n MA is an alkyl methacrylate monomer ranging from n = 2-12 carbons, of variable composition, and examined effects of polymer structure on the intracellular delivery of 3pRNA. Through in vitro screening of 30 polymers, we identified four lead carriers (4-50, 6-40, 8-40, and 10-40, where the first number refers to the alkyl chain length and the second number refers to the percentage of hydrophobic monomer) that packaged 3pRNA into ∼100-nm-diameter particles and significantly enhanced its immunostimulatory activity in multiple cell types. In doing so, these studies also revealed an interplay between alkyl chain length and monomer composition in balancing RNA loading, pH-responsive properties, and endosomal escape, studies that establish new structure-activity relationships for polymeric delivery of 3pRNA and other nucleic acid therapeutics. Importantly, lead carriers enabled intravenous administration of 3pRNA in mice, resulting in increased RIG-I activation as measured by increased levels of IFN-α in serum and elevated expression of Ifnb1 and Cxcl10 in major clearance organs, effects that were dependent on polymer composition. Collectively, these studies have yielded novel polymeric carriers designed and optimized specifically to enhance the delivery and activity of 3pRNA with potential to advance the clinical development of RIG-I agonists.

Mucosal Immunization with a pH-Responsive Nanoparticle Vaccine Induces Protective CD8+ Lung-Resident Memory T Cells.

Tissue-resident memory T cells (TRM) patrol nonlymphoid organs and provide superior protection against pathogens that commonly infect mucosal and barrier tissues, such as the lungs, intestine, liver, and skin. Thus, there is a need for vaccine technologies that can induce a robust, protective TRM response in these tissues. Nanoparticle (NP) vaccines offer important advantages over conventional vaccines; however, there has been minimal investigation into the design of NP-based vaccines for eliciting TRM responses. Here, we describe a pH-responsive polymeric nanoparticle vaccine for generating antigen-specific CD8+ TRM cells in the lungs. With a single intranasal dose, the NP vaccine elicited airway- and lung-resident CD8+ TRM cells and protected against respiratory virus challenge in both sublethal (vaccinia) and lethal (influenza) infection models for up to 9 weeks after immunization. In elucidating the contribution of material properties to the resulting TRM response, we found that the pH-responsive activity of the carrier was important, as a structurally analogous non-pH-responsive control carrier elicited significantly fewer lung-resident CD8+ T cells. We also demonstrated that dual-delivery of protein antigen and nucleic acid adjuvant on the same NP substantially enhanced the magnitude, functionality, and longevity of the antigen-specific CD8+ TRM response in the lungs. Compared to administration of soluble antigen and adjuvant, the NP also mediated retention of vaccine cargo in pulmonary antigen-presenting cells (APCs), enhanced APC activation, and increased production of TRM-related cytokines. Overall, these data suggest a promising vaccine platform technology for rapid generation of protective CD8+ TRM cells in the lungs.

Correction: The efficiency of cytosolic drug delivery using pH-responsive endosomolytic polymers does not correlate with activation of the NLRP3 inflammasome.

Correction for 'The efficiency of cytosolic drug delivery using pH-responsive endosomolytic polymers does not correlate with activation of the NLRP3 inflammasome' by Jessalyn J. Baljon et al., Biomater. Sci., 2019, DOI: 10.1039/c8bm01643g.

The efficiency of cytosolic drug delivery using pH-responsive endosomolytic polymers does not correlate with activation of the NLRP3 inflammasome.

Inefficient cytosolic delivery has limited the development of many promising biomacromolecular drugs, a long-standing challenge that has prompted extensive development of drug carriers that facilitate endosomal escape. Although many such carriers have shown considerable promise for cytosolic delivery of a diversity of therapeutics, the rupture or destabilization of endo/lysosomal membranes has also been associated with activation of the inflammasome with attendant risk of inflammation and toxicity. In this study, we investigated relationships between pH-dependent membrane destabilization, cytosolic drug delivery, and inflammasome activation using a series of well-defined poly[(ethylene glycol)-block-[(2-(dimethylamino)ethyl methacrylate)-co-(butyl methacrylate)] copolymers of variable second block composition and pH-responsive properties. We found that polymers that demonstrated the most potent membrane-destabilizing activity at early endosomal pH values in an erythrocyte hemolysis assay were most efficient at delivery of siRNA, yet tended to be associated with the least amount of NOD-like related protein 3 (NLRP3) inflammasome activation. By contrast, polymers that displayed minimal hemolysis activity and poor siRNA knockdown, and instead mediated lysosomal rupture likely due to a proton sponge mechanism, strongly induced NLPR3 inflammasome activation in a caspase- and cathepsin-dependent manner. Collectively, these findings reinforce the importance of early endosomal escape in minimizing inflammasome activation and also demonstrate the ability to tune the degree inflammasome activation via control of polymer structure with potential implications for design of vaccine adjuvants and immunotherapeutics.

Delivery of 5'-triphosphate RNA with endosomolytic nanoparticles potently activates RIG-I to improve cancer immunotherapy.

RNA agonists of the retinoic acid gene I (RIG-I) pathway have recently emerged as a promising class of cancer immunotherapeutics, but their efficacy is hindered by drug delivery barriers, including nuclease degradation, poor intracellular uptake, and minimal access to the cytosol where RIG-I is localized. Here, we explore the application of pH-responsive, endosomolytic polymer nanoparticles (NPs) to enhance the cytosolic delivery and immunostimulatory activity of synthetic 5' triphosphate, short, double-stranded RNA (3pRNA), a ligand for RIG-I. Delivery of 3pRNA with pH-responsive NPs with an active endosomal escape mechanism, but not control carriers lacking endosomolytic activity, significantly increased the activity of 3pRNA in dendritic cells, macrophages, and cancer cell lines. In a CT26 colon cancer model, activation of RIG-I via NP delivery of 3pRNA induced immunogenic cell death, triggered expression of type I interferon and pro-inflammatory cytokines, and increased CD8+ T cell infiltration into the tumor microenvironment. Consequently, intratumoral (IT) delivery of NPs loaded with 3pRNA inhibited CT26 tumor growth and enhanced the therapeutic efficacy of anti-PD-1 immune checkpoint blockade, resulting in a 30% complete response rate and generation of immunological memory that protected against tumor rechallenge. Collectively, these studies demonstrate that pH-responsive NPs can be harnessed to strongly enhance the immunostimulatory activity and therapeutic efficacy of 3pRNA and establish endosomal escape as a critical parameter in the design of carriers for immunotherapeutic targeting of the RIG-I pathway.

Therapeutically Active RIG-I Agonist Induces Immunogenic Tumor Cell Killing in Breast Cancers.

Cancer immunotherapies that remove checkpoint restraints on adaptive immunity are gaining clinical momentum but have not achieved widespread success in breast cancers, a tumor type considered poorly immunogenic and which harbors a decreased presence of tumor-infiltrating lymphocytes. Approaches that activate innate immunity in breast cancer cells and the tumor microenvironment are of increasing interest, based on their ability to induce immunogenic tumor cell death, type I IFNs, and lymphocyte-recruiting chemokines. In agreement with reports in other cancers, we observe loss, downregulation, or mutation of the innate viral nucleotide sensor retinoic acid-inducible gene I (RIG-I/DDX58) in only 1% of clinical breast cancers, suggesting potentially widespread applicability for therapeutic RIG-I agonists that activate innate immunity. This was tested using an engineered RIG-I agonist in a breast cancer cell panel representing each of three major clinical breast cancer subtypes. Treatment with RIG-I agonist resulted in upregulation and mitochondrial localization of RIG-I and activation of proinflammatory transcription factors STAT1 and NF-κB. RIG-I agonist triggered the extrinsic apoptosis pathway and pyroptosis, a highly immunogenic form of cell death in breast cancer cells. RIG-I agonist also induced expression of lymphocyte-recruiting chemokines and type I IFN, confirming that cell death and cytokine modulation occur in a tumor cell-intrinsic manner. Importantly, RIG-I activation in breast tumors increased tumor lymphocytes and decreased tumor growth and metastasis. Overall, these findings demonstrate successful therapeutic delivery of a synthetic RIG-I agonist to induce tumor cell killing and to modulate the tumor microenvironment in vivo Significance: These findings describe the first in vivo delivery of RIG-I mimetics to tumors, demonstrating a potent immunogenic and therapeutic effect in the context of otherwise poorly immunogenic breast cancers. Cancer Res; 78(21); 6183-95. ©2018 AACR.

Environmentally Triggerable Retinoic Acid-Inducible Gene I Agonists Using Synthetic Polymer Overhangs.

Retinoic acid-inducible gene I (RIG-I) is a cytosolic pattern recognition receptor (PRR) that potently activates antiviral innate immunity upon recognition of 5' triphosphorylated double-stranded RNA (pppRNA). Accordingly, RNA ligands of the RIG-I pathway have recently emerged as promising antiviral agents, vaccine adjuvants, and cancer immunotherapeutics. However, RIG-I is expressed constitutively in virtually all cell types, and therefore administration of RIG-I agonists causes risk for systemic inflammation and possible dose-limiting toxicities. Here, we establish proof-of-concept and initial design criteria for pppRNA prodrugs capable of activating the RIG-I pathway in response to specific environmental stimuli. We show that covalent conjugation of poly(ethylene glycol) (PEG) to the 3' end of the complementary strand, i.e., on the same side but opposite strand as the 5' triphosphate group, can generate a synthetic overhang that prevents RIG-I activation. Additionally, conjugation of PEG through a cleavable linker-here, a reducible disulfide bond-allows for removal of the synthetic overhang and restoration of immunostimulatory activity. Furthermore, we demonstrate that blockade of RIG-I activation via synthetic overhangs is dependent on PEG molecular weight, with a critical molecular weight between 550 and 1000 Da required to inhibit activity. Additionally, we demonstrate that blockade of RIG-I activity is conjugation site-dependent, as ligation of PEG to the opposite end of the RNA did not influence ligand activity. Collectively, this work demonstrates that conjugation of synthetic polymer overhangs to pppRNA through cleavable linkers is a viable strategy for the development of environmentally triggerable RIG-I-targeting prodrugs.