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Brain tumors remain especially challenging to treat due to the presence of the blood–brain barrier. The unique biophysical properties of nanomaterials enable access to the tumor environment with minimally invasive injection methods such as intranasal and systemic delivery. In this review, we will discuss approaches taken in NP delivery to brain tumors in preclinical neuro-oncology studies and ongoing clinical studies. Despite recent development of many promising nanoparticle systems to modulate immunologic function in the preclinical realm, clinical work with nanoparticles in malignant brain tumors has largely focused on imaging, chemotherapy, thermotherapy and radiation. Review of early preclinical studies and clinical trials provides foundational safety, feasibility and toxicology data that can usher a new wave of nanotherapeutics in application of immunotherapy and translational oncology for patients with brain tumors.

While promising, immunotherapy has yet to be fully unlocked for the preponderance of cancers where conventional chemoradiation reigns. This remains particularly evident in pediatric sarcomas where standard of care has not appreciably changed in decades. Importantly, pediatric bone sarcomas, like osteosarcoma and Ewing’s sarcoma, possess unique tumor microenvironments driven by distinct molecular features, as do rhabdomyosarcomas and soft tissue sarcomas. A better understanding of each malignancy’s biology, heterogeneity, and tumor microenvironment may lend new insights toward immunotherapeutic targets in novel platform technologies for cancer vaccines and adoptive cellular therapy. These advances may pave the way toward new treatments requisite for pediatric sarcomas and patients in need of new therapies.

Background: Since the preponderance of pediatric gliomas are mutationally 'bland,' immune checkpoint inhibitors are unlikely to mediate therapeutic benefit. Alternately, immunologic response can be induced de novo against pediatric gliomas with mRNA cancer vaccines. Messenger RNA represents a paradigm shift in vaccinology (i.e. COVID-19) given its flexibility, commercialization, and propensity to confer rapid protection with only a single vaccine. Objective: We sought to develop a new mRNA platform with an optimized backbone for insertion of both personalized and/or “off the shelf” (i.e. H3K27M) transcripts for rapid induction of anti-tumor activity against pediatric gliomas. Approach: We synthesized an mRNA backbone with optimized 5' and 3' UTRs for delivery of gene transcripts pertinent to pediatric brain tumors using a lipid-nanoparticle (NP) delivery vehicle. This vaccine utilizes a novel engineering design that layers tumor derived mRNA into a lipid-nanoparticle (NP) “onion-like” or multi-lamellar package. Results: We demonstrate immunogenicity of RNA-NPs delivering either personalized glioma mRNA or H3K27M mRNA. RNA-NPs localize to myeloid cells in murine KR158b brain tumors and activate dendritic cells that supplant regulatory intratumoral myeloid populations inducing antigenrecall response with long-term survivor benefit. Our optimized mRNA backbone yielded significantly improved anti-tumor efficacy compared with commercial backbones. We have shown this approach can be refined for co-delivery of immunomodulatory RNAs (i.e. GM-CSF) and/or delivery of siRNAs targeting immunoregulatory axes (PD-L1) in murine brain tumors (GL261). We have since established safety of RNA-NPs in acute/chronic murine GLP toxicity studies without cross-reactivity to normal-brain, and launched a large-animal canine brain tumor trial which demonstrated RNANPs to be feasible, safe and immunologically active. Conclusion: RNA-NPs reprogram the brain tumor microenvironment while inducing a gliomaspecific immune response. We have since received FDA-IND approval for first-in-human trials.

Purpose/Objective(s) Glioblastoma (GBM) is difficult to treat due to isolation behind the blood brain barrier and a notoriously immune-suppressive tumor microenvironment. RNA vaccines have recently shown great promise as activators of immune responses against viral pathogens but their efficacy in cancer is not well understood. Here, we describe a versatile, personalized mRNA-nanoparticle (RNA-NP) platform that can be customized to produce powerful anti-tumor immune activation, reprogram the brain tumor microenvironment, and enable tracking of particle localization to predict vaccine efficacy just two days after treatment. Materials/Methods Lipid mixtures (i.e., DOTAP, cholesterol) were complexed with mRNA encoding tumor antigens and evaluated for their ability to activate anti-tumor immune responses in vitro and in vivo. Particle localization after intravenous injection was assessed to identify constructs that enabled delivery of nucleic acids to intracranial tumors. Iron oxide nanoparticles (IONPs) were then incorporated into the cores of liposomes to enable particle tracking with MRI. Results Intravenous administration of RNA-NPs composed of DOTAP and RNA induced robust activation of innate immune cells resulting in prolonged survival in murine models of subcutaneous and intracranial melanoma. Inclusion of cholesterol in the lipid backbone enabled delivery of nucleic acids across the blood brain barrier and into tumor-associated myeloid cells in intracranial GL261 and KR158b tumors. These cholesterol-bearing liposomes not only activated innate immune cells in the tumor microenvironment (i.e., increased expression of CD80 and MHCII), but also enabled further manipulation of this compartment. Use of RNA-NPs to deliver siRNA targeting PD-L1 resulted in significant reduction in PD-L1 expression among tumor associated myeloid cells, leading to 37% long term survivorship in combination with systemic checkpoint blockade in an otherwise fatal model of GL261. IONPs were also incorporated into the cores of these particles to enable non-invasive tracking of particle localization with MRI. Preliminary experiments utilizing dendritic cells treated with iron-oxide-loaded RNA-NPs ex vivo demonstrate that a single dose of this therapy produces immunologic rejection of a murine melanoma model in which a dendritic cell vaccine currently in clinical trials yields no benefit. Furthermore, quantification of particle accumulation in lymph nodes with MRI correlates strongly with survival outcome (R =, indicating that tracking particle localization may be used to reliably differentiate responders from non-responders just two days after treatment. Conclusion RNA-NPs are a versatile platform for RNA delivery to immune cells. These particles are currently under investigation in canine trials with human clinical trials of our first-generation therapy beginning this year.

INTRODUCTION Glioblastoma (GBM) contains cell populations with distinct metabolic requirements, with fast-cycling cells harnessing aerobic glycolysis, and treatment-resistant slow-cycling cells (SCCs) preferentially engaging lipid metabolism. How the different tumor cells interact with immune cells and how this metabolic heterogeneity shapes the immune landscape in GBM has yet to be understood. OBJECTIVES The objectives are to unravel the various molecular signals and metabolic link that underlie the interaction of SCCs with the GBM microenvironment, in particular with the suppressive immune compartment, and to effectively target these interactions for better therapeutics. METHODS Multiple murine glioma cell lines were used to establish metabolic heterogeneity and communications, while various genetic and pharmacological approaches were applied to assess the effect of disrupting the metabolic interplay between SCCs and the immune system. RESULTS We determined that SCCs exhibit distinct metabolic dependencies, involving preferential lipid metabolism supported by enhanced fatty acid uptake. We also found that tumor progression is regulated by the interaction of SCCs with the immune system and established that SCCs recruit immune suppressive M2-like macrophages to the tumor microenvironment, which in turn work against tumor immune rejection by inhibiting T cell anti-tumor activity. The immune microenvironment shaped by SCCs is marked by specific metabolic features enhancing lipid exchange capacities that are exploited by SCCs to support their survival and functions. Importantly, disrupting lipid metabolic exchange sensitized tumors to chemotherapy. CONCLUSION Our results reveal that metabolic interactions between SCCs and tumor-associated macrophages within the GBM microenvironment play a critical role in the development of drug and immune resistant tumors. This study delineates these metabolic communications and assesses the potential therapeutic effect of disrupting these interactions to treat GBM. The insights generated from this project uncover fundamental principles of the emerging connections between the tumor microenvironment, cell metabolism, anti-tumor immunity, and associated therapeutic vulnerabilities.

Translation of nanoparticles (NPs) into human clinical trials for patients with refractory cancers has lagged due to unknown biologic reactivities of novel NP designs. To overcome these limitations, simple well-characterized mRNA lipid-NPs have been developed as cancer immunotherapeutic vaccines. While the preponderance of RNA lipid-NPs encoding for tumor-associated antigens or neoepitopes have been designed to target lymphoid organs, they remain encumbered by the profound intratumoral and systemic immunosuppression that may stymie an activated T cell response. Herein, we show that systemic localization of untargeted tumor RNA (derived from whole transcriptome) encapsulated in lipid-NPs, with excess positive charge, primes the peripheral and intratumoral milieu for response to immunotherapy. In immunologically resistant tumor models, these RNA-NPs activate the preponderance of systemic and intratumoral myeloid cells (characterized by coexpression of PD-L1 and CD86). Addition of immune checkpoint inhibitors (ICIs) (to animals primed with RNA-NPs) augments peripheral/intratumoral PD-1(+)CD8(+) cells and mediates synergistic antitumor efficacy in settings where ICIs alone do not confer therapeutic benefit. These synergistic effects are mediated by type I interferon released from plasmacytoid dendritic cells (pDCs). In translational studies, personalized mRNA-NPs were safe and active in a client-owned canine with a spontaneous malignant glioma. In summary, we demonstrate widespread immune activation from tumor loaded RNA-NPs concomitant with inducible PD-L1 expression that can be therapeutically exploited. While immunotherapy remains effective for only a subset of cancer patients, combination therapy with systemic immunomodulating RNA-NPs may broaden its therapeutic potency.

Cancer vaccines initiate antitumor responses in a subset of patients, but the lack of clinically meaningful biomarkers to predict treatment response limits their development. Here, we design multifunctional RNA-loaded magnetic liposomes to initiate potent antitumor immunity and function as an early biomarker of treatment response. These particles activate dendritic cells (DCs) more effectively than electroporation, leading to superior inhibition of tumor growth in treatment models. Inclusion of iron oxide enhances DC transfection and enables tracking of DC migration with magnetic resonance imaging (MRI). We show that T2*-weighted MRI intensity in lymph nodes is a strong correlation of DC trafficking and is an early predictor of antitumor response. In preclinical tumor models, MRI-predicted "responders" identified 2 days after vaccination had significantly smaller tumors 2-5 weeks after treatment and lived 73% longer than MRI-predicted "nonresponders". These studies therefore provide a simple, scalable nanoparticle formulation to generate robust antitumor immune responses and predict individual treatment outcome with MRI.

BACKGROUND The lack of appropriate preclinical murine glioblastoma models limits comprehensive toxicity/efficacy evaluation of investigational agents. To overcome this challenge, we evaluated the safety and activity of a new immunotherapeutic technology that we have pioneered (composed of tumor mRNA complexed into a custom lipid-nanoparticle formulation) in client-owned canines (pet dogs) diagnosed with malignant gliomas. OBJECTIVE/ METHODS Canine malignant gliomas were biopsied for generation of personalized tumor mRNA loaded into our custom lipid-nanoparticle (NP) vector. The patients received RNA-NPs intravenously beginning two weeks after their biopsy once weekly (x3) and no other anti-tumor therapeutic interventions. RESULTS Within a few hours after administration, tumor specific RNA-NPs elicited margination of peripheral blood mononuclear cells, which increased in the subsequent days/weeks post-treatment; suggesting that RNA-NPs mediate lymphoid honing of immune cell populations before egress. RNA-NPs also elicited increased: 1) serum interferon-α that spiked at 2 hours; 2) CD80 and MHCII on CD11c+ cells (demonstrating activation of peripheral DCs); and 3) interferon-γ + T-cells (i.e. activated T-cells). After receiving weekly RNA-NPs (×3), the canines had a steady course. Aside from low-grade fevers on the vaccination days, personalized tumor RNA-NPs (1x) were well tolerated with stable blood counts, chemistries, and renal/liver function tests. All patients assessed developed immunologic response with pseudoprogression or stable/smaller tumors by MRI. Although we have treated a small cohort, we have observed improvement in median/overall survival in all canine patients with terminal gliomas receiving RNA-NPs (compared with historical controls). CONCLUSION RNA-NPs were feasible, safe and immunologically active in client-owned canines with terminal gliomas. We have not appreciated significant toxicities in canines that would preclude investigation in humans at 1x dosing. Although these results need to be validated in larger canine data sets, these results suggest safety and activity of tumor specific RNA-NPs in canines with terminal gliomas.

BACKGROUND: Pediatric brain tumors are poorly immunogenic preventing current immunotherapies (i.e. checkpoint blockers) from having significant activity. Unlocking immunotherapy against these malignancies is predicated on reprogramming their immunogenicity. OBJECTIVE/METHODS: We developed a novel treatment platform, which leverages the use of clinically translatable lipid-nanoparticles (NPs) combined with tumor derived mRNA that simultaneously function as both vaccines and immunomodulating agents to reprogram the systemic/local microenvironments of pediatric brain cancers. RESULTS: Systemic tumor mRNA-NPs localize to lymphoid organs (i.e. liver, spleen lymph nodes) and to the intratumoral milieu, activating the immunologic microenvironment therein through release of type I interferon from plasmacytoid dendritic cells. This culminates in a CD4 and CD8 central memory T cell response against H3.1K27M murine gliomas. In immunologically resistant murine tumor models, RNA-NPs induce robust anti-tumor efficacy and mediate synergistic activity in settings where immune checkpoint inhibitors (i.e. anti-PD-L1 therapy) do not confer therapeutic benefit. We demonstrate safety of RNA-NPs in acute/chronic murine toxicity studies and in a client-owned canine (pet dog) diagnosed with a malignant glioma. In our first canine subject, RNA-NPs elicited activation of dendritic cells, release of type I interferon and activation of CD8+ T cells. CONCLUSION: RNA-NPs bypass MHC restriction and can be made readily available for all patients (and not only HLA specific haplotypes); they also provide a renewable antigen resource that can be used to continuously vaccinate patients for months/years after diagnosis. Through partnership with the Pacific Pediatric Neuro-Oncology Consortium (PNOC), we are in the final stages of FDA-IND submission before first-in-human trials.

BACKGROUND Molecular drivers of cancer immunogenicity in brain tumors are still being unraveled. While BATF3 expression, STING, and interferon response factors (IRFs) are necessary for cancer immunogenicity, the presence of type I interferon (IFN-I) is contextual having been reported to elicit both anti-tumoral and pro-tumoral effects. A better understanding of IFN-I signaling mechanisms is necessary to elucidate drivers of brain cancer immunogenicity and resistance. OBJECTIVE We sought to assess the role of IFN-I signaling in brain tumor immunogenicity and response to immune checkpoint inhibitors (ICIs) in ICI sensitive brain tumor models (i.e. GL261). We then sought to develop strategies to reset IFN-I signaling in ICI resistant brain tumor models (i.e. KR158b). METHODS To reset IFN-I signaling in immunologically ‘cold’ tumors unresponsive to ICIs, we developed lipid-nanoparticles (NPs) to deliver mRNA payloads to the brain tumor microenvironment (TME). RESULTS In immune-sensitive GL261 tumors, we showed that early release of IFN-I unlocks cancer immunogenicity and ICI response. Blockade of IFN-I during tumorigenesis (within 24h, but not days later) increases tumorigenicity and abrogates ICI activity in sensitive tumors. In ICI resistant KR158b tumors, we show that systemic administration of tumor-derived RNA-NPs localize to myeloid cells within the TME for simultaneous activation of multiple innate pathways including BATF3 (necessary for effector DCs), IRF5 (necessary for M2 to M1 macrophage reprograming), and IRF7 (necessary for IFN-I production). These RNA-NPs induce near-immediate release of IFN-I (within hours), reprogram the brain TME in an IFNAR1 (IFN-I receptor) dependent manner, and elicit significant anti-KR158b efficacy as a monotherapy. Following IFNAR1 blockade, RNA-NP mediated anti-tumor efficacy was abrogated. We demonstrated safety of tumor-specific RNA-NPs (derived from KR158b) in acute/chronic GLP toxicity studies without normal-brain cross-reactivity, and confirmed feasibility/safety and immunologic activity in large-animal studies. FUTURE DIRECTIONS We have since received FDA-IND approval for first-in-human trials (IND#BB-19304) in glioblastoma patients.

BACKGROUND Chimeric antigen receptor (CAR) modified T cell therapy is a promising treatment strategy for cancer therapy. We developed IL-8 receptor-linked CD70CAR (8R-70CAR), aiming to enhance T-cell intratumoral trafficking and persistence, guided by IL-8-secreting tumors. The preclinical results show that 8R-70CAR T cells induce complete tumor regression and long-lasting antitumor immunity, which necessitate further clinical development of this technology. OBJECTIVE To assess the toxicity of 8R-70CAR T cells, evaluate in vitro tumor recognition and expansion of the 8R-70CAR T cells, and develop clinically compliant SOPs for the CAR T cell manufacture. METHODS The m8R-70CAR (mouse), h8R-70CAR (human), and control CAR were constructed. Toxicology: C57BL/6J mice were intravenously injected with or without 2x106 m8R-70CAR T cells after total body irradiation (5Gy). The body weight, clinical signs, hematology, serum chemistry, serum cytokines, gross pathology, and histopathology of 12 vital organs were evaluated. Tumor recognition: T cells derived from GBM patients were transduced with h8R-70CAR and then co-cultured with CD70+ glioblastoma. INF-g production was measured. Ex vivo expansion: The G-Rex system was utilized for CAR T cell production, and the expansion of h8R-70CAR T cells was assessed by cell count. RESULTS No CAR T cell-related toxicity was observed in mouse study. In addition to a robust antitumor reactivity, the h8R-70CAR T cells present an auto-stimulative property that elicits greater ex vivo expansion compared to other CAR T cells tested. An approximately 1200-fold expansion was achieved during the 2 weeks CAR T cell production without additional stimulation, which allows production of the CAR T cells on a clinical trial scale using < 100mL of whole blood. CONCLUSION The 8R-70CAR T cells are effective, auto-stimulative, and safe. These properties are fundamental for successful clinical applications in cancer treatment. A phase I trial using the 8R-70CAR T cells in newly diagnosed GBM will be initiated soon.

Introduction: Immunotherapy is promising for patients with treatment-refractory head and neck squamous cell carcinoma (HNSCC). However, greater than 80% do not respond to checkpoint inhibitors, suggesting that the highly immunosuppressive immune microenvironment of HNSCC mediates therapeutic resistance. Our group has developed a novel treatment platform utilizing clinically translatable RNA-nanoparticles (NPs) to remodel the systemic and intratumoral HNSCC immune microenvironment to enable potent immunotherapeutic activity. Objective: The purpose of this study is to examine the immunologic effects and antitumor efficacy of tumor-derived RNA-NPs in murine models of oral cavity squamous cell carcinoma (OCSCC). Materials and Methods: Tumor-derived RNA-NPs were prepared by complexing in vitro transcribed mRNA from MOC1 cells (syngeneic murine oral cavity squamous cell carcinoma cell line) with nanoliposomes. These complexes were injected into MOC1 tumor-bearing C57BL/6 mice. Immunogenicity was determined from spleens and tumors by multiparameter flow cytometry and qPCR analysis. Efficacy was assessed based on subcutaneous tumor volume measurements. Results: Personalized tumor RNA-NPs mediated significant increases in systemic and tumor-infiltrating lymphocytes (TILs). Compared to control animals, RNA-NPs increased peripheral CD8+ T cells (p=0.0007) and decreased regulatory immune cells [CD4+CD25+FoxP3+ (Tregs)] (p=0.0043). WIthin the tumor microenvironment, RNA-NPs increased intratumoral CD3+CD8+ T cells (p =0.0278) and CD3+CD4+ T cells (p=0.0307). qPCR on MOC1 tumors revealed elevated expression of the effector cytokine interferon-γ (NP alone vs. RNA-NP; fold change 1.309 vs. 90.13) and reduced gene expression of arginase-1 (NP alone vs. RNA-NP; fold change 1.29 vs. 0.005), suggesting that RNA-NPs can attenuate the immunosuppressive microenvironment. Compared to control animals or mice receiving PD-L1 monotherapy, RNA-NPs mediated significantly greater antitumor efficacy based on subcutaneous tumor volume measurements (p Conclusion: RNA-NPs reprogram the peripheral and intratumoral HNSCC immune microenvironments and unlock antitumor activity in preclinical models. We have successfully generated tumor-derived RNA-NPs as a renewable resource that can be made readily available for HNSCC patients from a small tissue sample. FDA-IND enabling studies are currently under way in preparation for first-in-human trials. Citation Format: Natalie L. Silver, Rekha R. Garg, Hector R. Mendez-Gomez, Kristianna Fredenberg, Spencer Barnes, Frances L. Weidert, Peter Dziegielewski, Sayour J. Elias, Duane A. Mitchell. Personalized RNA nanoparticles remodel the HNSCC immunologic milieu to unlock immunotherapeutic activity [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A18.

BACKGROUND: In a randomized phase III study for recurrent glioblastoma (GBM), combination therapy with immune checkpoint inhibitors (ICIs) nivolumab and ipilimumab failed to improve overall survival. Since ICIs appear to be dependent on host-myeloid (and not tumor-cell) expression of PD-L1 (Tang et al. JCI, 2018), we sought to assess if tumor-mRNA nanoparticles (NPs) could prime the systemic and intratumoral GBM milieu with activated PD-L1+ host-myeloid cells sensitizing immunologically ‘cold’ tumors to immunotherapy. METHODS: We systemically administered personalized tumor-derived mRNA encapsulated in translatable lipid-NPs with excess positive charge for enhanced delivery and transfection of peripheral/intratumoral antigen presenting cells (APCs) in murine and large animal canine malignant glioma models. RESULTS: RNA-NPs activate systemic immunity within twenty-four hours, inducing significant increases in the percentage of CD11c+ myeloid-cells expressing PD-L1 and CD86. After only a single RNA-NP vaccine, the bulk of APCs within the spleen, liver, lymph nodes, bone marrow and tumor display an activated phenotype. These PD-L1+ APCs (CD11c+MHC-II+CD86+PD-L1+ cells from intracranial tumors) did not suppress immunity, but rather, heightened interferon-(IFN)-γ reactivity. Addition of ICIs (to animals primed with RNA-NPs) augmented peripheral/intratumoral PD-1+CD8+ cells and mediated synergistic anti-tumor efficacy in settings where ICIs alone did not confer therapeutic benefit. These synergistic effects were mediated by type–I-interferon released from PD-L1+ plasmacytoid dendritic-cells. In translational studies, personalized mRNA-NPs (from whole tumor-transcriptome) were safe and active in a client-owned canine with a spontaneous malignant glioma. In this patient, RNA-NPs elicited robust immunologic activity with increased percentages of activated PD-L1+ APCs and IFNγ+CD8+ cells. CONCLUSION: RNA-NPs elicit widespread immune activation concomitant with inducible PD-L1 expression on intratumoral APCs that can be therapeutically exploited. Since RNA-NPs bypass cost/complexity of cellular therapeutics, are amenable to central distribution, and can be made within days of tumor resection, these formulations can be expeditiously translated as biomodulators of GBM immunogenicity.

Cancer vaccines may be harnessed to incite immunity against poorly immunogenic tumors, however they have failed in therapeutic settings. Poor antigenicity coupled with systemic and intratumoral immune suppression have been significant drawbacks. RNA encoding for tumor associated or specific epitopes can serve as a more immunogenic and expeditious trigger of anti-tumor immunity. RNA stimulates innate immunity through toll like receptor stimulation producing type I interferon, and it mediates potent adaptive responses. Since RNA is inherently unstable, delivery systems have been developed to protect and deliver it to intended targets in vivo. In this review, we discuss liposomes as RNA delivery vehicles and their role as cancer vaccines.

While recurrent medulloblastoma (MB) remains almost uniformly fatal, our group has shown a trend (p=0.07) toward improved survival in patients receiving autologous tumor-RNA pulsed dendritic cells (DCs) coupled with ex vivo expanded total RNA activated T-cells. However, the advancement of autologous cellular therapeutics remains encumbered by significant cost, complexity and time to generation. To circumvent these challenges, we developed a novel treatment platform, which leverages the use of commercially available and clinically translatable nanoparticles (NPs) that can be combined with tumor-derived RNA for near-immediate induction of systemic immunity against MB. We identified a clinically translatable NP formulation for the delivery of RNA to antigen presenting cells for induction of anti-tumor T-cell immunity. When administered intravenously, RNA-NPs increased expression of co-stimulatory molecules on CD11c+ cells throughout reticuloendothelial organs and within the tumor microenvironment; this phenotype was dependent on type I interferon. Targeted inhibition of type I interferon signaling (via INFAR1-mAbs) abrogated anti-tumor immunity mediated by RNA-NPs. We enhanced the immunogenicity of this platform by simply combining mRNAs encoding for immunomodulatory molecules (i.e. HCV-PAMPs, GM-CSF) or by combining RNA-NPs with immune checkpoint inhibitors. Addition of checkpoint inhibitors to RNA-NPs mediated synergistic anti-tumor activity in settings where PD-1 or PD-L1 inhibition alone did not confer therapeutic benefit. In a pre-clinical cellular immunotherapy model targeting an anaplastic murine MB, RNA NPs superseded DCs in mediating anti-tumor activity. Based on these findings, we are exploring the preclinical safety, efficacy and immunologic effects of RNA-NPs targeting malignant brain tumors in canines (UF-IACUC#:201609430) before first in-human evaluation.

Crossing the blood–brain and the blood–cerebrospinal fluid barriers (BCSFB) is one of the fundamental challenges in the development of new therapeutic molecules for brain disorders because these barriers prevent entry of most drugs from the blood into the brain. However, some large molecules, like the protein transferrin, cross these barriers using a specific receptor that transports them into the brain. Based on this mechanism, we engineered a receptor/ligand system to overcome the brain barriers by combining the human transferrin receptor with the cohesin domain from Clostridium thermocellum, and we tested the hybrid receptor in the choroid plexus of the mouse brain with a dockerin ligand. By expressing our receptor in choroidal ependymocytes, which are part of the BCSFB, we found that our systemically administrated ligand was able to bind to the receptor and accumulate in ependymocytes, where some of the ligand was transported from the blood side to the brain side.