Covalent Polymer-RNA Conjugates for Potent Activation of the RIG-I Pathway.

RNA ligands of retinoic acid-inducible gene I (RIG-I) are a promising class of oligonucleotide therapeutics with broad potential as antiviral agents, vaccine adjuvants, and cancer immunotherapies. However, their translation has been limited by major drug delivery barriers, including poor cellular uptake, nuclease degradation, and an inability to access the cytosol where RIG-I is localized. Here this challenge is addressed by engineering nanoparticles that harness covalent conjugation of 5'-triphospate RNA (3pRNA) to endosome-destabilizing polymers. Compared to 3pRNA loaded into analogous nanoparticles via electrostatic interactions, it is found that covalent conjugation of 3pRNA improves loading efficiency, enhances immunostimulatory activity, protects against nuclease degradation, and improves serum stability. Additionally, it is found that 3pRNA could be conjugated via either a disulfide or thioether linkage, but that the latter is only permissible if conjugated distal to the 5'-triphosphate group. Finally, administration of 3pRNA-polymer conjugates to mice significantly increases type-I interferon levels relative to analogous carriers that use electrostatic 3pRNA loading. Collectively, these studies have yielded a next-generation polymeric carrier for in vivo delivery of 3pRNA, while also elucidating new chemical design principles for covalent conjugation of 3pRNA with potential to inform the further development of therapeutics and delivery technologies for pharmacological activation of RIG-I.

Structural optimization of siRNA conjugates for albumin binding achieves effective MCL1-directed cancer therapy.

The high potential of siRNAs to silence oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, divalent lipid-conjugated siRNAs are optimized for in situ binding to albumin to improve pharmacokinetics and tumor delivery. Systematic variation of the siRNA conjugate structure reveals that the location of the linker branching site dictates tendency toward albumin association versus self-assembly, while the lipid hydrophobicity and reversibility of albumin binding also contribute to siRNA intracellular delivery. The lead structure increases tumor siRNA accumulation 12-fold in orthotopic triple negative breast cancer (TNBC) tumors over the parent siRNA. This structure achieves approximately 80% silencing of the anti-apoptotic oncogene MCL1 and yields better survival outcomes in three TNBC models than an MCL-1 small molecule inhibitor. These studies provide new structure-function insights on siRNA-lipid conjugate structures that are intravenously injected, associate in situ with serum albumin, and improve pharmacokinetics and tumor treatment efficacy.

Engineering approaches for RNA-based and cell-based osteoarthritis therapies.

Osteoarthritis (OA) is a chronic, debilitating disease that substantially impairs the quality of life of affected individuals. The underlying mechanisms of OA are diverse and are becoming increasingly understood at the systemic, tissue, cellular and gene levels. However, the pharmacological therapies available remain limited, owing to drug delivery barriers, and consist mainly of broadly immunosuppressive regimens, such as corticosteroids, that provide only short-term palliative benefits and do not alter disease progression. Engineered RNA-based and cell-based therapies developed with synthetic chemistry and biology tools provide promise for future OA treatments with durable, efficacious mechanisms of action that can specifically target the underlying drivers of pathology. This Review highlights emerging classes of RNA-based technologies that hold potential for OA therapies, including small interfering RNA for gene silencing, microRNA and anti-microRNA for multi-gene regulation, mRNA for gene supplementation, and RNA-guided gene-editing platforms such as CRISPR-Cas9. Various cell-engineering strategies are also examined that potentiate disease-dependent, spatiotemporally regulated production of therapeutic molecules, and a conceptual framework is presented for their application as OA treatments. In summary, this Review highlights modern genetic medicines that have been clinically approved for other diseases, in addition to emerging genome and cellular engineering approaches, with the goal of emphasizing their potential as transformative OA treatments.

Albumin-binding RNAi Conjugate for Carrier Free Treatment of Arthritis.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are joint diseases that are associated with pain and lost quality of life. No disease modifying OA drugs are currently available. RA treatments are better established but are not always effective and can cause immune suppression. Here, an MMP13-selective siRNA conjugate was developed that, when delivered intravenously, docks onto endogenous albumin and promotes preferential accumulation in articular cartilage and synovia of OA and RA joints. MMP13 expression was diminished upon intravenous delivery of MMP13 siRNA conjugates, consequently decreasing multiple histological and molecular markers of disease severity, while also reducing clinical manifestations such as swelling (RA) and joint pressure sensitivity (RA and OA). Importantly, MMP13 silencing provided more comprehensive OA treatment efficacy than standard of care (steroids) or experimental MMP inhibitors. These data demonstrate the utility of albumin 'hitchhiking' for drug delivery to arthritic joints, and establish the therapeutic utility of systemically delivered anti-MMP13 siRNA conjugates in OA and RA. Editorial summary: Lipophilic siRNA conjugates optimized for albumin binding and "hitchhiking" can be leveraged to achieve preferential delivery to and gene silencing activity within arthritic joints. Chemical stabilization of the lipophilic siRNA enables intravenous siRNA delivery without lipid or polymer encapsulation. Using siRNA sequences targeting MMP13, a key driver of arthritis-related inflammation, albumin hitchhiking siRNA diminished MMP13, inflammation, and manifestations of osteoarthritis and rheumatoid arthritis at molecular, histological, and clinical levels, consistently outperforming clinical standards of care and small molecule MMP antagonists.

Core polymer optimization of ternary siRNA nanoparticles enhances in vivo safety, pharmacokinetics, and tumor gene silencing.

Gene silencing with siRNA nanoparticles (si-NPs) is promising but still clinically unrealized for inhibition of tumor driver genes. Ternary si-NPs containing siRNA, a single block NP core-forming polymer poly[(2-(dimethylamino)ethyl methacrylate)-co-(butyl methacrylate)] (DMAEMA-co-BMA, 50B), and an NP surface-forming diblock polymer 20 kDa poly(ethylene glycol)-block-50B (20kPEG-50B) have the potential to improve silencing activity in tumors due to the participation of both 50B and 20kPEG-50B in siRNA electrostatic loading and endosome disruptive activity. Functionally, single block 50B provides more potent endosomolytic activity, while 20kPEG-50B colloidally stabilizes the si-NPs. Here, we systematically explored the role of the molecular weight (MW) of the core polymer and of the core:surface polymer ratio on ternary si-NP performance. A library of ternary si-NPs was formulated with variation in the MW of the 50B polymer and in the ratio of the core and surface forming polymeric components. Increasing 50B core polymer MW and ratio improved si-NP in vitro gene silencing potency, endosome disruptive activity, and stability, but these features also correlated with cytotoxicity. Concomitant optimization of 50B size and ratio resulted in the identification of lead ternary si-NPs 50B4-DP100, 50B8-DP100, and 50B12-DP25, with potent activity and minimal toxicity. Following intravenous treatment in vivo, all lead si-NPs displayed negligible toxicological effects and enhanced pharmacokinetics and tumor gene silencing relative to more canonical binary si-NPs. Critically, a single 1 mg/kg intravenous injection of 50B8-DP100 si-NPs silenced the tumor driver gene Rictor at the protein level by 80% in an orthotopic breast tumor model. 50B8-DP100 si-NPs delivering siRictor were assessed for therapeutic efficacy in an orthotopic HCC70 mammary tumor model. This formulation significantly inhibited tumor growth compared to siControl-NP treatment. 50B8-DP100 si-NPs were also evaluated for safety and were well-tolerated following a multi-dose treatment scheme. This work provides new insight on ternary si-NP structure-function relationships and identifies core polymer optimization strategies that can yield safe si-NP formulations with potent oncogene silencing.

Structural Optimization of siRNA Conjugates for Albumin Binding Achieves Effective MCL1-Targeted Cancer Therapy.

The high potential for therapeutic application of siRNAs to silence traditionally undruggable oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, siRNAs were optimized for in situ binding to albumin through C18 lipid modifications to improve pharmacokinetics and tumor delivery. Systematic variation of siRNA conjugates revealed a lead structure with divalent C18 lipids each linked through three repeats of hexaethylene glycol connected by phosphorothioate bonds. Importantly, we discovered that locating the branch site of the divalent lipid structure proximally (adjacent to the RNA) rather than at a more distal site (after the linker segment) promotes association with albumin, while minimizing self-assembly and lipoprotein association. Comparison to higher albumin affinity (diacid) lipid variants and siRNA directly conjugated to albumin underscored the importance of conjugate hydrophobicity and reversibility of albumin binding for siRNA delivery and bioactivity in tumors. The lead conjugate increased tumor siRNA accumulation 12-fold in orthotopic mouse models of triple negative breast cancer over the parent siRNA. When applied for silencing of the anti-apoptotic oncogene MCL-1, this structure achieved approximately 80% MCL1 silencing in orthotopic breast tumors. Furthermore, application of the lead conjugate structure to target MCL1 yielded better survival outcomes in three independent, orthotopic, triple negative breast cancer models than an MCL1 small molecule inhibitor. These studies provide new structure-function insights on optimally leveraging siRNA-lipid conjugate structures that associate in situ with plasma albumin for molecular-targeted cancer therapy.

Dual Thermal- and Oxidation-Responsive Polymers Synthesized by a Sequential ROP-to-RAFT Procedure Inherently Temper Neuroinflammation.

This study is about multiple responsiveness in biomedical materials. This typically implies "orthogonality" (i.e., one response does not affect the other) or synergy (i.e., one increases efficacy or selectivity of the other), but an antagonist effect between responses may also occur. Here, we describe a family of very well-defined amphiphilic and micelle-forming block copolymers, which show both oxidative and temperature responses. They are produced via successive anionic ring-opening polymerization of episulfides and RAFT polymerization of dialkylacrylamides and differ only in the ratio between inert (N,N-dimethylacrylamide, DMA) and temperature-sensitive (N,N-diethylacrylamide, DEA) units. By scavenging Reactive Oxygen Species (ROS), these polymers are anti-inflammatory; through temperature responsiveness, they can macroscopically aggregate, which may allow them to form depots upon injection. The localization of the anti-inflammatory action is an example of synergy. An extensive evaluation of toxicity and anti-inflammatory effects on in vitro models, including BV2 microglia, C8D30 astrocytes and primary neurons, shows a link between capacity of aggregation and detrimental effects on viability which, albeit mild, can hinder the anti-inflammatory potential (antagonist action). Although limited in breadth (e.g., only in vitro models and only DEA as a temperature-responsive unit), this study suggests that single-responsive controls should be used to allow for a precise assessment of the (synergic or antagonist) potential of double-responsive systems.

A Reactive Oxygen Species-Scavenging 'Stealth' Polymer, Poly(thioglycidyl glycerol), Outperforms Poly(ethylene glycol) in Protein Conjugates and Nanocarriers and Enhances Protein Stability to Environmental and Biological Stressors.

This study addresses well-known shortcomings of poly(ethylene glycol) (PEG)-based conjugates. PEGylation is by far the most common method employed to overcome immunogenicity and suboptimal pharmacokinetics of, for example, therapeutic proteins but has significant drawbacks. First, PEG offers no protection from denaturation during lyophilization, storage, or oxidation (e.g., by biological oxidants, reactive oxygen species); second, PEG's inherent immunogenicity, leading to hypersensitivity and accelerated blood clearance (ABC), is a growing concern. We have here developed an 'active-stealth' polymer, poly(thioglycidyl glycerol)(PTGG), which in human plasma is less immunogenic than PEG (35% less complement activation) and features a reactive oxygen species-scavenging and anti-inflammatory action (∼50% less TNF-α in LPS-stimulated macrophages at only 0.1 mg/mL). PTGG was conjugated to proteins via a one-pot process; molar mass- and grafting density-matched PTGG-lysozyme conjugates were superior to their PEG analogues in terms of enzyme activity and stability against freeze-drying or oxidation; the latter is due to sacrificial oxidation of methionine-mimetic PTGG chains. Both in mice and rats, PTGG-ovalbumin displayed circulation half-lives up to twice as long as PEG-ovalbumin, but most importantly─and differently from PEG─without any associated ABC effect seen either in the time dependency of blood concentration, in the liver/splenic accumulation, or in antipolymer IgM/IgG titers. Furthermore, similar pharmacokinetic results were obtained with PTGGylated/PEGylated liposomal nanocarriers. PTGG's 'active-stealth' character therefore makes it a highly promising alternative to PEG for conjugation to biologics or nanocarriers.

Versatile Preparation of Branched Polylactides by Low-Temperature, Organocatalytic Ring-Opening Polymerization in N-Methylpyrrolidone and Their Surface Degradation Behavior.

We describe how the organocatalytic, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)-based lactide ring-opening polymerization can be effectively performed in a very polar solvent, N-methylpyrrolidone (NMP). Due to a low ceiling temperature, this "living" mechanism has been unreported to date, but we here demonstrate that through a combination of low temperature and repeated monomer additions (starve-fed process), this mechanism enables the generation of a plethora of multifunctional homo- and (stereo)block-poly(lactide)s (PLAs) with exquisite control of the molecular weight dispersity (typically D < 1.1) and topology (from linear through 4-, 6-, or 8-armed stars and up to ∼140 armed combs). They are scarcely obtainable or inaccessible through more classical synthetic methods due to the poor solubility of multifunctional initiators (polyols) in most organic solvents and monomer melts. In these precisely designed structures, branching significantly altered the nature of the materials' hydrolytic degradation, allowing them to acquire a pronounced surface character (as opposed to the bulk degradation of linear polymers). Finally, we have assessed the amenability of this method to in situ block copolymerization by using the tacticity of PLLA blocks in PLLA-b-PDLLA versus PDLLA-b-PLLA (L-LA polymerized before or after DL-LA) as a sensitive method to detect (stereochemical) defects.

"Tandem" Nanomedicine Approach against Osteoclastogenesis: Polysulfide Micelles Synergically Scavenge ROS and Release Rapamycin.

We show the first example of a synergic approach of oxidant (ROS) scavenging carrier and ROS-responsive drug release in the context of a potential therapy against osteoporosis, aiming to inhibit the differentiation of inflammatory cells into osteoclasts. In our "tandem" approach, a branched amphiphilic, PEGylated polysulfide (PPSES-PEG) was preferred over a linear analogue, because of improved homogeneity in the aggregates (spherical micelles vs mixture of wormlike and spherical), increased stability, and higher drug loading (up to ∼22 wt % of antiosteoclastic rapamycin). These effects are ascribed to the branching inhibiting crystallization in the polysulfide blocks. The ROS-scavenging micelles alone were already able to reduce osteoclastogenesis in a RAW 264.7 model, but the "drug" combination (the polymer itself + rapamycin released only under oxidation) completely abrogated the process. An important take-home message is that the synergic performance depended very strongly on the oxidant:oxidizable group molar ratio, a parameter to carefully tune in the perspective of targeting specific diseases.

Polyvalent Diazonium Polymers Provide Efficient Protection of Oncolytic Adenovirus Enadenotucirev from Neutralizing Antibodies while Maintaining Biological Activity In Vitro and In Vivo.

Oncolytic viruses offer many advantages for cancer therapy when administered directly to confined solid tumors. However, the systemic delivery of these viruses is problematic because of the host immune response, undesired interactions with blood components, and inherent targeting to the liver. Efficacy of systemically administered viruses has been improved by masking viral surface proteins with polymeric materials resulting in modulation of viral pharmacokinetic profile and accumulation in tumors in vivo. Here we describe a new class of polyvalent reactive polymer based on poly( N-(2-hydroxypropyl)methacrylamide) (polyHPMA) with diazonium reactive groups and their application in the modification of the chimeric group B oncolytic virus enadenotucirev (EnAd). A series of six copolymers with different chain lengths and density of reactive groups was synthesized and used to coat EnAd. Polymer coating was found to be extremely efficient with concentrations as low as 1 mg/mL resulting in complete (>99%) ablation of neutralizing antibody binding. Coating efficiency was found to be dependent on both chain length and reactive group density. Coated viruses were found to have reduced transfection activity both in vitro and in vivo, with greater protection against neutralizing antibodies resulting in lower transgene production. However, in the presence of neutralizing antibodies, some in vivo transgene expression was maintained for coated virus compared to the uncoated control. The decrease in transgene expression was found not to be solely due to lower cellular uptake but due to reduced unpackaging of the virus within the cells and reduced replication, indicating that the polymer coating does not cause permanent inactivation of the virus. These data suggest that virus activity may be modulated by the appropriate design of coating polymers while retaining protection against neutralizing antibodies.

Polymers for binding of the gram-positive oral pathogen Streptococcus mutans.

Streptococcus mutans is the most significant pathogenic bacterium implicated in the formation of dental caries and, both directly and indirectly, has been associated with severe conditions such as multiple sclerosis, cerebrovascular and peripheral artery disease. Polymers able to selectively bind S. mutans and/or inhibit its adhesion to oral tissue in a non-lethal manner would offer possibilities for addressing pathogenicity without selecting for populations resistant against bactericidal agents. In the present work two libraries of 2-(dimethylamino)ethyl methacrylate (pDMAEMA)-based polymers were synthesized with various proportions of either N,N,N-trimethylethanaminium cationic- or sulfobetaine zwitterionic groups. These copolymers where initially tested as potential macromolecular ligands for S. mutans NCTC 10449, whilst Escherichia coli MG1655 was used as Gram-negative control bacteria. pDMAEMA-derived materials with high proportions of zwitterionic repeating units were found to be selective for S. mutans, in both isolated and S. mutans-E. coli mixed bacterial cultures. Fully sulfobetainized pDMAEMA was subsequently found to bind/cluster preferentially Gram-positive S. mutans and S. aureus compared to Gram negative E. coli and V. harveyi. A key initial stage of S. mutans pathogenesis involves a lectin-mediated adhesion to the tooth surface, thus the range of potential macromolecular ligands was further expanded by investigating two glycopolymers bearing α-mannopyranoside and ß-galactopyranoside pendant units. Results with these polymers indicated that preferential binding to either S. mutans or E. coli can be obtained by modulating the glycosylation pattern of the chosen multivalent ligands without incurring unacceptable cytotoxicity in a model gastrointestinal cell line. Overall, our results allowed to identify a structure-property relationship for the potential antimicrobial polymers investigated, and suggest that preferential binding to Gram-positive S. mutans could be achieved by fine-tuning of the recognition elements in the polymer ligands.

A thermoresponsive and magnetic colloid for 3D cell expansion and reconfiguration.

A dual thermoresponsive and magnetic colloidal gel matrix is described for enhanced stem-cell culture. The combined properties of the material allow enzyme-free passaging and expansion of mesenchymal stem cells, as well as isolation of cells postculture by the simple process of lowering the temperature and applying an external magnetic field. The colloidal gel can be reconfigured with thermal and magnetic stimuli to allow patterning of cells in discrete zones and to control movement of cells within the porous matrix during culture.

Programmable polymer-DNA hydrogels with dual input and multiscale responses.

Combination switchable polymer-DNA hydrogels have been synthesized to respond to both a specific oligonucleotide recognition signal and a non-specific but biorelevant environmental trigger. The hydrogels exhibit rheological properties that can be modulated through interaction with complementary DNA strands and/or reduction. Furthermore, individual and combined oligonucleotide recognition and reduction responses allow control over pore sizes in the gel, enabling programmable release and transport of objects ranging from the nano- to micro-scale.