MRNA-based myeloid cell stimulation enhances therapeutic efficacy

Abstract

Disclosed are compositions comprising RNA, for example, an immune-reprogramming RNA such as an RNA encoding IRF8, an RNA encoding NIK, or both, encapsulated by lipids. Some embodiments further comprise RNA encoding an antigen. In various embodiments, the compositions are useful in stimulating the immune system, as vaccines, or as adjuvants to other treatments, such as anti-PD-1 blockade. Also disclosed are methods of treating cancer or infectious disease comprising administration of the composition.

Description

Docket No. 0050.2396-002 (MIT 25731)mRNA-based Myeloid Cell Stimulation Enhances Therapeutic EfficacyRELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 746,840, filed on January 17, 2025. This application also claims the benefit of U.S.Provisional Application No. 63 / 757,054, filed on February 11, 2025. The entire teachings of the above applications are incorporated herein by reference.INCORPORATION BY REFERENCE OF MATERIAL IN XML

[0002] This application incorporates by reference the Sequence Listing contained in the following extensible Markup Language (XML) file being submitted concurrently herewith:a) File name: 0050_2396_002_Sequence_Listing. xml; created January 16, 2026, 24,188 bytes in size.BACKGROUND

[0003] Cancer can develop numerous mechanisms of immune evasion, including immune cell exclusion, downregulation of tumor antigen presentation, and promotion of immunosuppressive immune cell phenotypes, among others. Immunotherapies aim to overcome this immune evasion by triggering immunostimulation or by reprogramming the immune system to generate anti-tumor immunity. These treatment paradigms have improved outcomes in many cancers, and there are now more than 60 FDA-approved immunotherapies. Unfortunately, many patients remain unresponsive to existing therapies. There remains a need, therefore, for continued development of immunotherapies to overcome this resistance to treatment and improve treatment responses.SUMMARY

[0004] Although immunotherapy has achieved long-term survival in a subset of cancer patients, its broader efficacy remains limited, primarily due to suboptimal priming of tumorspecific T cells. This limitation is often driven by an immunosuppressive tumor microenvironment characterized by limited presence of functional antigen-presenting cells (APCs) and inadequate activation of tumor-infiltrating lymphocytes. Disclosed herein, among other embodiments, are compositions comprising lipid nanoparticles (LNPs) utilized to deliver immune reprogramming mRNAs (IR-mRNAs) encoding the NF-KB inducing- 1 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)kinase (NIK) or interferon regulatory factor 8 (IRF8) for in situ reprogramming of immune cells. Specifically, these compositions comprising IR-mRNAs reprogram APCs in tumors, significantly increasing the presence of activated cDCls and immunostimulatory cytokines, resulting in robust priming of anti-tumor CD8+T cells. IR-mRNAs encapsulated in LNPs elicited durable antitumor responses in multiple syngeneic mouse tumor models via both intratumoral and intravenous delivery. Co-administration of IR- and antigen-mRNA elicited robust antigen-specific CD8+T cell responses (an about 10-fold increase), sustained longterm memory, and effectively prevented tumor growth in vaccinated mice. Additionally, adjuvanting influenza vaccines with compositions comprising IR-mRNAs significantly enhanced the humoral response by 5-fold and the cellular response by about 15-fold, underscoring their potential as versatile adjuvants for boosting adaptive immunity and improving therapeutic efficacy.

[0005] In certain aspects, provided herein is a composition comprising one or more of an RNA encoding nuclear factor kappa B (NF-KB) inducing kinase (NIK) and an RNA encoding interferon regulatory factor 8 (IRF8); and lipids encapsulating the RNA.

[0006] In some embodiments, the composition further comprises an RNA encoding an antigen.

[0007] In some embodiments, the antigen is a cancer antigen. In some embodiments, the cancer antigen is a neoantigen. In some embodiments, the cancer is colorectal cancer, bladder cancer, melanoma, lung cancer, prostate cancer, breast cancer, brain cancer, colon cancer, pancreatic cancer, ovarian cancer, and hepatocellular cancer, or lymphoma. In some embodiments, the cancer is colorectal cancer, bladder cancer, or melanoma.

[0008] In some embodiments, the antigen is an infectious disease antigen. In some embodiments, the infectious disease is COVID-19 or influenza. In some embodiments, the infectious disease antigen is a viral antigen. In some embodiments, the viral antigen is a SARS-CoV-2 spike or a hemagglutinin.

[0009] In some embodiments, the RNA encoding NIK comprises SEQ ID NO: 2 or 3. In some embodiments, the RNA encoding IRF8 comprises SEQ ID NO: 6 or 7.

[0010] In some embodiments, the RNA encoding IRF8, the RNA encoding NIK, or both, is a circular RNA. In some embodiments, the RNA encoding the antigen is a circular RNA. In some embodiments, the RNA encoding IRF8, the RNA encoding NIK, or both, comprises at least one of N1 -methylpseudouridine (NIMePsU), 5-methoxy-pseudouridine, psuedouridine, 5-methylcytidine, or a combination thereof. In some embodiments, the RNA- 2 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)encoding the antigen comprises at least one of N1 -methylpseudouridine (NIMePsU), 5-methoxy-pseudouridine, psuedouridine, 5-methylcytidine, or a combination thereof. In some embodiments, each uridine of the RNA encoding IRF8, the RNA encoding NIK, or both, is substituted with NIMePsU. In some embodiments, each uridine of the RNA encoding the antigen is substituted with NIMePsU.

[0011] In some embodiments, NIK comprises SEQ ID NO: 4. In some embodiments, IRF8 comprises SEQ ID NO: 8.

[0012] In some embodiments, NIK is a recombinant human NIK, IRF8 is a recombinant human IRF8, or both. In some embodiments, NIK lacks a negative-regulatory domain (NRD), a tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3)-binding domain, or both.

[0013] In some embodiments, the lipids form a lipid nanoparticle (LNP) encapsulating the RNA encoding IRF8, the RNA encoding NIK, the RNA encoding the antigen, or a combination thereof. In some embodiments, the lipids comprise one or more of: an ionizable lipid, a helper lipid, a structural lipid, and a polyethylene glycol (PEG)-modified lipid.

[0014] In some embodiments, the ionizable lipid comprises a spirocyclic diamine headgroup with branched tails.

[0015] In some embodiments, the LNP or one or more of the lipids localize the composition to the spleen.

[0016] In some embodiments, a protein corona of the LNP or one or more of the lipids comprises a protein that engages a receptor on a splenic macrophage, a splenic dendritic cell, or both. In some embodiments, the receptor is a C-type lectin receptor, a Complement receptor, or a scavenger receptor. In some embodiments, the C-type lectin receptors is dendritic cell-specific intercellular adhesion molecule-3 -grabbing non-integrin (DC-SIGN) or mannose, the Complement receptor is CR3 / 4, or the scavenger receptor is CD36 or SR-bl.

[0017] In some embodiments, a protein corona of the LNP or one or more of the lipids comprises an integrin-binding protein, a phagocytosis-associated protein, or both. In some embodiments, the integrin-binding protein or the phagocytosis-associated protein comprises a vitronectin, a prothrombin / coagulation factor, a fibrinogen, or a combination thereof.

[0018] In some embodiments, a protein corona of the LNP or one or more of the lipids comprises an opsonin. In some embodiments, the opsonin is selected from a thrombospondin-1, an ApoA-IV, or both.

[0019] In some embodiments, the ionizable lipid is AMG56, AMG511, AMG513, AMG514, AMG515, AMG516, AMG517, AMG518, AMG519, AMG520, AMG521,- 3 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)AMG522, AMG523, cKK-E12, or a combination thereof. In some embodiments, the ionizable lipid is AMG514.

[0020] In some embodiments, the LNP or one or more of the lipids localize the composition to the liver.

[0021] In some embodiments, a protein corona of the LNP or one or more of the lipids comprises albumin, ApoE, or both.

[0022] In some embodiments, the ionizable lipid is cKK-E12.

[0023] In some embodiments, the helper lipid is l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), l,2-dioleoyl-3 -dimethylammoniumpropane (DODAP), l,2-dioleoyl-sn-glycero-3 -phosphate (DOPA), l,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), l,2-dioleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DOPG), 1-palmitoyl-2-oleoyl-glycero-3 -phosphocholine (POPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), l-stearoyl-2-oleoyl-sn-glycero-3 -phosphocholine (SOPC), l,2-diphytanoyl-sn-glycero-3 -phosphocholine (4MEPC), or a combination thereof. In some embodiments, the helper lipid is DOPE.

[0024] In some embodiments, the structural lipid is cholesterol, Stigmasterol, betasitosterol, DC-cholesterol, 7-alpha-hydroxy cholesterol, 7 -beta-hydroxy cholesterol, 19-hydroxycholesterol, 20(s)-hydroxycholesterol, 24(s)-hydroxycholesterol, 25-hydroxycholesterol, fucosterol, campesterol, stigamstanol, or a combination thereof. In some embodiments, the structural lipid is cholesterol.

[0025] In some embodiments, the PEG-modified lipid is isdimyristoyl phosphatidylethanolamine polyethylene glycol (DMPE-PEG), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000), DSPE-PEG, DSG-PEG, DPPE-PEG, DOPE-PEG, DPG-PEG, or a combination thereof. In some embodiments, the PEG-modified lipid is DMPE-PEG or DMG-PEG 2000.

[0026] In some embodiments, the lipids have a molar composition of about 20-70% ionizable lipid, about 5-40% helper lipid, about 20-50% structural lipid, and about 0.5-5% PEG-modified lipid. In some embodiments, the lipids have a molar composition of 35 : 16 : 46.5 : 2.5 of ionizable lipid : DOPE : cholesterol : DMPE-PEG or DMG-PEG 2000. In some embodiments, a) the ionizable lipid and b) the RNA encoding IRF8, the RNA encoding NIK, the RNA encoding the antigen, or a combination thereof; are in a weight ratio of about 10:1.- 4 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)

[0027] In another aspect, the disclosure provides a method of inducing IL-12, IFN-a, IFN-P, IFN-y, CD86, IL-2, IL-4, IL-5, IL-6, CTSC, NLRC5, STAT3, CD40, CD80, CXCL9, or a combination thereof in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition.

[0028] In some embodiments, the subject is a human. In some embodiments, the composition is administered intratum orally. In some embodiments, the composition is administered intravenously. In some embodiments, the lipids induce expression of the RNA encoding IRF8, the RNA encoding NIK, the RNA encoding the antigen, or a combination thereof as mRNA in immune cells. In some embodiments, the immune cells are in a spleen.

[0029] In another aspect, the disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition.

[0030] In some embodiments, the method further comprises administering to the subject an anti-PD-1 checkpoint blockade. In some embodiments, the method further comprises administering to the subject an anti-PD-1 therapy, an anti-PD-Ll therapy, or an anti-CTLA4 therapy.

[0031] In some embodiments, the cancer is colorectal cancer, bladder cancer, melanoma, lung cancer, prostate cancer, breast cancer, brain cancer, colon cancer, pancreatic cancer, ovarian cancer, and hepatocellular cancer, or lymphoma. In some embodiments, the cancer comprises colorectal cancer, bladder cancer, or melanoma.

[0032] In some embodiments, the subject is a human. In some embodiments, the composition is administered intratum orally. In some embodiments, the composition is administered intravenously. In some embodiments, the lipids induce expression of the RNA as mRNA in immune cells. In some embodiments, the immune cells are in a spleen.

[0033] In certain aspects, provided herein is a vaccine comprising an RNA encoding an antigen and an RNA encoding interferon regulatory factor 8 (IRF8).

[0034] In some embodiments, the antigen is for a cancer. In some embodiments, the cancer is colorectal cancer or bladder cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is melanoma.

[0035] In some embodiments, the antigen is for an infectious disease, such as COVID-19. In some embodiments, the antigen is hemagglutinin or SARS-CoV-2 spike. In some embodiments, the infectious disease is influenza.- 5 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)

[0036] In some embodiments, one or more of the RNA encoding an antigen and the RNA encoding IRF8 is formulated in a lipid nanoparticle.

[0037] In other aspects, provided herein is a method of eliciting an immune response in a subject, the method comprising administering a therapeutically effective amount of an RNA encoding an antigen and a therapeutically effective amount of an RNA encoding interferon regulatory factor 8 (IRF8).

[0038] In some embodiments, the antigen is for a cancer. In some embodiments, the cancer is colorectal cancer or bladder cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is melanoma.

[0039] In some embodiments, the antigen is for an infectious disease, such as COVID-19. In some embodiments, the antigen is hemagglutinin or SARS-CoV-2 spike. In some embodiments, the infectious disease is influenza.

[0040] In some embodiments, one or more of the RNA encoding an antigen and the RNA encoding IRF8 is formulated in a lipid nanoparticle.

[0041] In some embodiments, the subject is an animal. In some embodiments, the subject is human.

[0042] In other aspects, the disclosure provides methods of treating cancer in a subject, the method comprising administering a therapeutically effective amount of an RNA encoding interferon regulatory factor 8 (IRF8). In some embodiments, the cancer is colorectal cancer or bladder cancer.

[0043] The compositions and methods disclosed herein can drive potent immune cell reprogramming to generate strong adaptive immune responses in the context of cancer immunotherapy and infectious disease vaccination with minimal toxicity. For example, intratumoral delivery directly reprograms immune cells, resulting in potent antitumor responses across multiple syngeneic cancer models following both intratumoral (IT) and intravenous (IV) administration, highlighting the versatility of the compositions for treating accessible lesions as well as metastatic or visceral malignancies. Notably, these responses were durable and conferred protection against tumor rechallenge, indicative of long-term antitumor immunity. Furthermore, co-administration of IR-mRNAs with anti-PD-1 immune checkpoint blockade (ICB) therapy significantly improved tumor regression and long-term survival in immunologically “cold” tumors. The compositions can be a complementary strategy to existing immune checkpoint blockades, which often fail due to insufficient preexisting antigen presentation and T cell priming.- 6 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)

[0044] The compositions can also be used in combination with chosen antigens as adjuvants to significantly augment immune responses for both cancer and infectious disease vaccines, as they are shown to provide both robust induction and sustained T-cell immunity. Notably, complete rejection of a tumor rechallenge demonstrating the durability and potency of the immune response resulting from treatment with the compositions.

[0045] Novel ionizable lipids were developed, designed to improve RNA delivery to splenic dendritic cells and macrophages, and significantly amplified immune responses induced by the compositions, resulting in effective tumor growth inhibition at lower doses. These findings underscore the importance of optimizing both adjuvant potency and delivery efficiency to maximize the therapeutic potential of cancer vaccines. Moreover, adjuvanted vaccines elicited significantly stronger humoral and cellular responses as compared to antigen alone. Another advantage of the compositions is the ability to precisely control the relative doses of antigen and adjuvant (IRF8 / NIK) RNA to enable reproducible responses across a range of antigens and providing a tunable strategy essential for shaping the desired adaptive immunity.

[0046] Treatment with the compositions did not result in sustained weight loss, behavioral abnormalities, or liver or spleen pathology, even at doses up to threefold higher than the therapeutic dose. Serum chemistry markers (ALT, AST) remained within normal ranges, further supporting a favorable tolerability profile. These findings indicate that the disclosed compositions and methods harness the immunostimulatory benefits of NIK and IRF8 while avoiding mechanisms associated with chronic pathway dysregulation.

[0047] Overall, the compositions and methods initiate cascaded signaling pathways that reprogram the tumor microenvironment and direct immune cell fate, ultimately enhancing therapeutic efficacy. Their capacity to improve antigen presentation and promote lymphocyte infiltration, effectively converting “cold” tumors into “hot”, positions them as strong candidates to complement existing immune checkpoint blockers in clinical settings.Moreover, the compositions and methods also demonstrate substantial potential to amplify antigen-specific responses in both cancer and infectious disease vaccines. In cancer vaccination, their ability to elicit unprecedented levels of antigen-specific CD8+T cells supports their use in personalized vaccines targeting patient-derived neoantigens.Additionally, by enhancing both humoral and cellular immunity, IR-mRNAs may serve as potent adjuvants for next-generation infectious disease vaccines, especially in vulnerable populations such as older adults or individuals with compromised immune systems.- 7 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0049] FIGs. 1A-C show interleukin 12 (IL-12) induction in dendritic cells. FIG. 1A shows bright field and fluorescent images. Myeloid cells were isolated from the bone marrow of IL-12-eYFP mice. Myeloid precursor cells were differentiated into dendritic cells (bone marrow-derived dendritic cells, z.e., BMDCs) using Flt3L and GM-CSF. The cells were then used for screens with different formulations. Aliquots of BMDCs received lipid nanoparticle (LNP) preparations with control, IRF8 or NIK mRNA. Scale bar = 50 pm. FIG. IB is a bar graph showing that LNPs with IRF8 or NIK mRNA led to high IL- 12 induction in roughly 30% of cells at the highest concentration tested (200 ng / lOOk cells). FIG. 1C is a bar graph showing that IL- 12 induction was the highest in the BMDCs treated with either NIK or IRF8 LNP preparations (n = 3 replicates; MFI, mean fluorescent intensity).

[0050] FIGs. 2A-B show in vitro activation of BMDCs by IRF8 mRNA. IRF8 mRNA was added at two different time points during the myeloid cell culture: at day 0 only or at day 0 and day 5. Then these two groups of BMDCs were analyzed at day 9 by flow cytometry to compare the expression of CD103+XCR1+ populations (type 1 conventional dendritic cells, / .<?., cDCl cells). FIG. 2A shows a representative flow gating strategy for quantifying cDCl populations as the percentage of total cells. WT, wild type untreated cells; SSC, side scatter. FIG. 2B shows populations of cDCl cells gated in CD103+XCR1+ for each group of BMDCs (lower panel) and a significant increase in cDCl population when the cells were treated with IRF8 on day 0 and day 5 (upper panel).

[0051] FIGs. 3A-C show expression of co-stimulatory molecules CD80 and CD86 as necessary second signal to ensure T cell activation. BL6 mice were injected intravenously with LNP_NIK (n = 3) and LNP_control (n = 3) mRNA at a dose of 5 pg per mouse. Spleen and inguinal lymph nodes (LN) were harvested from the mice after 48 hours. FIG. 3 A shows a representative flow gating strategy for quantifying the expression of CD80 and CD86 by flow cytometry gated in CD45+CD1 lc+ populations (n = 3 mice). F4 / 80, murine macrophage marker. FIGs. 3B and 3C are graphs showing the high expression of CD80 and CD86 in spleen (FIG. 3B) and LN (FIG. 3C) harvested from the mice that received NIK mRNA.- 8 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)

[0052] FIGs. 4A-E show in vivo efficacy in MC38 tumors. FIG. 4A is a graph that quantifies tumor growth in PBS control-treated mice. FIG. 4B is a graph that quantifies tumor growth in LNP NIK treated mice. FIG. 4C is a graph that quantifies tumor growth in LNP IRF8 treated mice. FIG. 4D is a survival curve of different mouse cohorts. FIG. 4E is a graph that shows tumor rejection in treated mice after rechallenging the surviving mice with MC3890 days post first dose. A remarkably long term memory response was elicited by both NIK and IRF8. I.V., intravenous.

[0053] FIGs. 5A-C show in vivo efficacy in MB49 tumors. FIG. 5A shows graphs that quantify tumor growth in different cohorts of mice. I.T., intratumoral. FIG. 5B is a graph that shows significant tumor suppression in the mice treated with either NIK or IRF8 by both administration routes. FIG. 5C is a survival curve of different mouse cohorts.

[0054] FIGs. 6A-B show quantification of peripheral blood T cells with ovalbumin (OVA) tetramer as a proportion of CD45+CD8+ T cells. BL6 mice were injected intravenously with LNP_NIK and LNP_OVA (5 pg NIK + 5 pg OVA per mouse) and LNP OVA (10 pg per mouse) as control at day 0 (prime dose) and again at day 12 (booster dose). Retro-orbital blood was drawn at Days 7, 19, 60, and 90 to quantify the peripheral blood T cells by OVA tetramer staining. FIG. 6A is a representative flow gating strategy for percentage of CD8 T cells in tetramer positive population FIG. 6B is a graph showing the marked difference in CD8 T cell population in the blood from mice treated with NIK and OVA compared to those with only OVA after booster dose. CD8 T cells were found to be persistently high in the treated group even after 90 days.

[0055] FIG. 7 is a graph showing that co-delivery of NIK or IRF8 mRNA significantly increased influenza hemagglutinin H3 -specific IgG titers. BALB / c mice (n = 5 per group) were immunized at day 0 with 0.1 pg H3 mRNA in combination with control, NIK or IRF8 mRNA. Sera were collected 2 weeks post-prime and assessed for H3 -specific IgG by enzyme-linked immunosorbent assay (ELISA).

[0056] FIGs. 8A-8B are a series of graphs showing levels of spike-specific IgG titers and antigen-specific T-cells in BALB / c mice (FIG. 8A) and C57BL / 6 mice (FIG. 8B). N = 5 mice per group were immunized at day 0 and day 21 (prime-boost regimen) with spike mRNA, a 1 : 1 combination of spike and IRF8 mRNA, and a 1 : 1 combination of spike and NIK mRNA with a total dose of 0.1 pg mRNA. Serum and Splenic immune cells were collected on day 35 and assessed for spike-specific IgG titers and antigen-specific T-cells using ELISpot assays. Both BALB / c (FIG. 8A) and C57BL6 (FIG. 8B) mice showed significant improvement in- 9 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)IgG titers in NIK and IRF8 adjuvanted formulations, while improved T cell responses were observed in BL6 mice only. This difference can be attributable to the weaker T-cell response generated by BALB / c mice, which are intrinsically Th-2 biased.

[0057] FIG. 9 is a schematic illustration showing reprogramming of the tumor microenvironment upon treatment with NIK or IRF8 mRNA.

[0058] FIGs. 10A-J. IR-mRNA Reprograming of BMDCs. Myeloid cells were isolated from the bone marrow of P40-IRES-eYFP and C57BL / 6J mice, treated with growth factors and IR-mRNA, then immune reprogramming was evaluated in the BMDCs. IL-12p40 eYFP reporter expression levels in treated BMDCs from IL-12p40-eYFP reporter mice are shown in FIG. 1 A. FIG. 10A is an image analysis quantifying IL-12p40-eYFP reporter expression following treatment of BMDCs with 20 ng of mRNA. Mean ± standard deviation (S.D.) shown, n = 3 wells (pooled from 5 mice). FIG. 10B shows expression of IFN-a and IFN-P in the cell culture supernatant following treatment of wild-type BMDCs with 20 ng of mRNA. Mean ± S.D. shown, n = 3 wells (pooled from 5 mice). FIG. 10C is a flow cytometry quantification of maturation markers in BMDCs from C57BL / 6J mice following treatment with 50 ng of mRNA. Mean ± S.D. shown, n = 3 wells (pooled from 5 mice). FIG. 10D shows a scheme for cDCl phenotyping of BMDCs following mRNA treatment at days 0, 5, and 9. FIG. 10E shows percentage cDCl cells after IR-mRNA treatment. Mean shown, n = 6 wells (pooled from 10 mice). FIG. 10F shows UMAPs showing sc-RNAseq data from BMDCs isolated from C57BL / 6J mice following IRF8 treatment. FIG. 10G shows feature plots of IL12p40 expression across cell clusters identified in FIG. 10F. Mean ± S.D. shown, n = 3 wells. FIGs. 10H-10J show monocole pseudotime reconstruction of DC progenitors, pDC, cDCl, and cDC2 populations following treatment of BMDCs with mRNA. Psuedotime analysis showing the developmental path of mRNA-treated BMDCs (FIG. 10H), cell-type labels overlaid on the pseudotime trajectory analysis (FIG. 101), and mRNA treatments overlaid on the pseudotime trajectory analysis (FIG. 10J). Statistical significance was analyzed using a two-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance (FIGs. 10B, 10C) or a one-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance (FIGs. 10A, 10E). Data are representative of three independent experiments (FIGs. 10A-10E), and two independent experiments (FIGs. 1 OF- 10 J).

[0059] FIGs. 11 A-E. IR-mRNA Active APCs In Vivo. In a strategy for evaluating APC activation, cKK-E12 mRNA-LNPs were injected intravenously into C57 / BL6J mice. Blood- 10 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)was collected 6 hours after injection. Flow analysis was performed on spleen and lymph node collected 48 hours after injection. FIG. 11 A shows serum cytokine concentrations 6 hrs. after administration of mRNA LNPs. Mean ± S.D. shown, n = 4 mice. FIGs. 1 IB and 11C show CD86 quantification in lymph node (FIG. 1 IB) and splenic (FIG. 11C) DCs 48 hrs. after administration of mRNA LNPs. Mean ± S.D. shown, n = 5 mice. CD86 quantification in lymph node (FIG. 1 ID) and splenic (FIG. 1 IE) macrophages 48 hrs. after administration of mRNA LNPs. Mean ± S.D. shown, n = 5 mice. Statistical analysis was performed using a mixed-effects model with the Geisser-Greenhouse correction and an uncorrected Fisher’s least significant difference, with individual variances computed for each comparison (FIG.11 A), or a one-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance (FIGs. 1 IB-1 IE). Data are representative of two independent experiments (FIGs. 1 IB-1 IE), and one independent experiment (FIG. 11 A).

[0060] FIGs. 12 A- J. IR-mRNA Treatment Initiates Immune Cell Activation and Recruitment in Tumors. For evaluation of tumor immunophenotype following mRNA LNP treatment, tumors were implanted into mice and intratumoral injections were administered at day 0. At day 1, tumor, spleen, and TdLN were collected to assess innate immunity (e.g., activation of DCs and lymphocytes). At day 7, tumor, spleen, and TdLN were collected to assess adaptive immunity (e.g., immune cell infiltration in tumors, expansion of effector T cells). FIG. 12A shows DC CD86 levels in TdLN, spleen, and tumor after treatment. Mean ± S.D. shown, n = 4 mice. FIG. 12B shows cDCl counts in the TdLN day one and day seven following treatment. Mean ± S.D. shown, n = 4 mice. 12C is a representative flow cytometry analysis of CD69 activation in y6 T cells in tumors on Day 1. FIGs. 12D and 12E are quantitative results showing percentage of CD69+ y6 T cells in the tumor on Day 1 (FIG. 12D) and number of CD69+ y6 T cells in the tumor on day 7 (FIG. 12E). Mean ± S.D. shown, n = 4 mice for LNP -treated groups n = 2 or 3 mice for PBS treated groups. FIG. 12F is a representative flow cytometry analysis of CD69 activation of NKT in tumors on Day 1. FIGs. 12G and 12H are quantitative results showing percentage of CD69+ NKT cells in the tumor on Day 1 (FIG. 12G) and number of CD69+ NKT cells in the tumor on Day 7 (FIG.12H). Mean ± S.D. shown, n = 4 mice for LNP -treated groups n = 2 or 3 mice for PBS treated groups. FIG. 121 shows CD4+and CD8+T cell counts in the tumors on day seven after treatment. Mean ± S.D. shown, n = 5 mice. FIG. 12J shows ratios of CD8 to Treg cells in the tumor on day seven following treatment. Mean ± S.D. shown, n = 3 mice for PBS group, 4 mice for FLuc group, and NIK / IRF8 groups. Statistical analysis was performed using one-- 11 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance. These data are representative of three independent experiments.

[0061] FIGs. 13A-G. IR-mRNA LNPs improve therapeutic outcomes with checkpoint inhibitors in aggressive tumor models. In an intratumoral treatment regimen for in vivo efficacy evaluation of NIK and IRF8 mRNA LNP therapy with MC38 tumors, C5BL / 6J mice were inoculated subcutaneously with 0.6 * 106MC38 cells at day 0. After the tumor reached about 100 mm3in size (e.g., at day 5), the lesion was injected intratumorally with a 5 pg dose of cKK-E12 mRNA-LNPs on days 7, 14, and 21. Control mice were sacrificed on day 30 and surviving mice were rechallenged on day 60. FIG. 13 A shows tumor volumes and FIG. 13B shows survival curves of different mouse cohorts, n = 9 mice for PBS, 10 mice for FLuc, 15 mice for IRF8, and 16 mice for NIK. FIG. 13C shows tumor growth curves following rechallenge of mice showing complete tumor rejection with MC38 cells on the opposite flank on day 60. FIG. 13D is a schematic illustrating the combination therapy approach using IV or IT administration of IR-mRNAs together with anti-PD-1 in MB49 tumor models. C57BL / 6J mice were SC inoculated with 0.2 * 106MB49 cells. When tumors reached about 100 mm3, mice received mRNA-LNPs (5 pg IT or 10 pg IV) in combination with anti-PD-1 antibody (10 mg / kg, administered intraperitoneally twice per week). Treatment groups included PBS (n = 5), anti-PD-1 alone (n = 7), and anti-PD-1 combined with FLuc, IRF8, or NIK mRNA-LNPs (n = 8 each). Tumor growth curves for each treatment group (FIG. 13E) and survival curves following IV (FIG. 13F) and IT (FIG. 13G) mRNA-LNP administration, n = 5 mice for PBS only, 7 mice for anti-PD-1 only, 8 mice for all other treatment groups. Statistical analysis was performed by the log-rank (Mantel-Cox) test (FIGs. 13B, 13F, 13G).

[0062] FIGs. 14A-H. IR-mRNAs generate systemic anti -turn or immunity exhibit therapeutic efficacy in metastatic tumors. The systemic treatment regimen for in vivo efficacy evaluation of NIK and IRF8 mRNA therapy in metastatic B16-F10 tumors was as follows: B6 albino mice were inoculated systemically (IV) with 1 * 106B16-F10-Fluc melanoma cells, followed by intravenous administration of a 10 pg dose of cKK-E12 mRNA-LNPs on day 5 and Day 9 post-tumor injection. Mice were sacrificed and lungs were harvested on day 15. B16-F10 tumor growth in lungs was monitored using IVIS imaging (FIG. 14B) and quantified (FIG. 14A). n = 10 mice per group. FIG. 14C shows representative H&E staining of treated and control lungs. In FIG. 14D, metastatic lung burden is shown as a percentage of lung tissue after NIK or IRF8 treatment, n = 10 mice. FIG. 14E shows representative immunofluorescence images of tumor infiltrating CD8+ T cells and FIG. 14F- 12 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)shows quantified results, n = 5 mice. FIG. 14G shows representative IHC images of lung tissue stained for Ki-67 and FIG. 14H shows quantified results, n = 5 mice. Statistical analysis was performed using a one-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance (FIGs. 14D, 14F, 14H).

[0063] FIGs. 15A-I. IR-mRNAs boost CD8+T cell response for cancer vaccines.C57BL / 6J mice were immunized at days 0 and 14 with OVA (10 pg), and OVA + FLuc / NIK / IRF8 (5 + 5 pg) mRNA-LNP. Blood samples were collected on Days 7, 28, 60, and 90 for quantification of OVA-specific CD8+T cells. Vaccinated mice were inoculated with 0.3 x 106B16-Ova cells subcutaneously on Day 90 and monitored for tumor growth. FIGs. 15A and 15B show representative flow cytometry analysis of the percentages of circulating OVA-specific CD8+T cells (FIG. 15 A) and quantified results (FIG. 15B). Mean ± S.D. shown, n = 5 mice. FIGs. 15C and 15D show tumor volume (FIG. 15C) and survival curves (FIG. 15D) following Bl 6-F 10 challenge. FIG. 15E is a schematic overview of the timeline for cancer vaccination using spleen-tropic LNPs (AMG514) and control LNPs (cKK-E12). Mice were immunized at days 0 and 14 with AMG514 (OVA, 3 pg), AMG 514 and cKK-E12 (1.5 pg OVA + 1.5 pg NIK) mRNA-LNP. Blood samples were collected on Day 7 and 28 for quantification of OVA-specific CD8+T cells, followed by B16-Ova cell inoculation for tumor challenge on Day 35. FIGs. 15F and 15G show tumor growth curves for different treatment groups (FIG. 15F) and survival curves (FIG. 15G) following tumor challenge, n = 5 mice. FIG. 15H and 151 show representative flow cytometry analysis of the percentages of circulating OVA-specific CD8+T cells (FIG. 15H) and quantified results (FIG. 151). Statistical significance was analyzed using a two-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance (FIGs. 15B, 151) and logrank (Mantel-Cox) test (FIG. 15D).

[0064] FIGs. 16A-E. IR-mRNAs boost the adaptive immune response for influenza vaccines. C57BL / 6J mice were vaccinated via intramuscular injection on days 0 and 14 with either H3 HA mRNA alone (1 pg) or H3 HA mRNA combined with a second mRNA as indicated (0.5 pg + 0.5 pg). FIGs. 16A-16C show Day 35 peptide pool stimulated splenocyte IFN-y ELISPOT images (FIG. 16 A), and quantified SFU for MHC-I (FIG. 16B) and MHC-II (FIG. 16C). Mean shown, n = 5 mice. FIG. 16D shows Day 35 HA binding antibody titers. Mean shown, n = 10 mice for H3, H3 + IRF8, and H3 + NIK, 5 for H3 + FLuc. FIG. 16E shows cytokine levels in the cell culture supernatant of peptide-stimulated splenocytes from vaccinated mice following vaccination. Mean ± S.D. shown, n = 5 mice. Statistical- 13 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)significance was analyzed using an ordinary one-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance (FIGs. 16B-16D), two-way analysis of variance mixed-effects model with the Geisser-Greenhouse correction and Tukey’s multiple comparisons test with individual variances computed for each comparison (FIG. 16E).

[0065] FIGs. 17A-G. Additional tolerability evaluation data. Schematic illustrating the treatment of C57BL / 6J mice with mRNA-LNPs, followed by blood and organ collection at 6 hours and Day 6 post-injection. FIG. 17A shows body weight measurement of the treated mice monitored up to 4 days. Mean ± S.D. shown, n = 3 mice for PBS and 4 mice for all others. FIGs. 17B-17F are graphs showing comparison of AST (FIG. 17B), ALT (FIG. 17C), serum albumin (FIG. 17D), alkaline phosphatase (FIG. 17E), and total bilirubin (FIG. 17F) levels in serum at 6 hour and day 6 post injection. Mean shown, n = 3-4 mice. FIG. 17G is a comparison of IL-1B, IL-6, TNF, CXCL1, CXCL2, GM-CSF, and IFN-y levels in serum at 6 hour and day 6 post injection. Mean ± S.D. shown, n = 3-4 mice. Statistical significance was analyzed using an unpaired t test with Welch correction with the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli.

[0066] FIGs. 18A-F. Additional tumor immune phenotyping characterization. FIGs. 18A-18B are Graphs showing CD86 levels in cDCl (FIG. 18A) and cDC2 (FIG. 18B) in TdLN, spleen, and tumor after IR-mRNA treatment. Mean ± S.D. shown, n = 4-5 mice. FIGs. 18C and 18D show cDC2 (FIG. 18C), pDC (FIG. 18D) counts in the TdLN in day one and day seven following treatment. Mean shown, n = 4-5 mice. FIGs. 18E and 18F show quantitative results of CD69 activated CD4+and CD8+T cells in tumor draining lymph nodes (FIG. 18E), and tumors (FIG. 18F) 7 days post treatment with mRNA-LNPs. Mean shown, n = 4-5 mice. Statistical analysis was performed using a one-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance.

[0067] FIGs. 19A-D. Spirocyclic di-amine ionizable lipid exhibit improved delivery to splenic APCs. The molecular structure of top-performing ionizable lipid AMG 514 is shown in FIG. 50A. FIG. 19A. In vitro transfection data for AMG lipids in DC2.4 cells after treatment with FLuc encapsulating LNPs dosed at 40 ng / well, shown in bar graph format. Mean ± s.d., n = 3 wells per group. RLU, relative luminescence unit. FIG. 19B.Quantification of FLuc protein expression in spleen following IV injection of Top 8 ionizable lipids. Mean ± S.D. shown. FIG. 19C. Liver and spleen expression for AMG514 and cKK-E12 (FLuc mRNA, dose = 1 pg, t = 6 hours). Mean ± S.D. shown. FIG. 19D. Schematic of- 14 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)the Ail4 / Cre mRNA mouse model, and flow cytometry quantification of tdTomato expressing cells in spleen, 48 hours after intravenous injection of Cre mRNA encapsulated in cKK-E12 and AMG514 (dose = 5 pg). Mean ± S.D. shown, n = 4-5 mice. Statistical significance was analyzed using a two-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance (FIG. 19B), an unpaired t test with Welch correction with the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (FIG. 19C), or an ordinary two-way analysis of variance with Sidak’s multiple comparisons tests (FIG. 19D).

[0068] FIGs. 20A-D. IR-mRNA Reprogramming of BMDCs into cDCl phenotype. Representative flow cytometry gating scheme for cDCl phenotyping of BMDCs from C57BL / 6J mice following treatment with PBS (FIG. 20A), Flue (FIG. 20B), IRF8 (FIG. 20C), or NIK (FIG. 20D).

[0069] FIG. 21. Flow cytometry gating strategy for myeloid cell analysis in TdLN. C57BL / 6 mice bearing established MB49 tumors were treated with mRNA-LNPs and sacrificed 24 hours post-treatment. Tumor-draining lymph nodes (TdLNs) were harvested, processed into single-cell suspensions, and stained with antibodies against indicated surface markers for flow cytometric analysis. Gating strategies for macrophages, plasmacytoid dendritic cells (pDCs), type I conventional DCs (cDCl), and type II conventional DCs (cDC2) are shown. Representative flow cytometry plots illustrate myeloid counts 24 hours following mRNA-LNP administration.

[0070] FIG. 22. Flow cytometry gating strategy for myeloid cell analysis in spleen. C57BL / 6 mice bearing established MB49 tumors were treated with mRNA-LNPs and sacrificed 24 hours post-treatment. Spleen was harvested, processed into single-cell suspensions, and stained with antibodies against indicated surface markers for flow cytometric analysis. Gating strategies for macrophages, plasmacytoid dendritic cells (pDCs), type I conventional DCs (cDCl), and type II conventional DCs (cDC2) are shown.Representative flow cytometry plots illustrate myeloid counts 24 hours following mRNA-LNP administration.

[0071] FIG. 23. Flow cytometry gating strategy for myeloid cell analysis in tumors. C57BL / 6 mice bearing established MB49 tumors were treated with mRNA-LNPs and sacrificed 24 hours post-treatment. Tumor was harvested, processed into single-cell suspensions, and stained with antibodies against indicated surface markers for flow cytometric analysis. Gating strategies for macrophages, plasmacytoid dendritic cells (pDCs),- 15 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)type I conventional DCs (cDCl), and type II conventional DCs (cDC2) are shown.Representative flow cytometry plots illustrate myeloid counts 24 hours following mRNA-LNP administration.

[0072] FIGs. 24A-E. Intravenous administration of IR-mRNALNPs inhibits tumor growth in MC38 tumor models. C57BL / 6J (n = 5 per group) mice were subcutaneously inoculated with 0.6 * 106MC38 cells. When tumors reached about 100 mm3(e.g., around day 5), mice received mRNA-LNPs (MD1 formulation, 0.5 mpk, 10 pg) intravenously (IV) on day 6 and day 9. FIGs. 24A-24C. Tumor growth curves for treatment group FLuc (FIG. 24A), IRF8 (FIG. 24B), and NIK (FIG. 24C). FIG. 24D. Survival curves following IR-mRNA treatment. FIG. 24E. Tumor volume on Day 15 after rechallenging the survived mice on Day 90. Statistical significance was analyzed using an ordinary one-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance.

[0073] FIGs. 25A-C. IR-mRNALNPs monotherapy inhibits tumor growth in aggressive MB49 tumor models. C57BL / 6J (n = 5 per group) mice were subcutaneously inoculated with 0.2 x 106MB49 cells. When tumors reached about 100 mm3, mice received mRNA-LNPs either intratumorally (IT) or intravenously (IV). Tumor growth curves for treatment group IRF8 - IT administration (FIG. 25 A), NIK - IV administration (FIG. 25B), and NIK - IT administration (FIG. 25C).

[0074] FIGs. 26A-B. IR-mRNAs boost antibody titers for influenza and SARS-CoV-2 antigens. Day 35 binding antibody titers following vaccination in BALB / cJ mice for IR-mRNA vaccines with influenza (H3) (FIG. 26A) and SARS-CoV-2 (spike) antigen (FIG. 26B). Dosing information: H3 / spike only (0.1 pg), H3 / spike + IRF8 / NIK (0.05 + 0.05 pg), n = 5 mice. The same indicated mRNA dose was used for both vaccine doses and doses were administered on days 0 and 21. Statistical significance was analyzed using an ordinary oneway analysis of variance with Sidak’s multiple comparisons test with a single pooled variance.

[0075] FIG. 27. IR-mRNAs induce cDCl reprogramming in BMDCs. UMAPs showing sc-RNAseq data from BMDCs isolated from C57BL / 6J mice following mRNA treatment. Feature plots show different cell clusters, with cDCl cells identified by expression of the Batf3 gene, a lineage-defining transcription factor for mature cDCls.

[0076] FIG. 28. sc-RNAseq analysis of immature DCs treated with IR-mRNA. Violin plots depicting the probability distribution of gene expression for maturation marks (CD80, CD86, CD83, CD40, and Ccr7), lineage-defining (Spil, Irf8, Ikzfl), antigen-processing- 16 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)(Nlrc5, Psmb9, Ctsc), IFN-signaling (Ifit3, Irf7, Stat2, Stat3), and immune-function (Ptprc) genes.

[0077] FIG. 29. Liver histopathology slides from IR-mRNALNP treated mice.Representative liver sections stained with H&E from mice 6 hrs. (left) and 6 days (right) following treatment with the indicated mRNA LNPs or PBS. LNP -treated mice show signs of single cell hepatocyte necrosis at 6 hrs. which has fully resolved by 6 days. (Scale bar = 200 pm).

[0078] FIG. 30. Spleen histopathology slides from IR-mRNALNP treated mice.Representative liver sections stained with H&E from mice 6 hrs. (left) and 6 days (right) following treatment with the indicated mRNA LNPs or PBS. LNP -treated mice show signs of extramedullary hematopoiesis at 6 hrs. which has fully resolved by 6 days. (Scale bar = 200 pm)

[0079] FIG. 31. IR-mRNAs induce cDCl reprogramming in BMDCs. Flow cytometry quantification of percentage cDCl cells after IR-mRNA treatment at various mRNA doses. Statistical significance was analyzed using a two-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance.

[0080] FIGs. 32A-C. Flow cytometry quantification of maturation markers in BMDCs from C57BL / 6J mice following mRNA treatment at different doses. NIK treatment upregulated both CD40 (18%+) (FIG. 32A) and CD86 (68%+) (FIG. 32B), whereas IRF8 treatment primarily upregulated CD86 (48%+) (FIG. 32B), with minimal effect on CD40 (FIG. 32A). In contrast, FLuc-transfected cells showed minimal marker expression.Additionally, MHC-II expression (FIG. 32C) was markedly increased following NIK (69%+) and IRF8 (53%+) treatment, while MHC-I levels remained unchanged. Mean ± S.D. shown, n = 3 wells. Statistical significance was analyzed using a two-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance.

[0081] FIGs. 33A-B. IR-mRNA stimulate IFN-I responses in vivo. Serum FIG. 33A shows IFN-oc and FIG. 33B shows IFN-[3 level 6 hours after administration of IR-mRNA LNPs. Mean ± S.D. shown, n = 3-4 mice. Statistical significance was analyzed using a oneway analysis of variance with Sidak’s multiple comparisons test with a single pooled variance.

[0082] FIGs. 34A-H. IR-mRNA Treatment Initiates Immune Cell Activation and Recruitment in Tumors. FIG. 34A. DC CD86 levels in TdLN, spleen, and tumor after treatment. Mean ± S.D. shown, n = 4 mice. FIG. 34B. cDCl counts in the TdLN day one and- 17 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)day seven following treatment. Mean ± S.D. shown, n = 4 mice. Percentage of CD69+ y6 T cells and NKT cells in tumors on Day 1 is shown in FIGs. 34C and 34E, respectively. FIGs. Quantitative results showing number of 76 T (FIG. 34F) and NKT (FIG. 34D) cells in the tumor on day 7. Mean ± S.D. shown, n = 4 mice. FIG. 32G. Ratio of CD8 to Treg cells in the tumor on day seven following treatment. Mean ± S.D. shown, n = 4 mice for PBS group, 5 mice for FLuc group, and 7 mice for NIK / IRF8 groups. FIG. 32H. CD4+and CD8+T cell counts in the tumors on day seven after treatment. Mean ± S.D. shown, n = 4 mice. Statistical analysis was performed using one-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance.

[0083] FIGs. 35A-F. Additional tumor immune phenotyping characterization. FIGs. 35 A, 35B. Graphs showing CD86 levels in cDCl (FIG. 35A) and cDC2 (FIG. 35B) in TdLN, spleen, and tumor after IR-mRNA treatment. Mean ± S.D. shown, n = 4 mice. cDC2 (FIG.35C) and pDC (FIG. 35D) counts in the TdLN in day one and day seven following treatment. Mean shown, n = 4 mice. FIGs. 35E, 35F. Quantitative results of CD69 activated CD4+and CD8+T cells in tumor draining lymph nodes (FIG. 35E), and tumors (FIG. 35F) 7 days post treatment with mRNA-LNPs. Mean shown, n = 4-5 mice. Statistical analysis was performed using a one-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance.

[0084] FIGs. 36A-B. Antibody depletion influences the efficacy of NIK and IRF8. Tumor growth curves of MC38 tumor-bearing mice treated intraperitoneally with depleting antibodies against CD8+(FIG. 36B) or CD4+(FIG. 36 A) T cells, followed by intratumoral administration of NIK or IRF8 mRNA-LNPs. Mean ± SEM shown, n = 5 mice. Statistical significance was analyzed using an unpaired t test with Welch correction with the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli.

[0085] FIGs. 37A-H. Immunophenotyping of tumor-infiltrating myeloid and lymphoid cells after IR-mRNA treatment. Flow cytometric quantification of number of CD 8 T cells in FIG. 37A, CD 4 T cells in FIG. 37B, Granulocytes in FIG. 37C, Ly6C high monocytes in FIG. 37D, Macrophages in FIG. 37E, cDCl in FIG. 37F, pDC in FIG. 37G, and cDC2in FIG. 37H in the MB49 tumor on day 7. Mean ± S.D. shown, n = 5 mice. Statistical analysis was performed using a one-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance.

[0086] FIGs. 38A-C. Dose-response studies of BMDC cytokine induction following IR-mRNA treatment. FIG. 38A. BMDCs from IL-12p40-eYFP reporter mice were treated with- 18 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)the indicated dose of IR-mRNA and then imaged via fluorescence microscopy to assess eYFP induction. Mean ± S.D. shown, n = 3 wells from 3 mice. FIGs. 38B, 38C. BMDCs from C57BL / 6J mice were treated with IR-mRNAs, the supernatant collected and characterized for levels of IFN-a (FIG. 38B), IFN-P (FIG. 38C). Mean ± S.D. shown, n = 3 wells from 3 mice. Statistical significance was analyzed using a two-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance.

[0087] FIGs. 39A-B. Combination of IR-mRNAs for influenza and cancer vaccines. C57BL / 6J mice were vaccinated on Days 0 and 14 with either H3 HA mRNA alone (1 pg), H3 HA mRNA combined with NIK or IRF8 mRNA (0.5 pg + 0.5 pg), or H3 HA mRNA (0.5 pg) combined with both NIK and IRF8 mRNAs (0.25 pg + 0.25 pg). FIG. 39A. Day 35 HA binding antibody titers. Mean ± S.D. shown, n = 5-10 mice. C5BL6 Mice immunized at days 0, and 14 with OVA (10 pg), and OVA + NIK or IRF8 (5 + 5 pg) and Ova (5 pg) + NIK and IRF8 (2.5 + 2.5 pg) mRNA. Blood samples were collected on Day 28 for quantification of OVA-specific CD8+ T cells. FIG. 39B. Percentage of circulating OVA-specific CD8+T cells assessed by flow cytometry. Mean ± S.D. shown, n = 5 mice. Statistical significance was analyzed using an ordinary one-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance.

[0088] FIG. 40. Quantification of mRNALNP immune cell transfection in the spleen. Cre mRNA encapsulated in cKK-E12 mRNA LNPs was dosed to Ail4 mice (dose = 5 pg) and after 48 hrs. and spleen was collected for flow cytometry analysis. Mean ± S.D. shown, n = 5 mice. Statistical significance was analyzed using a two-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance.

[0089] FIGs. 41A-B. AMG514 LNP characterization. FIG. 41 A. Normalized protein abundance from the corona of AMG514 LNPs. The protein corona formed on LNPs after incubation in serum is dominated by vitronectin (30.3%), prothrombin / coagulation factors (about 24%), and fibrinogen (9.3%). Lower-abundance but high-potency opsonins such as thrombospondin- 1 (1.0%), ApoE (1.0%), and ApoA-IV (6.9%) are also observed. FIG. 41B. Representative TNS assay curves for determining the apparent pKa of AMG514 LNPs.Apparent pKa was determined by fitting the data with a four-parameter logistic regression and obtaining the IC50.

[0090] FIG. 42. Flow cytometric characterization of protein expression in B16-F10 tumors following IT mRNA-LNP administration. 5 pg of tdTomato mRNA encapsulated in cKK-el2 LNPs were administered IT to C57BL / 6 mice bearing about 150 mm3subcutaneous- 19 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)GFP+B16-F10 and tumors were harvested 24 hours later for analysis. cKK-el2 LNPs transfected about 15% of tumor cells, about 14% of macrophages, about 20% of dendritic cells (cDCls, cDC2s, and pDCs), and about 5% of stromal cells, with minimal uptake in T cells and NK cells. These data indicate that intratumoral delivery of cKK-el2 LNPs results in predominant transfection of antigen-presenting cells (APCs), with additional uptake by tumor and stromal compartments. Mean ± S.D. shown, n = 5 mice.

[0091] FIGs. 43 A-C. IR-mRNA-induced DC activation in the lymph nodes. FIGs. 43 A-43C CD80 (FIG. 43 A), CD86 (FIG. 43B), and CD40 (FIG. 43C) quantification in lymph node DCs 48 hrs. after administration of mRNA LNPs. Mean ± S.D. shown, n = 5 mice. Statistical analysis was performed using a Sidak’s multiple comparisons test with a single pooled variance.

[0092] FIGs. 44A-B. Tumor-infiltrating eDC quantification in MB49 tumors. FIGs. 44A, 44B. Graphs showing cDCl (FIG. 44 A) and cDC2 (FIG. 44B) counts in the Tumor on day one and day seven following treatment. Mean shown, n = 4 mice. Statistical analysis was performed using a one-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance.

[0093] FIG. 45. Weight of lung tissues harvested from B16-F10 metastatic tumors on Day 15 after IR-mRNA treatment. Mean ± S.D. shown, n = 7-10 mice. Statistical analysis was performed using a Sidak’s multiple comparisons test with a single pooled variance.

[0094] FIGs. 46A-B. CD4+ (FIG. 46 A) and CD8+ (FIG. 46B) T cell counts in the tumors on day seven after treatment. Mean ± S.D. shown, n = 4 mice. Statistical significance was analyzed using a two-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance. These data are representative of three independent experiments.

[0095] FIG. 47. Representative fragment analyzer results for FLuc, IRF8, and NIK mRNAs.

[0096] FIGs. 48A-B. Additional data showing in vivo activation of DCs following IR-mRNA treatment. CD86 quantification in lymph node (FIG. 46A) and splenic (FIG. 46B) DCs 48 hrs. after administration of mRNA LNPs. Mean ± S.D. shown, n = 3 mice. Statistical analysis was performed using a one-way analysis of variance with Sidak’s multiple comparisons test with a single pooled variance.

[0097] FIG. 49. Scheme of a general procedure for the synthesis of branched lipid tails.- 20 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)

[0098] FIGs. 50A-C. AMG ionizable lipids AMG511, AMG513, AMG514, and AMG515 (FIG. 50A), AMG516, AMG517, AMG518, and AMG519 (FIG 50B), and AMG520, AMG521, AMG522, and AMG523 (FIG 50C).DETAILED DESCRIPTION

[0099] A description of example embodiments follows.Abbreviations

[0100] BMDC: bone marrow-derived dendritic cell

[0101] CD: cluster of differentiation

[0102] cDCl: type 1 conventional dendritic cell

[0103] cIAP: cellular inhibitor of apoptosis protein

[0104] DC: dendritic cell

[0105] FFL: firefly luciferase

[0106] Flt3L: Feline McDonough Sarcoma (Fms)-like tyrosine kinase 3 ligand

[0107] GM-CSF: granulocyte-macrophage colony-stimulating factor

[0108] H3: influenza hemagglutinin (HA) subtype H3

[0109] IgG: immunoglobulin G

[0110] IL-12-eYFP: enhanced yellow fluorescent protein-tagged interleukin 12

[0111] IL: interleukin

[0112] IRF8: interferon regulatory factor 8; also known as interferon consensus sequence binding protein 1 (ICSBP1)

[0113] LNP: lipid nanoparticle

[0114] MHCII: major histocompatibility complex II

[0115] NIK: NF-KP-inducing kinase; also known as nuclear factor kappa B inducing kinase or mitogen-activated protein kinase kinase kinase 14 (MAP3K14)

[0116] OVA: ovalbumin, a model antigen

[0117] PBS: phosphate-buffered saline

[0118] XCR1: XC-chemokine receptor 1Mouse and Cell Lines

[0119] BALB / c wild-type mice- 21 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)

[0120] BL6 (C57BL / 6) wild-type mice; also referred to as C57BL / 6J (lab code “J” for Jackson Labs, Bar Harbor, ME, USA)

[0121] IL-12-eYFP transgenic mice (C57BL / 6 background)

[0122] MB49 mouse (C57BL / 6) bladder carcinoma cell line (Cat. No. SCC148, Millipore Sigma, Burlington, MA, USA)

[0123] MC38 mouse (C57BL / 6) colon adenocarcinoma cell line (Cat. No. SCC172, Millipore Sigma, Burlington, MA, USA)

[0124] B16-F10 mouse (C57BL / 6J) melanoma cell line

[0125] B16-F10-Luc2 reporter-labeled mouse (C57BL / 6J) melanoma cell line

[0126] ^6.C -Gt(ROSA)26Sortml4(CAG~tdTomato)HzeIJ (Ail4D) reporter mouse modelIntroduction

[0127] Cancer can develop numerous mechanisms of immune evasion, including immune cell exclusion, downregulation of tumor antigen presentation, and promotion of immunosuppressive immune cell phenotypes, among others (Refs. 1-3). Immunotherapies aim to overcome this immune evasion by triggering immunostimulation or by reprogramming the immune system to generate anti-tumor immunity. These treatment paradigms have improved outcomes in many cancers, and there are now more than 60 FDA-approved immunotherapies (Ref. 4). Unfortunately, many patients remain unresponsive to existing therapies (Refs. 5,6). There remains a need, therefore, for continued development of immunotherapies to overcome this resistance to treatment and improve treatment responses.

[0128] mRNA lipid nanoparticle (LNP)-based therapeutics have emerged as a potent modality for the transient expression of proteins, for a range of disease applications (Ref. 7). These systems have the potential to address the unmet needs of existing immunotherapies by enabling transient expression of cytokines (Ref. 8). Transient delivery of mRNA-encoded effector molecules can augment adaptive immunity while mitigating toxicities from sustained exposure (Refs. 9, 10). For example, RNA encoding pro-inflammatory cytokines delivered either intratum orally (IL-23, IL-36g and OX40L cocktail) or systemically (IL- 12) have been used to generate anti-tumor responses (Refs. 11, 12). Similarly, co-delivery of IL-12 mRNA has shown to boost the immune response to SARS-CoV-2 vaccines in preclinical studies (Ref. 13). Despite this progress, effector-centric strategies have limitations: cytokine signals are often self-limiting when devoid of the context of additional signaling molecules (Refs. 14, 15). Secreted cytokines may also diffuse away from immune cells, thereby preventing- 22 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)appropriate local immune activation while simultaneously causing systemic adverse events (Refs. 16-18). While these previous studies have utilized secreted or transmembrane immune effectors, mRNA-LNPs also enable cytosolic expression of lineage-defining transcription factors and signaling kinases that control cell fate and activation states of immune cells. In contrast to cytokines and surface receptors, these intracellular factors can be used to simultaneously activate a diverse set of signaling cascades and reprogram immune cell phenotypes.

[0129] Given the potential advantages of intracellular factors, the studies disclosed herein sought to identify factors which could be used to reprogram myeloid cell subsets such as macrophages and dendritic cells (DCs), which orchestrate tumor-antigen presentation and priming of cytotoxic T cells (Refs. 2, 19). We identified two factors that could affect myeloid cell reprogramming - the NF-KB-inducing kinase (NIK) and interferon regulatory factor 8 (IRF8). NIK, a pivotal activator of the non-canonical NF-KB pathway that lies downstream of CD40 signaling, enhances DC-T-cell crosstalk and augments responses to checkpoint blockade (Refs. 2022). IRF8 is a lineage-defining factor required for cDCl development and antigen cross-presentation (Refs. 23-25). Both act upstream of multiple effector signals and can drive pro-inflammatory cytokine production and myeloid polarization toward antitumor states (Refs. 26, 27). Exploiting NIK therapeutically, however, has proven difficult. Prior approaches that rely on cellular inhibitor of apoptosis proteins (cIAPs) inhibition to stabilize NIK and trigger non-canonical NF-KB signaling have been limited by efficacy and toxicity, and there are no clinically deployed NIK agonists (Refs. 28, 29). Previous efforts to harness IRF8 have been more successful than those directed toward NIK; lentiviral delivery of IRF8 with other transcription factors (PU.1 / BATF3) has been shown to reprogram various cells into cDCl-like phenotypes, resulting in improved T cell priming and tumor regression in vivo (Refs. 30, 31). However, this strategy has only been demonstrated for ex vivo engineering or intratumoral administration, which is limited by the high cost of ex vivo DC vaccines and the invasive nature of intratumoral administration (Refs. 32, 33). Moreover, the use of lentiviral vectors in vivo presents challenges due to anti-vector immune responses and the resultant long-term expression of immunostimulatory genes (Refs. 34, 35). According to the present disclosure, it is hypothesized that mRNA-LNP delivery of NIK or IRF8, termed immune reprogramming mRNAs (IR-mRNAs), could overcome these limitations and enable productive myeloid cell reprogramming and immune responses.- 23 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)

[0130] Therefore, either NIK or IRF8 mRNA were formulated into LNPs; it was found that they induced strong antitumor efficacy across multiple syngeneic models via intravenous (IV) or intratumoral (IT) dosing and generated durable immune memory, evidenced by complete protection upon rechallenge. Additionally, to assess cancer vaccine potential, these IR-mRNAs were co-delivered with OVA mRNA (encoding a model antigen; see SEQ ID NO: 9) which significantly increased circulating OVA-specific CD8+T cells to approximately 25%. This response was also durable, persisting at least 90 days, and effectively prevented Bl 6-0 VA tumor growth in vaccinated mice. Furthermore, these IR-mRNAs markedly boosted adaptive immune responses when used with hemagglutinin (influenza A H3N2) and spike (SARS-CoV-2) antigens, highlighting their broad applicability for both cancer and infectious disease therapeutics. Mechanistic investigations reveal that these immune stimulants work by (i) activating APCs and reprogramming immature DCs into a cDCl phenotype, (ii) inducing expression of pro-inflammatory signals to recruit additional immune cells to the tumor microenvironment, and (iii) enhancing priming and activation of CD8+T cells (FIG. 9).

[0131] Overall, this approach contrasts with mRNA therapeutics that rely on delivering isolated effector molecules such as cytokines or co-stimulatory receptors. NIK and IRF8 instead serve as pleiotropic immune activators, engaging multiple distinct immune pathways through a single mRNA payload. This advancement in mRNA target design lays the foundation for next-generation therapies, potentially enabling diverse applications to enhance immune responses in contexts such as cancer immunotherapy and vaccination, with broad translational potential.Conclusions

[0132] Reprogramming of myeloid cells from an immature state to a mature, professional antigen-presenting phenotype has the potential to enhance immune responses against pathogenic or tumor-associated antigens, as demonstrated in the present disclosure. Delivery of mRNAs encoding intracellular cytosolic regulators remains largely underexplored for APC reprogramming. Here, mRNA LNPs were utilized to deliver the IR-mRNAs (NIK and IRF8) driving potent APC reprogramming to generate stronger adaptive immune responses in the context of cancer immunotherapy and infectious disease vaccination. Using flow cytometry and scRNA-seq, treatment of immature BMDCs with IR-mRNAs was demonstrated to drive their reprogramming toward a cDCl-like phenotype. This was evidenced by the upregulation- 24 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)of key maturation markers (CD40, CD80, CD86) and genes associated with antigen presentation and T cell activation (CTSC, NLRC5, STAT3, IL-12, IFN-a / p) (FIG. 10). IR-mRNA treatment also induced expression of pro-inflammatory cytokines, including IL-12 and type I interferons, which enhance T cell recruitment and activation. Importantly, RNA-seq revealed that IR-mRNAs upregulated lineage-defining transcription factors such as Batf3, Spil, and IRF8. In parallel, these cells exhibited increased expression of surface markers (MHC II, CD8, CD103, XCR1) characteristic of cDCls, indicating myeloid reprogramming into a cDCl-like state (FIG. 10, FIG. 27, FIG. 28). Although NIK and IRF8 initiate distinct signaling cascades, both ultimately amplify transcriptional programs associated with cDCl maturation, antigen processing, and type I IFN responsiveness. This convergence on a shared cross-presenting phenotype provides a mechanistic explanation for their similar antitumor effects. Thus, IR-mRNAs can act through complementary upstream pathways that funnel into a unified cDCl -driven immune response. When formulated into LNPs and administered to mice, these IR-mRNAs showed similar activity with minimal toxicity — a surprising result in view of the potency of the IR-mRNAs in stimulating immune activity. Much like the BMDC results, mice treated with IR-mRNA LNPs had increased levels of critical APCs and serum levels pro-inflammatory cytokines, indicating the in vivo activity of these IR-mRNAs (FIG.11, FIG. 18).

[0133] In syngeneic tumor-bearing mice, administration of IR-mRNAs induced robust APC activation and expanded the cDCl population within tumors and tumor-draining lymph nodes (TdLNs). This was accompanied by rapid recruitment of 76 T cells and NKT cells, followed by infiltration of CD8+T cells (FIG. 12). cKK-E12 mRNA-LNPs were taken up predominantly by APCs (about 35%), with additional transfection of tumor cells (about 15%) and stromal cells (about 5%). These data suggest that intratumoral delivery directly reprograms APC subsets, while transient NIK / IRF8 expression in tumor and stromal compartments may also contribute to remodeling of the TME (FIG. 42). Such immune remodeling resulted in potent antitumor responses across multiple syngeneic cancer models following both intratumoral (IT) and intravenous (IV) administration, highlighting the platform’s versatility for treating accessible lesions as well as metastatic or visceral malignancies. Notably, these responses were durable and conferred protection against tumor rechallenge, indicative of long-term antitumor immunity (FIG. 13, FIG. 14). Furthermore, coadministration of IR-mRNAs with anti-PD-1 immune checkpoint blockade (ICB) therapy significantly improved tumor regression and long-term survival in immunologically “cold”- 25 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)MB49 tumors (FIG. 13). These IR-mRNAs may, therefore, hold promise as a complementary strategy to existing immune checkpoint blockades, which often fail due to insufficient preexisting antigen presentation and T cell priming.

[0134] In addition to potentiating anti-tumor responses alone or in combination with immune checkpoint blockade, these IR-mRNAs can also be used in combination with chosen antigens as adjuvants to significantly augment immune responses for both cancer and infectious disease vaccines. Co-admini strati on of IR-mRNAs with OVA enhanced CD8+T cell responses, resulting in about 20% circulating antigen-specific CD8+T cells, one month post vaccination, with sustained response showing about 13% circulating T cells in the blood 3 months after vaccination, highlighting both robust induction and sustained T-cell immunity. Notably, all vaccinated mice completely rejected tumor rechallenge three months after the initial immunization, demonstrating the durability and potency of the immune response (FIG.15). To further enhance the efficacy of the cancer vaccine strategy, novel ionizable lipids (AMG 514) were developed, designed to improve mRNA delivery to splenic dendritic cells and macrophages (FIG. 19). AMG 514 significantly amplified vaccine-induced immune responses, resulting in effective tumor growth inhibition at lower doses. These findings underscore the importance of optimizing both adjuvant potency and delivery efficiency to maximize the therapeutic potential of cancer vaccines. Moreover, IR-mRNA adjuvanted in flu mRNA vaccines elicited significantly stronger humoral (about 5-fold) and cellular (about 15-fold) responses as compared to antigen alone (H3 mRNA) (FIG. 16). Another advantage of this mRNA-LNP platform is the ability to precisely control the relative doses of antigen and adjuvant (IRF8 / NIK) mRNA to enable reproducible responses across a range of antigens and providing a tunable strategy essential for shaping the desired adaptive immunity.

[0135] NIK activates the non-canonical NF-KB pathway downstream of receptors such as CD40 and BAFF-R, driving dendritic cell cross-priming and key immunostimulatory cytokines. Previously, it was shown that NIK loss in DCs eliminates anti-PD-1 efficacy and impairs CD8+T-cell priming, establishing NIK as essential for adaptive antitumor immunity (Refs. 21, 36). Although chronic or genetically sustained NIK overactivation can lead to aberrant non-canonical NF-KB signaling associated with inflammation and tumor progression, this biology fundamentally differs from the transient and tightly regulated expression achieved through mRNA-LNP delivery (Ref. 81). Under physiological conditions, NIK protein is continuously targeted for degradation and accumulates only upon specific receptor engagement or pharmacologic NIK stabilization (e.g., cellular inhibitor of apoptosis- 26 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)protein (cIAP) inhibition). Thus, transient NIK expression from mRNA closely resembles normal physiological kinetics rather than the chronic, pathological activation linked to oncogenesis (Ref. 82). Consistent with this, NIK and IRF8 mRNA-LNP vaccination did not result in sustained weight loss, behavioral abnormalities, or liver or spleen pathology (FIG.29-FIG. 30). Even at doses up to threefold higher than the therapeutic dose, NIK-treated mice showed weight curves comparable to Flue mRNA-LNP controls and fully recovered by Day 4. Serum chemistry markers (ALT, AST) remained within normal ranges, further supporting a favorable tolerability profile (FIG. 17). Moreover, NIK and IRF8 mRNA-LNPs induced robust therapeutic activity across three tumor models (MC38, MB49, and B16-F10) using both systemic and intratumoral routes. Mice achieving complete tumor regression (FIG.13, FIG. 15) were monitored for up to one year and exhibited no delayed toxicity or behavioral abnormalities. These findings indicate that the disclosed approach harnesses the immunostimulatory benefits of NIK and IRF8 while avoiding mechanisms associated with chronic pathway dysregulation.

[0136] Overall, IR-mRNAs encoding cytosolic immune-reprogramming factors initiate cascaded signaling pathways that reprogram the tumor microenvironment and direct immune cell fate, ultimately enhancing therapeutic efficacy. Their capacity to improve antigen presentation and promote lymphocyte infiltration, effectively converting “cold” tumors into “hot”, positions them as strong candidates to complement existing immune checkpoint blockers in clinical settings. Moreover, IR-mRNAs also demonstrate substantial potential to amplify antigen-specific responses in both cancer and infectious disease vaccines. In cancer vaccination, their ability to elicit unprecedented levels of antigen-specific CD8+T cells supports their use in personalized vaccines targeting patient-derived neoantigens.Additionally, by enhancing both humoral and cellular immunity, IR-mRNAs may serve as potent adjuvants for next-generation infectious disease vaccines, especially in vulnerable populations such as older adults or individuals with compromised immune systems.Example Advantages

[0137] The efficacy of NIK and IRF8 mRNA LNPs as vaccine adjuvants and immunotherapies is surprising for several reasons, as discussed in detail below.

[0138] Chronic overexpression of NIK has frequently been discussed as an expression pattern associated with the formation of Tregcells, tumor proliferation, and cancer- 27 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)progression. The formation of cytotoxic CD8+T cells and tumor regression in response to transient, mRNA-induced overexpression of NIK is therefore unexpected. See Ref. 99.

[0139] It has been previously shown that lentiviral-driven expression of the combination of IRF8, PU.l, and BATF3 can lead to anti -tumor responses. However, it was unexpected that IRF8 would work alone. See Ref. 30.

[0140] The level of T cell response we observe in response to vaccination was also unexpected. The contemporary T-cell cancer adjuvanted vaccines typically utilize a much higher dose to generate a strong and durable T cell response (ranging from 15 pg to 40 pg whereas, in the results disclose herein, a response was achieved at a dose as low as 3-5 pg). See Refs. 66, 71.

[0141] Agonist immunotherapies such as these have frequently been limited by toxicity. It was surprising that the disclosed IRF8 and NIK mRNA LNPs did not have any higher toxicity in mouse models than was observed with the control (FLuc) mRNA LNPs. See Refs.59, 61, and 60.

[0142] Additionally, many previous RNA / DNA encoded immune stimulants have been administered locally (i.e. intratumorally or intramuscularly). Surprisingly, intravenous administration was effective without dose-limiting toxicity.

[0143] Most prior approaches have utilized combinations of secreted or membrane-bound factors, therefore it was unclear if cytosolic effector factors such as NIK and IRF8 would be effective as immunotherapies. See Refs. 11 and 100.

[0144] Ref. 101 demonstrates the co-loading of mRNAs with antigen mRNA, which then function as adjuvants for vaccination. This study shows that not all mRNA sequences tested (constitutively active TAK1, TRAM, IRF3, IRF7, and MyD88) can induce sustained T cell immune responses. The authors of Ref. 101 then demonstrated that a STING mutant mRNA can function as an adjuvant. Similar to this, the authors of Ref. 102 used a cGAS mutant mRNA as an adjuvant. Prior studies have independently validated the cGAS / STING pathway for its adjuvant activities in boosting CD8 T cell responses following vaccination. There is also published evidence that IRF3 and IRF7, both transcription factors, fail in this regard, making it more surprising that IRF8, which was never tested in prior studies, works as a potent immune activator and possible vaccination adjuvant, as disclosed herein.

[0145] Ref. 103 demonstrates that the macrophage expression of IRF8 can enhance tumor growth by triggering the exhaustion of CD8 T cells. IRF8 is necessary for tumor-associated macrophage antigen presentation to cytotoxic lymphocytes, which allows sub-optimal antigen- 28 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)presentation to CD8 T cells, resulting in their functional exhaustion. Data from this paper suggests that the expression of IRF8 in the tumor microenvironment, particularly among macrophages, may be detrimental to anti-tumor immune responses. This contrasts with the results observed herein, thus making it surprising that IRF8 mRNA treatments are anti -turn or.Example Embodiments

[0146] Disclosed herein are mRNA sequences encoding for certain transcription factors (NIK and IRF8) that trigger novel immunostimulatory pathways in myeloid cells. This approach enhances cancer and infectious disease therapeutics and vaccines.

[0147] Accordingly, in one aspect, the disclosure provides a composition comprising a lipid nanoparticle containing as active ingredient an IRF8 mRNA sequence or active fragment thereof. In another aspect, the disclosure provides a composition comprising a lipid nanoparticle containing as active ingredient a NIK mRNA sequence or active fragment thereof. In another aspect the disclosure provides a composition comprising a lipid nanoparticle containing IRF8 and NIK mRNA sequences or active fragment thereof. In any of the above aspects, the mRNA sequence(s) is / are optimized for mRNA codon usage and secondary structure.

[0148] In yet another aspect, the disclosure provides a method of stimulating or boosting an immune response in a subject or inducing IL-12 expression in a subject comprising administering to the subject any of one the foregoing compositions in an amount effective to stimulate or boost the immune response in the subject or induce IL- 12 expression in the subject.

[0149] In a further aspect, the disclosure provides a method of activating BMDCs, stimulating expression of CD80 and CD86 to induce T cell activation, or increasing CD8 T cells in a subject in need thereof comprising administering to the subject a composition of any of one the foregoing compositions in an amount sufficient to activate the BMDCs, stimulate expression of CD80 and CD86 to induce T cell activation or increase CD8 T cells in the subject.

[0150] Also disclosed herein are methods of treatment. These include a method of treating colorectal cancer or bladder cancer in a subject comprising administering a composition of any of one the foregoing compositions to the subject in an amount effective to treat the cancer.- 29 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)

[0151] In certain aspects, provided herein is a vaccine comprising an RNA encoding an antigen and an RNA encoding interferon regulatory factor 8 (IRF8).

[0152] In some embodiments, the antigen is for a cancer. In some embodiments, the cancer is colorectal cancer or bladder cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is melanoma.

[0153] In some embodiments, the antigen is for an infectious disease, such as COVID-19. In some embodiments, the antigen is hemagglutinin or SARS-CoV-2 spike. In some embodiments, the infectious disease is influenza.

[0154] In some embodiments, one or more of the RNA encoding an antigen and the RNA encoding IRF8 is formulated in a lipid nanoparticle.

[0155] In other aspects, provided herein is a method of eliciting an immune response in a subject, the method comprising administering a therapeutically effective amount of an RNA encoding an antigen and a therapeutically effective amount of an RNA encoding interferon regulatory factor 8 (IRF8).

[0156] In some embodiments, the antigen is for a cancer. In some embodiments, the cancer is colorectal cancer or bladder cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is melanoma.

[0157] In some embodiments, the antigen is for an infectious disease, such as COVID-19. In some embodiments, the antigen is hemagglutinin or SARS-CoV-2 spike. In some embodiments, the infectious disease is influenza.

[0158] In some embodiments, one or more of the RNA encoding an antigen and the RNA encoding IRF8 is formulated in a lipid nanoparticle.

[0159] In some embodiments, the subject is an animal. In some embodiments, the subject is human.

[0160] In other aspects, the disclosure provides methods of treating cancer in a subject, the method comprising administering a therapeutically effective amount of an RNA encoding interferon regulatory factor 8 (IRF8). In some embodiments, the cancer is colorectal cancer or bladder cancer.

[0161] In certain aspects, provided herein is a vaccine comprising an RNA encoding an antigen and an RNA encoding interferon regulatory factor 8 (IRF8). In some embodiments, a vaccine comprises an RNA encoding NF-KP-inducing kinase (NIK).

[0162] In some embodiments, a vaccine comprises an IRF8 RNA, e.g., a mouse IRF8 RNA or a human IRF8 RNA. In some embodiments, a vaccine comprises a codon-optimized- 30 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)IRF8 RNA. In some embodiments, a mouse codon-optimized IRF8 RNA is encoded by DNA having the sequence of SEQ ID NO: 5. In some embodiments, a vaccine comprises a codon-optimized human IRF8 RNA having the sequence of SEQ ID NO: 6 or 7.

[0163] In some embodiments, a vaccine comprises a NIK RNA, e.g., a mouse NIK RNA or a human NIK RNA. In some embodiments, a vaccine comprises a codon-optimized NIK RNA. In some embodiments, a mouse codon-optimized NIK RNA is encoded by DNA having the sequence of SEQ ID NO:1. In some embodiments, a vaccine comprises a codon-optimized human NIK RNA having the sequence of SEQ ID NO: 2 or 3.

[0164] In some embodiments, an RNA is a messenger RNA (mRNA) or a self-amplifying RNA (saRNA; / .<?., a self-replicating RNA). In some embodiments, an RNA comprises one or more modified nucleosides, e.g., N1 -methylpseudouridine (CAS No.: 13860-38-3).

[0165] As used herein, the term “vaccine” refers to any composition, e.g., any pharmaceutical composition, capable of promoting (e.g., eliciting) an immune response to an antigen in a subject. In some embodiments, a vaccine is capable of improving a subject’s immunity to a disease. In some embodiments, a vaccine is capable of treating or preventing a disease in a subject. For example, in some embodiments, a vaccine is capable of treating cancer by targeting a cancer antigen or a cancer neoantigen.

[0166] In some embodiments, the antigen is associated with a disease. In some embodiments, the disease is a cancer, an infectious disease, a genetic disease, an autoimmune disease, an inflammatory disease, a degenerative disease, or the like.

[0167] In some embodiments, the antigen is for a cancer. In some embodiments, the cancer is colorectal cancer or bladder cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is melanoma.

[0168] In some embodiments, the antigen is a cancer antigen. In some embodiments, the antigen is a cancer neoantigen.

[0169] In some embodiments, the antigen is for an infectious disease. In some embodiments, the infectious disease is a viral disease. In some embodiments, the infectious disease is a bacterial disease.

[0170] In some embodiments, the infectious disease is COVID-19.

[0171] In some embodiments, the antigen is a viral antigen. In some embodiments, the antigen is a glycoprotein. In some embodiments, the antigen facilitates viral binding (e.g., docking) to a host cell (e.g., an animal cell). In some embodiments, the antigen is hemagglutinin, SARS-CoV-2 spike, or a variant thereof. In some embodiments, the- 31 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)hemagglutinin is Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hll, H12, H13, H14, H15, Hl 6, Hl 7, Hl 8, or a variant thereof. In some embodiments, the SARS-CoV-2 spike contains at least one mutation, for example, one or more of D614G, N439K, Y453F, A69-70, E484K, K444E, G446V, L452R, F490S, S477G, and the like. In some embodiments, the antigen is neuraminidase (e.g., exo-a-sialidase, CAS No., 9001-67-6). In some embodiments, the neuraminidase is Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, or a variant thereof.

[0172] In some embodiments, the infectious disease is influenza.

[0173] In some embodiments, one or more of the RNA encoding an antigen and the RNA encoding IRF8 is formulated in a lipid nanoparticle.

[0174] In other aspects, provided herein is a method of eliciting an immune response in a subject, the method comprising administering a therapeutically effective amount of an RNA encoding an antigen and a therapeutically effective amount of an RNA encoding interferon regulatory factor 8 (IRF8).

[0175] In some embodiments, the antigen is for a cancer. In some embodiments, the cancer is colorectal cancer or bladder cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is melanoma.

[0176] In some embodiments, the antigen is for an infectious disease.

[0177] In some embodiments, the infectious disease is COVID-19. In some embodiments, the antigen is hemagglutinin or SARS-CoV-2 spike.

[0178] In some embodiments, the infectious disease is influenza.

[0179] In some embodiments, one or more of the RNA encoding an antigen and the RNA encoding IRF8 is formulated in a lipid nanoparticle.

[0180] In some embodiments, the subject is an animal. In some embodiments, the animal is selected from humans, dogs, monkeys, pigs, rats, mice, hamsters, rabbits, cats, chickens, pigs, cattle, sheep, goats, and horses. In some embodiments, the subject is human. In some embodiments, the RNA encoding an antigen, the RNA encoding interferon regulatory factor 8 (IRF8), or both comprise a human, dog, monkey, pig, rat, mouse, hamster, rabbit, cat, chicken, pig, cattle, sheep, goat, or horse RNA sequence, or a combination thereof. In some embodiments, the RNA encoding an antigen, the RNA encoding interferon regulatory factor 8 (IRF8), or both comprise a variant or modification of a human, dog, monkey, pig, rat, mouse, hamster, rabbit, cat, chicken, pig, cattle, sheep, goat, or horse RNA sequence, or a combination thereof. In some embodiments, the RNA encoding an antigen, the RNA- 32 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)encoding interferon regulatory factor 8 (IRF8), or both comprise a codon-optimized sequence.

[0181] In some aspects, the disclosure provides methods of treating cancer in a subject, the method comprising administering a therapeutically effective amount of an RNA encoding interferon regulatory factor 8 (IRF8). In some embodiments, the therapeutically effective amount of an RNA encoding IRF8 is administered in combination with one or more additional therapeutic agents. In some embodiments, the therapeutically effective amount of an RNA encoding IRF8 is administered as a monotherapy.Example Agents

[0182] In contrast to prior mRNA therapeutic targets, disclosed herein is the expression of the immune-activating kinase (NIK) and the transcription factor interferon regulatory factor 8 (IRF8) as vectors for immunotherapy. Specifically, prior receptor pathways used for mRNA therapy are from the Tumor Necrosis Factor Receptor (TNF-R) family, including CD40 / CD40L, CD27 / CD70, and OX40 / OX40L. These pathways can activate the non-canonical NFKP pathway, which has been demonstrated to be important in regulating antitumor immunity. Based on the disclosed data relating to enforced NIK expression through mRNA delivery and the resulting immune stimulation, NIK may be a master signaling node of immune signaling. Specifically, powerful immune activation effects were observed, especially regarding adaptive CD8 T cell response from NIK-adjuvanted vaccination with a single factor, as opposed to combination mRNA formulations. This can enable lower dosing of mRNAs as we can establish strong immune stimulation with only one mRNA.Furthermore, the disclosed approach is distinguished by targeting a key immune signaling node, as opposed to direct cytokine or co-stimulatory receptor / ligand expression. Expression of NIK leads to both enhancement of co-stimulation receptors such as CD80 and CD86 and cytokines such as IL- 12 from one therapeutic intervention.

[0183] IRF8 is a transcription factor essential for developing type 1 conventional dendritic cells (cDCl). cDCl are an important antigen-presenting cell type that initiates and supports CD8 T cell responses. Recent evidence demonstrates that cDCl requires IRF8 beyond development and that IRF8 can reinforce the expression of key anti-tumor signals such as IL-12 and CXCL9. Indeed, consistently high expression levels of IRF8 may be needed for optimum immune stimulation. IRF8 mRNA containing lipid nanoparticles have been developed, which can raise cellular IRF8 levels as a therapeutic intervention. This- 33 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)approach is the first to leverage IRF8 as a therapeutic. IRF8 mRNA enhances dendritic cell (DC) expression of IL-12 and the output of cDCl-like cells from in vitro culture, raising the prospect of developing IRF8-enhanced DC vaccines. Likewise, IRF8 mRNA treatments could trigger substantial anti-tumor immunity and boost vaccination responses.RNA

[0184] In some embodiments, the RNA encoding IRF8, the RNA encoding NIK, or both, is a circular RNA. In some embodiments, the RNA encoding the antigen is a circular RNA. For example, a composition can comprise one or more circular RNAs (e.g., species of circular RNAs), wherein each circular RNA comprises one or more of NIK RNA, IRF8 RNA, and antigen RNA. In some embodiments, NIK, IRF8, and the antigen are encoded on separate RNAs. In some embodiments, two or more of NIK, IRF8, and the antigen are encoded on the same RNA.

[0185] In some embodiments, at least one uridine of the one or more of the RNAs disclosed herein is replaced with, i.e., comprises, a modified uridine, e.g., Nl-methylpseudouridine (NIMePsU). In some embodiments, the RNA is modified with at least one NIMePsU. In some embodiments, the RNA is fully modified with NIMePsU, i.e., each uridine of the RNA is an NIMePsU. In some embodiments, the RNA encoding IRF8, the RNA encoding NIK, or both, comprises at least one of N1 -methylpseudouridine (NIMePsU), 5-methoxy-pseudouridine, psuedouridine, 5-methylcytidine, or a combination thereof. In some embodiments, the RNA encoding the antigen comprises at least one of Nl-methylpseudouridine (NIMePsU), 5-methoxy-pseudouridine, psuedouridine, 5-methylcytidine, or a combination thereof.

[0186] In some embodiments, each uridine of the RNA encoding IRF8, the RNA encoding NIK, or both, is substituted with NIMePsU. In some embodiments, each uridine of the RNA encoding the antigen is substituted with NIMePsU. In such examples, RNA is considered to be fully modified by NIMePsU or fully derivatized with NIMePsU. As used herein, substitution of uracil (e.g., naturally occurring uracil of an analog thereof) with NIMePsU or another modified nucleobase refers to substitution with respect to a wild-type or unmodified nucleic acid sequence or design. For example, a nucleic acid described by a certain sequence may be originally synthesized to contain NIMePsU rather than uracil. In- 34 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)another example, a nucleic acid comprising uracil can be amplified such that the amplification product comprises NIMePsU, or primarily NIMePsU, rather than uracil.

[0187] In some embodiments, NIK is a recombinant human NIK, IRF8 is a recombinant human IRF8, or both. In some embodiments, NIK lacks a negative-regulatory domain (NRD), a tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3)-binding domain, or both. In some embodiments, NIK lacks an NRD; see, for example, Ref. 104. In some embodiments, NIK lacks a TRAF3 -binding domain; see, for example, Ref. 105.

[0188] Recombinant human RNA disclosed herein encodes one or more functional domains of the encoded protein product. For example, a recombinant human RNA can encode the functional domains of NIK or IRF8, as well as a tag or label, a signal sequence, or the like.

[0189] In some embodiments, the RNA is modified (e.g., with modified a modified nucleobase). In some embodiments, the RNA contains a stabilizing variant that renders the protein product transcribed therefrom resistant to degradation, e.g., by removing a ubiquitination site. In some embodiments, the RNA contains a variant or other structural modification (e.g., insertion, deletion, or rearrangement) that confers increased or constitutive activity. In some embodiments, the RNA contains a variant that increases its affinity for a binding partner, e.g., a DNA motif or another protein. In some embodiments, the RNA contains a variant that decreases its affinity for a negative regulator.NIK

[0190] In some embodiments, a composition comprises at least an RNA encoding nuclear factor kappa B (NF-KB) inducing kinase (NIK). An RNA encoding NIK is an example of an IR-mRNA. In some embodiments, the RNA can comprise SEQ ID NO: 2 or 3. In some embodiments, at least one uridine of the RNA is replaced with, or comprises, a modified uridine, e.g., N1 -methylpseudouridine (NIMePsU). In some embodiments, the RNA is modified with at least one NIMePsU. In some embodiments, the RNA is fully modified with NIMePsU, i.e., each uridine of the RNA comprises NIMePsU (SEQ ID NO: 3). In some embodiments, the NIK comprises SEQ ID NO:4. When the RNA encoding NIK is translated, the translation product can comprise, for example, SEQ ID NO:4. In some embodiments, the NIK lacks a negative-regulatory domain (NRD); see, for example, Ref. 104.- 35 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)IRF8

[0191] In some embodiments, a composition comprises at least an RNA encoding interferon regulatory factor 8 (IRF8). An RNA encoding IRF8 is an example of an IR-mRNA. In some embodiments, the RNA can comprise SEQ ID NO: 6 or 7. In some embodiments, at least one uridine of the RNA is replaced with, or comprises, a modified uridine, e.g., N1 -methylpseudouridine (NIMePsU). In some embodiments, the RNA is modified with at least one NIMePsU. In some embodiments, the RNA is fully modified with NIMePsU, i.e., each uridine of the RNA comprises NIMePsU (SEQ ID NO: 7). In some embodiments, the NIK comprises SEQ ID NO: 8. When the RNA encoding NIK is translated, the translation product can comprise, for example, SEQ ID NO: 8.Lipid NanoparticlesLipids

[0192] In certain aspects, provided herein is a composition comprising one or more of an RNA encoding nuclear factor kappa B (NF-KB) inducing kinase (NIK) and an RNA encoding interferon regulatory factor 8 (IRF8); and lipids encapsulating the RNA. In some embodiments, the composition further comprises an RNA encoding an antigen. In some embodiments, the composition further comprises two or more RNAs encoding two or more antigens.

[0193] In some embodiments, the lipids form a lipid nanoparticle (LNP) encapsulating the RNA encoding IRF8, the RNA encoding NIK, the RNA encoding the antigen, or a combination thereof. For example, a composition can comprise an LNP encapsulating one or more of a NIK RNA, and IRF8 RNA, and an antigen RNA. In other examples, a composition can comprise multiple LNPs (e.g., species of LNPs), wherein each of the LNPs encapsulates different RNA or RNAs.

[0194] In some embodiments, the LNP or one or more of the lipids localize the composition to the spleen. In some embodiments, one or more of the lipids localize the composition to the spleen.

[0195] In some embodiments, the LNP or one or more of the lipids has a protein corona. As used herein, “corona” or “protein corona” refers to proteins that associate with an LNP or a lipid, e.g., a particular type of lipid in the LNP formulation. Proteins form the corona as they interact with the LNP or lipid. Such interactions can be spontaneous and can be the- 36 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)result of affinity between the protein and LNP or lipid. Proteins within the corona can influence the effective physiochemical properties of the LNP or lipid, which, in turn, can influence therapeutic properties of compositions comprising the LNP or lipid, e.g., localization sites. See also, Ref. 107.

[0196] In some embodiments, the PEG-modified lipid is DMPE-PEG or DMG-PEG 2000. Compositions comprising DMPE-PEG or DMG-PEG 2000 (DMG-PEG2k) are disclosed in the Examples herein. DMPE-PEG and DMG-PEG 2000 are considered to have similar properties; accordingly, different LNP formulations that comprise DMPE-PEG or DMG-PEG 2000, but which are otherwise the same, are considered to be interchangeable in the intended applications, e.g., therapeutic uses.

[0197] In some embodiments, the lipids have a molar composition of about 20-70% ionizable lipid, about 5-40% helper lipid, about 20-50% structural lipid, and about 0.5-5% PEG-modified lipid. In some embodiments, the lipids have a molar composition of 35 : 16 : 46.5 : 2.5 (i.e., 35%, 16%, 46.5%, and 2.5%) of ionizable lipid : DOPE : cholesterol : DMPE-PEG or DMG-PEG 2000.

[0198] In some embodiments, a lipid nanoparticle (LNP) comprises at least one of (i) an ionizable lipid, (ii) a helper lipid; (iii) a structural lipid, and (iv) a PEG-modified lipid, wherein the LNP is capable of delivering the RNA of the composition to cell (e.g., an immune cell present in a subject) or tissue e.g., a spleen present in a subject), such that the RNA translated. Examples of ionizable lipids and LNPs are set forth, for example, in Example 9 herein, and in: US20250205158A1 (Biodegradable Lipids and Formulations for Delivery of mRNA); US20240226302A1 (Biodegradable Lipids and Formulations for Intramuscular, In Vitro, and Ex Vivo Transfection of mNRA); WO2020237227 (Circular RNA Compositions and Methods); and WO2025188636 (Ionizable Lipids for messenger RNA Delivery).

[0199] In some embodiments, the composition disclosed herein comprises the ionizable lipid ckk-el2. In some embodiments, LNPs disclosed herein comprise the ionizable lipid ckk-e!2.- 37 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)ckk-el2 (CAS No.: 1432494-65-9)

[0200] In some embodiments, the composition disclosed herein comprises the ionizable lipid AMG511 (FIG. 50A). In some embodiments, LNPs disclosed herein comprise the ionizable lipid AMG511 (FIG. 50A).

[0201] In some embodiments, the composition disclosed herein comprises the ionizable lipid AMG513 (FIG. 50A). In some embodiments, LNPs disclosed herein comprise the ionizable lipid AMG513 (FIG. 50A).

[0202] In some embodiments, the composition disclosed herein comprises the ionizable lipid AMG514 (FIG. 50A). In some embodiments, LNPs disclosed herein comprise the ionizable lipid AMG514 (FIG. 50A).

[0203] In some embodiments, the composition disclosed herein comprises the ionizable lipid AMG515 (FIG. 50A). In some embodiments, LNPs disclosed herein comprise the ionizable lipid AMG515 (FIG. 50A).

[0204] In some embodiments, the composition disclosed herein comprises the ionizable lipid AMG516 (FIG. 50B). In some embodiments, LNPs disclosed herein comprise the ionizable lipid AMG516 (FIG. 50B).

[0205] In some embodiments, the composition disclosed herein comprises the ionizable lipid AMG517 (FIG. 50B). In some embodiments, LNPs disclosed herein comprise the ionizable lipid AMG517 (FIG. 50B).- 38 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)

[0206] In some embodiments, the composition disclosed herein comprises the ionizable lipid AMG518 (FIG. 50B). In some embodiments, LNPs disclosed herein comprise the ionizable lipid AMG518 (FIG. 50B).

[0207] In some embodiments, the composition disclosed herein comprises the ionizable lipid AMG519 (FIG. 50B). In some embodiments, LNPs disclosed herein comprise the ionizable lipid AMG519 (FIG. 50B).

[0208] In some embodiments, the composition disclosed herein comprises the ionizable lipid AMG520 (FIG. 50C). In some embodiments, LNPs disclosed herein comprise the ionizable lipid AMG520 (FIG. 50C).

[0209] In some embodiments, the composition disclosed herein comprises the ionizable lipid AMG521 (FIG. 50C). In some embodiments, LNPs disclosed herein comprise the ionizable lipid AMG521 (FIG. 50C).

[0210] In some embodiments, the composition disclosed herein comprises the ionizable lipid AMG522 (FIG. 50C). In some embodiments, LNPs disclosed herein comprise the ionizable lipid AMG522 (FIG. 50C).

[0211] In some embodiments, the composition disclosed herein comprises the ionizable lipid AMG523 (FIG. 50C). In some embodiments, LNPs disclosed herein comprise the ionizable lipid AMG523 (FIG. 50C).

[0212] In some embodiments, the composition disclosed herein comprises the ionizable lipid AMG56. In some embodiments, LNPs disclosed herein comprise the ionizable lipid AMG56.5024769. vlDocket No. 0050.2396-002 (MIT 25731)

[0213] In some embodiments, the helper lipid is l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; CAS No. 816-94-4), l,2-dioleoyl-3 -trimethylammonium -propane (DOTAP; e.g., CAS Nos.132172-61-3 or 144189-73-1), l,2-dioleoyl-3 -dimethylammonium -propane (DODAP; CAS No. 127512-29-2), l,2-dioleoyl-sn-glycero-3 -phosphate (DOPA; CAS No. 108392-02-5), l,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS; CAS No. 90693-88-2), 1,2-dioleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DOPG; CAS No. 67254-28-8), l-palmitoyl-2-oleoyl-glycero-3 -phosphocholine (POPC; CAS No. 26853-31-6), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE; CAS No. 26662-94-2), l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC; CAS No. 56421-10-4), l,2-diphytanoyl-sn-glycero-3-phosphocholine (4MEPC; CAS No. 207131-40-6), or a combination thereof.

[0214] Some embodiments relate to lipid nanoparticles for delivery of a vaccine. Some suitable LNPs, LNP components, methods of formulating payloads, including, for example, proteins and / or nucleic acids, into LNPs, as well as methods of contacting cells or tissues in vitro, ex vivo, or in vivo, with payloads formulated into LNPs, are disclosed herein, and additional suitable LNPs, LNP components, methods of formulating payloads, including, for example, proteins and / or nucleic acids, into LNPs, as well as methods of contacting cells or tissues will be apparent to the skilled artisan in view of the present disclosure and the knowledge in the art. Non-limiting examples of suitable LNPs, LNP components, formulation methods and methods of contacting cells or tissues include those disclosed in Technov et al., ACS Nano 2021, 15, 16982-17015; Finn et al., Cell Rep. 2018 Feb 27;22(9):2227-2235; Gillmore et al., N Engl J Med 2021; 385:493-502; Yan etal., Biomater Sci. 2021 Sep 14;9(18):6001-6011; Kazemian etal., Mol Pharm. 2022 Jun 6;19(6):1669-1686; Mohammadian Farsani et al., Heliyon. 2024 Jan 1 l;10(2):e24606; Raguram et al., Cell.2022 Jul 21;185(15):2806-2827; Ma etal., Chembiochem. 2023 May 2;24(9):e202200801; Kowalski et al., Mol Ther. 2019 Apr 10;27(4):710-728; Madigan et al., Nat Rev Drug Discov. 2023 Nov;22(l l):875-894; Aziz et al., J Biomater Sci Polym Ed. 2023 Feb;34(3):398-418; Leung et al., Adv Genet. 2014;88:71-110; Mok et al., Biochimica et Biophysica Acta, 1999;1419(2): 137-150; Eldrige, etal., Lipid Nanoparticles: Production, Characterization, and Stability. Springer 2014, ISBN: 3319107100; Prakash et al., Adv Drug Deliv Rev. 2022 May; 184: 114197; PCT application publications WO 2020 / 206231, WO 2015 / 095340, WO 2017 / 173054, WO 2020 / 219876, WO 2015 / 035136, WO 2010 / 144740, WO 2015 / 199952, WO 2017 / 075531, and WO 2018 / 081480; U.S. application publications- 40 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)US 2004 / 0142025, US 2007 / 0042031, and US 2020 / 0385721; and US Patents Nos. US 7,745,651; US 7,799,565; US 7,901,708; US 8,058,069; US 8,158,601; US 8,492,359; US 8,642,076; US 8,822,668; US 9,005,654; US 9,006,417; US 9,139,554; US 9,364,435; US 9,404,127; US 9,415,109; US 9,518,272; US 9,593,077; US 9,682,139; US 9,878,042; US 9,999,673; US 10,723,692; US 10,941,395; and US 11,141,378.

[0215] In some embodiments, lipid nanoparticles include a helper lipid, an ionizable lipid, a sterol, or an agent, or a pharmaceutically acceptable salt of any of the foregoing, or a combination thereof.

[0216] In some embodiments, at least 10 mole percent (mol %) (e.g., at least 20 mol %, at least 30 mol %, at least 35 mol %, at least 40 mol %, at least 50 mol %, etc.) of the lipids of the lipid nanoparticle are ionizable lipids. In some embodiments, from about 10 mol % to about 80 mol % (e.g, about 10 mol % to about 70 mol %, about 10 mol % to about 60 mol %, about 20 mol % to about 60 mol %, about 20 mol % to about 50 mol %, about 20 mol % to about 45 mol %, about 20 mol % to about 40 mol %, about 25 mol % to about 40 mol %, about 30 mol % to about 40 mol %, etc.) of the lipids of the lipid nanoparticle are ionizable lipids. In some embodiments, about 35 mol % of the lipids of the lipid nanoparticle are ionizable lipids.

[0217] In some embodiments, at least 5 mol % (e.g, at least 10 mol %, at least 15 mol %, at least 20 mol %, at least 25 mol %, etc.) of the lipids of the lipid nanoparticle are helper lipids. In some embodiments, from about 5 mol % to about 50 mol % (e.g., about 5 mol % to about 40 mol %, about 10 mol % to about 40 mol %, about 15 mol % to about 40 mol %, about 15 mol % to about 35 mol %, about 20 mol % to about 35 mol %, about 20 mol % to about 30 mol %, about 25 mol % to about 30 mol %, etc.) of the lipids of the lipid nanoparticle are helper lipids. In some embodiments, about 28 mol % of the lipids of the lipid nanoparticle are ionizable lipids.

[0218] In some embodiments, the lipid nanoparticle comprises ionizable lipid and agent, where the weight ratio of ionizable lipid to agent (e.g., nucleic acid, such as mRNA) is from about 20:1 to about 2:1 (e.g., about 15:1 to about 2:1, about 15:1 to about 3:1, about 15:1 to about 4:1, about 15:1 to about 5:1, about 14:1 to about 6:1, about 13:1 to about 7:1, about 12: 1 to about 8:1, about 11:1 to about 9:1, etc.). In some embodiments, the ratio of ionizable lipid to agent is about 10:1.

[0219] Non-limiting examples of ionizable lipids and helper lipids include lipids and salts thereof having one, two, three, or more fatty acid or fatty alkyl chains and a pH-titratable- 41 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)amino head group (e.g., an alkylamino or dialkylamino head group). Ionizable lipids and helper lipids are typically protonated (i.e., positively charged) at a pH below the pKaof the lipid and is substantially neutral at a pH above the pKa. In some embodiments, the ionizable lipid is l,l'-[[2-[4-[2-[[2-[Z>z 2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-l-piperazinyl]ethyl]imino]Zh -2-dodecanol (C12-200), 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione (ckk-e-12) or 5-(((3-(dibutylamino)propyl)amino)methyl)-6-hydroxyundecane- 1,11 -diyl (9Z,9’Z,12Z,12’Z)-bis(octadeca-9,12-dienoate) (IR-117-17).

[0220] In some embodiments, the helper lipid is a quaternary ammonium, phosphatidylethanolamine, phosphatidylserine, or phosphatidylcholine derivative, or a combination thereof.

[0221] In some embodiments the LNPs comprise an ionizable lipid. In some embodiments, the ionizable lipids contain one or more groups which is ionic at physiological pH but may have no charge at a certain pH outside of the range of physiological pH values. Typically, the ionizable lipids of the present disclosure are cationic ionizable lipids which have a cationic charge at physiological pH values. The ionizable cationic group may contain one or more protonatable amines which are able to form a cationic group at physiological pH. The ionizable lipid compound may also further comprise one or more lipid components such as two or more fatty acids with C1-C30 alkyl or alkenyl carbon groups. In some embodiments of the composition of the present application, the ionizable cationic lipids refer to lipid and lipid-like molecules with nitrogen atoms that can acquire charge (pKa). These molecules with amino groups typically have between 2 and 6 hydrophobic chains, often alkyl or alkenyl such as C1-C30 alkyl or alkenyl groups, but may have at least 1 or more than 6 tails.

[0222] The ionizable lipid component of the lipid nanoparticle may comprise between 1 and 99 mole percent of the lipid molecules in the nanoparticle. In other embodiments, the ionizable lipid component may comprise between 5 and 90 mole percent of the lipid molecules in the lipid nanoparticles. In some embodiments the ionizable lipid component comprises between 10 and 75, 20 and 75, 20 and 60, or 20 and 55 mole percent of the lipid molecules in the lipid nanoparticles.

[0223] In some embodiments, the lipid nanoparticle comprises sterol (e.g., cholesterol) in an amount of from about 0 mol% to about 30 mol% (e.g., about 0 mol% to about 25 mol%, about 0 mol% to about 24 mol%, about 0 mol% to about 23 mol%, about 0 mol% to about 22 mol%, about 0 mol% to about 21 mol%, about 0 mol% to about 20 mol%, about 0 mol% to- 42 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)about 19 mol%, about 0 mol% to about 18 mol%, about 0 mol% to about 17 mol%, about 0 mol% to about 16 mol%, about 0 mol% to about 15 mol%, about 5 mol% to about 15 mol%, etc.). In some embodiments, the sterol is in an amount of about 10 mol%.

[0224] As used herein, “sterols” refer to steroids and steroid derivatives, where the term “steroid” is a class of compounds with a four ring 17 carbon cyclic structure which can further comprises one or more substitutions including alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acyl groups, or a double bond between two or more carbon atoms. In one aspect, the ring structure of a steroid comprises three fused cyclohexyl rings and a fused cyclopentyl ring as in the structure of cholesterol.

[0225] In some embodiments, a sterol suitable in the present disclosure is a derivative of cholestane. A cholestane derivative includes one or more non-alkyl substitution on the fused ring system. In some embodiments, the cholestane or cholestane derivative is a cholestene or cholestene derivative.

[0226] Sterols that may be suitable in the present disclosure include, but are not limited to cholesterol, sitosterol, stigmasterol, fucosterol, spinasterol, brassicasterol, ergosterol, cholestanone, cholestenone, coprostanol, cholesteryl-2’ -hydroxy ethyl ether, cholesteryl-4’-hydroxybutyl ether and the like. In some embodiments, the sterol of the present disclosure is a phytosterol. In some embodiments, the sterol of the present disclosure is cholesterol.

[0227] The sterol component of the lipid nanoparticle of the present disclosure may comprise of between 1 and 99 mole percent of the lipid molecules in the nanoparticle. In other embodiments, the Sterol component may comprise of between 1 and 75 mole percent of the lipid molecules in the lipid nanoparticles. In some embodiments the Sterol component comprises between 5 and 75, 5 and 60, 10 and 50, or 15 and 45 mole percent of the lipid molecules in the lipid nanoparticles.

[0228] In some embodiments, the lipid nanoparticle comprises an agent. In some embodiments the agent is a nucleic acid. Nucleic acid agents can be double-stranded or single-stranded. In addition, nucleic acid agents can be linear or circular nucleic acid molecules. In some embodiments the nucleic acid is DNA, RNA, or a non-natural nucleic acid. The RNA agent may be RNA encoding a peptide or a non-coding RNA. The RNA agent may be mRNA, siRNA, miRNA, an enhancer RNA, long noncoding RNA, and the like. In particular embodiments, the agent is a mRNA. The nucleic acid may regulate gene expression, encode a peptide, or be an aptamer, among other possibilities. In some embodiments, a nucleic acid has a length of about 15 to about 30 nucleotides, for example,- 43 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)about 20-25 nucleotides (e.g., 20, 21, 22, 23, 24 or 25 nucleotides). In some embodiments, a nucleic acid has a length of greater than about 30 nucleotides, such as, for example, about 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 750, 1000 or more nucleotides.

[0229] The lipid particles of the present disclosure may comprise one or more agents. (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 agents).

[0230] In some embodiments, the lipid nanoparticles in the formulation have an encapsulation efficiency of at least about 60% (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, etc.) following nebulization of the formulation. In some embodiments, the encapsulation efficiency is from about 60% to about 95% (e.g., about 65% to about 95%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 95%, about 80% to about 90%, about 85% to about 90%, etc.) following nebulization of the formulation. As used herein, encapsulation efficiency refers to the amount of an agent encapsulated within particles (e.g., LNPs) of a composition, expressed as a percent of the total amount of agent. In some embodiments the encapsulation efficiency is greater than 50%. In some embodiments the encapsulation efficiency is greater than 60%. In some embodiments the encapsulation efficiency is greater than 70%. In some embodiments the encapsulation efficiency is greater than 80%. In some embodiments the encapsulation efficiency is greater than 90%. In some embodiments is greater than 91%. In some embodiments the encapsulation efficiency is greater than 93%. In some embodiments the encapsulation efficiency is greater than 95%. In some embodiments the encapsulation efficiency is greater than 97%. In some embodiments the encapsulation efficiency is greater than 98%. In some embodiments the encapsulation efficiency is greater than 99%. In some embodiments, the encapsulation efficiency is as determined by the modified Quant-iT RiboGreen RNA assay (Thermo Fisher), e.g., as described in Walsh C. etal. Methods Mol Biol. 2014;1141:109-20 and the Exemplification.

[0231] In some embodiments, the lipid nanoparticles in the formulation have an average diameter of less than 200 nm (e.g., less than 190 nm, less than 180 nm, less than 170 nm, less than 160 nm, less than 150 nm, etc.) following nebulization of the formulation. In some embodiments, the lipid nanoparticles in the formulation have an average diameter of from about 100 nm to about 200 nm (e.g., about 100 nm to about 190 nm, about 100 nm to about 180 nm, about 100 nm to about 170 nm, about 100 nm to about 160 nm, about 100 nm to- 44 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)about 150 nm, about 100 nm to about 140 nm, about 100 nm to about 130 nm, about 100 nm to about 120 nm, etc.) following nebulization of the formulation.

[0232] Formulations described herein may be administered parenterally (including subcutaneously, intramuscularly, intravenously and intradermally), by inhalation, nasally, orally, topically, rectally, buccally, vaginally or via an implanted reservoir. The terms “parenteral” and “parenterally,” as used herein, include subcutaneous, intracutaneous, intravenous, intramuscular, intratumoral, intraocular, intravitreal, intraarticular, intra-arterial, intra-synovial, intrasternal, intrathecal, intralesional, intrahepatic, intraperitoneal, intralesional and intracranial injection or infusion techniques. In some aspects, a formulation described herein is administrable intravenously and / or intraperitoneally. In some aspects, a formulation described herein is administrable by inhalation.

[0233] Formulations described herein can also be administered subcutaneously, intraperitoneally, intratumorally or intravenously, e.g., in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that can be employed are mannitol, dextrose, water, phosphate buffered saline, Ringer's solution, lactated Ringer's solution and isotonic sodium chloride solution. Commonly used surfactants such as Tweens or Spans and / or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulation.- 45 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)Example Pharmaceutical Compositions / Administration

[0234] In some embodiments, the methods disclosed herein further comprise administering to the subject one or more additional therapies. In some embodiments, an additional therapy can be an anti-PD-1 checkpoint blockade, such as an anti-PD-1 therapy or an anti-PD-Ll therapy. In some embodiments, an additional therapy can be another immune checkpoint blockade, such as an anti-CTLA4 therapy. In some embodiments, an additional therapy can be chemotherapy, radiation, therapy with a biologic, or other therapy administered according to standards of care.

[0235] In some embodiments, the cancer is colorectal cancer, bladder cancer, melanoma, lung cancer, prostate cancer, breast cancer, brain cancer, colon cancer, pancreatic cancer, ovarian cancer, and hepatocellular cancer, or lymphoma. In some embodiments, the cancer is prostate cancer; see also, Ref. 106.

[0236] In some embodiments, the subject is a human. In some embodiments, the composition is administered intratum orally. In some embodiments, the composition is administered intravenously. In some embodiments, the lipids induce expression of the RNA as mRNA in immune cells. In some embodiments, the immune cells are in a spleen.

[0237] In embodiments of the present disclosure, the vaccines described herein may be provided in compositions, e.g., pharmaceutical compositions.

[0238] Therefore, in some embodiments, the disclosure also relates to compositions, e.g., compositions comprising a vaccine, e.g., a vaccine comprising an mRNA encoding an antigen and an mRNA encoding interferon regulatory factor 8 (IRF8), and a pharmaceutically acceptable carrier. In one aspect, the present disclosure provides pharmaceutical compositions comprising an effective amount of a vaccine described herein and a pharmaceutically acceptable excipient. Pharmaceutical compositions of the present disclosure may comprise a vaccine as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, excipients or diluents.

[0239] In some embodiments, a pharmaceutically acceptable carrier can be an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to the subject.

[0240] A pharmaceutically acceptable carrier can include, but is not limited to, a buffer, excipient, stabilizer, or preservative. Examples of pharmaceutically acceptable carriers are solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, such as salts,- 46 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)buffers, saccharides, antioxidants, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifying agents, or combinations thereof. The amounts of pharmaceutically acceptable carrier(s) in the pharmaceutical compositions may be determined experimentally based on the activities of the carrier(s) and the desired characteristics of the formulation, such as stability and / or minimal oxidation.

[0241] In some embodiments, such compositions may comprise buffers such as acetic acid, citric acid, histidine, boric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, Tris buffers, HEPPSO, HEPES, neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, sucrose, mannose, or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); antibacterial and antifungal agents; and preservatives.

[0242] In certain embodiments, compositions of the present disclosure can be formulated for a variety of means of parenteral or non-parenteral administration. In one embodiment, the compositions can be formulated for infusion or intravenous administration. Compositions disclosed herein can be provided, for example, as sterile liquid preparations, e.g., isotonic aqueous solutions, emulsions, suspensions, dispersions, or viscous compositions, which may be buffered to a desirable pH. Formulations suitable for oral administration can include liquid solutions, capsules, sachets, tablets, lozenges, and troches, powders liquid suspensions in an appropriate liquid and emulsions.

[0243] In one aspect, the disclosure relates to administering a therapeutically effective amount of a composition comprising a vaccine described herein for the treatment of a subject having, or at risk of developing, a disease or disorder, e.g., cancer. In another aspect, the disclosure relates to administering a therapeutically effective amount of a composition comprising a vaccine described herein for the treatment of a subject having an infectious disease, or for the prevention of a subject from becoming infected with an infectious disease.

[0244] The term “cancer” refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. See e.g., Stedman’s Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990. Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer;- 47 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi’s sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett’s adenocarcinoma); Ewing’s sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g. , head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non- Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenstrom’s macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B -lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T- cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease);- 48 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastoma a.k.a. Wilms’ tumor, renal cell carcinoma); liver cancer (e.g, hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g, bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Paget’s disease of the penis and scrotum); pineal oma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget’s disease of the vulva).

[0245] Pharmaceutical compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated or prevented. The quantity and frequency of administration will be determined by such factors as the condition of the subject, and the type and severity of the subject’s disease, although appropriate dosages may be determined by clinical trials.- 49 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)

[0246] The terms “treat” or “treatment” refer to therapeutic treatment wherein the object is to slow down (lessen) an undesired physiological change or disease, or provide a beneficial or desired clinical outcome during treatment. Beneficial or desired clinical outcomes include alleviation of symptoms, diminishment of extent of disease, stabilized ( / .<?., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and / or remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if a subject was not receiving treatment. Those in need of treatment include those subjects already with the undesired physiological change or disease as well as those subjects prone to have the physiological change or disease.

[0247] The terms “prevent” and “prevention” refer to a prophylactic measure wherein the object is to reduce or eliminate infection with an infectious disease, symptoms of an infection, and / or results of an infection in a subject. Prevention includes alleviation of symptoms and / or severity of infection, diminishment of extent of sequelae, stabilized ( / .<?., not worsening) state of disease, amelioration or palliation of the disease state, and / or remission (whether partial or total), whether detectable or undetectable. Those in need of prevention include those subjects already with the infectious disease as well as those subjects prone to becoming infected with the infectious disease.

[0248] A “therapeutically effective amount” or “effective amount”, used interchangeably herein, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of a therapeutic or a combination of therapeutics to elicit a desired response in the individual. Example indicators of an effective therapeutic or combination of therapeutics that include, for example, improved well-being of the patient, reduction of disease burden, arrested or slowed progression of disease, and / or absence of progression of disease to other locations in the body.

[0249] As used herein, the term “subject” refers to an animal. The terms “subject” and “patient” may be used interchangeably herein. As such, a “subject” includes a human that is being treated for a disease, or prevention of a disease, such as a patient.

[0250] In some embodiments, the methods and vaccines described herein may be used to treat an animal subject belonging to any classification, such as any member of the class Mammalia. As used herein, “subject” includes humans, domestic animals, such as laboratory- 50 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)animals (e.g., dogs, monkeys, pigs, rats, mice, hamsters, etc.), household pets (e.g., cats, dogs, rabbits, etc.) and livestock (e.g., chickens, pigs, cattle (e.g., a cow, bull, steer, or heifer), sheep, goats, horses, etc.), and non-domestic animals. In some embodiments, a subject is a mammal (e.g., a non-human mammal). In some embodiments, a subject is a human.

[0251] Delivery systems useful in the context of embodiments of the disclosed compositions and methods may include time-released, delayed release, and sustained release delivery systems such that the delivery of the compositions occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. The composition can be used in conjunction with other therapeutic agents or therapies. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician, and may be particularly suitable for certain composition embodiments disclosed herein.

[0252] Release delivery systems include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polyesteramides, polyorthoesters, polycaprolactones, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer systems that are lipids including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di- and tri-glycerides; sylastic systems; peptide based systems; hydrogel release systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. In some embodiments, lipid nanoparticles or polymers are used as delivery vehicles for vaccines, e.g., therapeutic and prophylactic vaccines, described herein, including delivery of mRNA to tissues.

[0253] In certain embodiments, the administration of the compositions may be carried out in any manner, e.g., by parenteral or nonparenteral administration, including by aerosol inhalation, injection, infusions, ingestion, transfusion, implantation or transplantation. For example, the compositions described herein may be administered to a patient trans-arterially, intradermally, subcutaneously, intratumorally, intramedullary, intranodally, intramuscularly, by intravenous (i.v.) injection, intranasally, intrathecally or intraperitoneally. In one aspect, the compositions of the present disclosure are administered intravenously. In one aspect, the compositions of the present disclosure are administered to a subject by intradermal or subcutaneous injection. The compositions may be injected, for instance, directly into a tumor, lymph node, tissue, organ, or site of infection.- 51 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)

[0254] In one embodiment, administration may be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or longer. Repeated courses of treatment are also possible, as is chronic administration. The repeated administration may be at the same dose or at a different dose.

[0255] In some embodiments, the compositions may be administered according to the disclosed methods by maintenance therapy, such as, e.g., once a week for a period of 6 months or more.

[0256] In one embodiment, cells can transiently express the mRNA described herein (e.g., mRNA encoding an antigen, mRNA encoding IRF8, or both) for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after introduction. Transient expression of the mRNA can be affected by the method of delivery. In one embodiment, the mRNA is transduced into the cell by electroporation. In one embodiment, the mRNA is introduced into the cell by lipid transfection methods known in the art.

[0257] In some embodiments, a vaccine as described herein may be used in combination with other known agents and therapies. Administered “in combination”, as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject’s treatment, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disease and before the disease has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery”. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.- 52 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)

[0258] In further embodiments, a composition described herein may be used in a treatment regimen in combination with surgery, radiation, chemotherapy, antibodies, or other agents.EXAMPLESEXAMPLE 1Initial Studies

[0259] Tremendous strides have been made in applying mRNA technology for vaccination and therapeutics and offer hope in the more resilient oncological and infectious disease space. Yet, many hurdles remain, therapeutic efficacy being one of the biggest.Disclosed herein are mRNA encoding for certain transcription factors which trigger novel immunostimulatory pathways in myeloid cells. This approach tremendously enhances cancer and infectious disease therapeutics and vaccines.

[0260] mRNA-based vaccines for COVID-19 have been successful, and now been safely administered to billions of people, highlighting their potential for clinical translation. Lipid nanoparticles are clinically advanced delivery materials for mRNA payloads. A major challenge to generate a strong immune response is to enable effective antigen presentation by myeloid cells, resulting in higher therapeutic efficacy. This is particularly important for cancer vaccines where the mRNA neoantigen approach is only marginally beneficial in large trials (Ref. 86). Hence, it is critical to develop new strategies which can enhance immunostimulation, and neoantigen presentation.

[0261] Currently, immune stimulatory mRNAs focus mainly on encoding cytokines or co-stimulatory molecules. RNA encoding pro-inflammatory cytokines delivered either intratum orally (IL-23, IL-36y, and OX40L cocktail) or systemically (IL- 12) have demonstrated limited to moderate efficacy in generating anti-tumor responses in both clinical and preclinical settings. Alternatively, ex-vivo engineering of dendritic cells (DCs) with TriMix mRNA (CD40L, TLR4-a and CD70) has been evaluated in clinical trials for patients with breast cancer and late-stage melanoma with limited efficacy (Refs. 11, 83). Regardless of delivery method (protein or RNA), cytokine-based therapies have met limited clinical success to date. One reason for the limited response to these agents is the context with which the signal is sensed, and that cytokine signals are self-limiting when devoid of context.Similarly, targeting cell surface receptors / ligands induce immunostimulation which is limited- 53 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)to the signaling cascades from this small set of receptors. Moreover, deficiency in cytokine signaling regulation can result in runaway inflammation and tissue damage resulting from autoimmunity. In contrast to prior mRNA therapeutic targets, disclosed herein is expression of the immune-activating kinase (NIK) and the transcription factor interferon regulatory factor 8 (IRF8) as vectors for immunotherapy in myeloid cells (Refs. 21, 84). Expression of these novel targets remarkably enhances immunostimulation. Presented herein are the first mRNA therapeutics for (i) NIK and (ii) IRF8; and a demonstration of how this approach is remarkably effective in boosting ensuing immune response for cancer and infectious disease. Therapeutic expression of NIK was determined to enhance DC co-stimulation and CD8 T cell responses. Subsequently, strong anti-tumor immune response was elicited in several mouse cancer models. Furthermore, co-delivery of NIK mRNA with model (ovalbumin) or influenza antigen encoding mRNA was determined to potently enhance vaccination responses by both boosting antigen-specific CD8 T cells and antibody titers. These results thus demonstrate the clinical utility of NIK-activating therapies to augment immune responses in cancer and infectious disease settings. Further, IRF8 mRNA enhanced DC expression of IL-12 and the output of cDCl-like cells from in vitro culture, raising the prospect of developing IRF8-enhanced DC vaccines. Likewise, IRF8 mRNA treatments could trigger substantial antitumor immunity and boost vaccination responses. This is the first approach leveraging mRNA-based expression of NIK and IRF8 as a therapeutic. Moreover, the disclosed studies demonstrating that mRNA-encoded NIK activation boosts anti-cancer immunity are groundbreaking, especially considering that NIK has previously been negatively associated with cancer outcomes. Simultaneously, IRF8-based therapeutics offer a unique platform to facilitate antigen-presentation that translates into enhanced CD8 T cell responses in either cancer or infectious disease states. Overall, these strategies significantly differ from other mRNA-based therapeutics focused on effector factors such as cytokines and co-stimulatory receptors. Therefore, the present disclosure proposes that factors such as NIK and IRF8 are pleiotropic immune stimulants, which activate multiple independent immune pathways using singular mRNA payloads. This advancement in mRNA target design will serve as cornerstone to generate next-generation of therapies, which will allow manifold applications to boost immunity in settings such as cancer and vaccination with broad biological applications.- 54 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)Design and Synthesis of mRNA sequences

[0262] Sequences encoding mouse NIK and IRF8 were fully optimized for mRNA codon usage and secondary structure using mRNAid, an open-source platform for therapeutic mRNA optimization, design and visualization (Ref. 85). Gene blocks for these sequences were purchased from Genewiz and cloned into an in vitro transcription vector containing a T7 polymerase promoter sequence, a CLEANCAP® AG site that enables transcription of a polyadenylated, translationally competent, immune-evasive 5'-m7G Cap-1 structure, and an encoded poly A tail of 100 nucleotides. After ligation into expression vectors, mRNAs were produced using HISCRIBE® T7 High Yield RNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions.N1 -methylpseudouridine modified mRNA was synthesized using the HISCRIBE® T7 High Yield RNA Synthesis Kit (New England Biolabs) according to the manufacturer’s instructions. mRNA length was verified using an Agilent Fragment Analyzer (Agilent Technologies, Santa Clara, CA, USA). SEQ ID NO: 1 is the NIK coding sequence and SEQ ID NO: 5 is the IRF8 coding sequence.Synthesis of Lipid Nanoparticles (LNPs)

[0263] LNPs for in vitro and in vivo studies were prepared by microfluidic mixing.Briefly, the lipid components were dissolved in ethanol and, separately, mRNA encoding for NF-KP inducing kinase (NIK), IRF8 transcription factor (IRF8) or firefly luciferase (Flue) was diluted into a solution of 10 mM citrate pH 3.0 at a concentration of 133 ng / pL. The two solutions were mixed in a 3:1 water / ethanol volume ratio. LNPs used in these studies were formulated with a molar composition of (35:16:46.5:2.5) using ckk-el2 ionizable lipid (CAS No.: 1432494-65-9), DOPE (l,2-Dioleoyl-sn-glycero-3-PE; CAS No.: 4004-05-1) as a helper lipid, cholesterol, and DMPE-PEG (polyethylene glycol (PEG)-modified (PEGylated) 1,2-Dimyristoyl-sn-glycero-3-PE (DMPE); DMPE CAS No.: 998-07-2), respectively.In vitro efficacy of NIK and IRF8 mRNA to induce IL- 12 secretion in primary myeloid cells

[0264] NIK, IRF8 and Flue mRNA were first investigated for their ability to induce IL- 12 in bone-marrow derived dendritic cells, one measure of immunostimulation of these cells. For these experiments, neoantigen mRNA had not yet been co-introduced, simply to enable identification of the native ability of each mRNA for DC stimulation. Interestingly, the LNP- 55 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)by itself had no immunostimulatory effects. Briefly, myeloid cells were isolated from the bone marrow of IL-12-eYFP mice. Myeloid precursor cells were differentiated into dendritic cells (DC) using granulocyte-macrophage colony-stimulating factor (GM-CSF) and Fms-like tyrosine kinase 3 ligand (Flt3L) and then used for screens with MD1 formulated NIK, IRF8 and firefly luciferase (FFL; control) mRNA. Highest IL-12 induction was observed in NIK and IRF8 mRNA treated cells in a dose responsive manner, with minimal IL-12 induction observed upon treatment with FFL mRNA (FIGs. 1 A-C; n = 3 replicates).Enhancing type 1 conventional dendritic cell (cDCl) population in vitro through treatment with IRF8 mRNA

[0265] Next, the ability of IRF8 mRNA delivery to regulate primary dendritic phenotype in vitro was investigated. LNPs encapsulating IRF8 mRNA were added at two different time points during the myeloid cell culture: at day 0 only and at day 0 and day 5, followed by flow cytometry analysis on day 9, where cells were stained for CD103+XCR1+ markers to identify cDCl population (FIGs. 2A-B). A significant increase in the cDCl phenotype was observed. Notably, about 80% of the myeloid cell population exhibited cDCl phenotype after treatment with 350 ng IRF8 dosed on day 0 and day 5.In vivo activation of NIK to activate primary dendritic cells

[0266] Next, the ability of NIK mRNA to activate dendritic cells in the lymphoid tissues after systemic administration of LNPs was investigated. These LNPs were formulated with a molar composition of (35:16:46.5:2.5) using ckk-el2 ionizable lipid, DOPE as a helper lipid, cholesterol, and DMPE-PEG, respectively. mRNA loaded LNPs were intravenously administered in mice (n = 3) at a dose of 5 pg. Spleen and inguinal lymph nodes were harvested from the mice after 48 hours and expression of activation markers in lymphoid (spleen, FIG. 3B; and lymph node, FIG. 3C) dendritic cells was analyzed using flow cytometry (gating strategy for CD45+CD1 lc+ populations (n = 3 mice) shown in FIG. 3 A). NIK mRNA treated mice demonstrated high expression of co-stimulatory markers (CD80 and CD86), exhibiting their ability to activate DCs.- 56 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)In vivo anti-tumor efficacy of LNPs formulated with mRNA encoding NIK and IRF8

[0267] BL6 mice were implanted with MC38 tumor cells on day 0 and which were allowed to grow to about 100 mm3solid flank tumors. Mice were then treated with an intravenous (i.v.) injection of MD1-NIK LNPs, MD1-IRF8 LNPs (0.5 milligrams per kilogram (mpk) / 10 pg of mRNA) or PBS controls (n = 5) on day 6 and day 9. Mice were sacrificed when tumors had grown to a volume greater than about 1500 mm3. LNP NIK and LNP IRF8 significantly reduced tumor growth (FIGs. 4A-E) after intravenous administration in mice. By day 15, tumors in 3 of 5 mice treated with LNP NIK (FIG. 4B) and LNP IRF8 (FIG. 4C) were completely eradicated. The remaining 2 of 5 treated mice showed significantly slower tumor growth, prolonging survival as compared to untreated mice. On the other hand, tumors rapidly progressed in PBS treated mice, which were sacrificed when the tumor reached a volume of about 1000 mm3(FIG. 4A). These results led to a 60% longterm survival of treated animals, with all control -treated animals dead 27 days after tumor implantation (FIG. 4D). All the surviving mice were re-challenged with MC38 tumor cells in the opposite flank 90 days post first injection. 100% of the mice in NIK i.v. and about 70% of the mice in IRF8 i.v. treated groups rejected tumor growth completely, exhibiting long term memory response (FIG. 4E).

[0268] Next, the anti -tumor potency of LNP NIK and LNP IRF8 was investigated in another tumor model through different routes of administration. BL6 mice received MB49 tumors on day 0 which were allowed to grow to about 100 mm3established tumors. On day 5, mice were treated with i.v. administered LNP_NIK (n = 5; 10 pg / mouse) or LNP_control mRNA and PBS as control (n = 5). Simultaneously, another mouse cohort received intratumoral (i.t.) administration of LNP NIK and LNP IRF8. Mice were sacrificed when tumors had grown to a volume greater than 2000 mm3. By day 15, tumors in LNP treated mice treated showed significantly slower tumor growth (FIGs. 5 A-B), prolonging survival as compared to control groups. Tumors rapidly progressed in control mice, which were sacrificed when the tumor reached a volume of about 1500 mm3. These results led to a 100% long-term survival of treated animals, with all control-treated animals dead 20 days after tumor implantation (FIG. 5C).- 57 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)NIK and IRF8 mRNA as adjuvants for cancer and infectious disease vaccines

[0269] Given the role played by NIK (NF-KP inducing kinase) and IRF8 pathways in stimulating adaptive immunity as demonstrated herein, the ability of these TFs to act as adjuvants for boosting cellular and humoral immune response was investigated. BL6 mice were treated with intravenously administered LNPs encapsulating mRNA encoding ovalbumin (OVA; LNP_OVA, 5 pg) in combination with LNP_NIK (5 pg) at day 0 (prime dose) and again at day 12 (booster dose). Mice treated with LNP OVA (10 pg) and PBS were used as controls. Retro-orbital blood was drawn three times to quantify the peripheral blood T cells by OVA tetramer staining. Quantification of peripheral blood T cells positive for OVA tetramer as a proportion of CD45+CD8+ T cells showed that, notably, LNP NIK boosted the ensuing cellular response in mice as evidenced by increased blood CD8+T-cell population after both prime and boost dosing. Notably, co-delivery of OVA and NIK mRNA induced stronger cellular response which persisted until day 90, highlighting the potential of this strategy to generate stronger and durable immunity against cancer (FIGs. 6A-B).

[0270] Next, the ability of NIK and IRF8 mRNA to boost humoral response was investigated. BALB / c mice were vaccinated with influenza hemagglutinin (HA) H3 influenza mRNA in combination with NIK or IRF8 mRNA (n = 5 per group) at a dose of 0.1 pg, followed by blood collection at day 14 to evaluate IgG titers by enzyme-linked immunosorbent assay (ELISA). Mice vaccinated with FFL mRNA and H3 alone were used as controls (n = 5). Co-delivery of NIK or IRF8 mRNA significantly increased the H3-specific IgG titers indicating the potential of this strategy to develop more potent vaccines for infectious diseases (FIG. 7).EXAMPLE 2In vitro efficacy of NIK and IRF8 mRNA in primary myeloid cells leading to activation of innate immunity pathways and cell phenotype differentiation.

[0271] NIK and IRF8 act upstream of multiple effector pathways and are associated with increased pro-inflammatory cytokine production (Refs. 22, 25). Therefore, to initially evaluate the immunostimulatory potential of IR-mRNAs, the IR-mRNAs were formulated with MESSENGERMAX® transcription reagent (Mmax) and administered to immature bone-marrow-derived dendritic cells (BMDCs). Activation of various immune signaling pathways was then examined. To evaluate IL-12 induction, BMDCs from p40-IRES-eYFP reporter mice were used, which express eYFP under the control of the IL-12p40 promoter.- 58 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)IL-12p40 reporters are widely used as validated surrogates for IL-12p70 secretion, owing to the well-established correlation between IL-12p40 expression and IL-12p70 production (Refs. 36-38). Significantly higher eYFP levels were observed in both IR-mRNA-treated cells relative to the control firefly luciferase (Flue) mRNA treatment, indicating sequencespecific upregulation of IL-12p40 (FIG. 1A, FIG. 10A, and FIG. 38A). IL-12 is involved in T helper type 1 (Thl) cell differentiation and maturing cytotoxic T cell anti -turn or immunity (Ref. 39), therefore, this enhanced secretion of IL-12 may indicate that these IR-mRNAs strengthen the ability of DCs to activate cell-mediated immunity (Ref. 21). Stimulation of type I interferon (IFN-I) production was also assessed following treatment with NIK and IRF8, as IFN-I is another important signal for T cell priming (Ref. 40). Transfection of wildtype C57BL / 6J BMDCs with IR-mRNAs resulted in significantly higher IFN-I secretion compared to Flue mRNA, further confirming their immunostimulatory potential.Interestingly, NIK induced the release of both IFN-a and IFN-P, whereas IRF8 primarily stimulated IFN-a production (FIG. 10B, FIG. 38B, FIG. 38C). Notably, NIK elicited a stronger interferon response than IRF8, consistent with previous reports identifying NIK as a central signaling node in the non-canonical NF-KB pathway (Ref. 41). Overall, these results demonstrate the ability of these IR-mRNAs to induce the expression of key T cell priming signals.

[0272] Next, the potential of IR-mRNAs to reprogram immature DCs into mature, T cell priming phenotypes was further evaluated using both flow cytometry and single-cell RNA sequencing (scRNA-seq). IR-mRNAs were again formulated with Mmax and administered to immature BMDCs from wild-type C57BL / 6J mice. Flow cytometry revealed a significant increase in the proportion of activated DCs as evidenced by significant increases in CD86 and MHC-II (FIG. 10C, FIG. 32). Furthermore, the proportion of cDCl cells significantly increased following IR-mRNA treatment (FIG. 10D, FIG. 10E, and FIG. 31). Notably, IRF8 transfection resulted in about 77% of the total population adopting a cDCl phenotype, representing an about 4.3-fold increase compared to PBS and an about 3.3-fold increase compared to Flue controls, consistent with IRF8’s role in driving cDCl lineage commitment (Ref. 42). NIK mRNA treatment also enhanced cDCl differentiation, possibly due to the increased production of type-I IFNs and IL-12, both of which are known to support cDCl development (Refs. 23, 43). Consistent with flow cytometry results, the scRNA-seq results demonstrated an expansion of cDCl-like cells marked by Batf3 expression (FIG. 27; see also, Refs. 24, 44). A similar shift in phenotype was observed in IR-mRNA-treated BMDCs- 59 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)characterized by significant increases in IL- 12, CD40, CD80, CD83, CD86, and Ccr7 expression within this population with scRNAseq (FIG. 10F, FIG. 10G, and FIG. 28).Interestingly, an increase in CD40 / 80 transcription levels was not reflected in flow cytometry studies (FIG. IOC), suggesting transcriptional activation does not always translate directly into surface protein accumulation (see also, Ref. 45). scRNA-seq analysis of immature DCs revealed upregulation of transcription factors associated with cDCl development, such as Spiland Irf8, upon IR-mRNA treatment (see also, Refs. 46, 47). Concurrently, genes involved with antigen processing and cross-presentation, including Ctsc, Nlrc5, and Psmb9, were also elevated (see also, Ref. 48). Upregulation of Ifit3, Irf7, Stat2, and Stat3 indicated activation of type I IFN signaling following IR-mRNA treatment (FIG. 28; see also, Ref. 49). Collectively, these findings highlight the developmental, antigen-processing, and immunostimulatory axes that define cDCl function (see also, Ref. 46). cDCls are critical for the cross-priming of CD8+T cells and play a central role in cancer vaccine efficacy and T cell-mediated immunity, suggesting these IR-mRNAs are capable of promoting an antitumor phenotype within BMDCs (see also, Refs. 44, 50).

[0273] Finally, a Monocle psuedotime analysis was performed on the scRNA-seq data to determine whether cell reprogramming was occurring through maturation pathways or cell fate conversion (Ref. 51). The reconstructed trajectory with an origin in pre-DCs bifurcates into the two conventional dendritic cell lineages: cDCl and cDC2 (FIG. 10H, FIG. 101). A third branch also emerges from the same progenitor population, leading to a clearly segregated pDC cluster (FIG. 10H, FIG. 101). Importantly, the pDC branch diverges away from both cDCl and cDC2 and does not intersect or merge with either of the conventional DC lineages. This pattern is consistent with the known developmental separation between pDCs and eDCs. Then, when experimental conditions were overlaid (FIG. 10J), the PBS and Flue controls remained localized to early cDCl pseudotime states, whereas IRF8 and NIK treatments shifted cells toward progressively later pseudotime along the cDCl maturation branch. Together, these results demonstrate that IRF8 and NIK promote advancement along the endogenous pre-DC to cDCl maturation axis, rather than inducing fate conversion from cDC2s or pDCs into cDCls, or altering overall lineage identity. These results demonstrate that delivery of IR-mRNAs can reprogram DCs into a cDCl phenotype and induce activation and expression of key co-stimulatory surface signals, which are expected to enhance T cell priming and activation (see also, Ref. 52).- 60 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)EXAMPLE 3In Vivo Assessment of Immune Reprogramming and Toxicity upon Systemic Administration of mRNA-adjuvants

[0274] After confirming that these IR-mRNAs can activate APCs in vitro, their ability to stimulate IFN-I expression and APC activation was tested in vivo. NIK, IRF8, and Flue mRNAs were formulated individually into LNPs using a previously reported formulation containing the ionizable lipid cKK-E12, l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), cholesterol, and l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG), as this formulation has previously been shown to give high levels of mRNA expression in the liver and antigen-presenting cells (Refs. 53, 54). mRNA LNPs were then administered intravenously (IV) and their immunostimulatory effects were assessed. Six hours after LNP administration, IFN-I levels were significantly higher in IR-mRNA-treated mice relative to both control mRNA and PBS-treated mice (FIG. 11 A, FIG. 33). In accordance with the treatment of BMDCs, NIK stimulated higher levels of IFN-P than IRF8 while both mRNAs yielded similar levels of IFN-a.

[0275] Given that IFN-I induction enhances DC expression of MHC-II and costimulatory molecules (Ref. 55), the ability of the IR-mRNAs to upregulate antigen-presenting cell (APC) activation markers in lymphoid tissues following systemic administration of LNPs was evaluated. Treated mice exhibited elevated expression of co-stimulatory markers (CD86) on eDCs isolated from the spleen and lymph nodes. In the spleen, CD86 was upregulated in approximately 30% of APCs in NIK- and IRF8-treated mice, compared to only about 2% in Flue (control) mRNA-treated mice. In the lymph nodes, IRF8 treatment led to increased expression of CD40, CD80, and CD86, whereas NIK treatment primarily upregulated CD86 (FIG. 44, FIG. 48). Additionally, both NIK- and IRF8-treated mice demonstrated a marked increase in Ml-polarized macrophages (about 30-35% in the spleen and about 10% in the lymph nodes), relative to Flue-treated controls (about 2.5% in both tissues). In agreement with the in vitro observations, these studies demonstrate that IR-mRNA LNPs function to reprogram key APCs toward a pro-inflammatory, mature phenotype which is critical for potentiating antitumor immune responses (see also, Refs. 52, 56).

[0276] Although these IR-mRNA LNPs induce immune activation, systemic delivery of cytokines and mRNA-LNP therapies may be associated with acute immune and hepatic toxicities. Therefore, a thorough toxicological evaluation was conducted to identify any potential IR-mRNA-specific toxicity. Mice were intravenously administered 1.5 mg / kg of- 61 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)mRNA (Flue, NIK, or IRF8) formulated into cKK-E12 LNPs and monitored for 6 days for changes in body weight, biochemical markers, and histopathology. All mRNA-LNP -treated mice exhibited a similar, transient decrease in body weight at 6 hours, which normalized to PBS-treated levels by day 4. Similarly, moderate hepatotoxicity, indicated by elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and reduced serum albumin at 6 hours, was observed across all treatment groups, including Flue controls. These changes largely resolve by day 6, with modest residual reductions in serum albumin for mRNA LNP treated groups (FIG. 17b-FIG. 17f; see also, Ref. 57). Systemic upregulation of pro-inflammatory cytokines (GM-CSF, IFN-y, IL-ip, TNF, IL-6, and CXCL2) was detected in NIK- and IRF8-treated mice at 6 hours (FIG. 11 A) but returned to baseline levels by day 6 (FIG. 17g). Histopathological evaluation of the liver and spleen showed no significant differences between both IR-mRNAs and the control mRNA treated mice (FIG.29 and FIG. 30). Overall, these results demonstrate that LNP-mediated delivery of NIK and IRF8 mRNA causes only transient and minimal systemic and hepatic inflammation, comparable to the Flue control mRNA. This is particularly promising compared to immunocytokine- or adjuvant-based cancer therapies, which often exhibit substantial toxicity and limit clinical translation (Refs. 58-61). Given the established clinical use of mRNA-LNP platforms, these findings further support the translational potential of NIK and IRF8 mRNA-based immunotherapies.EXAMPLE 4Reprogramming of tumor microenvironment from NIK, IRF8 immunotherapy.

[0277] Next, the effect of IR-mRNA LNPs on the immune microenvironment within tumors was examined. For these initial evaluations, the MB49 bladder was selected because its comparatively low baseline immunogenicity provides a stringent setting to characterize treatment-driven immune remodeling (Ref. 62). Therefore, MB49 syngeneic tumors were implanted subcutaneously and once the tumors reached about 100 mm3in size, IR-mRNA LNPs (0.25 mg / kg) was dosed intratumorally and the tumor, spleen, and tumor-draining lymph nodes were collected on days 1 and 7 post-injection to evaluate phenotypic and cellular changes associated with innate and adaptive immune responses, respectively.Following NIK and IRF8 mRNA-LNP treatment, mild activation of both cDCl and cDC2 populations in tumors and tumor-draining lymph nodes was observed in the IRF8-treated- 62 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)group, as indicated by increased CD86 expression relative to Flue treatment (FIG. 12, FIG.18a, FIG. 18b). NIK treatment, in contrast, showed significant CD86 upregulation in the tumor only, with insignificant upregulation in the draining lymph node. Twenty-four hours post-IRF8 treatment, cDCl populations were elevated in tumor-draining lymph nodes (TdLNs) compared to controls. By day 7, both NIK and IRF8 treatments increased cDCl abundance in TdLNs, with IRF8 having a more pronounced effect (FIG. 12B). Interestingly, cDC2 expansion in tumors at day 7 was observed only in IRF8-treated mice, with no change seen at 24 hours or in TdLNs for either treatment. Plasmacytoid DCs (pDCs) remained unchanged across all groups (FIG. 18, FIG. 35, FIG. 44). This is consistent with IRF8’s role as a transcription factor essential for cDCl development, and the ability of both IRF8 and NIK to stimulate Type I IFNs that recruit immune cells to tumors and TdLNs (Refs. 23, 24, 43). Notably, the selective expansion and activation of cDCls is very useful, as they are important drivers of antitumor CD8+T cell priming (Ref. 44).

[0278] The activation of both innate and adaptive lymphocytes was then assessed following NIK and IRF8 mRNA-LNP treatment (see also, Refs. 63, 64). Both natural killer T (NKT) and gamma delta (yb) T cells, which can kill tumor cells independently of classical MHC recognition, showed increased CD69 expression in tumors and tumor-draining lymph nodes (TdLNs) 24 hours after treatment (FIG. 12, FIG. 34). This early activation was accompanied by greater accumulation of NKT and yb T cells in tumors compared to PBS and control mRNA groups. In addition to innate lymphocyte activation, NIK and IRF8 treatment enhanced adaptive immunity. Tumor-infiltrating CD8+T cells showed increased CD69 expression in both treatment groups, while CD4+T cells were activated only in NIK -treated tumors (FIG. 18e, FIG. 18f). In TdLNs, no significant activation was observed in CD4+T cells, whereas CD8+T cells showed activation only in the IRF8-treated group and not in the NIK-treated group. By day 7, both NIK and IRF8 treatments increased infiltration of effector and memory CD8+T cells in tumors compared to controls. NIK treatment also elevated CD4+T cell numbers, whereas IRF8 did not affect CD4+T cell infiltration (FIG. 121, FIG. 46). Notably, both treatments resulted in a high CD8+ / Treg ratio, indicating robust antitumor immunity without increasing immunosuppressive Tregs. These results demonstrate that the innate immune reprogramming by NIK and IRF8 initiates anti-tumor adaptive immunity.- 63 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)EXAMPLE 5Elimination of Established Tumors and Generation of Long-Lasting Systemic Immunity by mRNA-LNPs

[0279] Given the ability of IR-mRNA LNPs to reprogram the tumor immune microenvironment, the therapeutic efficacy of these treatments was next evaluated in syngeneic tumor models. For these studies, both systemic and intratumoral delivery strategies were explored to compare their efficacy. Systemic delivery is clinically preferred due to its ease and for its ability to target both primary and metastatic tumors and induce systemic immunity (Refs. 65, 66). However, systemic drug exposure at higher doses may cause adverse reactions. In contrast, intratumoral administration can be more invasive, but it may reduce systemic therapeutic exposure while simultaneously providing higher levels within the tumor and surrounding lymphoid tissues for improved antigen trafficking (Refs. 67-69). For initial therapeutic evaluations, IR-mRNAs were tested in two established subcutaneous MC38 (colorectal) and MB49 (bladder) syngeneic models, to demonstrate broad applicability across tumor types.

[0280] For further evaluation in the MC38 model, tumors were subcutaneously implanted in C57BL / 6J mice and weekly intratumoral injections of IR-mRNA LNPs or controls administered for three weeks. Mice treated with NIK or IRF8 mRNA LNPs (0.25 mg / kg) showed significant tumor regression, while control groups receiving PBS or Flue mRNA failed to control tumor growth. By day 20, complete tumor response was observed in 11 / 15 IRF8-treated and 11 / 16 NIK-treated mice, with the remaining showing delayed progression and prolonged survival. In contrast, control tumors rapidly progressed, reaching about 1500 mm3, necessitating euthanasia. This resulted in 73% (IRF8) and 69% (NIK) long-term survival, with all control mice deceased by day 27 (FIG. 13). Surviving mice rechallenged on the opposite flank at day 60 demonstrated robust memory responses, with 91% (NIK) and 82% (IRF8) rejecting tumors completely. Intravenous administration of NIK or IRF8 mRNA (0.5 mg / kg, two doses on days 6 and 9) similarly suppressed tumor growth, with 60% of mice in both groups achieving complete regression by day 15 (FIG. 24). This led to 60% long-term survival, compared to 0% in controls. Upon rechallenge at day 90, 100% of NIK- and 67% of IRF8-treated mice rejected tumor growth, confirming durable immune memory via systemic LNP administration as well. CD8+T-cell depletion fully abolished the therapeutic efficacy of NIK and IRF8 mRNA, with tumors progressing similarly to PBS controls. In contrast, CD4+T-cell depletion had minimal impact, and treated mice responded comparably to wild-type- 64 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)controls. These results indicate that CD8+T cells are the principal mediators of the antitumor effect (FIG. 36).

[0281] Next, the co-treatment of IR-mRNA LNPs with anti-PD-1 antibody therapy was investigated to assess whether checkpoint inhibition could enhance the therapeutic efficacy of newly primed systemic T cell responses (Ref. 70). For these studies, C57BL / 6J mice bearing MB49 tumors were treated with IR-mRNA LNPs via both intravenous and intratumoral administration in combination with intraperitoneally administered anti-PD-1 checkpoint blockade (i.e., an anti-PD-1 antibody; anti-mouse PD-1 antibody (CD279), Catalog No. BP0146, Bio X Cell, Lebanon, NH, USA). Untreated mice (PBS), mice treated with anti-PD-1 alone, and mice treated with a control mRNA-LNPs (Flue) in combination with anti-PD-1 were used as controls. Anti-PD-1 monotherapy induced only modest delays in tumor growth and morbidity compared to PBS. The combination with Flue LNPs did not result in any additional tumor inhibition (FIG. 13E). In contrast, both NIK and IRF8 mRNA-LNP treatments led to significant tumor growth suppression across all treated mice, with complete tumor responses observed with IRF8 and NIK treatment both intratumorally and intravenously (FIG. 13F, FIG. 13G). Notably, even in the absence of anti-PD-1 co-treatment, NIK and IRF8 monotherapies demonstrated potent anti-tumor activity, although they failed to induce complete responses in any animals (FIG. 25). These results indicate that IR-mRNA LNP treatments can elicit robust anti-tumor responses in aggressive, immunotherapy refractory tumor models and hold promise for achieving durable therapeutic benefit when combined with checkpoint blockade.

[0282] Next, the ability of IR-mRNA LNPs to treat a metastatic model of cancer in mice was evaluated. U106luciferase-expressing B16-F10 melanoma cells were injected to establish lung metastases, and tumor growth was monitored via bioluminescence imaging. One week after tumor engraftment, mice received intravenous injections of LNPs encapsulating NIK, IRF8, or tdTomato (control) mRNA on days 5 and 9 (FIGs. 14A-H). Encouragingly, NIK- and IRF8-treated mice exhibited significantly reduced tumor growth compared to control groups (FIG. 14A, FIG. 14B). On day 15, the lungs were harvested to quantify tumor nodules and metastatic burden in the lung tissue via histopathology (FIG. 14 d). Both NIK and IRF8 treatments remarkably reduced lung metastases, with NIK achieving greater efficacy (FIG. 14D). Correspondingly, lung weight was significantly lower in treated groups (FIG. 45), reflecting reduced tumor burden. Furthermore, immunohistochemistry studies revealed 5.8-fold (NIK) and 4.5-fold (IRF8) increase in cytotoxic CD8+T cell- 65 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)infiltration in the tumors as compared to controls (FIG. 14E, FIG. 14F). Tumor cell proliferation was reduced about 12.4-fold (NIK) and about 5-fold (IRF8) compared to control mRNA (FIG. 14G, FIG. 14H). These findings support the hypothesis disclosed herein, namely, that these IR-mRNA LNPs not only promote dendritic cell activation but subsequently induce effector T cell responses which suppress tumor growth. Overall, systemic NIK and IRF8 mRNA LNPs elicited robust antitumor immunity, highlighting their therapeutic potential with metastatic and non-superficial tumors.EXAMPLE 6NIK and IRF8 mRNAs as adjuvants for cancer vaccines

[0283] After establishing that immune-reprogramming mRNAs enhance systemic antitumor immunity and improve checkpoint inhibitor therapy, it was investigated whether these mRNAs could generate durable, functional memory T cells capable of preventing or delaying tumor growth. Since systemic delivery of NIK and IRF8 mRNA promotes IFN signaling and dendritic cell maturation in lymphoid tissues, as disclosed in the foregoing Examples, it was hypothesized that co-delivery with a neoantigen-encoding mRNA could enhance antigenspecific effector and memory T cell responses capable of tumor rejection.

[0284] C57BL / 6J mice were immunized with PBS, OVA mRNA alone (10 pg), or OVA mRNA co-delivered with either NIK, IRF8 or fLuc (control) mRNA at a 1:1 ratio, keeping the total RNA dose constant (10 pg). Vaccinations were conducted using a prime-boost regimen on days 0 and 14. Blood was collected from mice on Day 7, Day 28 to evaluate the priming and expansion of antigen specific CD8+T cells, followed by monitoring of blood CD8+T cells on Day 60 and Day 90 to assess lasting memory response (FIGs. 15A-I). On day 7, after the prime dose, there was no significant increase in CD8+T cell blood count for IRF8 (6.15%) or fLuc (4.15%) groups, co-delivery with NIK showed marginally higher CD8+T cell counts (about 7.7%). After the boost dose, however, there were substantially higher antigen-specific blood CD8+T cell counts in mice that received adjuvanted vaccines. NIK and IRF8 showed 20% and 15% tetramer-positive CD8+T cells, respectively, significantly higher than OVA alone (5.6%) or fLuc co-administered mice (4.7%) (FIG. 15A, FIG. 15B). Following vaccination, we assessed the longevity of this immune memory. Two months after first dose, both NIK and IRF8 immunized mice showed about 4-fold higher circulating CD8+T cells as compared to controls (OVA, fLuc) treated mice. Antigen-specific CD8+T cells- 66 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)remained persistent even 3 months after vaccination with NIK and IRF8, showing 12 and 13% tetramer-positive CD8+T cells, respectively. In contrast, control mice showed minimal remaining CD8+T cells by this time point (FIG. 15 A, FIG. 15B). Potential synergy between NIK and IRF8 was also investigated by co-administering NIK + IRF8 + OVA, but an improvement in the generation of OVA-specific CD8+T cells was not observed (FIG. 39).

[0285] The protective efficacy of this prophylactic vaccination was then evaluated using a B16-OVA subcutaneous melanoma model. 90 days after the first dose, OVA-expressing B16-F10 cells were subcutaneously implanted in the immunized mice. OVA (alone) and fLuc (co-administered) treated mice showed a slight delay in tumor growth; however, it did not significantly improve their survival probability compared to unvaccinated mice (FIG. 15C, FIG. 15D). Both NIK and IRF8-treated mice showed complete rejection in 5 / 5 treated mice (FIG. 15C, FIG. 15D). These results indicate that these IR-mRNAs can adjuvant prophylactic cancer vaccines to enhance the generation of long-lived, functional CD8+T cells.EXAMPLE 7Lymphoid- Targeted Delivery of NIK and IRF8 Enhances their Cancer Vaccine Adjuvant Effects

[0286] Although these experiments demonstrated the potential of NIK and IRF8 as cancer vaccine adjuvants, the LNPs used for this experiment primarily yield mRNA expression in the liver; accordingly, it was hypothesized that cancer vaccine potency could be further enhanced by combining the adjuvant effects of the IR-mRNAs with targeted RNA delivery to lymphoid organs such as the spleen, where APCs are in proximity to T cells (see also, Refs. 66, 71). In order to identify a LNP formulation which targets mRNA delivery to the spleen, a combinatorial library of 100 ionizable lipids was screened; the combinatorial library was previously reported (Ref. 72). fLuc mRNA was formulated into LNPs with these ionizable lipids, injected intravenously, and its biodistribution was illuminated via bioluminescent imaging. From the initial screen, the ionizable lipid AMG56 was identified, which gave relatively specific and potent mRNA expression in the spleen. This ionizable lipid consists of a spirocyclic diamine headgroup with branched tails (FIGs. 19A-D). To further improve mRNA delivery to splenic APCs, an additional 20 ionizable lipids were synthesized by varying the tail length. The lipids were screened to identify the most potent vehicles for splenic delivery (see Example 9 for synthesis details). All of these lipids demonstrated enhanced spleen delivery compared to the standard control LNP (cKK-E12),- 67 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)with AMG514 achieving about 4-fold higher delivery (FIG. 19B, FIG. 19C). Both cKK-E12 and AMG514 mediated mRNA transfection in splenic dendritic cells and macrophages (FIG.19D), though AMG514 showed significantly greater transfection efficiency in these key antigen-presenting cell populations. To investigate the mechanism of spleen targeting of AMG514, the LNP protein corona was investigated following incubation with plasma. These LNPs were found to have enriched levels of integrin-binding and phagocytosis-associated proteins on their surface, including vitronectin (30.3%), prothrombin / coagulation factors (about 24%), and fibrinogen (9.3%), which collectively engage receptors abundantly expressed on splenic macrophages and dendritic cells (Refs. 73-75). Additional lower-abundance but high-potency opsonins — such as thrombospondin- 1 (1.0%) and apolipoprotein A-IV (ApoA-IV) (6.9%) — further support scavenger-receptor-mediated internalization by myeloid cells (FIG. 41a; see also, Refs. 76, 77). Together, this corona profile promotes rapid uptake by phagocytic immune populations and accounts for the predominant localization of AMG514 LNPs to the spleen and their efficient delivery to macrophages and dendritic cell subsets. Notably, this pattern differs from liver-tropic LNPs, whose coronas are typically dominated by apolipoprotein E (ApoE) and albumin — components largely absent here.Consistent with this distinction, AMG514 LNPs exhibit a pKa of about 7.5, markedly higher than the about 6.5 pKa characteristic of liver-tropic formulations (FIG. 41b; see also, Ref. 78).

[0287] Next, it was investigated whether these spleen-tropic LNPs could enhance adaptive immune responses by co-delivering immune-reprogramming mRNAs and neoantigen-encoding OVA mRNA to the splenic lymphoid compartments. C57BL / 6J mice were immunized with PBS, AMG514-formulated OVA mRNA alone (3 pg), OVA mRNA co-delivered with NIK mRNA at a 1 : 1 ratio (total RNA dose 3 pg), formulated using either AMG514 or cKK-E12 (FIG. 15E). A lower dose was selected for these experiments to evaluate the potential for improved splenic delivery to increase potency. Blood was collected from vaccinated mice on day 7 and day 28 to assess the priming and expansion of antigen specific CD8+T cells. Encouragingly, AMG514 formulated NIK + OVA generated about 15% tetramer positive CD8+T cells, significantly higher than cKK-E12 formulated NIK + OVA (about 6%) (FIG. 15H, FIG. 151). Additionally, NIK co-delivery once again showed superior CD8+T cell priming relative to OVA-only vaccinated mice.

[0288] Given the strong T cell responses from AMG514 LNPs with NIK mRNA (FIG.15H, FIG. 151), vaccinated mice were challenged with B16-OVA tumor cells on Day 35 and- 68 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)monitored for tumor growth. AMG514 OVA-only immunized mice showed delayed tumor growth compared to untreated PBS controls and one mouse showed complete tumor rejection (FIG. 15G). Mice vaccinated with NIK + OVA showed significantly higher delays in tumor growth and higher rejection rates. cKK-E12 LNPs gave complete rejection in 3 / 5 treated mice, while AMG514 LNPs showed an increased, though not statistically significant, complete rejection in 5 / 5 mice (FIG. 15G). These studies demonstrate the utility of an optimized mRNA delivery vehicle in combination with these immune reprogramming mRNAs to enhance tumor-specific immune responses.EXAMPLE 8IR-mRNAs for adjuvanted infectious disease mRNA vaccines

[0289] Given the performance of NIK and IRF8 as cancer vaccine adjuvants disclosed in the foregoing Examples, it was hypothesized that co-delivery of either NIK or IRF8 mRNA with a viral antigen-encoding mRNA could extend the application of these IR-mRNAs to infectious disease vaccines. mRNA vaccines for infectious diseases have emerged as a transformative platform, exemplified by their rapid development and success against COVID-19 (Ref. 79). mRNA vaccines are adjuvanted by both the mRNA and lipid nanoparticles (LNPs), which can activate innate immune pathways to enhance adaptive immunity (Ref. 10). For example, the BNT162b2 COVID-19 vaccine has been shown to activate the MDA5 and IFNAR1 signaling pathways in antigen-presenting cells (APCs), driving the resultant cellular and humoral immunity (Ref. 80). While these formulations can drive antigen-specific immune responses, there remains opportunities to further enhance the potency of mRNA vaccines for infectious diseases. IR-mRNAs induce type I IFN secretion and IL-12 production (FIG. 10), while simultaneously promoting DC maturation. Together, these effects drive a Th 1 -type response that enhances the generation of robust adaptive immunity (Ref. 39).

[0290] To evaluate the potential of the IR-mRNAs to adjuvant infectious disease mRNA vaccines, BALB / cJ mice were vaccinated with hemagglutinin (H3) (A / Tasmania / 503 / 2020 H3N2) mRNA alone or combined with IRF8, NIK, or a control (fLuc) mRNA using a primeboost regimen on days 0 and 21. Antibody titers were measured to assess humoral responses. Similarly, mice were vaccinated with SARS-CoV-2 spike mRNA (encoding a spike protein; see SEQ ID NO: 10) under the same conditions. In both antigen models, adjuvanted vaccines- 69 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)(with NIK or IRF8) elicited a 4-fold increase in IgG titers compared to unadjuvanted vaccines, while the fLuc control showed no enhancement (FIG. 26).

[0291] Building on these results, vaccination studies were further performed in C57BL / 6J mice, a Thl-biased model. Mice were again vaccinated with H3 mRNA in cKK-E12 LNPs either alone or with cKK-E12 LNPs encoding IRF8, NIK, or fLuc mRNA. On day 35, adjuvanted vaccines induced about 4.5-fold (IRF8) and about 4.9-fold (NIK) higher antibody titers than controls (FIG. 16D). Potential synergy was also investigated between NIK and IRF8 by co-administering NIK + IRF8 + H3 mRNAs, but an improvement in the generation of H3-binding antibodies was not observed (FIG. 39). IFN-y enzyme-linked immunosorbent spot (ELISpot) analysis of peptide-stimulated splenocytes revealed a robust Thl-biased cellular response, with MHC-I peptide pools eliciting higher responses than MHC-II. Like the cancer vaccine results, NIK and IRF8 adjuvanted vaccines induced substantially stronger CD8+T cell responses, with an about 13.3-fold and about 14.4-fold increase in the number of MHC-I spots, respectively, compared to the control and unadjuvanted groups (FIG. 16A-FIG.16C). Additionally, the cytokines secreted from the peptide-stimulated splenocytes were analyzed, and upregulation of a diverse range of Thl- and Th2-associated cytokines was observed, including IL-2, IL-4, IL-5, and IL-6 (FIG. 16f), providing further confirmation of the strong cellular responses induced by IR-mRNA adjuvanting. Overall, these results demonstrate extension of NIK and IRF8 to infectious disease vaccines to enhance their efficacy.EXAMPLE 9: Lipid Nanoparticle Formulation, Synthesis, and Characterization

[0292] The methods of Example 9 relate to Examples 2-8.Lipid nanoparticle formulation

[0293] l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE; CAS No.: 4004-05-1) (Avanti, Alabaster, AL, USA), Cholesterol (Millipore-Sigma, Burlington, MA, USA), and l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k; CAS No. 160743-62-4) (Avanti) stocks were prepared by dissolving at 10 mg / mL in ethanol. Ionizable lipids (e.g., ckk-el2, AMG511, AMG513-523) were dissolved at 40 mg / mL in ethanol. The lipid phases were prepared using a previously reported formulation summarized in Table 1. Separately, the aqueous phases for formulations were made by preparing a 133 ng / pL solution of mRNA in 10 mM citrate, pH 3.0. The LNPs were then formulated using a- 70 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)herringbone microfluidic mixing channel, mixing the aqueous and lipid phases in a 3 : 1 volume ratio at a total flow rate of 1.2 mL / min.Table 1. Formulations used for LNPs

[0294] Following formulation, LNPs were exchanged into PBS using 100 kilodalton (kDa) cutoff Amicon centrifugal filters (Millipore-Sigma). Briefly, LNPs were diluted at least 4* with PBS then concentrated, repeating 3 times before concentrating to the final desired volume such that the final pH was 7 - 7.5.LNP characterization

[0295] mRNA concentration and encapsulation was determined using a Quant-IT® RiboGreen® ultrasensitive fluorescent nucleic acid stain assay. LNPs were diluted in either native Tris EDTA (TE) buffer (ThermoFisher, Waltham, MA, USA) or denaturing TE buffer + 0.5% Triton-X 100 (Millipore-Sigma) to measure the “free” and total mRNA, respectively.50 pL of these unknowns were added to a black 96-well plate. Separately, standard curves ranging from 2 ng / pL - 0 ng / pL were prepared in each of these buffers, and 50 pL were added to the same 96-well plate. Finally, Quant-IT® RiboGreen® reagent (ThermoFisher) was diluted 1 :200 in TE buffer, and 100 pL of this solution was added to each of the wells. The well plate was then mixed at 400 revolutions per minute (rpm) with a plate shaker for 5 mins, and the fluorescence was read with an excitation / emission of 485 / 535. LNPs were measured in triplicate, and standards were measured in duplicate. RNA concentration in each of the unknown wells was calculated by fitting the standard curves with a linear trendline. Encapsulation efficiency (%EE) was calculated according to(total RNA) - (free RNA)%EE = - - — - - x 100.total RNA

[0296] All dosing was performed based on the total RNA concentration.- 71 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)Table 2. Representative LNP Characterization DataGeneral synthetic methods

[0297] Anhydrous triethylamine, pyridine, EtOH, MeOH, CH3CN, THF, DMF and CH2CI2 were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). All other reagents were purchased from commercial sources and were used without further purification unless noted otherwise. Synthetic chemistry scale up services for selective chemical intermediates were obtained from TCG Lifesciences. All reactions were carried out under a positive pressure of argon atmosphere and monitored by TLC on Silica Gel G-25 UV254 (0.25 mm) unless stated otherwise. Spots were detected under UV light and / or by iodine, ethanolic solution of phosphomolybdic acid, or kMnO-r Column flash chromatography was performed using Teledyne Isco CombiFlash® instrument equipped with UV detector, an evaporative light scattering detector (ELSD) and prepacked silica gel, alumina or reverse phase C18 silica gel columns. Crude products were mixed thoroughly with celite and loaded into empty cartridges supplied by Teledyne Isco.General LC-MS analysis method

[0298] Liquid chromatography tandem mass spectroscopy (LC-MS / MS; abbreviated LC-MS) was carried out on a Waters (Milford, MA, USA) BioAccord® mass spectrometer equipped with an Acquity® Premier UPLC® Ultra Performance Liquid Chromatography technology operated by UNIFI (an application programming interface (API); software version 3.1.0.16). Waters bridged ethylene hybrid (BEH) C18 column (2.1 mm x 50 mm, 1.7 pm particle size) at 40 °C, using 0.1% formic acid in water (solvent A) and 20% isopropanol in acetonitrile with 0.1% formic acid (solvent B) at 0.6 mL / min. Sample chamber was kept at 8 °C. The following linear gradient was employed: (time (min), % B) 0, 20; 3, 95; 9, 95; 9.1, 20; 15, 20. UV spectra were monitored at dual wavelengths of 254 and 214 nm. Mass spectra were acquired in positive ion mode with a capillary voltage of 1.5 kV, a sample cone voltage of 30 V. The cone gas flow was set to 50 L / h and desolvation gas flow was 600 L / h.Desolvation temperature and source temperature were set to 550 and 120 °C, respectively. The acquisition range was m / z 50-2000. The scan acquisition rate was 1 Hz.- 72 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)Lipid tail synthesis

[0299] The general synthetic procedure for the branched lipid tails is shown in FIG. 51. First Steglich esterification between alkene-alcohols and branched fatty acids. To 0.2 M solution of 1 eq. of branched acid (Millipore-Sigma) in anhydrous di chloromethane (DCM) was added 1.5 eq. of alkene-alcohol (Millipore- Sigma or TCI (Portland, OR, USA)), 0.05 eq. of 4-(Dimethylamino)pyridine (DMAP; CAS No. 1122-58-3) (Millipore-Sigma), and 1.1 eq. of 3 -(3 -Dimethylaminopropyl)- 1-ethyl-carbodiimide hydrochloride (EDC-HC1; CAS No. 25952-53-8) (Chem-Impex, Wood Dale, IL, USA). The reaction was allowed to proceed at 25 °C for 16-18 hrs. then quenched with a saturated solution of NaHCOs in water. This mixture was added to a separatory funnel and the aqueous phase extracted twice with DCM then dried with Na2SO4, filtered, and rotovapped to dryness. The crude product was then purified via silica column chromatography with hexanes / ethyl acetate.

[0300] Alkene-esters were then epoxidized via the Prilezhaev reaction. To 1 eq. of a 0.2 M alkene-ester solution in DCM was added 1.1 eq. of meta-chloroperoxybenzoic acid (mCPBA; CAS No. 937-14-4) (Millipore- Sigma) at 25 °C. Reaction progress was monitored via TLC and stopped upon completion after about 3-5 hrs. The reaction was then quenched with a solution of Na2S20s in water. The reaction was then filtered and rotovapped to dryness. The crude material was then purified via silica column chromatography with hexanes / ethyl acetate.General procedure for synthesis of the ionizable lipids

[0301] A microwave vial (0.2-0.5 ml) was charged with the epoxy ester (one equivalent more than the number of reactive amines) followed by a 0.42 M solution of the headgroup amine in isopropanol. The total reaction volume was made up to 250 pl. The microwave vial was then sealed and heated to 90 °C for 1 hour. Once the reaction was judged complete by LC-MS, the reaction mixture was directly loaded on a RediSep® gold silica gel chromatographic column and purified using dichloromethane (solvent A) and a mixture (solvent B) of dichloromethane, methanol, and ammonium hydroxide (25:7:1) on CombiFlash® NextGen 300+ instrument. The fractions containing the desired lipid were concentrated and dried under vacuum. The purity of all AMG lipids was390% as judged by LC-MS. All AMG lipids thus generated were obtained as inseparable diastereomeric mixtures.- 73 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)Spectral characterizations of the AMG ionizable lipidsAMG511

[0302] 'H NMR (500 MHz, CDCh) 64.07 (t, J= 6.6 Hz, 4H), 3.63 (dddt, J= 17.9, 10.6, 7.2, 3.7 Hz, 2H), 2.76 (q, J= 7.6 Hz, 1H), 2.64 - 2.48 (m, 5H), 2.37 - 2.18 (m, 8H), 1.61 (qt, J= 12.5, 6.9 Hz, 15H), 1.45 (tq, J= 14.0, 5.3 Hz, 7H), 1.37 - 1.16 (m, 70H), 0.89 (t, J= 6.7 Hz, 12H).

[0303] 13C NMR (126 MHz, CDCh) 6 176.7, 67.9, 66.2, 64.5, 64.1, 62.0, 53.3, 51.4, 45.9, 39.9, 38.0, 37.9, 35.0, 35.0, 32.6, 31.9, 31.7, 29.8, 29.6, 29.5, 29.5, 29.5, 29.3, 29.2, 28.7, 27.5, 27.4, 26.0, 25.7, 25.6, 22.7, 22.6, 14.1, 14.1.

[0304] HRMS (ESI) m / z 989.9257 (calculated for C62H121N2O6 [M+H]+989.9225)

[0305] LC-MS purity: 92%AMG513

[0306] 'H NMR (500 MHz, CDCh) 64.07 (t, J= 6.6 Hz, 4H), 3.63 (dddt, J= 17.9, 10.7, 7.2, 3.8 Hz, 2H), 2.76 (dt, J= 8.9, 7.1 Hz, 1H), 2.64 - 2.46 (m, 5H), 2.28 (dddd, J= 36.2, 16.2, 6.5, 4.0 Hz, 8H), 1.69 - 1.56 (m, 15H), 1.53 - 1.40 (m, 5H), 1.35 (d, J= 8.9 Hz, 16H), 1.27 (s, 33H), 0.89 (dt, J= 7.2, 4.0 Hz, 12H).

[0307] 13C NMR (126 MHz, CDCh) 6 176.7, 67.9, 66.1, 64.5, 64.1, 62.0, 53.3, 51.4, 45.9, 39.9, 38.0, 37.9, 35.0, 35.0, 32.6, 32.5, 31.9, 31.9, 31.7, 31.7, 29.7, 29.6, 29.5, 29.3, 29.2, 29.2, 28.7, 27.5, 27.4, 25.9, 25.6, 25.6, 22.7, 22.6, 14.1, 14.1.

[0308] HRMS (ESI) m / z 933.8623 (calculated for C58H113N2O6 [M+H]+933.8599)

[0309] LC-MS purity: 97%AMG514

[0310] 'H NMR (500 MHz, CDCh) 64.74 (d, J= 0.8 Hz, 1H), 4.07 (t, J= 6.6 Hz, 4H), 3.73 - 3.60 (m, 3H), 2.87 -2.75 (m, 1H), 2.70 -2.51 (m, 4H), 2.44 - 2.21 (m, 7H), 1.63 (tddd, J= 21.2, 15.7, 10.1, 5.1 Hz, 12H), 1.53 - 1.32 (m, 18H), 1.26 (s, 40H), 0.93 - 0.81 (m, 12H).

[0311] 13C NMR (126 MHz, CDCh) 6 176.7, 74.8, 67.7, 66.1, 64.5, 64.1, 62.1, 53.4, 51.5, 45.9, 39.9, 37.7, 37.6, 34.9, 34.9, 32.6, 32.5, 32.5, 31.9, 31.7, 31.7, 31.7, 29.6, 29.5, 29.5, 29.4, 29.4, 29.4, 29.4, 29.4, 29.3, 29.2, 29.2, 28.7, 27.5, 27.4, 25.9, 25.6, 25.5, 22.7, 22.7, 22.6, 14.1, 14.1.- 74 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)

[0312] HRMS (ESI) m / z 905.8259 (calculated for C56H109N2O6 [M+H]+905.8286)

[0313] LC-MS purity: 98%AMG515

[0314] 'H NMR (500 MHz, CDCI3) 64.07 (td, J= 6.5, 1.7 Hz, 3H), 3.74 - 3.47 (m, 2H), 2.74 (dt, J= 8.8, 7.0 Hz, 1H), 2.66 - 2.46 (m, 5H), 2.35 - 2.13 (m, 6H), 1.76 - 1.50 (m, 12H), 1.49 - 1.34 (m, 9H), 1.25 (d, J= 4.5 Hz, 41H), 0.87 (td, J= 7.0, 1.6 Hz, 12H).

[0315] 13C NMR (126 MHz, CDCI3) 6 176.6, 67.7, 66.0, 64.4, 63.9, 61.9, 53.3, 51.4, 45.8, 45.8, 45.8, 39.9, 39.9, 38.0, 37.9, 34.5, 34.5, 32.6, 32.5, 32.5, 32.5, 32.4, 31.9, 31.9, 31.8, 31.7, 31.7, 31.7, 31.7, 31.7, 31.6, 29.6, 29.5, 29.5, 29.4, 29.4, 29.4, 29.4, 29.3, 29.3, 29.3, 29.3, 29.3, 29.2, 29.2, 29.2, 29.2, 29.1, 28.8, 27.5, 27.5, 27.4, 27.4, 27.4, 27.4, 22.7, 22.7, 22.6, 22.6, 22.6, 22.6, 22.6, 22.6, 22.5, 22.1, 22.1, 14.1, 14.1, 14.1, 14.0.

[0316] HRMS (ESI) m / z 849.7686 (calculated for C52H101N2O6 [M+H]+849.7660)

[0317] LC-MS purity: 98%AMG516

[0318] 'H NMR (500 MHz, CDCI3) 64.07 (t, J= 6.6 Hz, 4H), 3.70 - 3.55 (m, 1H), 2.75 (dt,J= 8.9, 7.1 Hz, 1H), 2.63 -2.45 (m, 2H), 2.35 - 2.16 (m, 7H), 1.69 - 1.51 (m, 8H), 1.51 - 1.38 (m, 4H), 1.37 - 1.15 (m, 82H), 0.89 (td, J= 7.0, 1.3 Hz, 12H).

[0319] 13C NMR (126 MHz, CDCI3) 6 176.7, 67.9, 66.2, 64.5, 64.1, 62.0, 53.3, 51.5, 45.9, 39.9, 38.0, 37.9, 35.0, 35.0, 32.6, 31.9, 31.9, 29.8, 29.7, 29.6, 29.6, 29.5, 29.5, 29.5, 29.5, 29.5, 29.4, 29.4, 29.3, 29.3, 29.3, 29.3, 29.3, 29.3, 29.2, 29.2, 28.7, 27.5, 26.0, 25.7, 25.6, 22.7, 22.7, 22.7, 14.1.

[0320] HRMS (ESI) m / z 1074.0193 (calculated for C68H133N2O6 [M+H]+1074.0164)

[0321] LC-MS purity: 97%AMG517

[0322] 'H NMR (500 MHz, CDCI3) 64.06 (t, J= 6.6 Hz, 4H), 3.62 (dddt, J= 17.5, 10.4, 6.9, 3.7 Hz, 2H), 2.75 (dt, J= 9.0, 7.1 Hz, 1H), 2.67 - 2.46 (m, 3H), 2.27 (dddd, J= 35.3, 16.1, 6.1, 3.8 Hz, 7H), 1.60 (qq, J= 11.8, 7.0 Hz, 11H), 1.51 - 1.39 (m, 4H), 1.29 (d, J= 39.6 Hz, 87H), 0.88 (td, J= 7.0, 1.3 Hz, 12H).- 75 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)

[0323] 13C NMR (126 MHz, CDC13) 6 176.7, 67.9, 66.1, 64.5, 64.1, 62.0, 53.3, 51.4, 45.8, 39.9, 38.0, 37.9, 35.0, 35.0, 32.6, 31.9, 31.9, 29.7, 29.6, 29.6, 29.5, 29.5, 29.5, 29.5, 29.3, 29.3, 29.3, 29.3, 29.3, 29.2, 28.7, 27.5, 27.5, 25.9, 25.6, 25.6, 22.7, 22.7, 14.1.

[0324] HRMS (ESI) m / z 1045.9873 (calculated for C66H129N2O6 [M+H]+1045.9851)

[0325] LC-MS purity: 96%AMG518

[0326] 'H NMR (500 MHz, CDCI3) 64.07 (t, J= 6.6 Hz, 4H), 3.62 (dddt, J= 18.1, 10.6, 7.1, 3.6 Hz, 2H), 2.80 -2.68 (m, 1H), 2.64 -2.44 (m, 4H), 2.36 - 2.15 (m, 6H), 1.68 - 1.53 (m, 9H), 1.53 - 1.33 (m, 17H), 1.26 (s, 58H), 0.88 (t, J= 6.8 Hz, 12H).

[0327] 13C NMR (126 MHz, CDCI3) 6 176.7, 67.8, 66.1, 64.5, 64.1, 62.0, 53.3, 51.5, 45.8, 39.9, 38.0, 37.9, 34.9, 34.9, 32.5, 31.9, 31.9, 29.6, 29.6, 29.5, 29.5, 29.5, 29.4, 29.3, 29.3, 29.3, 28.7, 27.5, 25.9, 25.6, 25.5, 22.7, 22.7, 14.1.

[0328] HRMS (ESI) m / z 1017.9566 (calculated for C64H125N2O6 [M+H]+1017.9538)

[0329] LC-MS purity: 98%AMG519

[0330] 'H NMR (500 MHz, CDCI3) 64.09 (t, J= 6.5 Hz, 4H), 3.64 (ddp, J= 17.4, 10.5, 3.6 Hz, 2H), 3.47 (s, 2H), 2.83 - 2.71 (m, 1H), 2.55 (dtd, J= 22.7, 12.5, 6.1 Hz, 4H), 2.40 -2.19 (m, 6H), 1.72 - 1.52 (m, 14H), 1.52 - 1.35 (m, 11H), 1.27 (s, 62H), 0.89 (td, J= 7.0, 1.5 Hz, 12H).

[0331] 13C NMR (126 MHz, CDCh) 6 176.7, 67.7, 66.0, 64.4, 64.0, 61.9, 53.3, 51.5, 45.8, 39.9, 38.0, 37.8, 34.6, 34.5, 32.5, 31.9, 31.9, 29.6, 29.6, 29.5, 29.5, 29.4, 29.4, 29.3, 29.2, 28.9, 27.5, 22.7, 22.7, 22.2, 22.1, 14.1.

[0332] HRMS (ESI) m / z 961.8942 (calculated for C60H117N2O6 [M+H]+961.8912)

[0333] LC-MS purity: 96%AMG520

[0334] 'H NMR (500 MHz, CDCh) 64.07 (t, J= 6.6 Hz, 4H), 3.63 (dddt, J= 18.1, 10.7, 7.2, 3.7 Hz, 2H), 3.55 - 3.29 (m, 3H), 2.75 (dt, J= 8.9, 7.1 Hz, 1H), 2.67 - 2.47 (m, 5H), 2.40 - 2.16 (m, 8H), 1.75 - 1.51 (m, 14H), 1.45 (ddtd, J= 13.2, 8.5, 5.5, 3.0 Hz, 6H), 1.39 -1.15 (m, 48H), 0.89 (td, J= 7.0, 3.0 Hz, 12H).- 76 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)

[0335] 13C NMR (126 MHz, CDC13) 6 176.7, 67.9, 66.2, 64.5, 64.1, 62.0, 53.3, 51.4, 45.8, 45.8, 39.9, 38.0, 37.9, 35.0, 35.0, 32.6, 32.3, 31.7, 31.7, 29.8, 29.7, 29.5, 29.5, 29.2, 28.7, 27.4, 26.0, 25.7, 25.6, 22.6, 22.6, 22.6, 22.6, 14.1, 14.1, 14.0, 13.9.

[0336] HRMS (ESI) m / z 877.7953 (calculated for C54H105N2O6 [M+H]+877.7973)

[0337] LC-MS purity: 97%AMG521

[0338] 'H NMR (500 MHz, CDCI3) 64.07 (t, J= 6.6 Hz, 4H), 3.63 (dddt, J= 18.0, 10.8, 7.3, 3.8 Hz, 2H), 3.51 (dd, J= 10.4, 5.4 Hz, OH), 2.83 - 2.70 (m, 1H), 2.67 - 2.45 (m, 5H), 2.41 - 2.13 (m, 8H), 1.60 (tq, J= 12.4, 5.9 Hz, 16H), 1.53 - 1.39 (m, 6H), 1.30 (dtdd, J = 32.9, 20.8, 7.3, 3.9 Hz, 44H), 0.89 (td, J= 6.9, 2.7 Hz, 12H).

[0339] 13C NMR (126 MHz, CDCI3) 6 176.7, 67.9, 66.2, 64.5, 64.1, 62.0, 53.3, 51.5, 45.8, 39.9, 38.0, 37.9, 35.0, 35.0, 32.6, 32.3, 31.7, 31.7, 29.7, 29.7, 29.7, 29.5, 29.5, 29.2, 29.2, 28.7, 27.4, 26.0, 25.6, 25.6, 22.7, 22.6, 22.6, 22.6, 14.1, 14.1, 14.0, 14.0.

[0340] HRMS (ESI) m / z 849.7683 (calculated for C52H101N2O6 [M+H]+849.7660)

[0341] LC-MS purity: 97%AMG522

[0342] 'H NMR (500 MHz, CDCI3) 64.08 (t, J= 6.7 Hz, 4H), 3.63 (dddt, J= 17.9, 10.7, 7.2, 3.9 Hz, 2H), 2.76 (dt, J= 8.9, 7.1 Hz, 1H), 2.67 - 2.47 (m, 4H), 2.42 - 2.17 (m, 7H), 1.70 - 1.53 (m, 14H), 1.45 (ddddd, J= 13.2, 11.3, 8.5, 5.8, 2.6 Hz, 5H), 1.40 (s, OH), 0.89 (td, J= 7.0, 2.7 Hz, 12H).

[0343] 13C NMR (126 MHz, CDCI3) 6 176.7, 67.9, 66.1, 64.5, 64.1, 62.0, 53.3, 51.4, 45.8, 45.8, 39.9, 38.0, 37.9, 35.0, 35.0, 32.6, 32.3, 31.7, 31.7, 29.7, 29.7, 29.6, 29.2, 29.2, 28.7, 27.4, 25.9, 25.6, 25.6, 22.6, 22.6, 22.6, 22.6, 14.1, 14.1, 14.0, 14.0.

[0344] HRMS (ESI) m / z 821.7368 (calculated for C50H97N2O6 [M+H]+821.7347)

[0345] LC-MS purity: 98%AMG523

[0346] 'H NMR (500 MHz, CDCI3) 64.08 (t, J= 6.6 Hz, 3H), 3.64 (dddt, J= 17.8, 10.5, 7.0, 3.8 Hz, 2H), 2.86 - 2.70 (m, 1H), 2.70 - 2.44 (m, 4H), 2.41 - 2.13 (m, 6H), 1.62 (dtd, J =- 77 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)21.8, 9.0, 4.7 Hz, 12H), 1.46 (ddddd, J= 13.3, 11.2, 8.6, 6.2, 2.9 Hz, 3H), 1.40 (s, 17H), 0.90 (td, J= 7.0, 2.7 Hz, 12H).

[0347] 13C NMR (126 MHz, CDC13) 6 176.7, 67.9, 67.8, 66.1, 66.1, 64.4, 64.1, 64.1, 62.0, 53.3, 45.8, 39.9, 38.0, 37.9, 35.0, 35.0, 34.9, 32.6, 32.3, 31.7, 29.7, 29.7, 29.4, 29.2, 29.2, 28.7, 28.7, 27.4, 25.9, 25.6, 25.6, 25.6, 25.5, 22.6, 22.6, 22.6, 22.6, 14.1, 14.0.

[0348] HRMS (ESI) m / z 793.7019 (calculated for C48H93N2O6 [M+H]+793.7034)

[0349] LC-MS purity: 96%EXAMPLE 10: Methods

[0350] The methods of Example 10 relate to Examples 2-8.Animal studies

[0351] All animal studies were approved by the Massachusetts Institute of Technology (MIT) and / or Massachusetts General Hospital (MGH) Institutional Animal Care and Use Committee (IACUC; animal protocol no. 2021N000272), and were consistent with local, state, and federal regulations as applicable. Animals were housed in a pathogen-free environment with a 12-hour day / night cycle with access to food and water ad libitum. All mice were 6 - 8 weeks old at the start of experiments and were sourced from Jackson Labs (Bar Harbor, ME, USA). C57BL / 6J were used for all tumor experiments (except Bl 6-F 10 metastatic tumors) and for HA vaccination experiments. B6. 29-Ill2btmI ILkyA mice were utilized for IL-12 eYFP reporter assays. B6(Cg)-Tyrc 2JA mice were used for B16-F10 metastatic experiments. BALB / cJ mice were utilized for SARS-CoV-2 vaccination experiments.IVIS® In Vivo Imaging

[0352] For bioluminescent imaging, D-Luciferin Potassium Salt (Revvity, Waltham, MA, USA) in PBS was injected intraperitoneally at a dose of 200 mg / kg. 15 minutes after injection, mice were anesthetized with isoflurane for live imaging or sacrificed for organ collection and ex vivo imaging.- 78 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)Endotoxin testing

[0353] Prior to injection, all LNPs used in vaccine experiments were screened for endotoxin contamination using the PyroGene® Recombinant Factor C Endpoint Fluorescent Assay (Lonza, Walkersville, MD, USA), following the manufacturer’s protocol.RNA synthesis

[0354] Hemagglutinin (A / Tasmania / 503 / 2020) (HA) RNA was generously provided by Sanofi.

[0355] The mouse and human NIK and IRF8 sequences were obtained via PCR from sequence optimized constructs in plasmids (GeneWiz). Sequences were codon optimized using the open-source mRNAid tool for therapeutic mRNA optimization, design and visualization (Ref. 85). The SARS-CoV-2 spike (B.1.617.2) open reading frame (ORF) was obtained as a linear gene block (Integrated DNA Technologies, Coralville, IA, USA). The tdTomato ORF was obtained from Addgene (Watertown, MA, USA) plasmid #54642 using PCR. The firefly luciferase ORF was obtained from pGL3 (Promega, Madison, WI, USA) using PCR. These ORFs were cloned into a custom mRNA plasmid containing a T7 promoter with a CleanCap AG site, 573’ UTRs, and a 100-nucleotide poly A tail. Plasmids were transformed into NEB 5-alpha F’ / 1E. Coli (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. Plasmids were purified using mini or midi-preps (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions. Sequences were verified using nanopore sequencing. Plasmids were then linearized using 10 U of NsiL HF (New England Biolabs) per 1 pg of DNA in rCutSmart buffer (New England Biolabs) for 1 hr. at 37 °C. In vitro transcription (IVT) was then performed using the HiScribe® T7 High Yield RNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA) using the manufacturer’s instructions for synthesis of CleanCap mRNA. Where indicated, a modified HiScribe® kit with a temperature stabilized Hi-T7 RNA polymerase (New England Biolabs) was used to produce low-dsRNA mRNA according to the manufacturer’s instructions. Nl-methylpseudouridine triphosphate (NIMePsU) (TriLink, San Diego, CA, USA) was substituted for uridine triphosphate (UTP) in all reactions to produce fully modified mRNA. Following IVT, template DNA was removed by adding 4 U of DNAse I (New England Biolabs) per 1 pg of template to the IVT reaction and incubating at 37 °C for 15 mins. The mRNA was then purified using the Monarch RNA cleanup kit (500 pg) (New England- 79 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)Biolabs). The molecular weight of the RNA was verified using a Fragment Analyzer (Agilent Technologies, Santa Clara, CA, USA).Immunization studies

[0356] LNPs were formulated as described above with HA (A / Tasmania / 503 / 2020), fLuc, NIK, or IRF8 mRNA. All vaccination doses were normalized to a total mRNA dose of 1 pg; in cases where HA was co-administered with another mRNA, LNPs comprising HA mRNA and LNPs comprising the other mRNA were mixed 50 / 50 weight / weight (w / w) (0.5 pg HA mRNA LNPs + 0.5 pg fLuc / NIK / IRF8 mRNA LNPs). All LNPs were injected IM in the quadricep with an injection volume of 50 pL. All vaccination experiments followed a prime-boost dosing regimen with doses given on day 0 and day 21. On day 35, mice were sacrificed, and blood was collected via cardiac puncture, and their spleens were collected for further characterization.Cell Culture

[0357] B16-F10 and B16-F10-Luc2 were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). B16-OVA, MC38, and MB49 were purchased from Millipore-Sigma. These cells were routinely screened for murine pathogens and mycoplasma contamination. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 4.5 g / L D-glucose, GlutaMAX® dipeptide, L-alanyl-L-glutamine, and 10% fetal bovine serum (FBS) (ThermoFisher).

[0358] BMDCs were cultured by isolating bone marrow from the femur and tibia of mice. Bone marrow cells were then cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with GlutaMAX® (ThermoFisher), 10% FBS (ThermoFisher), 100 U / mL penicillin (ThermoFisher), 100 pg / mL streptomycin (ThermoFisher), 50 pM P-mercaptoethanol (Millipore, Burlington, MA, USA), 200 ng / mL Flt3L-Fc (R&D Systems, Minneapolis, MN, USA), and 20 ng / mL GM-CSF (R&D Systems). On day 9, non-adherent cells were harvested and used immediately.IFN-I ELISAs

[0359] Serum and BMDC cell culture supernatant IFN-a and IFN-P were measured using enzyme-linked immunosorbent assay (ELISA) kits (Biolegend, San Diego, CA, USA) according to the manufacturer’s instructions.- 80 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)Binding antibody ELISAs

[0360] Clear, flat-bottomed, high-binding 96-well plates were coated with either spike (B.1.617.2) (Bio-Techne, Minneapolis, MN, USA) or hemagglutinin (provided as a gift from Sanofi, Morristown, MD, USA). Plates were coated overnight at 4 °C by adding 100 pL of a 1 pg / mL antigen solution in 100 mM carbonate buffer, pH 9.6, to each well. The coated plates were then washed with ELISA wash buffer (Biolegend) and blocked for 1 hr. at room temperature (RT) with 150 pL of 1% BSA in PBS (Miltenyi Biotec, Gaithersburg, MD, USA). The plate was then washed again, and 100 pL of serially diluted serum samples were then added to the plate and incubated at room temperature (RT) for 1 hr. Next, the plate was washed, and the secondary goat anti-mouse IgG, horseradish peroxidase (HRP) antibody (ThermoFisher) was diluted 1:3,000. 100 pL of this solution was added to each well and incubated at RT for 1 hr. After incubation, the plate was washed and 100 pL of 3, 3’, 5,5’-tetramethylbenzidine (TMB) chromogen solution (ThermoFisher) was added to each well and incubated for 30 mins. Finally, the HRP reaction was stopped by the addition of 100 pL of 0.5 M H2SO4. The absorbance of each well was immediately read at 450 nm with 600 nm as a reference using an Infinite M200 Pro (Tecan, Mannedorf, Switzerland) plate reader. The resultant data were then fit with a 4-parameter logistic (4-PL) sigmoidal curve, and the endpoint titers were taken as the point at which this curve crossed four times the absorbance of the blank wells.IFN-yELISpot

[0361] Spleens were dissociated into single-cell suspensions by mashing through a 70 pm strainer (Corning, Corning, NY, USA). Cells were pelleted and red blood cells lysed using red blood cell (RBC) lysis buffer (Biolegend) for 5 minutes on ice. The RBC lysis reaction was stopped by adding 4 volumes of PBS + 1% bovine serum albumin (BSA). Cells were then pelleted, resuspended in PBS + 1% BSA, and counted using a Countess® 3 automated cell counter (ThermoFisher).

[0362] Mouse IFN-y ELISpots (BD Biosciences, Franklin Lakes, NJ, USA) were performed according to the manufacturer’s instructions. Briefly, ELISpot plates were coated with the capture antibody overnight at 4°C, washed, and then blocked for 2 hours at RT using complete media: 10% FBS (ThermoFisher) in RPMI 1640 + GlutaMAX® (ThermoFisher). After blocking, splenocytes were plated at the indicated density in 100 pL of complete media. Separately, Hemagglutinin (A / Tasmania / 503 / 2020) major histocompatibility complex class I- 81 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)and II (MHC-I and MHC-II) restricted peptide pools were obtained as a gift from Sanofi and were dissolved in dimethyl sulfoxide (DMSO) at 0.2 mg / mL. These peptide pools were diluted 1 : 100 in complete media, and 100 pL of this solution was added to the cells in the ELISpot plate (final peptide concentration 1 pg / mL). Cells were stimulated for 16 hrs. the supernatant was then collected for further analysis, and spots were developed on the plate according to the manufacturer’s instructions. Plates were allowed to dry overnight and then imaged on an ImmunoSpot Analyzer (C.T.L., Shaker Heights, OH, USA).LNP biodistribution and expression characterizationFlow cytometry

[0363] Lymph nodes, tumors, and spleens were dissociated by using Micro Tissue Homogenizers (Kimble Biomasher® II closed system tissue grinder). Tumors were digested by the addition of Collagenase IV at 0.2 mg / mL in RPMI 1640 followed by vigorous shaking at 37°C for 45 min. The digested tumor tissues, lymph node, and spleen were filtered through a 70 pm cell strainer and resuspended in protein-free PBS. For the spleen, red blood cells (RBCs) were lysed with RBC lysis solution (Biolegend).

[0364] Following dissociation, cells were stained for 30 minutes at 4 °C with AquaAmine Live Dead Fixable viability stain (Thermo Fisher), then Fc blocked (Miltenyi), and stained with the antibodies of interest at 4 °C for 30 minutes. Following staining, cells were washed twice and then fixed with 4.2% paraformaldehyde (PF A) (BD Biosciences) at 4 °C for 15 mins. Finally, cells were stored in PBS + 1% BSA (Miltenyi) before analysis and analyzed within 2 days on Attune NxT Flow Cytometer (Invitrogen, Waltham, MA, USA), and BD FACS Symphony A3 (BD Biosciences). Compensation was performed using UltraComp eBeads® compensation beads (ThermoFisher). Data was analyzed using FlowJo VI 0.Representative gating for flow cytometry studies is shown in FIG. 20-FIG. 23. List of Antibodies is shown in Table 3.Flow cytometry gating

[0365] All gating strategies included doublet cell exclusion gates (FSC-A / SSC-A and SSC-A / SSC-H; FSC, forward scatter; SSC; side scatter; A, area; H, height) and a dead cell exclusion gate (eFluor 506 low or negative with Fixable viability dye).

[0366] Gating strategy for myeloid cell populations:- 82 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)Lineage cocktail: B220, CD 19, NK1.1, CD3eDCs: Lineages-, CD45+, F4 / 80low, CDllc+, MHCII+cDCL Lineages-, CD45+, F4 / 80low, CDllc+, MHCII+, XCR1+CD103+ cDCL Lineages-, CD45+, F4 / 80low, CDllc+, MHCII+, XCR1+, CD103+ CD8+ cDCL Lineages-, CD45+, F4 / 80low, CDllc+, MHCII+, XCR1+, CD8+ cDC2: Lineages-, CD45+, F4 / 80low, CDllc+, MHCII+, XCR1-, CDllb+, Sirpa+ pDC: Lineages-, CD45+, F4 / 80low, CDllc-, MHCII-, Siglec-H+

[0367] Gating strategy for T cells:Lineage cocktail: CDllb, B220, Ly6GT cells: Lineages-, CD45+, CD3e+CD8+ T: Lineages-, CD45+, CD3e+, CD8a+CD4+ T: Lineages-, CD45+, CD3e+, CD4+Tregs: Lineages-, CD45+, CD3e+, CD4+, FoxP3+NKT: Lineages-, CD45+, CD3e+, ybTCR-, CD49b+ybT: Lineages-, CD45+, CD3e+, Y8TCR+, CD49b-

[0368] Gating strategy for naive and memory T cells:Lineage cocktail: CDllb, B220, Ly6GNaive T: Lineages-, CD45+, CD3e+, CD8a+ / CD4+, CD62Lhi, CD44lowCentral memory T (CM T): Lineages-, CD45+, CD3e+, CD8a+ / CD4+, CD62Lhi, CD44hiEffector memory T (EM T): Lineages-, CD45+, CD3e+, CD8a+ / CD4+, CD62Llow, CD44hiTable 3 : Flow Cytometry staining antibodies- 83 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)- 84 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)Ail4 reporter mice transfection analysis

[0369] B6.Cg-( R(ROSA)26Sortml4(CAG~tdTomato)HzeIJ (Ail4D) reporter mice were intravenously injected with AMG514 and cKK-E12 LNPs encapsulating Cre recombinase mRNA (0.25 mg kg1per mouse; TriLink). At 72 hours post-injection, spleen and liver tissues were harvested and were digested by the addition of Collagenase IV at 0.2 mg / mL in RPMI 1640, followed by vigorous shaking at 37°C for 45 min. The digested tissues were passed through a 70 pm cell strainer, and the cell suspensions were centrifuged and resuspended in staining buffer. Cells were incubated with antibodies against lineagespecific markers in staining buffer for 30 min at 4 °C, followed by flow cytometric analysis as described above.Histology

[0370] Tissues were harvested and fixed for 24 hrs. in 10% neutral buffered formalin at 4 °C. Following fixing, tissues were washed with deionized water and stored in 70% ethanol. Tissues were then cut in the transverse direction along their midplane, dehydrated and paraffin embedded. 4 pm-thick sections were then mounted on slides and deparaffinized.- 85 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)These were then either subjected to hematoxylin and eosin (H&E) staining or further processing for immunohistochemistry (IHC).

[0371] For IHC, the deparaffinized tissues were subjected to heat-mediated antigen retrieval in pH 6 citrate buffer at 97 °C for 20 minutes. Sections then underwent endogenous peroxidase deactivation for 10 minutes, followed by blocking for 30 minutes. After blocking, the primary antibody was added for 60 minutes, the slides were washed, and the secondary antibody was added for 30 minutes. After washing, staining was performed with DAB substrate (3,3 ’-diaminobenzidine) for 5 minutes and followed by counterstaining with hematoxylin. Slides were then mounted and scanned using a slide scanner with a 40X objective.Tumor inoculation and tumor therapy

[0372] Tumor cell lines were detached from culture flasks and resuspended in PBS at an appropriate concentration for tumor inoculation. Subcutaneous tumors were inoculated by injecting cells into the flank of mice. Metastatic tumors were inoculated by intravenous tail vein injection. Subcutaneous tumor volumes were measured by caliper and their volumes calculated according to the formula: V=0.5 x L x I / ! / 2, where V is volume, L is length, and W is width.Luminex

[0373] Multiplexing analysis was performed using the Luminex™ 200 system flowbased bead reader (Luminex, Waltham, MA, USA) by Eve Technologies Corp (Calgary, AB, Canada). All samples were diluted 1:1 with PBS + 1% BSA (Miltenyi Biotec). Samples were analyzed using Eve Technologies’ Mouse High Sensitivity 18-Plex Discovery Assay® (MilliporeSigma, Burlington, Massachusetts, USA) according to the manufacturer's protocol. The 18-plex consisted of GM-CSF, IFNy, IL-la, IL-ip, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12(p70), IL- 13, IL- 17 A, KC / CXCL1, LIX, MCP-1, MIP-2 and TNFa. Assay sensitivities of these markers range from 0.06 - 9.06 pg / mL for the 18-plex. Serum collected 6 hrs. after vaccination was analyzed using Eve Technologies’ Mouse Focused 10-Plex Discovery Assay® (MilliporeSigma, Burlington, Massachusetts, USA) according to the manufacturer's protocol. The 10-plex consisted of GM-CSF, IFNy, IL-ip, IL-2, IL-4, IL-6, IL-10, IL-12p70, MCP-1, and TNFa. Assay sensitivities of these markers range from 0.4 - 10.9 pg / mL for the- 86 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)10-plex. Individual analyte sensitivity values are available in the MilliporeSigma MILLIPLEX® (multiplex assays for Luminex instruments) Multiplex Assay User Guide.RNA SequencingBulk RNA Sequencing and Analysis

[0374] Total RNA were then purified from BMDCs using RNeasy® RNA purification Plus Mini Kit [Catalog number 74134] and stored at -80C until being submitted for RNA sequencing. RNA samples were cleaned using 2x solid phase reverse immobilization (SPRI) RNAClean beads and quality was assessed using an Advanced Analytical Technologies, Inc. (AATI) Fragment Analyzer (Agilent). Around 30ng of RNA was prepared into Nextgeneration sequencing (NGS) libraries using NEBNext Poly(A) mRNA isolation module run at 1 :5 dilution on an SPT Labtech (Royston, UK) Mosquito-HV liquid handler followed by preparation with NEBNext Ultrall Directional RNA library prep kit for Illumina modified to run at 1:10 dilution on a MosquitoHV (see also, Ref. 87) and amplified with custom primers replacing the Illumina P5 / P7 sequences with Singular S1 / S2 sequences using 16 cycles amplification. Completed libraries were validated on an AATI Fragment Analyzer, pooled, and sequenced 50nt paired end on a Singular G4 high-throughput, in situ spatial multiomics platform.

[0375] Following the sequencing, the RNA-seq data was then analyzed. RNA-seq data was used to quantify transcripts from the GRCm39 mouse assembly with the Ensembl version 113 annotation using the nf-core / maseq workflow revision 3.18.0 (Ref. 88). Gene level summaries were prepared from the star salmon quantitation using tximport version 1.32.0 (Ref. 89) running under R version 4.4.1 (Ref. 91) with tidyverse version 2.0.0 (Ref.90). Differential expression analysis was done with DESeq2 version 1.44.0 (Refs. 92, 93) using apeglm log fold change shrinkage (Ref. 94). Preranked Gene Set Enrichment Analysis (Ref. 95) was done using javaGSEA version 4.3.3 with msigDb version v2024.1 (Ref. 96) mouse gene sets.Single-Cell RNA Sequencing and Analysis

[0376] To provide more comprehensive and unbiased view of immunotherapeutic response across different cells within BMDCs. Single cells were generated by BMDCs cells following day 9 culturing were counted on a Luna dual fluorescence cell counter. 3' RNA- 87 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)libraries were prepared from about 5k cells on using 10X Genomics (Pleasanton, CA, USA) 3'RNA (GEM-X) chemistry on a ChromiumX using 12 cycles of PCR. Final libraries were sequenced on either an Illumina NovaSeq6000 on an SP flowcell (26 forward, 90nt reverse) or an Element AVITI high output flowcell (43 forward, 50 reverse). Output data was processed using 10X Genomics cell ranger using default configuration.Sample collection and data organization

[0377] scRNAseq data was processed. Single-cell RNA sequencing (scRNA-seq) data were generated on the 10X Genomics Chromium platform from four independent mouse samples which were annotated as “FFL,” “PBS,” “NIK,” and “IRF8,” respectively.Ambient RNA correction

[0378] To account for ambient RNA contamination, each raw 10X count matrix was loaded into SoupX (Ref. 97). An initial Seurat (v5.2.0) (Ref. 98) object was created from the raw counts, normalized, and subjected to principal component analysis (PC A) (using the top 2,000 variable features) and Louvain clustering via principal components 1-10. Clusters were transferred back into the SoupX object with setClusters(), and contamination fractions were estimated using autoEstCont(). Cleaned counts were obtained via adjustCounts() and served as input for all subsequent analyses.Seurat preprocessing and doublet detection

[0379] Cleaned count matrices were imported into Seurat with min. cells = 3 and min. features = 200. The percentage of mitochondrial reads (percent.mt) was calculated from genes beginning with “mt-.” Data were log-normalized, the top 2,000 variable features were identified, and the data were scaled. PC A was then performed, retaining the first 30 components, followed by uniform manifold approximation and projection (UMAP) embedding, neighborhood graph construction, and Louvain clustering at a resolution of 0.4. Logz-transformed feature and unique molecular identifier (UMI) counts were added to metadata as 12.n_feature and 12.n_count.

[0380] Expected doublet rates for each sample were computed from a Poisson model:= / riVcells 100001=pNcells 10000Doublet Rate=l-e- - e-Ule- Doublet Rate=l-e- - e- e-- 88 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)

[0381] DoubletFinder was applied via a parameter sweep over principal components one through ten to identify optimal pK values and to estimate homotypic doublet proportions. Each object was run twice, first with the raw expected doublet count and then adjusted for homotypic doublets yielding two metadata columns (renamed to DF. classifications. first and DF. classifications. second) and one pANN score column (renamed to pANN). Doublet-annotated Seurat objects were saved for downstream merging

[0382] The four doublet-annotated Seurat objects were merged into a single object (seurat. merge) using merge(), with cell barcodes prefixed by sample identifiers. Individual sample objects were then removed to conserve memory. The merged object was log-normalized, variable features were recalculated across all samples, and data were scaled. PCA on the combined data was followed by elbow-plot inspection to confirm that thirty components captured the primary sources of variation. UMAP embeddings were generated on these components, and clustering was again performed at resolution 0.4.

[0383] Cluster composition was examined by computing contingency tables of cluster identity versus sample condition and by visualizing the UMAP embedding split by condition and by doublet classification to ensure that flagged doublets did not form artifactual clusters.Quality control and selection of singlets

[0384] Cells annotated as “Doublet” under the DF. classifications. second column were removed to produce a singlet-only object (seurat. singlet). Additional quality filters excluded cells with mitochondrial percentages above 25% and those exhibiting low library complexity, defined as logio(genes) / logio(UMIs) < 0.75. Density and scatter plots of UMI count and gene count guided thresholds of at least 500 UMIs and 200 genes per cell. The resulting high-quality singlet object (seurat. singlet.filt) was saved for integration.Data integration and embedding

[0385] To correct for residual batch effects among the four conditions, the filtered singlet object was integrated using Seurat’s canonical-correlation analysis (CCA)-based method. The RNA assay was designated the default, and the first thirty principal components served as anchors. After integration, all data layers were joined back into a single assay via JoinLayers(). A new nearest-neighbor graph and clustering at resolution 0.4 were computed on the integrated reduction, and a UMAP embedding specific to the CCA reduction (“umap.cca”) was generated for downstream visualization.- 89 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)Cluster annotation and cell-type assignment

[0386] Canonical marker genes for dendritic cell subsets, cDCls, cDC2s, plasmacytoid DCs, DC_progenitors, Macrophages, Basophils, and Neutrophils were visualized with violin plots split by condition to confirm expression patterns. Numeric cluster identities (0-18) were then renamed to biologically meaningful labels (for example, clusters 0, 2,9, and 13 as “cDCls,” clusters 3, and 12 as “DC_progenitors,” clusters 1, 4, and 15 as “cDC2s,” and so forth). Clusters labeled “Debris” were excluded by subsetting, and a new metadata column, celltype, was populated with these annotated labels.

[0387] To link cluster annotations back to marker gene discovery, a lookup table mapping numeric clusters to cell types was drawn from the metadata and joined to the results of FindAllMarkers. This enriched marker table, annotated with cell-type assignments, was written to an Excel file. Proportions of each cell type by condition were calculated and visualized via UMAP plots split by condition and colored by celltype.Software and reproducibility

[0388] To ensure a consistent computational environment, the entire workflow was run inside a Singularity container built from the Docker image docker: / / yannvrb56 / r441seurat5signac, which encapsulated R (v4.4.1), Seurat 5.2.0 and all dependencies. Other key packages included SoupX (vl.6.2) for ambient RNA correction, DoubletFinder (v2.0.4) for doublet detection, ggplot2 and patchwork for visualization, and openxlsx / readxl / writexl for file I / O.Statistical Analysis

[0389] Statistical analysis was performed using Graphpad Prism.NON-PATENT REFERENCES

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Nat Commun. 2025 Sep 30;16(l):8699. doi: 10.1038 / s41467-025-63726-2. PMID: 41027853; PMCID:PMC12485112.Table 4. Summary of sequences of the Sequence Listing.INCORPORATION BY REFERENCE; EQUIVALENTS

[0391] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0392] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.- 99 - 5024769. vl

Claims

DocketNo. 0050.2396-002 (MIT 25731)CLAIMSWhat is claimed is:

1. A composition comprising:a) one or more of:i) an RNA encoding interferon regulatory factor 8 (IRF8); and ii) an RNA encoding nuclear factor kappa B (NF-KB) inducing kinase (NIK); andb) lipids encapsulating the RNA.

2. The composition of claim 1, further comprising an RNA encoding an antigen.

3. The composition of claim 2, wherein the antigen is a cancer antigen.

4. The composition of claim 3, wherein the cancer antigen is a neoantigen.

5. The composition of claim 3, wherein the cancer is colorectal cancer, bladder cancer, melanoma, lung cancer, prostate cancer, breast cancer, brain cancer, colon cancer, pancreatic cancer, ovarian cancer, and hepatocellular cancer, or lymphoma.

6. The composition of claim 5, wherein the cancer is colorectal cancer, bladder cancer, or melanoma.

7. The composition of claim 2, wherein the antigen is an infectious disease antigen.

8. The composition of claim 7, wherein the infectious disease is COVID-19 or influenza.

9. The composition of claim 7, wherein the infectious disease antigen is a viral antigen.

10. The composition of claim 9, wherein the viral antigen is a SARS-CoV-2 spike or a hemagglutinin.

11. The composition of claim 1, wherein the RNA encoding IRF8 comprises SEQ ID NO:6 or 7.

12. The composition of claim 1, wherein the RNA encoding NIK comprises SEQ ID NO:2 or 3.- 100 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)13. The composition of claim 1, the RNA encoding IRF8, wherein the RNA encoding NIK, or both, is a circular RNA.

14. The composition of claim 2, wherein the RNA encoding the antigen is a circular RNA.

15. The composition of claim 1, wherein the RNA encoding IRF8, the RNA encoding NIK, or both, comprises at least one of N1 -methylpseudouridine (NIMePsU), 5- methoxy-pseudouridine, psuedouridine, 5-methylcytidine, or a combination thereof.

16. The composition of claim 2, wherein the RNA encoding the antigen comprises at least one of N1 -methylpseudouridine (NIMePsU), 5-methoxy-pseudouridine, psuedouridine, 5-methylcytidine, or a combination thereof.

17. The composition of claim 1, wherein each uridine of the RNA encoding IRF8, the RNA encoding NIK, or both, is substituted with NIMePsU.

18. The composition of claim 2, wherein each uridine of the RNA encoding the antigen is substituted with NIMePsU.

19. The composition of claim 1, wherein IRF8 comprises SEQ ID NO: 8.

20. The composition of claim 1, wherein NIK comprises SEQ ID NO: 4.

21. The composition of claim 1, wherein IRF8 is a recombinant human IRF8, NIK is a recombinant human NIK, or both.

22. The composition of claim 1, wherein NIK lacks a negative-regulatory domain (NRD), a tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3)-binding domain, or both.

23. The composition of claim 1 or 2, wherein the lipids form a lipid nanoparticle (LNP) encapsulating the RNA encoding IRF8, the RNA encoding NIK, the RNA encoding the antigen, or a combination thereof.

24. The composition of claim 20, wherein the lipids comprise one or more of:a) an ionizable lipid;- 101 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)b) a helper lipid;c) a structural lipid; andd) a polyethylene glycol (PEG)-modified lipid.

25. The composition of claim 24, wherein the ionizable lipid comprises a spirocyclic diamine headgroup with branched tails.

26. The composition of claim 24, wherein the LNP or one or more of the lipids localize the composition to the spleen.

27. The composition of claim 24, wherein a protein corona of the LNP or one or more of the lipids comprises a protein that engages a receptor on a splenic macrophage, a splenic dendritic cell, or both.

28. The composition of claim 27, wherein the receptor is a C-type lectin receptor, a Complement receptor, or a scavenger receptor.

29. The composition of claim 28, wherein the C-type lectin receptors is dendritic cellspecific intercellular adhesion molecule-3 -grabbing non-integrin (DC-SIGN) or mannose, the Complement receptor is CR3 / 4, or the scavenger receptor is CD36 or SR-bl.

30. The composition of claim 24, wherein a protein corona of the LNP or one or more of the lipids comprises an integrin-binding protein, a phagocytosis-associated protein, or both.

31. The composition of claim 30, wherein the integrin-binding protein or the phagocytosis-associated protein comprises a vitronectin, a prothrombin / coagulation factor, a fibrinogen, or a combination thereof.

32. The composition of claim 24, wherein a protein corona of the LNP or one or more of the lipids comprises an opsonin.

33. The composition of claim 32, wherein the opsonin is selected from a thrombospondin- 1, an ApoA-IV, or both.- 102 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)34. The composition of claim 24, wherein the ionizable lipid is AMG56, AMG511, AMG513, AMG514, AMG515, AMG516, AMG517, AMG518, AMG519, AMG520, AMG521, AMG522, AMG523, cKK-E12, or a combination thereof.

35. The composition of claim 24, wherein the ionizable lipid is AMG514.

36. The composition of claim 24, wherein the LNP or one or more of the lipids localize the composition to the liver.

37. The composition of claim 24, wherein a protein corona of the LNP or one or more of the lipids comprises albumin, ApoE, or both.

38. The composition of claim 24, wherein the ionizable lipid is cKK-E12.

39. The composition of claim 24, wherein the helper lipid is l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), l,2-dioleoyl-3- dimethylammonium -propane (DODAP), l,2-dioleoyl-sn-glycero-3 -phosphate (DOPA), l,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dioleoyl-sn- glycero-3-phospho-(l'-rac-glycerol) (DOPG), l-palmitoyl-2-oleoyl-glycero-3- phosphocholine (POPC), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (POPE), l-stearoyl-2-oleoyl-sn-glycero-3 -phosphocholine (SOPC), 1,2-diphytanoyl- sn-glycero-3 -phosphocholine (4MEPC), or a combination thereof.

40. The composition of claim 24, wherein the helper lipid is DOPE.

41. The composition of claim 24, wherein the structural lipid is cholesterol, Stigmasterol, beta-sitosterol, DC-cholesterol, 7-alpha-hydroxycholesterol, 7-beta- hydroxy cholesterol, 19-hydroxy cholesterol, 20(s)-hydroxy cholesterol, 24(s)- hydroxycholesterol, 25-hydroxycholesterol, fucosterol, campesterol, stigamstanol, or a combination thereof.

42. The composition of claim 24, wherein the structural lipid is cholesterol.

43. The composition of claim 24, wherein the PEG-modified lipid is isdimyristoyl phosphatidylethanolamine polyethylene glycol (DMPE-PEG), 1,2-dimyristoyl-rac-- 103 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)glycero-3 -methoxypolyethylene glycol-2000 (DMG-PEG 2000), DSPE-PEG, DSG- PEG, DPPE-PEG, DOPE-PEG, DPG-PEG, or a combination thereof.

44. The composition of claim 24, wherein the PEG-modified lipid is DMPE-PEG or DMG-PEG 2000.

45. The composition of claim 24, wherein the lipids have a molar composition of about 20-70% ionizable lipid, about 5-40% helper lipid, about 20-50% structural lipid, and about 0.5-5% PEG-modified lipid.

46. The composition of claim 24, wherein the lipids have a molar composition of 35 : 16 :46.5 : 2.5 of ionizable lipid : DOPE : cholesterol : DMPE-PEG or DMG-PEG 2000.

47. The composition of claim 24, wherein:a) the ionizable lipid, andb) the RNA encoding IRF8, the RNA encoding NIK, the RNA encoding the antigen, or a combination thereof,are in a weight ratio of about 10:1.

48. A method of inducing IL-12, IFN-a, IFN-P, IFN-y, CD86, IL-2, IL-4, IL-5, IL-6, CTSC, NLRC5, STAT3, CD40, CD80, CXCL9, or a combination thereof in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1-22.

49. The method of claim 48, wherein the subject is a human.

50. The method of claim 48, wherein the composition is administered intratum orally.

51. The method of claim 48, wherein the composition is administered intravenously.

52. The method of claim 48, wherein the lipids induce expression of the RNA encoding IRF8, the RNA encoding NIK, the RNA encoding the antigen, or a combination thereof as mRNA in immune cells.

53. The method of claim 52, wherein the immune cells are in a spleen.- 104 - 5024769. vlDocket No. 0050.2396-002 (MIT 25731)54. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1-22.

55. The method of claim 54, wherein the method further comprises administering to the subject an anti-PD-1 checkpoint blockade.

56. The method of claim 54, wherein the method further comprises administering to the subject an anti-PD-1 therapy, an anti-PD-Ll therapy, or an anti-CTLA4 therapy.

57. The method of claim 54, wherein the cancer is colorectal cancer, bladder cancer, melanoma, lung cancer, prostate cancer, breast cancer, brain cancer, colon cancer, pancreatic cancer, ovarian cancer, and hepatocellular cancer, or lymphoma.

58. The method of claim 54, wherein the cancer comprises colorectal cancer, bladder cancer, or melanoma.

59. The method of claim 54, wherein the subject is a human.

60. The method of claim 54, wherein the composition is administered intratum orally.

61. The method of claim 54, wherein the composition is administered intravenously.

62. The method of claim 61, wherein the lipids induce expression of the RNA as mRNA in immune cells.

63. The method of claim 62, wherein the immune cells are in a spleen.

64. A vaccine comprising:a) an RNA encoding an antigen; andb) an RNA encoding interferon regulatory factor 8 (IRF8).

65. The vaccine of claim 64, wherein the antigen is for a cancer.

66. The vaccine of claim 65, wherein the cancer is colorectal cancer or bladder cancer.

67. The vaccine of claim 65, wherein the cancer is a solid tumor.

68. The vaccine of claim 65, wherein the cancer is melanoma.- 105 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)69. The vaccine of claim 64, wherein the antigen is for an infectious disease.

70. The vaccine of claim 69, wherein the infectious disease is COVID-19.

71. The vaccine of claim 69, wherein the antigen is hemagglutinin or SARS-CoV-65 spike.

72. The method of claim 69, wherein the infectious disease is influenza.

73. The vaccine of claim 64, wherein one or more of the RNA encoding an antigen and the RNA encoding IRF8 is formulated in a lipid nanoparticle.

74. A method of eliciting an immune response in a subject, the method comprising administering:a) a therapeutically effective amount of an RNA encoding an antigen; and b) a therapeutically effective amount of an RNA encoding interferon regulatory factor 8 (IRF8).

75. The method of claim 74, wherein the antigen is for cancer.

76. The method of claim 75, wherein the cancer is colorectal cancer or bladder cancer.

77. The method of claim 75, wherein the cancer is a solid tumor.

78. The method of claim 75, wherein the cancer is melanoma.

79. The method of claim 74, wherein the antigen is for an infectious disease.

80. The method of claim 79, wherein the infectious disease is COVID-19.

81. The method of claim 79, wherein the antigen is SARS-CoV-2 spike or hemagglutinin.

82. The method of claim 79, wherein the infectious disease is influenza.

83. The method of claim 74, wherein one or more of the RNA encoding an antigen and the RNA encoding IRF8 is formulated in a lipid nanoparticle.

84. The method of claim 74, wherein the subject is an animal.- 106 - 5024769. vlDocketNo. 0050.2396-002 (MIT 25731)85. The method of claim 74, wherein the subject is human.

86. A method of treating cancer in a subject, the method comprising administering a therapeutically effective amount of an RNA encoding interferon regulatory factor 8 (IRF8).

87. The method of claim 86, wherein the cancer is colorectal cancer or bladder cancer.- 107 - 5024769. vl

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