We tested whether indomethacin or rosiglitazone treatment could rejuvenate spatial ability and hippocampal neurogenesis in aging rats. Young (4 mo;
= 30), middle-aged (12 mo;
= 31), and aged (18 mo;
...= 31) male Fischer 344 rats were trained and then tested in a rapid acquisition water maze task and then fed vehicle (500 μl strawberry milk), indomethacin (2.0 mg/ml), or rosiglitazone (8.0 mg/ml) twice daily for the remainder of the experiment. A week after drug treatment commenced, the rats were given 3 daily BrdU (50 mg/kg) injections to test whether age-related declines in neurogenesis were reversed. One week after the final BrdU injection (~2.5 weeks after the 1st water maze session), the rats were trained to a find novel hidden water maze platform location, tested on 15 min and 24 h probe trials and then killed 24 h later. During the first water maze session, young rats outperformed aged rats but all rats learned information about the hidden platform location. Middle-aged and aged rats exhibited better memory probe trial performances than young rats in the 2nd water maze session and indomethacin improved memory probe trial performances on the 2nd vs. 1st water maze session in middle-aged rats. Middle-aged rats with more new neurons had fewer phagocytic microglia and exhibited better hidden platform training trial performances on the 2nd water maze session. Regardless of age, indomethacin increased new hippocampal neuron numbers and both rosiglitazone and indomethacin increased subependymal neuroblasts/neuron densities. Taken together, our results suggest the feasibility of studying the effects of longer-term immunomodulation on age-related declines in cognition and neurogenesis.
BackgroundDiffuse midline glioma (DMG) is a universal fatal glial brain cancer in children. We tested our novel multilamellar mRNA lipid particle aggregate vaccine (RNA-LPA, IND19304—Sayour),1 a ...tumor-agnostic treatment platform that encapsulates tumor specific RNA and delivers the payload in a highly immunogenic fashion, as an approach to treating this currently incurable cancer.MethodsUsing the K2 DMG model,2 we implant H3K27M-expressing DMG cells into the 4th ventricle of P1-P3 neonatal C57BL/6 mice. RNA-LPA generated from predicated human H3K27M epitopes or total-tumor mRNA are administered intravenously beginning at day 35. We performed multiparameter 3D geospatial fluorescent microscopy to characterize mRNA transduction. Immunologic responses to treatment were evaluated by multiparameter flow cytometry, microscopy, and cytokine profiling.ResultsMice developed clinical neurological signs of disease by day 30–35. RNA-LPAs targeting human H3K27M epitopes were found to be immunogenic in wild-type mice. Intriguingly, nonspecific enhanced green fluorescent protein (eGFP)-RNA-LPAs resulted in statistically significant survival benefits compared to mice treated with empty LPs. However, tumor-specific RNA-LPAs (either H3K27M-specific or total tumor mRNA-derived) also enhanced survival and additionally resulted in a subset of mice with long-term survival. This survival benefit was observed despite the development of clinical hydrocephalus in mice treated with RNA-LPAs. 3D microscopy established that tumors demonstrated invasive disease and microvascular erosion in mice. We found that mRNA transduces fibroblastic reticular cells (FRCs) in the spleen and lymph nodes, prompting widespread immune activation. Treatment with RNA-LPA led to massive increases in production inflammatory cytokines (i.e. TNF-α) and chemokines (i.e. CCL2), which led to recruitment of the majority of circulating monocytes and lymphocytes to secondary lymphoid organs.ConclusionsRNA-LPAs extend survival in our highly aggressive DMG model, including curative outcomes in cohorts treated with either total tumor or H3K27M RNA-LPs. These data suggest that RNA-LPs are capable of stimulating host adaptive immune responses against established DIPG tumors. Signs of hydrocephalus in treated mice may indicate pseudoprogression due to immunologic response, yet mice were frequently able to survive this development. Future studies will further characterize the immunologic response in these mice and support expansion of our existing IND for a multi-institutional phase I clinical trial for children with DMG, who currently have no curative options.AcknowledgementsWe appreciate funding from the ChadTough Defeat DIPG Foundation and the DIPG/DMG Research Funding Alliance. John Ligon and Elias Sayour contributed equally and are co-senior authors.ReferencesMendez-Gomez H, DeVries A, Castillo P, Stover B, Qdaisat S, Von Roemling C, Ogando-Rivas E, Weidert F, McGuiness J, Zhang D, Chung MC, Li D, Zhao C, Marconi C, Campaneria Y, Chardon-Robles J, Grippin A, Karachi A, Thomas N, Huang J, Milner R, Sahay B, Sawyer WG, Ligon JA, Silver N, Simon E, Cleaver B, Wynne K, Hodik M, Molinaro A, Guan J, Kellish P, Doty A, Lee J-H, Carrera-Justiz S, Rahman M, Gatica S, Mueller S, Prados M, Ghiaseddin A, Mitchell DA, Sayour EJ. mRNA aggregates harness danger response for potent cancer immunotherapy. medRxiv. 2023:2023.03.12.23287108. doi: 10.1101/2023.03.12.23287108.Misuraca KL, Cordero FJ, Becher OJ. Pre-Clinical Models of Diffuse Intrinsic Pontine Glioma. Front Oncol. 2015;5:172. doi: 10.3389/fonc.2015.00172. PubMed PMID: 26258075; PMCID: PMC4513210.Ethics ApprovalWork approved under UF IACUC 202200000375
Abstract
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 antigen-recall 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 RNA-NPs to be feasible, safe and immunologically active.
Conclusion
RNA-NPs reprogram the brain tumor microenvironment while inducing a glioma-specific immune response. We have since received FDA-IND approval for first-in-human trials (IND#BB-19304) in pediatric patients with high-grade gliomas (PNOC020 study, NCT04573140).
Abstract
BACKGROUND
High-grade gliomas are an aggressive subset of CNS tumors in need of novel treatment strategies. However, tumor heterogeneity, the immunosuppressive tumor microenvironment (TME) ...and the lack of immunogenic tumor specific targets have limited the translational power of many immunotherapies for high-grade gliomas. The promising application of mRNA vaccines has the potential to restore immunosurveillance through peripheral and intratumoral reprograming of the TME.
METHODS
Our lab has developed a lipid particle multilamellar aggregate (RNA-LPA) vaccine that encapsulates tumor specific RNA and delivers the payload to the immune system in a highly immunogenic fashion, similar to that of a virus. We sought to assess anti-tumor activity across preclinical murine models and investigate the mechanisms for response.
RESULTS
We demonstrated the immune activation potential of RNA-LPAs encoding for glioma associated antigens (i.e., pp65 and H3K27M) in preclinical murine models. We also showed activity of personalized RNA-LPA in canine and human studies (NCT04573140) for patients with high-grade gliomas. Exogenous RNA is recognized by pathogen recognition receptors (PRRs) which trigger a type I interferon (IFN) response and proinflammatory cytokine production. While most RNA based vaccines activate an innate immune response primarily through toll-like receptor 7 (TLR7), we reveal that RNA-LPAs activate the innate immune system through retinoic acid-inducible gene 1 protein (RIG-I). As a result of PRR signaling, the type I IFN and cytokine/chemokine response (IL-6, G-CSF, TNF-alpha, CXCL9, CXCL10, CCL2, CCL4) mediates massive recruitment of activated dendritic cell and activated T cell in lymphoreticular organs where antigen presenting cells and cytolytic T cell colocalize to initiate an adaptive immune response against glioma specific antigens.
CONCLUSION
RNA-LPAs are a novel platform immunotherapy that initiate anti-tumor immunity against malignant brain tumors through noncanonical activation of intracellular PRRs and widespread recruitment of peripheral blood mononuclear cells to sites of RNA-LPA localization.
Abstract
BACKGROUND
DIPG remains a uniformly fatal disease in dire need of new therapies. There are currently no systemic therapies with proven efficacy against these tumors. To overcome these ...barriers, we developed a systemic mRNA lipid particle (LP) vaccine that localizes to tumors and reticuloendothelial (RE) organs to modulate both innate and adaptive immunity against poorly immunogenic tumors like DIPG.
OBJECTIVE
We sought to assess whether RNA-LPs encoding for model antigens (e.g. H3K27M) would reprogram innate immunity and simultaneously elicit sustained adaptive immunity against diffuse midline glioma (DMG).
RESULTS
RNA-LP encoding for model antigens elicit massive recruitment of nearly all monocytes and lymphocytes to RE organs secondary to a danger response mediated by release of orchestrated cytokines (IL-12, TNF-α, IFN-α) and chemokines (CCL2, CCL4, CXCL9-10). This corresponds to increases in absolute numbers of activated DCs and T cells in the RE organs of mice and reprogramming the glioma tumor microenvironment in canines (pet dogs with spontaneous disease). In neonatal mice inoculated midline with established murine K2 gliomas, we observed that RNA-LP vaccines encoding for H3K27M (beginning at ~day 30) elicit significant long-term survivorship, which appears curative in the bulk of animals treated. While H3K27M specific constructs appeared superior, we observed improved survivorship with irrelevant mRNA species (i.e. pp65 and GFP) suggesting these tumors to be particularly sensitive to innate immune modulation. In mice with DMGs, we observed clinical symptomatology of edema/hydrocephalus followed by improvement in many animals suggesting pseudoprogression from intratumoral inflammation and remodeling.
CONCLUSION
RNA-LPs reprogram the tumor microenvironment of poorly immunogenic tumors in effect making them ‘hot.’ Treatment responses in murine models of advanced DMGs are encouraging. We are advancing a final RNA-LP formulation for FDA-IND submission and early phase clinical trials for DIPG through multi-institutional consortia.
Abstract
BACKGROUND: Diffuse Intrinsic Pontine Glioma (DIPG) remains uniformly recalcitrant and thus necessitates development of novel targeted therapies. The histone mutation in H3K27M is conserved ...in the preponderance of DIPG patients and may be exploited as a neoepitope for cancer immunotherapy. We have developed a novel treatment platform, which leverages the use of clinically translatable nanoparticles (NPs) combined with H3K27M mRNA neoantigens for in vivo activation of dendritic cells and generation of DIPG specific T cells. OBJECTIVE/METHODS: Since neoantigens have been shown to mediate immunologic response through MHC class II, we sought to identify MHCII restricted epitopes spanning the H3.1K27M junction for development of RNA-NP vaccines against DIPG. RESULTS: We identified MHCII restricted H3.1K27M epitopes not present in the wild-type sequence. We constructed a 25-mer sequence (75 amino acids) spanning the range of epitopes, before cloning it into our custom pGEM-4z plasmid containing a poly A tail and 5’ UTR with a T7 promoter for amplification of MHC-II restricted H3.1K27M mRNA. We complexed this RNA with our custom NP formulation and investigated its immunogenicity in C57Bl/6 mice. Unlike MHC-I restricted epitopes (i.e. OVAlbumin’s SINFEKL epitope), MHC-II restricted H3K27M epitopes elicited significant increases in activated CD4 and CD8 central memory T cells. We demonstrate that this central memory phenotype correlates with anti-tumor efficacy and long-term survival outcomes in murine tumor models. CONCLUSION: RNA-NPs can be made readily available for all DIPG patients (and not only HLA specific haplotypes) providing a renewable antigen resource that can be used to continuously vaccinate patients for months/years after diagnosis. Herein, we identify MHCII restricted H3.1K27M epitopes for activation of immunologic central memory necessary for long-lived anti-tumor efficacy.
Abstract BACKGROUND Diffuse Midline Glioma (DMG) is an inoperable pediatric brain tumor with insufficient treatment options. Our group has developed a novel treatment modality for this disease and ...others, using mRNA vaccines consisting of tumor derived antigens in a unique lipid-nanoparticle (NP). METHODS We have treated mice, canines, and human patients with primary brain tumors to define local immunotherapuetic treatment response. We treated our neonatally implanted DMG model with NPs generated from total-tumor mRNA beginning day 31 and used MRI, MR spectroscopy, suprerspectral imaging, and immunofluorescence to visualize and quantify treatment effects. RESULTS In canine glioma patients enrolled in clinical trial, we visualized radiographically enlarging tumors of animals that ultimately became long-term survivors, supporting the concept of a process by which lesion size increases in the absence of true disease progression. In our clinical trials investigating NPs as a treatment for human and canine glioma, we found features of both reactive gliosis and traditional pseudoprogression (immune infiltration), suggesting a new radiographic diagnosis, thus named paraprogression. In mice bearing DMG, NP therapy led to long-term survivors, despite the development of hydrocephalus. We were able to visualize treatment response during the course of NP therapy using MRI. CONCLUSIONS These results are rapidly translatable to aid clinical decision making in patients with brain tumors treated with immunotherapy. Future studies will aim to mitigate effect of paraprogression without compromising benefit of NP therapy.
Abstract
BACKGROUND
Diffuse Intrinsic Pontine Glioma (DIPG) is uniformly fatal. Upon diagnosis, DIPG cannot be safely debulked and systemic therapies have unproven benefit. Immunotherapy can overcome ...these obstacles, but objective responses are rare; and cancer antigens, even when tumor specific (i.e. H3K27M), are usually poorly immunogenic.
OBJECTIVE
We sought to develop a murine model of diffuse midline glioma (DMG) that better recapitulates human disease. In these models, we tested different iterations of mRNA loaded nanoparticle (NP) vaccines for their therapeutic effect. APPROACH: We implanted murine gliomas (expressing H3K27M mutation) midline into developing neonatal brains. We manufactured different mRNA constructs encoding for foreign proteins (e.g. GFP), H3K27M or wildtype H3K27 and evaluated immunogenicity and anti-tumor efficacy. These mRNA vaccines were layered into lipid particles (RNA-NP) and administered intravenously when animals became acutely symptomatic (similar to when patients are diagnosed).
RESULTS
Surprisingly, all mRNA constructs tested were sufficient to elicit anti-tumor efficacy against DMGs. This was true for both antigen specific (H3K27M) and non-antigen specific constructs (GFP, H3K27 wildtype). While H3K27M encoding RNA-NPs were superior to H3K27 wildtype encoding RNA-NPs (p=0.057), the adaptive immune effects appear marginal relative to the innate immune responses generated by all RNA-NP constructs. These innate responses are characterized by induction of type I interferon response from plasmacytoid DCs. Interestingly, these effects could not be recapitulated in murine models of adult type glioblastoma (KR158b) which required antigen specificity for induction of anti-tumor efficacy.
CONCLUSION
These data suggest that pediatric gliomas may be particularly sensitive to innate immunity. We have optimized development of a target H3K27M mRNA construct that balances innate and adaptive bi-directional immune induction. We are completing FDA-IND enabling data to support translation of H3K27M loaded RNA-NPs into human clinical trials.
Cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Here, we create “onion-like” multi-lamellar RNA lipid particle aggregates (LPAs) to ...substantially enhance the payload packaging and immunogenicity of tumor mRNA antigens. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for Toll-like receptor engagement in immune cells, systemically administered RNA-LPAs activate RIG-I in stromal cells, eliciting massive cytokine/chemokine response and dendritic cell/lymphocyte trafficking that provokes cancer immunogenicity and mediates rejection of both early- and late-stage murine tumor models. In client-owned canines with terminal gliomas, RNA-LPAs improved survivorship and reprogrammed the TME, which became “hot” within days of a single infusion. In a first-in-human trial, RNA-LPAs elicited rapid cytokine/chemokine release, immune activation/trafficking, tissue-confirmed pseudoprogression, and glioma-specific immune responses in glioblastoma patients. These data support RNA-LPAs as a new technology that simultaneously reprograms the TME while eliciting rapid and enduring cancer immunotherapy.
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•RNA-LPAs mimic dangerous emboli for lymphoreticular entrapment and systemic immunity•Systemic immunity resets both the peripheral and intratumoral milieu via IFNAR1/RIG-I•RNA-LPAs are safe and effective tumor re-modulators in canines with spontaneous gliomas•RNA-LPAs reprogram the TME and elicit adaptive immunity in human GBM patients
Systemically administered mRNA aggregates (RNA-LPA) transfect lymphoreticular organs, inducing a massive cytokine/chemokine response that rapidly reprograms the tumor microenvironment while mobilizing dendritic cells/lymphocytes to elicit rapid and enduring cancer immunotherapy.
Abstract
BACKGROUND
Although mRNA vaccines have been deployed with great success against COVID-19, unlocking this technology against glioblastoma will necessitate new lipid-nanoparticle formulations ...that overcome cancer tolerance and immunosuppression.
OBJECTIVE/METHODS
We sought to develop a novel mRNA vaccine system to make tolerogenic tumor antigens appear more dangerous through use of unmodified nucleosides (pathogen associated molecular patterns, PAMPs) and highly cationic lipid shells that elicit a systemic damage response against cancer antigens.
RESULTS
We developed a novel vaccine formulation that increases payload packaging of tumor amplified mRNA into multilamellar (onion-shaped) particles for systemic (intravenous) administration. We demonstrate significant immunogenicity and efficacy of multilamellar RNA-NPs in syngeneic murine models for high-grade glioma (KR158b-pp65), and diffuse midline glioma (H3K27M DMG). Remarkably, RNA-NPs significantly improve median survival outcomes of DMG bearing mice beginning therapy at endpoint (Day 35 after midline intracranial implantation). Unlike prototypical mRNA vaccines that activate endosomal toll-like receptors (i.e. TLR7), multilamellar RNA-NPs elicit immunologic response predominantly through intracellular pathogen recognition receptors (RIG-I); long-term survival benefits from RNA-NPs were completely abrogated in RIG-I knockout mice. In canines (pet dogs) with spontaneous gliomas, RNA-NPs elicit massive recruitment/activation of peripheral blood mononuclear cells (PBMCs) which correlate with their trafficking into lymphoreticular organs (in follow-up murine studies). In canines receiving neoadjuvant RNA-NPs, prior to glioma biopsy, we see significant reprogramming of the glioma microenvironment with increased gene signatures for antigen processing/presentation, interferon signaling and cytotoxicity. Upon translation into human clinical trials for glioblastoma patients (NCT04573140), RNA-NPs elicit rapid (within hours) release of cytokines (e.g. IL-1, IL-6, IL-12 TNF-α, interferons) and chemokines (e.g. MIP1α, MCP-1, IP-10), which correlate with mobilization of PBMCs and activation of dendritic cells/CD8 lymphocytes.
CONCLUSION
First-in-human application of systemic multilamellar RNA-NP vaccines results in significant biologic effects and rapid immunologic reprogramming.
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