The development of ionizable mRNA-lipid nanoparticle (mRNA-LNP) nucleic acid carriers facilitated the clinical translation of the Coronavirus 2019 (COVID-19) mRNA vaccines BNT162b2 and mRNA-1273. Here, we discuss insights into rational improvements to mRNA vaccines, focusing on LNP modifications for mRNA-LNP biodistribution control, miRNA-based biodistribution control of encoded transcripts, and precision adjuvantation strategies.

AbstractAdjuvants, materials added to vaccines to enhance the resulting immune response, are important components of vaccination that are many times overlooked. While vaccines always include an antigen to tell the body what to vaccinate to, of equal importance the adjuvant provides the how, a significant factor in producing a complete response. The adjuvant space has been slow to develop with the first use of an adjuvant in a licensed vaccine occurring in the 1930s, and remaining the only adjuvant in licensed vaccines for the next 80 years. However, with vaccination at the forefront of protection against new and complex pathogens, it is important to consider all components when designing an effective vaccine. Here we summarize the adjuvant space in licensed vaccines as well as the novel adjuvant space in clinical trials with a specific focus on the materials utilized and their resulting impact on the immune response. We discuss five major categories of adjuvant materials: aluminum salts, nanoparticles, viral vectors, TLR agonists, and emulsions. For each category, we delve into the current clinical trials space, the impact of these materials on vaccination, as well as some of the ways in which they could be improved. Adjuvants present an exciting opportunity to improve vaccine responses and stability, this review will help inform about the current progress of this space.Translational impact statementIn the aftermath of the COVID‐19 pandemic, vaccines for infectious diseases have come into the spotlight. While antigens have always been an important focus of vaccine design, the adjuvant is a significant tool for enhancing the immune response to the vaccine that has been largely underdeveloped. This article provides a broad review of the history of adjuvants and, the current vaccine adjuvant space, and the progress seen in adjuvants in clinical trials. There is specific emphasis on the material landscape for adjuvants and their resulting mechanism of action. Looking ahead, while the novel vaccine adjuvant space features exciting new technologies and materials, there is still a need for more to meet the protective needs of new and complex pathogens.

Vaccination is an important strategy for the prevention of infectious diseases worldwide. Adjuvants can be incorporated in vaccine formulations to enhance the resultant immune response and subsequently confer more robust protection upon natural infection. While adjuvants have exciting potential to improve vaccination, the landscape of materials employed in clinical adjuvants is small and its expansion is needed to facilitate vaccine development against current and future infectious diseases. This study introduces the first ionic liquid (IL) adjuvant comprised of choline and sorbic acid (ChoSorb) to produce an antigen-specific cellular as well as humoral immune response against multiple antigens. The abilities of ChoSorb as a vaccine adjuvant is evaluated and characterized through material analysis, innate immune responses, and adaptive responses to both a model and clinical grade antigen. With the robust immune responses generated by ChoSorb and the accompanying mechanistic insights, this study introduces ILs as a new class of adjuvant materials for future vaccine design.

Proteins are among the most common therapeutics for the treatment of diabetes, autoimmune diseases, cancer, and metabolic diseases, among others. Despite their common use, current protein therapies, most of which are injectables, have several limitations. Large proteins such as monoclonal antibodies (mAbs) suffer from poor absorption after subcutaneous injections, thus forcing their administration by intravenous injections. Even small proteins such as insulin suffer from slow pharmacokinetics which poses limitations in effective management of diabetes. Here, a deep eutectic-based delivery strategy is used to offer a generalized approach for improving protein absorption after subcutaneous injections. The lead formulation enhances absorption of mAbs after subcutaneous injections by ≈200%. The same composition also improves systemic absorption of subcutaneously injected insulin faster than Humalog, the current gold-standard of rapid acting insulin. Mechanistic studies reveal that the beneficial effect of deep eutectics on subcutaneous absorption is mediated by their ability to reduce the interactions of proteins with the subcutaneous matrix, especially collagen. Studies also confirm that these deep eutectics are safe for subcutaneous injections. Deep eutectic-based formulations described here open new possibilities for subcutaneous injections of therapeutic proteins.

Therapeutic neural stem cells (tNSCs) are a promising new platform for the treatment of glioblastoma (GBM). tNSCs exhibit a characteristic known as tumor tropism, in which they can migrate towards distant GBM foci via cytokine signaling. Complementarily, genetic engineering of NSCs may be performed to turn the cells into drug-producing therapeutics. Together, this results in NSCs that act as targeted drug delivery vehicles that can seek out and kill invasive GBM lesions post-resection. However, one limitation of this cell therapy platform is that tNSCs delivered directly into the GBM resection cavity are rapidly cleared. We hypothesized that the commercially available, FDA-approved hemostat FLOSEAL® may be used as a drug delivery system for improving cell persistence in the brain, thus resulting in improved therapeutic efficacy. It was found that tNSCs encapsulated in FLOSEAL® were detectable in the brain for over 95 days in mice, a drastic improvement compared to directly injected cells and cells encapsulated in other existing hemostat systems, which persisted 2 weeks or less. However, two in vivo efficacy studies of tNSCs encapsulated in FLOSEAL® yielded contrasting results. While the FLOSEAL®-tNSC system was significantly more efficacious against a GBM8 tumor model in mice compared to directly injected tNSCs, it was not significantly more effective against a U87 tumor model. This could be due to a variety of factors, including the tumor type (diffuse vs. solid for GBM8 and U87, respectively) and negative impacts of FLOSEAL® on tNSC markers of proliferation, migration, drug production, and anti-apoptosis. While FLOSEAL® is a promising material for the delivery of tNSCs in the treatment of post-operative GBM, alternative systems that allow for improved persistence while maintaining the therapeutic activity of the cells would be optimal for long-term treatment with tNSCs.

Induced neural stem cells (iNSCs) have emerged as a promising therapeutic platform for glioblastoma (GBM). iNSCs have the innate ability to home to tumor foci, making them ideal carriers for antitumor payloads. However, the in vivo persistence of iNSCs limits their therapeutic potential. We hypothesized that by encapsulating iNSCs in the FDA-approved, hemostatic matrix FLOSEAL®, we could increase their persistence and, as a result, therapeutic durability. Encapsulated iNSCs persisted for 95 days, whereas iNSCs injected into the brain parenchyma persisted only 2 weeks in mice. Two orthotopic GBM tumor models were used to test the efficacy of encapsulated iNSCs. In the GBM8 tumor model, mice that received therapeutic iNSCs encapsulated in FLOSEAL® survived 30 to 60 days longer than mice that received nonencapsulated cells. However, the U87 tumor model showed no significant differences in survival between these two groups, likely due to the more solid and dense nature of the tumor. Interestingly, the interaction of iNSCs with FLOSEAL® appears to downregulate some markers of proliferation, anti-apoptosis, migration, and therapy which could also play a role in treatment efficacy and durability. Our results demonstrate that while FLOSEAL® significantly improves iNSC persistence, this alone is insufficient to enhance therapeutic durability.

The mucus barrier lining the gastrointestinal tract poses a significant barrier to the oral delivery of macromolecular drugs. Successful approaches to overcoming this barrier have primarily focused on reducing drug and carrier interactions with mucus or disrupting the mucus layer directly. Choline-based ionic liquids (ILs) such as choline geranate and choline glycolate (CGLY) have recently been shown to be effective in enhancing the intestinal absorption of macromolecules such as insulin and immunoglobulin (IgG), respectively. Herein, the use of choline-based ILs as mucus-modulating agents for safely improving drug penetration through mucus is described. Choline-based ILs significantly increase the diffusion rates of cationic dextrans through mucin solution. Choline-maleic acid (CMLC 2:1) enhances the diffusion of 4 kDa cationic dextran in mucin solution by more than fourfold when compared to phosphate-buffered saline control. Choline-based ILs also reduce mucus viscosity without significantly impacting the native mucus gel structure. In vitro studies in a mucus-secreting coculture model with Caco-2 and HT29MTX-E12 cells further demonstrate the effectiveness of ILs in improving transport of cationic molecules in the presence of secreted mucus. This work demonstrates the potential for choline-based ionic liquids to be used as nondestructive mucus-modulating agents for enabling enhanced oral delivery of macromolecular drugs.

Engineered neural stem cells (NSCs) have recently emerged as a promising therapy. Acting as a tumor-homing drug-delivery system, NSCs migrate through brain tissue to seek out primary and invasive tumor foci. NSCs can deliver therapeutic agents, such as TNFα-related apoptosis-inducing ligand, directly to the tumor and suppress glioblastoma (GBM) in murine models. While the mainstays for evaluating NSC migration and efficacy have been two-dimensional chemotaxis assays and mouse models, these low-throughput and small-scale systems limit our ability to implant and track these cells for human translation. To circumvent these challenges, we developed a three-dimensional culture system using a matrix of poly-l-lactic acid 6100 microfibers suspended in agar. These bioinspired brain matrices were used to model tumor growth, NSC migration, and efficacy of NSC therapy at small and human scale. Kinetic fluorescent imaging confirmed growth of tumors in both small and human-sized bioinspired brain matrix. Tumors proliferated 50-fold and 3-fold for GBM and human metastatic breast cancer, respectively, over 7 days. We next explored the impact of tumor location on NSC migration. When NSCs were implanted 2 mm lateral from the tumor foci, NSCs colocalized with the GBM within 7 days. In models of multifocal disease, NSCs were found to colocalize with multiple tumors, preferentially migrating to tumor foci closest to the site of NSC implantation. Lastly, therapeutic NSCs were implanted at increasing distances (0, 2, 5, or 10 mm) laterally from GBM foci to investigate the effects of distance on NSC efficacy. Serial imaging showed reduced fluorescence at tumor sites, implicating GBM apoptosis across all distances. NSCs coinjected with tumor induced a near-complete response in <10 days, while NSCs implanted 10 mm laterally from the tumor induced a near-complete response by day 30. Lastly, GBM foci were established in each hemisphere of the model and control or therapeutic NSCs were implanted adjacent to tumor cells in the right hemisphere. Kinetic imaging showed that NSC therapy attenuated progression of GBM foci, while GBM cells treated with control NSC expanded rapidly over 21 days. In conclusion, we developed a new bioinspired model that supports growth of human brain cancer cells and enables rapid tracking of NSC therapy. Impact statement Tumor-homing and tumor-killing-engineered neural stem cell (NSC) therapies have shown immense promise in both preclinical and clinical trials. However, as cell therapies continue to evolve, cost-effective and high-throughput screening assays are needed to assess the proliferation, migration, and efficacy of these cells. In this study, we developed a bioinspired brain matrix for the evaluation of engineered NSCs. Importantly, this matrix is easy to fabricate, scalable, and allows for sterile real-time, noninvasive imaging using our custom bioreactor. We then utilized the bioinspired brain matrix system to answer key questions around the tumor-homing migration and efficacy of engineered NSC therapies that are challenging to address with traditional models.

Abstract BACKGROUND Induced neural stem cells (iNSCs) have emerged as a promising therapeutic platform for glioblastoma (GBM). iNSCs have the innate ability to home to tumor foci, making them ideal carriers for anti-tumor payloads. However, iNSC persist for only two weeks in the murine GBM tumor resection cavity. We hypothesized that, by encapsulating iNSCs in a scaffold matrix, we could increase both the persistence of the cells the therapeutic durability. METHODS iNSCs expressing TRAIL were encapsulated in a gelatin-thrombin matrix; fibrinogen was used to polymerize the matrix. SEM was used to explore interactions between iNSCs and the scaffold matrix. To evaluate persistence, iNSCs encapsulated in the matrix were implanted into mock resection cavities of athymic nude mice and followed via BLI. To study the impacts of encapsulation on iNSC efficacy, athymic nude mice were implanted with U87 or GBM8 tumors. Tumors were then resected, and iNSCs encapsulated in the matrix were implanted; tumor volume was monitored via BLI. RESULTS SEM images showed homogeneous distribution of iNSCs throughout the matrix; iNSCs were completed encased in the fibrin clot component of the matrix and did not adhere to gelatin. In vivo, encapsulated iNSCs persisted for nearly 100 days whereas iNSCs directly injected into the brain parenchyma persisted < 20 days. Using mice bearing GBM8 tumors, animals treated with a high dose of therapeutic encapsulated iNSCs survived ~60 days longer than animals treated with non-therapeutic cells. A similar trend was observed in animals inoculated with U87 tumors. While not statistically significant, 25% of mice treated with iNSCs encapsulated in the gelatin-thrombin matrix survived longer than those treated with iNSCs encapsulated in a fibrin-only matrix, suggesting additional benefit due to the gelatin component. FUTURE DIRECTIONS Prospective experiments will explore the impact of the scaffold on iNSC phenotype, including proliferation, differentiation, and migration markers.

Adjuvants play a critical role in the design and development of novel vaccines. Despite extensive research, only a handful of vaccine adjuvants have been approved for human use. Currently used adjuvants are mostly composed of components that are non-native to the human body, such as aluminum salt, bacterial lipids, or foreign genomic material. Here, a new ionic-liquid-based adjuvant is explored, synthesized using two metabolites of the body, choline and lactic acid (ChoLa). ChoLa distributes the antigen efficiently upon injection, maintains antigen integrity, enhances immune infiltration at the injection site, and leads to a potent immune response against the antigen. Thus, it can serve as a promising safe adjuvant platform that can help to protect against pandemics and future infectious threats.

The rapid clearance of intravenously administered nanoparticles (NPs) from the bloodstream is a major unsolved problem in nanomedicine. Here, we describe the first use of biocompatible protein-avoidant ionic liquids (PAILs) as NP surface modifiers to reduce opsonization. An ionic liquid choline hexenoate, selected for its aversion to serum proteins, was used to stably coat the surface of poly(lactic-co-glycolic acid) (PLGA) NPs. Compared with bare PLGA and poly(ethylene glycol)-coated PLGA particles, the PAIL-PLGA NPs showed resistance to protein adsorption in vitro and greater retention in blood of mice at 24 hours. Choline hexenoate redirected biodistribution of NPs, with preferential accumulation in the lungs with 50% of the administered dose accumulating in the lungs and <5% in the liver. Lung accumulation was attributed to spontaneous attachment of the PAIL-coated NPs on red blood cells in vivo. Overall, ionic liquids are a promising class of materials for NP modification for biomedical applications.

Immune checkpoint inhibitors have been successful across several tumor types; however, their efficacy has been uncommon and unpredictable in glioblastomas (GBM), where <10% of patients show long-term responses. To understand the molecular determinants of immunotherapeutic response in GBM, we longitudinally profiled 66 patients, including 17 long-term responders, during standard therapy and after treatment with PD-1 inhibitors (nivolumab or pembrolizumab). Genomic and transcriptomic analysis revealed a significant enrichment of PTEN mutations associated with immunosuppressive expression signatures in non-responders, and an enrichment of MAPK pathway alterations ( PTPN11 , BRAF ) in responders. Responsive tumors were also associated with branched patterns of evolution from the elimination of neoepitopes as well as with differences in T cell clonal diversity and tumor microenvironment profiles. Our study shows that clinical response to anti-PD-1 immunotherapy in GBM is associated with specific molecular alterations, immune expression signatures, and immune infiltration that reflect the tumor’s clonal evolution during treatment.

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults. Afflicted patients have a median survival rate of only 15 months, and patient survival statistics have remained stagnant for over three decades. Current standard of care includes maximal safe tumor resection, chemotherapy, and radiation. Nevertheless, GBM is aggressive, making recurrence and deeper tumor infiltration inevitable. Neural stem cells transdifferentiated from a skin biopsy, i.e. induced neural stem cells (iNSCs), have the innate ability to home to tumors, and, when engineered with cytotoxic proteins, can actively kill cancer cells. However, direct injection of these cells into the hostile immune environment of the tumor resection cavity results in an accelerated clearance rate and therefore a shortened therapeutic window. To combat this clearance issue, we investigated the FDA-approved hemostatic matrix, Floseal, as a cell delivery platform to increase iNSC persistence. In vitro, SEM imaging showed homogeneous iNSC distribution throughout Floseal. Using our surgical resection model of GBM in mice, we delivered iNSCs into the post-surgical cavity in Floseal and by direct injection to model current clinical delivery strategies. Serial kinetic imaging showed that human iNSC persistence delivered into the cavity in Floseal persisted over 90 days, while cells directly injected into the brain parenchyma persist less than 20 days. When we investigated the impact on tumor kill, we found the increase in persistence increased survival of GBM-bearing mice more than 30 days compared to control cells. Light-sheet microscopy showed wild-type neural stem cells migrate into invasive GBM-8 tumors in the contralateral hemisphere, and we are now using this approach to validate the homing of iNSCs delivered via Floseal. Administration of iNSCs encapsulated in the biocompatible Floseal matrix offers a promising, clinically-translatable therapeutic strategy for GBM.

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults. Afflicted patients have a median survival rate of only 15 months, and patient survival statistics have remained stagnant for over three decades. Current standard of care includes maximal safe tumor resection, chemotherapy, and radiation. Nevertheless, GBM is aggressive, making recurrence and deeper tumor infiltration inevitable. Neural stem cells transdifferentiated from a skin biopsy, i.e. induced neural stem cells (iNSCs), have the innate ability to home to tumors, and, when engineered with cytotoxic proteins, can actively kill cancer cells. However, direct injection of these cells into the hostile immune environment of the tumor resection cavity results in an accelerated clearance rate and therefore a shortened therapeutic window. To combat this clearance issue, we investigated the FDA-approved hemostatic matrix, Floseal, as a cell delivery platform to increase iNSC persistence. In vitro, SEM imaging showed homogeneous iNSC distribution throughout Floseal. Using our surgical resection model of GBM in mice, we delivered iNSCs into the post-surgical cavity in Floseal and by direct injection to model current clinical delivery strategies. Serial kinetic imaging showed that human iNSC persistence delivered into the cavity in Floseal persisted over 90 days, while cells directly injected into the brain parenchyma persist less than 20 days. When we investigated the impact on tumor kill, we found the increase in persistence increased survival of GBM-bearing mice more than 30 days compared to control cells. Light-sheet microscopy showed wild-type neural stem cells migrate into invasive GBM-8 tumors in the contralateral hemisphere, and we are now using this approach to validate the homing of iNSCs delivered via Floseal. Administration of iNSCs encapsulated in the biocompatible Floseal matrix offers a promising, clinically-translatable therapeutic strategy for GBM.

In the version of this article originally published, the graph in Extended Data Fig. 2c was a duplication of Extended Data Fig. 2b. The correct version of Extended Data Fig. 2c is now available online.