Modifying the inflammatory state of immune cells in vivo by regulating the cellular activity state.

Abstract

To provide a method for treating cancer and autoimmune diseases by in vivo transforming immunosuppressive tumor-associated macrophages (TAMs) that support cancer growth and metastasis into highly activated tumoricidal macrophages.SOLUTION: Provided is a method for altering the activation state of macrophages within a tumor in vivo, comprising administering to a tumor site nanoparticles comprising a poly(β)-amino ester core and a polyglutamic acid coating, in which in vitro transcribed mRNA encoding (i) one or more interferon regulatory factors (IRFs) and (ii) IKKβ are encapsulated within the core, the activation state of the macrophages being altered from an inactivated state to an activated state.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 618,908, filed on 18 January 2018, and the said patent is thereby incorporated by reference as if it were entirely described herein.

[0002] Statement regarding sequence listings The sequence listing relating to this application is provided in text format instead of as a hard copy and is incorporated herein by reference. The name of the text file containing the sequence listing is 18-073-WO-PCTsequenceListing_ST25.txt. The text file is 145kb, was created on January 14, 2019, and submitted electronically via EFS-Web.

[0003] This disclosure provides a system and method for in vivo regulating the activation state of immune cells. This system and method can be used to convert immunosuppressive macrophages that support cancer growth and metastasis into highly activated tumor-killing macrophages. [Background technology]

[0004] Several adverse physiological conditions are associated with either immune system activation (e.g., autoimmune disorders) or immunosuppression (e.g., cancer). For example, macrophages are important immune effector cells that infiltrate cancer tissue in large numbers. However, in the tumor microenvironment, macrophages undergo a switch from an activated, tumor-killing state to an immunosuppressive phenotype that actually promotes tumor growth and metastasis. Pollard, Nat Rev Cancer 4, pp. 71-78 (2004); Mantovani et al., Nat Rev Clin Oncol (2017).

[0005] Understanding that immunosuppressed macrophages in the tumor microenvironment promote cancer growth and metastasis has led to considerable effort in developing therapies targeting immunosuppressed tumor-associated macrophages (TAMs). Many efforts to address TAMs have focused on killing them to mitigate immunosuppression in the tumor microenvironment. However, this approach simply results in TAMs being replaced by newly arriving macrophages in the tumor microenvironment. Furthermore, even when some TAMs are successfully killed, most therapeutic agents developed to date have failed to penetrate sufficiently into the tumor microenvironment. While some small molecule drugs and antibodies have shown some success, these approaches have suppressed all macrophages in the body, resulting in dangerous side effects. (Bowman & Joyce, Immunotherapy 6, pp. 663-666 (2014)). Therefore, as everyone affected by cancer understands, there is a great need for more effective treatment strategies with fewer side effects. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Pollard, Nat Rev Cancer 4, pp. 71-78 (2004) [Non-Patent Document 2] Mantovani et al., Nat Rev Clin Oncol (2017) [Non-Patent Document 3] Bowman & Joyce, Immunotherapy 6, pp. 663-666 (2014) [Overview of the Initiative] [Means for solving the problem]

[0007] This disclosure provides systems and methods for modulating the function of immune cells in vivo. In certain embodiments, these systems and methods are used to reverse the immunosuppressive tumor-supporting state of tumor-associated macrophages (TAMs) to transform these TAMs into highly activated macrophages that kill tumor cells. Thus, the systems and methods disclosed herein aim not merely to kill TAMs, but rather to redirect their activity from tumor-promoting to tumor-destroying. In certain embodiments, these systems and methods are used as therapeutic agents to induce the killing of cancer cells and / or to reduce or prevent the growth or development of new cancer cells. From the data disclosed herein, it is evident that these systems and methods can completely eradicate and suppress ovarian cancer, a cancer type that is notoriously difficult to control.

[0008] The systems and methods disclosed herein can provide mechanisms for altering the immunosuppressive state in a tumor and reconstructing the tumor microenvironment. In these embodiments, the reconstructed tumor microenvironment can make the tumor more sensitive to companion treatments such as vaccines, chimeric antigen receptor (CAR) therapy, and / or chemotherapy.

[0009] Importantly, the systems and methods disclosed herein can be used locally in the tumor microenvironment, eliminating the need to rely on systemic treatments that disrupt immune system homeostasis as a whole. Furthermore, certain embodiments have been optimized to successfully infiltrate the tumor microenvironment.

[0010] Certain embodiments modify the activation state of immune cells in vivo by utilizing particles that deliver nucleotides encoding activation regulators, such as transcription factors. Specific useful particles have a positively charged nucleus and a neutral or negatively charged surface and deliver nucleotides encoding the transcription factor interferon regulator (IRF5) in combination with the kinase IKKβ. A specific size of <130 nm ensures tumor invasion. Furthermore, the particles may contain TAM-targeting ligands that direct more selective uptake of the particles by the TAM. For example, a TAM expresses the cell surface receptor CD206, and this receptor can be targeted by including mannose on the particle surface.

[0011] Many of the figures submitted herein would be better understood if they were in color. The application considers color versions of the figures as part of the original submission and reserves the right to present color images of the figures in subsequent proceedings. [Brief explanation of the drawing]

[0012] [Figure 1A] Schematic diagram illustrating the genetic conversion of tumor-associated macrophages (TAMs) into tumor-killing cells using targeted mRNA nanoparticles. (Figure 1A) Injectable nanoparticles have been developed to deliver in vitro transcribed mRNA encoding the M1 polarization transcription factor as a novel method for rationally reprogramming TAMs for therapeutic purposes without causing systemic toxicity. The first planned clinical application, designed to treat ovarian cancer patients using repeated intraperitoneal injections of mRNA nanoparticles, is illustrated. [Figure 1B] Schematic diagram illustrating the genetic conversion of tumor-associated macrophages (TAMs) into tumor-killing cells using targeted mRNA nanoparticles. (Figure 1B) Schematic diagram illustrating the genetic reprogramming of brain TAMs into tumor-killing macrophages using targeted mRNA nanoparticles. [Figure 2A]Nanoparticles carrying mRNA encoding IRF5 and IKKβ can imprint an inflammatory M1-like phenotype. (Figure 2A) Design of macrophage-targeting polymer NPs assembled using mRNA encoding key regulators of macrophage polarization. The particles consist of a PbAE-mRNA polyplex core coated with a layer of PGA-dimannose, which directs the particles to a target mannose receptor (CD206) expressed by M2-like macrophages. Synthetic mRNA encapsulated in the NPs is also depicted, which has been engineered to encode a reprogramming transcription factor. [Figure 2B] Nanoparticles carrying mRNA encoding IRF5 and IKKβ can imprint an inflammatory M1-like phenotype. (Figure 2B) Transmission electron microscope images of a group of NPs (scale bar 200 nm) and a single NP (inset, scale bar 50 nm). [Figure 2C] Nanoparticles carrying mRNA encoding IRF5 and IKKβ can imprint an inflammatory M1-like phenotype. (Figure 2C) NP size distribution, measured using NanoSight NS300 instrument. [Figure 2D] Nanoparticles carrying mRNA encoding IRF5 and IKKβ can imprint an inflammatory M1-like phenotype. (Figure 2D) The NPs showed high transfection (46%) of bone marrow-derived macrophages (BMDMs) after 1 hour of exposure. [Figure 2E] Nanoparticles carrying mRNA encoding IRF5 and IKKβ can imprint an inflammatory M1-like phenotype. (Figure 2E) Gene transfer efficiency into bone marrow-derived macrophages (BMDM) measured by flow cytometry 24 hours after particle transfection. [Figure 2F] Nanoparticles carrying mRNA encoding IRF5 and IKKβ can imprint an inflammatory M1-like phenotype. (Figure 2F) Relative viability of NP-transfected and untransfected macrophages (assessed by staining with Annexin V and PI). Ns; not significant. [Figure 2G]Nanoparticles carrying mRNA encoding IRF5 and IKKβ can program an inflammatory M1-like phenotype. (Fig. 2G) Expression kinetics of codon-optimized IRF5 mRNA (blue, left Y-axis) and endogenous IRF5 mRNA (black, right Y-axis) measured by qRT-PCR. n = 3 at each time point. [Figure 2H] Nanoparticles carrying mRNA encoding IRF5 and IKKβ can program an inflammatory M1-like phenotype. (Fig. 2H) Timeline depicting the NP transfection protocol and the culture conditions of BMDMs used in Figs. 2I–2K. [Figure 2I] Nanoparticles carrying mRNA encoding IRF5 and IKKβ can program an inflammatory M1-like phenotype. (Fig. 2I) Gene expression profiles of IRF5 / IKKβ NP-transfected macrophages compared to signature M1 cells stimulated with the Toll-like receptor 6 agonist MPLA. Results are depicted as a volcano plot showing the distribution of fold changes in gene expression. M1 signature genes are shown. The P value for overlap between IRF5 / IKKβ NP-transfected macrophages and the M1 signature gene set was determined by GSEA. [Figure 2J] Nanoparticles carrying mRNA encoding IRF5 and IKKβ can program an inflammatory M1-like phenotype. (Fig. 2J) Heatmap of M1 signature gene expression in macrophages cultured in IL-4 versus cells cultured in IL-4 and transfected with IRF5 / IKKβ NPs. [Figure 2K] Nanoparticles carrying mRNA encoding IRF5 and IKKβ can program an inflammatory M1-like phenotype. (Fig. 2K) Box-and-whisker plots showing the mean counts and S.E.M for the indicated genes. [Figure 3]In vitro screening of the effects of different members of the interferon regulatory factor (IRF) family (delivered with or without their activating kinases) on the phenotype of mouse macrophages. Bone marrow-derived macrophages (BMDMs) from C57BL / 6 were incubated in M-CSF-conditioned medium and transfected with (1) control GFP, (2) mouse IRF5, (3) mouse IRF5 and IKKβ kinase that phosphorylates IRF5, (4) mouse IRF8 and IKKβ kinase, (5) mouse IRF8 K310R, a mutant of IRF8 with a conversion from Lys-310 to Arg (K310R) (White et al., J Biol Chem. June 24, 2016), or (6) mRNA-PBAE NPs carrying synthetic mRNA encoding mouse IRF7 / 3(5D). This fusion protein contains the DNA-binding domain (DBD) and constitutively active domain (CAD) of IRF-7, as well as the nuclear export signal (NES) and IRF association domain of IRF3 (Lin et al., Molecular and Cellular Biology. 18.5, 1998). Two days after NP transfection, cells were collected for flow cytometry analysis for the TAM-related macrophage marker Egr2 and the activated macrophage marker CD38. Based on this in vitro screening, NPs co-delivering mRNAs encoding mIRF5 and IKKβ kinase were selected for the remaining in vitro and therapeutic in vivo experiments described herein. [Figure 4A] Repeated intraperitoneal injection of mRNA nanocarriers delivering IRF5 and IKKβ genes into macrophages more than doubles the mean survival time of mice bearing disseminated ovarian cancer. (Figure 4A) Timeline and dosing schedule. Arrows indicate the time of I.P. injection. [Figure 4B] Repeated intraperitoneal injection of mRNA nanocarriers delivering IRF5 and IKKβ genes into macrophages more than doubles the mean survival time of mice bearing disseminated ovarian cancer. (Figure 4B) Serial bioluminescence imaging of tumor growth in control and treated mice. [Figure 4C]Repeated intraperitoneal injection of mRNA nanocarriers delivering IRF5 and IKKβ genes into macrophages more than doubled the average survival time of mice with disseminated ovarian cancer. (Figure 4C) Kaplan-Meier survival curves for treated versus control mice. Statistical analysis was performed using log-rank tests. [Figure 4D] Repeated intraperitoneal injection of mRNA nanocarriers delivering IRF5 and IKKβ genes into macrophages more than doubled the average survival time of mice with disseminated ovarian cancer. (Figure 4D) Flow cytometry quantification of in vivo transfection rates in different immune cell subpopulations 48 hours after a single ip administration of GFP mRNA-carrying D-mannose coated NPs as a control: macrophages (CD45+, CD11b+, MHCII+, CD11c-, Ly6C- / low, Ly6G-), monocytes (CD45+, CD11b+, MHCII+, CD11c-, Ly6C+, Ly6G-), neutrophils (CD45+, CD11b+, MHCII+, CD11c-, Ly6G+), CD4+ T cells (CD45+, TCR-β chain+, CD4+, CD8-), CD8+ T cells (CD45+, TCR-β chain+, CD4-, CD8+), and natural killer cells (CD45+, TCR-β chain-, CD49b+). [Figure 4E] Repeated intraperitoneal injection of mRNA nanocarriers delivering IRF5 and IKKβ genes into macrophages more than doubled the average survival time of mice with disseminated ovarian cancer. (Figure 4E) Flow cytometry analysis of macrophage phenotype in the peritoneum of mice with disseminated ID8 ovarian cancer. Animals were treated with four doses of IRF5 / IKKβ NP or PBS. [Figure 4F] Repeated intraperitoneal injection of mRNA nanocarriers delivering IRF5 and IKKβ genes into macrophages more than doubled the average survival time of mice with disseminated ovarian cancer. (Figure 4F) Box plots summarizing the relative percentage (left panel) and absolute number (right panel) of Ly6C-, F4 / 80+, and CD206+ (M2-like) macrophages. [Figure 4G]Repeated intraperitoneal injection of mRNA nanocarriers delivering IRF5 and IKKβ genes into macrophages more than doubled the average survival time of mice with disseminated ovarian cancer. (Figure 4G) Corresponding numbers for Ly6C-, F4 / 80+, and CD206-(M1-like) macrophages. [Figure 4H] Repeated intraperitoneal injection of mRNA nanocarriers delivering IRF5 and IKKβ genes into macrophages more than doubled the average survival time of mice with disseminated ovarian cancer. (Figure 4H) Representative hematoxylin and eosin-stained sections of ovarian tumor-infiltrating mesentery isolated from PBS control (upper panel) or IRF5 / IKKβ NP-treated animals (lower panel; scale bar 100 μm). A 10x magnified view of a representative malignant lesion is shown on the right (scale bar 50 μm). [Figure 4I] Repeated intraperitoneal injection of mRNA nanocarriers delivering IRF5 and IKKβ genes into macrophages more than doubled the average survival time of mice with disseminated ovarian cancer. (Figure 4I) Luminex assay measured cytokines produced by peritoneal macrophages isolated from each treatment group. CD11b+, F4 / 80+ peritoneal macrophages were isolated by fluorescence-activated cell sorting and cultured ex vivo. After 24 hours, the cell culture supernatant was collected. In parallel experiments, FACS-sorted CD11b+, F4 / 80+ peritoneal macrophages were directly analyzed by pRT-PCR to determine the expression levels of four major regulators of the macrophage phenotype (serpin B2, Retnla, Ccl11, and Ccl5). [Figure 4J]Repeated intraperitoneal injection of mRNA nanocarriers delivering IRF5 and IKKβ genes into macrophages more than doubled the average survival time of mice with disseminated ovarian cancer (Figure 4I). Luminex assay measured cytokines produced by peritoneal macrophages isolated from each treatment group. CD11b+, F4 / 80+ peritoneal macrophages were isolated by fluorescence-activated cell sorting and cultured ex vivo. After 24 hours, the cell culture supernatant was collected. In a parallel experiment, FACS-sorted CD11b+, F4 / 80+ peritoneal macrophages were directly analyzed by pRT-PCR to determine the expression levels of four major regulatory factors of the macrophage phenotype (serpin B2, Retnla, Ccl11, and Ccl5). The results are summarized in the box plot in Figure 4J. [Figure 5A] The macrophage-programmed mRNA nanocarrier is highly biocompatible and safe for repeated administration. (Figure 5A) In vivo distribution of macrophage-targeted IRF5 / IKKβ NPs following ip administration. NP-delivered (codon-optimized) mRNA was detected by qPCR 24 hours after a single injection of particles containing 50 μg of mRNA. [Figure 5B] The macrophage-programmed mRNA nanocarrier is highly biocompatible and safe for repeated administration. (Figure 5B) Schematic diagram of the experimental timeline. 24 hours after the last dose, mice were euthanized by CO2 inhalation. Blood was collected in heparin-coated tubes via posterior orbital hemorrhage for serological chemistry and complete blood count. Autopsies were performed for histological analysis of the liver, spleen, pancreas, mesentery and reticularis, stomach, and bladder. [Figure 5C]Macrophage programming mRNA nanocarriers are highly biocompatible and safe for repeated administration. (Figure 5C) Representative hematoxylin and eosin-stained sections of various organs isolated from control or NP-treated animals. Scale bar, 100 μm. Lesions found in NP-treated animals are shown and described here based on comparative pathologist analysis. Relevant findings for each numbered image are: [1] Separate lesions of cellular infiltrations predominantly composed of mononuclear cells mixed with a small number of granulocytes; mild extramedullary hematopoiesis. [2] Mild to moderate hepatocyte enlargement in a few localized, broad areas. [3] Moderate myeloid (predominant), erythrocyte and meganuclear cell hyperplasia in the red medulla. [4] Mild hypocellularity in the white medulla. [5] Presence of moderate multifocal infiltrations of macrophages, lymphocytes, plasma cells and granulocytes in the mesentery. [6] Mild to moderate infiltration of macrophages mixed with lymphocytes, plasma cells and granulocytes; mild dissociation and disappearance of acinars; mild diffuse disappearance of zymogen granules from acinar cells. [7] Dense aggregates of lymphocytes mixed with macrophages around adipose tissue. [8] Mild multifocal vacuolar degeneration of parietal and primary cells in the gastric mucosa. [Figure 5D] Macrophage programming mRNA nanocarriers are highly biocompatible and safe for repeated administration. (Figure 5D) Serum chemistry and blood cell counts. [Figure 5E] Macrophage programming mRNA nanocarriers are highly biocompatible and safe for repeated administration. (Figure 5E) Luminex assay measurement of serum IL-6 cytokines 4 or 8 days after single intravenous injection of IRF5 / IKKβ NPs. [Figure 5F] Macrophage programming mRNA nanocarriers are highly biocompatible and safe for repeated administration. (Figure 5F) Luminex assay measurement of serum TNF-α cytokines 4 or 8 days after single intravenous injection of IRF5 / IKKβ NP. [Figure 6A]Intravenously administered IRF5 / IKKβ nanoparticles can control tumor metastasis in the lungs. (Figure 6A) In vivo distribution of macrophage-targeted IRF5 / IKKβ NPs following IV administration. Codon-optimized mRNA was measured by qPCR 24 hours after a single IV injection of particles containing 50 μg of mRNA. [Figure 6B] Intravenously administered IRF5 / IKKβ nanoparticles can control tumor metastasis in the lungs. Lung metastasis was established in C57BL / 6 albino mice by injection of 1 × 10⁶ B16F10 firefly luciferase-expressing melanoma cells via the tail vein. After 7 days, the animals were randomly assigned to either the IRF5 / IKKβ NP treatment group, the control GFP NP group, or the PBS control group. (Figure 6B) Timeline and administration plan. [Figure 6C] Intravenously administered IRF5 / IKKβ nanoparticles can control tumor metastasis in the lungs. Lung metastasis was established in C57BL / 6 albino mice by injecting 1 × 10⁶ B16F10 firefly luciferase-expressing melanoma cells via the tail vein. After 7 days, the animals were randomly assigned to either the IRF5 / IKKβ NP treatment group, the control GFP NP group, or the PBS control group. (Figure 6C) Confocal microscopy of healthy lungs (left panel) and B16F10 tumor-infiltrated lungs (right panel). Infiltrating macrophage populations fluoresce green. [Figure 6D] Intravenously administered IRF5 / IKKβ nanoparticles can control tumor metastasis in the lungs. Lung metastasis was established in C57BL / 6 albino mice by injecting 1 × 10⁶ B16F10 firefly luciferase-expressing melanoma cells via the tail vein. After 7 days, the animals were randomly assigned to either the IRF5 / IKKβ NP treatment group, the control GFP NP group, or the PBS control group. (Figure 6D) Serial bioluminescent tumor imaging. [Figure 6E]Intravenously administered IRF5 / IKKβ nanoparticles can control tumor metastasis in the lungs. Lung metastasis was established in C57BL / 6 albino mice by injection of 1 × 10⁶ B16F10 firefly luciferase-expressing melanoma cells via the tail vein. After 7 days, animals were randomly assigned to either the IRF5 / IKKβ NP treatment group, the control GFP NP group, or the PBS control. (Figure 6E) Kaplan-Meier survival curves by treatment group. ms represents the median survival time. Statistical analysis was performed using the log-rank test, with P<0.05 considered significant. [Figure 6F] Intravenously administered IRF5 / IKKβ nanoparticles can control tumor metastasis in the lungs. Lung metastasis was established in C57BL / 6 albino mice by injection of 1 × 10⁶ B16F10 firefly luciferase-expressing melanoma cells via the tail vein. After 7 days, the animals were randomly assigned to either the IRF5 / IKKβ NP treatment group, the control GFP NP group, or the PBS control group. (Figure 6F) Representative images (top row) and micrographs of lungs containing B16F10 melanoma metastases representing each group following 2 weeks of treatment. [Figure 6G] Intravenously administered IRF5 / IKKβ nanoparticles can control tumor metastasis in the lungs. Lung metastasis was established in C57BL / 6 albino mice by injecting 1 × 10⁶ B16F10 firefly luciferase-expressing melanoma cells via the tail vein. After 7 days, the animals were randomly assigned to either the IRF5 / IKKβ NP treatment group, the control GFP NP group, or the PBS control group. (Figure 6G) Number of lung tumor lesions. [Figure 6H] Intravenously administered IRF5 / IKKβ nanoparticles can control tumor metastasis in the lungs. Lung metastasis was established in C57BL / 6 albino mice by injecting 1 × 10⁶ B16F10 firefly luciferase-expressing melanoma cells via the tail vein. After 7 days, the animals were randomly assigned to either the IRF5 / IKKβ NP treatment group, the control GFP NP group, or the PBS control group. (Figure 6H) Phenotypic characterization of monocyte / macrophage populations during bronchoalveolar lavage from each treatment group. [Figure 6I]Intravenously administered IRF5 / IKKβ nanoparticles can control tumor metastasis in the lungs. (Figure 6I) Summary of relative percentages of suppressed and activated macrophages. [Figure 7A] Macrophage reprogramming improves radiotherapy outcomes in gliomas. (Figure 7A) T2 MRI scans and histological staining following the onset of PDGFβ-driven glioma in RCAS-PDGF-B / Nestin-Tv-a;Ink4a / Arf- / -;Pten- / - transgenic mice 21 days post-induction. [Figure 7B] Macrophage reprogramming improves the outcomes of radiotherapy in gliomas. (Figure 7B) Confocal microscopy of CD68+TAM infiltrating the glioma margin. Scale bar, 300 μm. [Figure 7C] Macrophage reprogramming improves radiotherapy outcomes in gliomas. (Figure 7C) Flow cytometry analysis of macrophage (F4 / 80+, CD11b+) populations in healthy brain tissue versus glioma. [Figure 7D] Macrophage reprogramming improves the outcomes of radiotherapy in gliomas. (Figure 7D) Kaplan-Meier survival curves of mice with established gliomas treated with IRF5 / IKKβ as monotherapy. Timeline and administration schedule are shown above. ms, median survival time. Statistical analysis was performed using log-rank tests, with P<0.05 considered statistically significant. [Figure 7E] Macrophage reprogramming improves radiotherapy outcomes in gliomas. (Figure 7E) Kaplan-Meier survival curves of mice with established gliomas treated with IRF5 / IKKβ in combination with brain tumor radiotherapy. Timeline and administration schedule are shown above. ms, median survival time. Statistical analysis was performed using log-rank tests, with P<0.05 considered statistically significant. [Figure 7F] Macrophage reprogramming improves the outcomes of radiotherapy in gliomas. (Figure 7F) Serial bioluminescence imaging of tumor progression. [Figure 8A]IVT mRNA-supported nanoparticles encoding human IRF5 / IKKβ efficiently reprogram human macrophages. (Figure 8A) Timeline and culture conditions for differentiating human THP-1 mononuclear cell line into inhibitory M2-like macrophages. [Figure 8B] IVT mRNA-supported nanoparticles encoding human IRF5 / IKKβ efficiently reprogram human macrophages. (Figure 8B) Bioluminescence imaging of M2-differentiated THP1-Lucia cells cultured in 24 wells and transfected with indicated concentrations of NPs supporting human IRF5 / IKKβ mRNA versus control GFP mRNA. IRF-induced Lucia luciferase levels were determined 24 hours after transfection using Quanti-Luc. [Figure 8C] IVT mRNA-supported nanoparticles encoding human IRF5 / IKKβ efficiently reprogram human macrophages. (Figure 8C) Summary of bioluminescence counts. [Figure 8D] IVT mRNA-supported nanoparticles encoding human IRF5 / IKKβ efficiently reprogram human macrophages. (Figure 8D) Differences in M1 macrophage marker CD80 in IL-1β cytokine secretion. [Figure 8E] IVT mRNA-supported nanoparticles encoding human IRF5 / IKKβ efficiently reprogram human macrophages. (Figure 8E) Difference in surface expression (Figure 8E) M1-macrophage marker CD80. [Figure 9] A list of antibodies used in the bone marrow and lymphocyte immunophenotype panels described in Example 1. [Figure 10-1]Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-2] Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-3] Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-4]Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-5] Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-6] Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-7]Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-8] Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-9] Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-10]Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-11] Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-12] Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-13]Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-14] Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-15] Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-16]Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-17] Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-18] Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-19]Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-20] Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-21] Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 10-22]Exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure are provided. The identities of SEQ ID NOs: 1-44, 110, and 111 are shown in Figure 11. In particular, with respect to SEQ ID NO: 15, the fusion protein includes the DBD (DNA binding domain) and CAD (constitutive activity domain) of mouse IRF7, as well as the NES (nuclear export signaling) and IRF-association domain of mouse IRF3. The IRF-association domain contains Asp mutations in four serine and one threonine residues in the N-terminal region, giving rise to constitutive activation and transposition of the fusion protein (see Lin R et al., (1998), above). In particular, with respect to SEQ ID NO: 17, the SUMO (small ubiquitin-like modifier) ​​binding site in mouse IRF8 is lysine (K) 310. SUMO2 / 3 binding prevents IRF-8 from binding to and activating IRF8-responsive genes. Mutations at the K310 residue prevent SUMO from binding to IRF8, increasing IRF8-specific gene transcription by 2 to 5 times (Chang TH et al., The Journal of Immunology (2012) 189(7): pp. 3548-3556). [Figure 11-1] This provides a list of example protein sequences and coding nucleotide sequences. [Figure 11-2] This provides a list of example protein sequences and coding nucleotide sequences. [Modes for carrying out the invention]

[0013] Several adverse physiological conditions are associated with either immune system activation (e.g., autoimmune disorders) or immunosuppression (e.g., cancer). For example, macrophages are important immune effector cells that infiltrate cancer tissue in large numbers. However, in an immunosuppressive tumor environment, macrophages undergo a switch from an activated, tumor-killing state to an immunosuppressive phenotype that promotes tumor growth and metastasis. These tumor-associated immunosuppressed macrophages (TAMs) are associated with poor prognosis (Komohara Y et al., (2014) Cancer Science 105(1): pp. 1-8). TAMs induce angiogenesis, lymphocyte formation, and stromal remodeling. TAMs also play an important role in promoting tumor invasion and metastasis through the secretion of enzymes plasmin, uPA, matrix metalloproteinases (MMPs), and cathepsin B (Komohara, Y et al., (2016) Advanced drug delivery reviews 99: pp. 180-185; Gocheva V et al., (2010) Genes Dev 24: pp. 241-255; Wang R et al., (2011) Lung Cancer 74: pp. 188-196). Apart from mediating tumor growth and progression, TAMs can interact with other immune cells to suppress innate and adaptive anti-tumor immune responses.

[0014] Some small molecule drugs focus on blocking the localization of TAM precursor cells to tumors by targeting pathways involved in cell recruitment or proliferation (i.e., inhibitors of the CSF-1 / CSF-1R pathway (Pyon;teck et al., Nat Med 19, pp. 1264-1272 (2013); Tap et al., N Engl J Med 373, pp. 428-437 (2015)) or the CCL2 pathway (Nywening et al., Lancet Oncol 17, pp. 651-662 (2016))). These approaches require repeated systemic exposure to high doses of small molecule drugs. Furthermore, clinical trials of these drugs have shown poor responses when not combined with cytoreductive therapy. Nywening et al., Lancet Oncol 17, pp. 651-662 (2016); Butowski et al., Neuro Oncol 18, pp. 557-564 (2016). Furthermore, these small molecule approaches do not actively promote macrophage antitumor activity.

[0015] Conventional nanocarriers such as liposomes have been formulated with bisphosphonates or other antiproliferative agents that systemically destroy macrophages within tumors (i.e., liposomal clodronates) (Fritz et al., Front Immunol 5, 587 (2014)). Oncolytic viruses have also been used to deliver siRNA and indirectly promote TAM phagocytosis by deactivating immune evasion pathways within tumors (Chao et al., Curr Opin Immunol 24, pp. 225-232 (2012)). However, macrophages destroyed using these approaches are spontaneously replaced by newly arriving macrophages, which is also immunosuppressive.

[0016] Antibodies that induce functional activation of TAMs are being developed. These approaches utilize antibodies that target specific antigen types within tumors. Mantovani et al., Nat Rev Clin Oncol (2017). However, the success of these antibodies is limited by their low tumor penetration and heterogeneous distribution. Thurber et al., Adv Drug Deliv Rev 60, pp. 1421-1434 (2008). They have also not addressed tumor escape variants that lack the antigens targeted by the antibodies.

[0017] None of the approaches described herein directly and effectively reprogram TAMs to become activated tumor-killing macrophages as disclosed herein. The systems and methods disclosed herein are remarkably innovative because they enable the reprogramming of TAMs to become tumor-clearing macrophages while simultaneously reducing tumor-promoting TAM load. Currently, there are no other methods that enable physicians to rationally reprogram TAMs for these therapeutic purposes. Mantovani et al., Nat Rev Clin Oncol (2017); Gabrilovich & Nagaraj, Nat Rev Immunol 9, pp. 162-174 (2009). The methods themselves can provide therapeutic effects in the treatment of tumors. By modulating and reconfiguring the tumor microenvironment, the disclosures make tumors more sensitive to other types of treatment such as vaccines, immunotherapy (e.g., CAR), and / or chemotherapy.

[0018] Certain embodiments utilize particles that deliver to cells nucleotides encoding genes that encode activation regulators, such as transcription factors (e.g., interferon regulators (IRFs)) and / or kinases (e.g., IKKβ). These activation regulators modulate macrophage polarization (Figure 1). Macrophage polarization is a highly dynamic process that macrophages undergo as their physiological activity changes. As shown, in most tumors, TAMs exhibit an immunosuppressed phenotype, which can result in an “M2” phenotype. In contrast, activated macrophages can exhibit an “M1” phenotype, which results in the killing of tumor cells. Certain embodiments disclosed herein reverse the polarization of tumor-promoting TAMs into tumor-killing macrophages. This effect alleviates the immunosuppressive environment within the tumor by inducing inflammatory cytokines, activating other immune cells, and phagocytosing tumor cells.

[0019] Macrophage activation is the process of changing the phenotype or function of macrophages from (i) an inactivated state to an activated state; (ii) an inactivated state to an activated state; (iii) an activated state to a further activated state; or (iv) an inactivated state to an inactivated state. The inactivated state refers to an immunosuppressed phenotype that promotes tumor growth and metastasis. The inactivated state means that macrophages do not promote tumor growth or metastasis, nor do they promote the killing of tumor cells. The activated state means that macrophages exhibit tumor-killing activity. In certain embodiments, the activated state results in the M1 phenotype, which is described more fully below. In certain embodiments, the inactivated state results in the M2 phenotype, which is also described more fully below.

[0020] Macrophage inactivation is a process that changes the phenotype or function of a macrophage from (i) an activated state to a less activated state; (ii) an activated state to an inactivated state; (iii) an activated state to an inactivated state; or (iv) an inactivated state to an inactivated state. In certain embodiments, the inactivated state is M2. In certain embodiments, the activated state is M1.

[0021] In certain embodiments, one advantage of the disclosed system and method is that the inflammation induced by the treatment remains localized at the treatment site, thus saving the patient from systemic toxicity. To achieve this advantage, locally injected particles target TAMs in the tumor environment, (2) deliver nucleotides that selectively reprogram signaling pathways that control macrophage polarization, and (3) become completely degradable locally via physiological pathways (Sahin et al., Nat Rev Drug Discov 13, pp. 759-780 (2014)).

[0022] Achieving high expression of exogenous nucleotides in solid tumors is a challenge in vivo. Prior to this disclosure, conventional nanocarrier-based nucleotide delivery systems, such as viruses or liposomes, have been limited by their restricted diffusion within tumor tissue, Jain & Stylianopoulos, Nat Rev Clin Oncol 7, pp. 653-664 (2010). To circumvent this barrier, certain embodiments utilize nanoparticles (NPs) with enhanced diffusivity so that the NPs deliver nucleotides to a large population of TAMs within the tumor. Certain embodiments utilize NPs with a size <130 nm that bear a neutral surface charge. Certain embodiments may further include targeting ligands bound to the surface of the NPs. For example, dimannose can be bound to the NP surface to enable more selective targeting to the mannose receptor (CD206) expressed on the surface of TAM cells. Other TAM cell surface receptors that can be targeted include early proliferation response protein 2 (Egr2), CD163, CD23, interleukin (IL) 27RA, CLEC4A, CD1a, CD1b, CD93, CD226, IL13-Ra1, IL-4r, IL-1R type II, decoy IL-1R type II, IL-10r, macrophage scavenger receptors A and B, Ym-1, Ym-2, low-density receptor-associated protein 1 (LRP1), IL-6r, CXCR1 / 2, and PD-L1.

[0023] In certain embodiments, the systems and methods disclosed herein involve administering particles to a subject requiring particle administration. The particles are directed to macrophages present in the tumor of the subject and are designed to be internally transported by the macrophages. After internal transport, the particles further deliver one or more nucleotides having sequences encoding IRF5 and IKKβ. The one or more nucleotides modify the macrophages to express IRF5 and IKKβ. Without being bound by theory, IKKβ kinase activates the IRF5 transcription factor by phosphorylation. The activated IRF5 then triggers the expression of inflammatory cytokines including type I interferon (IFN) genes, tumor necrosis factor (TNF), IL-6, IL-12, and IL-23, as well as tumor suppressor factors. In M2 macrophages to which one or more nucleotides encoding IRF5 and IKKβ have been internally translocated, the expression of the aforementioned genes via IRF5 action results in a phenotypic or functional conversion of the macrophage from an M2 phenotype to an M1 phenotype, thereby enabling the macrophage to kill or otherwise trigger the destruction of tumor cells, thereby making it possible to treat cancer. In certain embodiments, the particles are internally translocated into macrophages by phagocytosis. In certain embodiments, the particles are internally translocated into macrophages by ligand-mediated endocytosis (e.g., CD-206-mediated endocytosis). In certain embodiments, delivery of particles containing the IRF5 and IKKβ genes into macrophages involves, for example, (1) binding to the macrophage, (2) internal translocation of the particles by the macrophage, (3) escape from intracellular vesicles into the cytoplasm after internal translocation, (4) release of one or more nucleotides, (5) the nucleotides being transported into the nucleus of the macrophage, and (6) delivery of genes to be transcribed for expression of IRF5 and IKKβ.

[0024] The aspects of this disclosure are now described in further detail as follows: (1) macrophages and macrophage phenotypes; (2) cellular pathways affecting macrophage polarization; (3) nucleotides encoding activation regulators; (4) particles delivering nucleotides; (5) targeting ligands for more selective nucleotide delivery; (6) pharmaceutical compositions comprising the particles; (7) methods of use; and (8) experimental examples.

[0025] (1) Macrophages and macrophage phenotypes. Macrophages are white blood cells of the immune system that differentiate from bone marrow-derived monocytes. Macrophages are characterized by their phagocytic activity and antigen-presenting ability. Macrophages play an important role in both innate and adaptive immune responses. Phenotypically, macrophages express the surface marker F4 / 80 (Ly71) and may also express other surface markers such as CDllb (Macl), CDllc, CD14, CD40, or CD68.

[0026] Macrophages play a crucial role in both innate and adaptive immunity by activating T lymphocytes. In cancer, macrophages are one of the large populations of invasive leukocytes associated with solid tumors (Gordon S & Taylor PR (2005) Nature Reviews Immunology 5(12): pp. 953-964). Macrophages can be recruited to the tumor site from surrounding tissues or by the tumor itself through the secretion of chemotactic molecules. Depending on their phenotype, macrophages participate in the immune response against tumors in a polarized manner. "Phenotype" is used herein to refer to the physical or biochemical characteristics of a cell as a result of the interaction of genotype and environment, and may include cellular function.

[0027] Macrophages that activate Th1 T lymphocytes often exhibit an inflammatory response and are characterized by M1 polarization or a “classical activation” phenotype. Activated macrophages (i.e., M1 macrophages or macrophages with the M1 phenotype), also known as “killer macrophages,” inhibit cell proliferation, cause tissue damage, mediate resistance to pathogens, and possess potent tumor-killing activity. These macrophages can increase the expression of mediators responsible for antigen presentation and co-stimulation; when neutrophil infiltration into tumor areas is promoted, neutrophil-targeted tumor regression results. The M1 phenotype can also be demonstrated by increased antigen presentation compared to a suitable control state. In certain embodiments, the M1 phenotype can be demonstrated by M1 macrophage production of reactive oxygen species (ROS) and nitric oxide (NO). NO has antiproliferative effects essential for defense against pathogens and abnormal cells such as tumor cells. In certain embodiments, the M1 phenotype can be demonstrated by a pro-inflammatory state that induces Th1 immunity through the production of cytokines such as IL-12. In certain embodiments, activated macrophages are classically activated macrophages that can phagocytose pathogens.

[0028] In addition to function, the M1 phenotype can also be demonstrated by surface markers expressed by macrophages; factors, proteins, or compounds produced by macrophages upon polarization; or genes induced by macrophages during polarization. M1 polarization can result in a phenotype demonstrated by the expression of CD80, CD86, iNOS, cytokine signaling suppressor 3 (SOCS3), TNFα, IL-1, IL-6, IL-12, IL-23, type I IFN, CXCL1, CXCL2, CXCL3, CXCL5, CXCL8, CXCL9, and CXCL10. In certain embodiments, the M1 phenotype includes increased expression of CD80. In certain embodiments, the M1 phenotype includes CD206-, MHCII+, CD11c-, and CD11b+.

[0029] On the other hand, macrophages that activate Th2 T lymphocytes often exhibit an anti-inflammatory response and are frequently shown to have an "M2" phenotype. Inactivated macrophages (i.e., M2 macrophages or macrophages with the M2 phenotype), also known as "repair macrophages," are involved in metazoa parasite containment, cell proliferation, tissue repair, tumor progression, anti-inflammatory pathways, and immunosuppression. The M2 phenotype can reduce antigen presentation and phagocytosis compared to a suitable control state. The M2 phenotype can be demonstrated by the expression of, for example, arginase 1 (Arg1 (arginase activity is associated with proliferative effects and tissue repair responses), IL-10, TGF-β, PPArγ, KLF4, CD206 (MRC1), Dectin-1 (signaling non-TLR pattern recognition receptor), DC-SIGN (type C lectin), scavenger receptor A, scavenger receptor B-1, CD163 (high affinity scavenger receptor for hemoglobin-haptoglobin complexes), chemokine receptors CCR2, CXCR1, and CXCR2, YM1 (chitinase 3-like 3), and Fizz1; as well as the secretion of chemokines CCL17, CCL22, and CCL24. In certain embodiments, inactivated macrophages promote metastasis and / or resistance to chemotherapy. In certain embodiments, the M2 phenotype can be demonstrated by the expression of CD206+, MHCII-, CD11c+, and CD11b low Includes.

[0030] Table 1 provides specific combinations of criteria that can be used to distinguish between M1 phenotypes and M2 phenotypes (including sub-phenotypes named M2a, M2b, M2c, and M2d).

[0031] [Table 1]

[0032] Assays for evaluating macrophage phenotypes can utilize different molecular signatures specific to M1 or M2 phenotypes. A commonly recognized marker profile for M1 macrophages is CD80+, while M2- macrophages can be considered CD163+. Therefore, these markers can be evaluated by performing flow cytometry. Moving macrophages closer to the M1 type and away from the M2 type can also be evaluated by measuring the increase in the IL-12 / IL-10 ratio or the CD163- / CD163+ macrophage ratio. In certain embodiments, M1 vs. M2 morphology can be evaluated by light microscopy. In certain embodiments, phagocytic assays may be used in conjunction with other assays to evaluate whether macrophages are M1 type or M2 phenotype. Phagocytic assays of different macrophage populations may be performed by incubating the phagocytic entity with macrophages at a concentration consistent with the normalized total surface area per cell. The phagocytic entity may be added to the macrophage culture. The phagocytic entity may be labeled, for example, with a fluorescent label. The phagocytic index may be determined by the median total fluorescence intensity measured per macrophage. Quantification of phagocytosis may be performed, for example, by flow cytometry. Tumor cell killing assays may also be utilized. In certain embodiments, the M1 phenotype includes reduced expression of signature M2 macrophage genes, including serpin B2 (an inhibitor of urokinase-type plasminogen activator), CCL2 (CC-motif chemokine ligand 2), CCL11 (CC-motif chemokine ligand 11), and Retnla (resistin-like alpha; Fizz1). In certain embodiments, the M1 phenotype includes increased expression of M1 differentiation genes, including CCL5 (CC-motif chemokine ligand 5).

[0033] Gene expression (e.g., M1 expression of CD80, CD86 and / or other genes mentioned above) can be measured by assays well known to those skilled in the art. Methods for measuring gene expression include NanoString nCounter® expression assay (NanoString Technologies, Inc., Seattle, WA), Northern blotting, dot blotting, microarrays, sequential gene expression analysis (SAGE), RNA-seq, and quantitative RT-PCR. Methods for measuring gene expression products, such as protein levels, include ELISA (enzyme-linked immunosorbent assay), Western blotting, FACS, radioimmunoassay (RIA), sandwich assay, fluorescence insight hybridization (FISH), immunohistochemistry, immunoelectrophoresis, immunoprecipitation, and immunofluorescence using detection reagents such as antibodies or protein binders.

[0034] (2) Cellular pathways influencing macrophage polarization. Macrophage polarization toward activation or inactivation phenotypes arises from interactions between macrophages and several different molecules or environments. For example, M1 macrophage polarization is triggered by stimuli including Toll-like receptor (TLR) ligands (e.g., lipopolysaccharide (LPS), muramyl dipeptide, lipoteichoic acid, imiquimod, CpG), IFNγ, TNFα, and macrophage colony-stimulating factor (GM-CSF). M2 polarized macrophages can be divided into subsets depending on the stimulus that induces polarization: the M2a subset is induced by IL-4, IL-13, or fungal and helminthic infections; M2b is induced by IL-1 receptor ligand, immune complexes, and LPS; M2c is induced by IL-10, TGF-β, and glucocorticoids; and M2d is induced by IL-6 and adenosine. M2 macrophage polarization can also be triggered by IL-21, GM-CSF, complement components, and apoptotic cells. Macrophage polarization is also regulated by local microenvironmental conditions such as hypoxia.

[0035] The aforementioned molecules and environment influence macrophage polarization by triggering different intracellular signaling pathways, including transcription factors. Transcription factors involved in both M1 and M2 polarization include IRF, signaling and transcriptional activator (STAT), SOCS3 protein, and activated B cell nuclear factor kappa light chain enhancer (NFκB). Mitogen-activated protein kinase (MAPK) also plays a role in directing macrophage function towards either the M1 or M2 phenotype.

[0036] The IRF / STAT pathway is activated by stimuli such as IFN and TLR signaling, as discussed above, and polarizes macrophages to the M1 active state via STAT1. On the other hand, stimuli such as IL-4 and IL-13 divert macrophages to the M2 active state via STAT6 (Sica A & Bronte V (2007) J Clin Invest 117: pp. 1155-1166). Therefore, these signaling events promote inflammatory immune responses and tumor-killing activity in the case of M1 macrophage polarization, or immunosuppressive pro-tumor responses in the case of M2 macrophage polarization.

[0037] Several intracellular molecules involved in the induction of the M1 phenotype include the G protein-coupled receptor P2Y(2)R, which plays a role in inducing NO via NOS2 (Eun SY et al., (2014) Int Immunopharmacol 18: pp. 270-276); SOCS3, which activates the NFκB / PI-3 kinase pathway to produce NO (Arnold CE et al., (2014) Immunology 141: pp. 96-110); and the growth and differentiation factor activin A, which promotes the M1 marker and downregulates IL-10 (Sierra-Filardi E et al., (2011) Blood 117: pp. 5092-5101).

[0038] Other intracellular molecules involved in inducing the M1 phenotype include IRFs. IRFs are a group of transcription factors with diverse roles, including the virus-mediated activation of IFNs, as well as the regulation of cell growth, differentiation, apoptosis, and immune system activity. Members of the IRF family are characterized by a conserved N-terminal DNA-binding domain containing tryptophan (W) repeats.

[0039] IRF5 is a transcription factor that possesses a helix-turn-helix DNA-binding motif and mediates virus- and IFN-induced signaling pathways. IRF5 acts as a molecular switch that controls whether macrophages promote or inhibit inflammation. IRF5 activates type I IFN genes, inflammatory cytokines including TNF, IL-6, IL-12, and IL-2, tumor suppressor factors, and Th1 and Th17 responses. IRF5 is encoded by the human IRF5 gene (OMIM ID 607218) located on chromosome 7q32. Several isoforms / transcriptional variants of IRF5 are recognized. In certain embodiments, the isoforms of human IRF5 include isoform 1 (UniProt commissioned Q13568-1, SEQ ID NO: 1), isoform 2 (UniProt commissioned Q13568-2, SEQ ID NO: 2), isoform 3 (UniProt commissioned Q13568-3, SEQ ID NO: 3), isoform 4 (UniProt commissioned Q13568-4, SEQ ID NO: 4), isoform 5 (UniProt commissioned Q13568-5, SEQ ID NO: 5), and isoform 6 (UniProt commissioned Q13568-6, SEQ ID NO: 6). In certain embodiments, the isoforms of human IRF5 include isoform 1 encoded by the nucleotide sequence shown in SEQ ID NO: 23, isoform 2 encoded by the nucleotide sequence shown in SEQ ID NO: 24, isoform 3 encoded by the nucleotide sequence shown in SEQ ID NO: 25, isoform 4 encoded by the nucleotide sequence shown in SEQ ID NO: 26, isoform 5 encoded by the nucleotide sequence shown in SEQ ID NO: 27, and isoform 6 encoded by the nucleotide sequence shown in SEQ ID NO: 28. In certain embodiments, mouse IRF5 contains the amino acid sequence shown in SEQ ID NO: 7. In certain embodiments, mouse IRF5 is encoded by the nucleotide sequence shown in SEQ ID NO: 29. M1 macrophages have been shown to upregulate IRF5.

[0040] IRF1 and IRF8 also play crucial roles in the development and function of myeloid cells, including the activation of macrophages by pro-inflammatory signals such as IFNγ, Dror N et al., (2007) Mol Immunol. 44(4):338-346. In certain embodiments, human IRF1 includes the amino acid sequence shown in SEQ ID NO: 8. In certain embodiments, human IRF1 is encoded by the nucleotide sequence shown in SEQ ID NO: 30. In certain embodiments, mouse IRF1 includes the amino acid sequence shown in SEQ ID NO: 12. In certain embodiments, mouse IRF1 is encoded by the nucleotide sequence shown in SEQ ID NO: 34. In certain embodiments, human IRF8 includes the amino acid sequence shown in SEQ ID NO: 11. In certain embodiments, human IRF8 is encoded by the nucleotide sequence shown in SEQ ID NO: 33. In certain embodiments, mouse IRF8 includes the amino acid sequence shown in SEQ ID NO: 16. In certain embodiments, mouse IRF8 is encoded by the nucleotide sequence shown in SEQ ID NO: 38.

[0041] IRF3 is a homolog of IRF1 and IRF2. IRF3 contains several functional domains, including the NES, DBD, C-terminal IRF-associating domain, and several regulatory phosphorylation sites. Upon serine / threonine phosphorylation, IRF3 is found in an inactive cytoplasmic form that forms a complex with the transcriptional co-agent CREB-binding protein. This complex then translocates to the nucleus and activates the transcription of IFN-α and -β, as well as other interferon-inducible genes. In certain embodiments, the isoforms of human IRF3 include isoform 1 (UniProt commissioned Q14653-1), isoform 2 (UniProt commissioned Q14653-2), isoform 3 (UniProt commissioned Q14653-3), isoform 4 (UniProt commissioned Q14653-4), and isoform 5 (UniProt commissioned Q14653-5). In certain embodiments, human IRF3 isoform 1 contains the amino acid sequence shown in SEQ ID NO: 9. In certain embodiments, human IRF3 isoform 1 is encoded by the nucleotide sequence shown in SEQ ID NO: 31. In certain embodiments, mouse IRF3 includes the amino acid sequence shown in SEQ ID NO: 13. In certain embodiments, mouse IRF3 is encoded by the nucleotide sequence shown in SEQ ID NO: 35.

[0042] IRF7 has been shown to play a role in the transcriptional activation of type I IFN genes. In certain embodiments, human IRF7 isoform 1 includes isoform A (UniProt commissioned Q92985-1), isoform B (UniProt commissioned Q92985-2), isoform C (UniProt commissioned Q92985-3), and isoform D (UniProt commissioned Q92985-4). In certain embodiments, human IRF7 isoform A includes the amino acid sequence shown in SEQ ID NO: 10. In certain embodiments, human IRF7 isoform A is encoded by the nucleotide sequence shown in SEQ ID NO: 32. In certain embodiments, mouse IRF7 includes the amino acid sequence shown in SEQ ID NO: 14. In certain embodiments, mouse IRF7 is encoded by the nucleotide sequence shown in SEQ ID NO: 36.

[0043] One or more IRF mutants that contribute to IRF activation may also be used. For example, phosphorylation mimic mutants of human mutant 3 / mutant 4 of IRF5 (isoform 4, SEQ ID NO: 4) in which amino acid residues S425, S427, S430, and S436 are replaced with phosphorylation-mimicking residues such as aspartic acid residues (Chen W et al., (2008) Nat Struct Mol Biol. 15(11): pp. 1213-1220); and phosphorylation mimic mutants of human mutant 5 of IRF5 (isoform 2, SEQ ID NO: 2) in which amino acid residues T10, S158, S309, S317, S451, and / or S462 are replaced with phosphorylation-mimicking residues such as aspartic acid residues (Chang Foreman HC et al., see above); Mutations from residues S156, S158, and T160 of human IRF5 isoform a (mutant 1, isoform 3, SEQ ID NO: 3) and isoform b (mutant 2, isoform 1, SEQ ID NO: 1) to phosphorylation-mimicking residues such as aspartic acid residues for constitutive nuclear accumulation of IRF5 (Lin R et al., (2005) J Biol Chem 280(4):3088~3095); and IRF3 phosphorylation-mimicking mutants in which the amino acid residue S396 of IRF3 is replaced with phosphorylation-mimicking residues such as aspartic acid (Chen W et al., see above). In certain embodiments, the mouse IRF7 / IRF3 fusion protein contains Asp(D) mutations in four serine and one threonine residues in the IRF3-associating domain (SEQ ID NO: 15), constitutive activation and translocation of the fusion protein (Lin R et al., (1998) see above; Lin et al., (2000) Molecular and Cellular Biology 20: pp. 6342-6353). In certain embodiments, the mouse IRF7 / IRF3 fusion protein containing D mutations in four serine and one threonine residues in the IRF3-associating domain is encoded by the nucleotide sequence shown in SEQ ID NO: 37. In certain embodiments, the mouse IRF8 mutant contains a substitution of lysine (K) with arginine (R) at amino acid residue 310 (SEQ ID NO: 17).In certain embodiments, a mouse IRF8 mutant containing a K-R substitution at amino acid residue 310 is encoded by the nucleotide sequence shown in SEQ ID NO: 39. Small ubiquitin-like modifiers (SUMOs), which primarily bind to IRF8 at K310, inhibit the activation of IRF8-responsive genes. Centrin-specific protease 1 (SENP1) targets SUMO2 / 3. SENP1 activity "de-SUMOlated" IRF8 (and other proteins), causing IRF8 to change from an inhibitor to an activator of M1 macrophage differentiation (directly and through transactivation activity). Preventing SUMO from binding to IRF8 by the K310 mutation results in a 2-5-fold increase in IRF8-specific gene transcription (see Chang TH et al., (2012), above).

[0044] Certain embodiments of this disclosure include engineered IRF transcription factors. In certain embodiments, the engineered IRF transcription factor includes an IRF that lacks a functional self-repressive domain and is therefore insensitive to feedback inactivation (Thompson et al., (2018) Front Immunol 9:2622). For example, human IRF5 with a 2-3-fold increase in activity can be obtained by deleting amino acids 489-539 of the human IRF5 protein (Barnes et al., (2002) Mol Cell Biol 22:5721-5740). In certain embodiments, deleting or muting the self-repressive domain of IRF4, a transcription factor that promotes the M2 phenotype, can create a more active IRF4 in the context of treating autoimmune diseases. In certain embodiments, the self-repressive domain of the IRF can be found at the carboxyl terminus of the IRF protein. In certain embodiments, the engineered IRF transcription factor comprises an IRF lacking one or more functional nuclear export signals (NES) that capture the IRF in the nucleus and thus enhance transcription. For example, nuclear accumulation of human IRF5 can be achieved by mutating the NES of human IRF5 by replacing two leucine residues with alanine (L157A / L159A) (Lin et al., (2000) Molecular and Cellular Biology 20: pp. 6342-6353). In certain embodiments, the engineered IRF transcription factor comprises a fusion of one or more IRFs, a fusion of one or more IRF fragments, and a fusion of mutated IRFs.

[0045] NFκB is also an important transcription factor involved in macrophage M1 activation. NFκB regulates the expression of numerous inflammatory genes, including TNFα, IL1B, cyclooxygenase 2 (COX-2), IL-6, and IL12p40. NFκB activity is regulated via activation of inhibitors of the kappa B kinase (IKK) trimer complex (two kinases, IKKα and IKKβ, and the regulatory protein, IKKγ). When upstream signals converge at the IKK complex, these signals first phosphorylate to activate IKKβ kinase, which then phosphorylates the inhibitory molecule, the kappa B inhibitor (I-κB). This leads to proteasomal degradation of I-κB, releasing the NFκB p65 / p50 heterodimer from the NFκB / I-κB complex. The NFκB p65 / p50 heterodimer then translocates to the nucleus and binds to the promoters of inflammatory genes.

[0046] IKKβ is an activating kinase for NFκB and other transcription factors such as IRF5. IKKβ also phosphorylates several other signaling pathway components, including FOXO3, NCOA3, BCL10, IRS1, NEMO / IKBKG, NFκB subunits RELA and NFKB1, as well as IKK-related kinases TBK1 and IKBKE. In certain embodiments, the isoforms of human IKKβ include isoform 1 (UniProt O14920-1, SEQ ID NO: 18), isoform 2 (UniProt O14920-2, SEQ ID NO: 19), isoform 3 (UniProt O14920-3, SEQ ID NO: 20), and isoform 4 (UniProt O14920-4, SEQ ID NO: 21). In certain embodiments, the isoforms of human IKKβ include isoform 1 encoded by the nucleotide sequence shown in SEQ ID NO: 40, isoform 2 encoded by the nucleotide sequence shown in SEQ ID NO: 41, isoform 3 encoded by the nucleotide sequence shown in SEQ ID NO: 42, and isoform 4 encoded by the nucleotide sequence shown in SEQ ID NO: 43. In certain embodiments, mouse IKKβ includes the amino acid sequence shown in SEQ ID NO: 22. In certain embodiments, mouse IKKβ is encoded by the nucleotide sequence shown in SEQ ID NO: 44.

[0047] This disclosure provides the co-expression of an IRF transcription factor together with one or more molecules that can activate IRF, resulting in TAM reprogramming to an activated state for tumor killing. In certain embodiments, co-expression strategies include co-expression of IRF5 and IKKβ; co-expression of IRF5 and TANK-binding kinase 1 (TBK-1), TNF receptor-associating factor 6 (TRAF6) adapter, receptor-interacting protein 2 (RIP2) kinase, and / or NFκB kinase ε (IKKε) (Chang Foreman HC et al., (2012) PLoS One 7(3):e33098); co-expression of IRF5 and protein kinase DNA-PK (Ryzhakov G et al., (2015) J of Interferon & Cytokine Res 35(2):pp. 71-78); co-expression of IRF5 and protein kinase tyrosine kinase BCR-ABL (Massimo M et al., (2014) Carcinogenesis 35(5):pp. 1132-1143); and co-expression of IRF5 or IRF8 and one or more components of the COP9 signalosome (Korczeniewska This includes J et al., (2013) Mol Cell Biol 33(6):1124-1138; and Cohen H et al., (2000) J Biol Chem 275(50):39081-39089).

[0048] In certain embodiments, the teachings of this disclosure can be applied to the management of conditions triggered by hyperimmune activation (e.g., autoimmune diseases). Macrophages play a crucial role in autoimmune diseases such as systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, and Sjögren's syndrome (Ushio et al., World J Immunol 2017;7(1):1-8). Therefore, cellular pathways supporting the immunosuppressive M2 phenotype are also described.

[0049] The activation regulator involved in the induction of the M2 phenotype is Kruppel-like factor 4 (KLF-4). KLF-4 works in conjunction with STAT6 to induce M2 genes such as Arg-1, CD206 (Mrc1, mannose receptor), Fizz1 (resistin-like α), and peroxisome proliferator-activated receptor gamma (PPArγ), while inhibiting M1 genes such as TNFα, Cox-2, CCL5, and iNOS. The nuclear receptor PPARγ has been shown to regulate genes involved in the oxidative metabolism and activation of the M2 phenotype (Odegaard JI et al., (2007) Nature 447: pp. 1116-1120).

[0050] The cytokine IL-21 mediates M2 polarization by reducing NOS2 expression and increasing STAT3 phosphorylation (Li SN et al., (2013) Mediators Inflamm 2013, pp. 548073).

[0051] IRF4 negatively modulates TLR signaling in a MyD88-independent manner, driving the M2 phenotype (Satoh T et al., (2010) Nat Immunol 11, pp. 936-944). In certain embodiments, human IRF4 is UniProt contracted Q15306. BMP-7 also induces M2 polarization in vitro via activation of the SMAD-PI3K-Akt-mTOR pathway (Rocher C et al., (2013) Plos One 8:e84009).

[0052] The transcription factor glucocorticoid-induced leucine zipper (GILZ). GILZ is a dexamethasone-inducible gene that mediates glucocorticoid (GC) action in various cell types, and this gene can induce the repressive M2 macrophage phenotype. GILZ expression is rapidly and ubiquitously induced by GC, and the protein product interacts with known transcription factors such as NF-κB, Raf-1, TORC2, AP-1, Ras, and C / EBP to inhibit the expression of pro-inflammatory genes. Therefore, GILZ is thought to be able to mimic the therapeutic anti-inflammatory effects of GC while avoiding adverse effects (Ronchetti, S. et al., Front Endocrinol (Lausanne) 2015; 6:170). In certain embodiments, GILZ is human GILZ with the amino acid sequence shown in SEQ ID NO: 110. In certain embodiments, GILZ is human GILZ encoded by the nucleotide sequence shown in SEQ ID NO: 111.

[0053] As shown, hypoxia also affects macrophage polarization through the hypoxia-inducible factors HIF-1α and HIF-2α. HIF-1α regulates NOS2 expression and supports the emergence of the M1 phenotype, while HIF-2α regulates Arg1 expression and supports the emergence of the M2 phenotype (Takeda N et al., (2010) Genes Dev 24: pp. 491-501).

[0054] [Table 2]

[0055] (3) Nucleotides. In this disclosure, nucleotides encoding genes that regulate the activity state are delivered to immune cells. “Genes” means nucleotide sequences that encode activation regulators. This definition includes various sequence polymorphisms, mutations, and / or sequence variants, such modifications which do not affect the function of the activation regulators. The term “genes” may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. The term may further include all introns and other DNA sequences spliced ​​from mRNA transcripts, along with variants arising from alternative splicing sites. Nucleotide sequences encoding activation regulators can be RNA that directs the expression of the activation regulators. These nucleotide sequences include RNA sequences that are translated into proteins in certain embodiments. In certain embodiments, those skilled in the art will recognize that DNA sequences containing thymine (T) bases can be equivalent to mRNA sequences having the same sequence, except that the T bases are replaced by uracil (U) bases. Nucleotide sequences include both full-length nucleotide sequences and incomplete-length sequences derived from full-length proteins. The sequences may also include degenerate codons of one or more native sequences that can be introduced to provide codon selection in specific immune cells. Gene sequences encoding activation regulators described herein are available in publicly accessible databases and publications. "Encoding" refers to the characteristic of a sequence of nucleotides, such as plasmids, genes, cDNA, or mRNA, that serves as a template for the synthesis of an activation regulator.

[0056] In certain embodiments, the nucleotides include synthetic mRNA. In certain embodiments, the synthetic mRNA is manipulated to increase intracellular stability using 5'-capping. By using multiple different 5'-cap structures, a 5'-cap of the synthetic mRNA molecule can be created. For example, the anti-reverse cap analog (ARCA) cap contains a 5'-5'-triphosphate guanine-guanine bond, where one guanine contains an N7 methyl group and a 3'-O-methyl group. The synthetic mRNA molecule may be capped post-transcriptionally using an enzyme that causes the creation of a 5'-cap structure. For example, recombinant vaccinia virus capping enzyme and recombinant 2'-O-methyltransferase enzyme can produce a standard 5'-5'-triphosphate bond between the most 5' terminal nucleotide of the mRNA and a guanine nucleotide, where the guanine contains an N7 methylation and the last 5'-nucleotide contains a 2'-O-methyl group that creates a Cap1 structure. This results in a cap with even higher translational capacity and cellular stability, as well as reduced activation of inflammatory cytokines.

[0057] In certain embodiments, other modifications to the synthetic mRNA to reduce immunogenicity, promote mRNA stability, and / or facilitate mRNA translation may include the 5'- and 3'-terminal untranslated regions (UTRs), the Kozak translation initiation sequence in the 5'UTR, a modified ribonucleoside, and / or a poly-A tail. In certain embodiments, the modified ribonucleoside may include pseudouridine (ψ), 5-methylcytidine (5mC), N6-methyladenosine (m6A), 2-thiouridine (2sU), 5-methoxyuridine (5moU), and N-1-methylpsoiduridine (m1ψ). In certain embodiments, the UTR may include the alpha- and / or betaglobin UTR. In certain embodiments, the synthetic mRNA is produced by PCR amplification from the corresponding DNA plasmid by the 5'UTR. 100~250The process involves creating a DNA template containing the coding DNA sequence of a desired protein having an overhang. The DNA template can then be used to produce mRNA by an in vitro transcription reaction. During in vitro transcription, a 5' cap structure (e.g., ARCA), a modified ribonucleoside, and / or a 3' poly(A) tail can be incorporated. Several in vitro transcription systems are commercially available, including, for example, the MEGAscript T7 transcription kit (ThermoFisher Scientific, Waltham, MA), the Riboprobe® System T7 (Promega, Madison, WI), the AmpliScribe® T7 high-yield transcription kit (Epicentre, Madison, WI), and the HiScribe® T7 in vitro transcription kit (New England Biolabs, Ipswich, MA). In certain embodiments, synthetic mRNA can be synthesized by companies synthesizing nucleic acids (e.g., TriLink Biotechnologies, San Diego, CA).

[0058] Synthetic mRNA or other nucleotides may be circularized. Such nucleotides may be circulated or concatemerized to create a translatable molecule that facilitates the interaction between poly(A)-binding proteins and 5'-terminal binding proteins. The mechanism of circulation or concatemerization may occur through at least three different pathways: 1) chemical, 2) enzymatic, or 3) ribozyme-catalyzed. The newly formed 5'- / 3'- bond may be intramolecular or intermolecular.

[0059] In the first pathway, the 5'-terminus and 3'-terminus of the nucleotide may contain chemically reactive groups that, when brought close together, form a new covalent bond between the 5'-terminus and 3'-terminus of the molecule. The 5'-terminus may contain an NHS ester reactive group, and the 3'-terminus may contain a 3'-amino-terminus nucleotide, so that in an organic solvent, the 3'-amino-terminus nucleotide at the 3'-terminus of the nucleotide molecule undergoes nucleophilic attack on the 5'-NHS ester moiety to form a new 5'- / 3'-amide bond.

[0060] In a second pathway, a T4 RNA ligase may be used to enzymatically link a 5'-phosphorylated nucleotide molecule to the 3'-hydroxyl group of the nucleic acid to form a new phosphodiester bond. In an example reaction, 1 μg of nucleic acid molecules can be incubated with 1 to 10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) at 37°C for 1 hour according to the manufacturer's protocol. The ligation reaction may occur in the presence of a split oligonucleotide that can base-pair both the juxtaposed 5'- and 3'- regions to support the enzymatic ligation reaction.

[0061] In the third pathway, the 5'- or 3'-end of the cDNA template encodes a ligase ribozyme sequence such that, during in vitro transcription, the resulting nucleotide molecule contains an active ribozyme sequence capable of ligating the 5'-end of the nucleic acid molecule to the 3'-end of the nucleic acid molecule. The ligase ribozyme may be derived from a group I intron, hepatitis delta virus, hairpin ribozyme, or selected by SELEX (Systematic Evolution of Ligands by Exponential Enrichment). The ribozyme-ligase reaction may take 1 to 24 hours at a temperature of 0 to 37°C.

[0062] In certain embodiments, the nucleotides include plasmids, cDNA, or mRNA that can contain, for example, sequences (e.g., genes) for expressing activation regulators. Suitable plasmids include standard plasmid vectors and minicircle plasmids that can be used to deliver genes to lymphocytes. The nucleotides (e.g., minicircle plasmids) may further contain any additional sequence information to facilitate transient expression in modified cells. For example, the nucleotides may include promoters such as general promoters, tissue-specific promoters, cell-specific promoters, and / or cytoplasm-specific promoters. As shown, promoters and plasmids (e.g., minicircle plasmids) are generally well known in the art and can be prepared using conventional techniques.

[0063] In certain embodiments, the nucleotide encoding IRF is used in combination with one or more additional nucleotides encoding other IRFs. In certain embodiments, the nucleotide encoding IRF is used in combination with one or more additional nucleotides encoding other IRFs and the nucleotide encoding IKKβ. In certain embodiments, the nucleotide encoding IRF is used in combination with the nucleotide encoding IKKβ in ratios of 0.5:1, 1:1, 2:1, 3:1, 4:1, and 5:1. In certain embodiments, the nucleotide encoding IRF is used in combination with the nucleotide encoding IKKβ in a ratio of 3:1.

[0064] Certain embodiments can deliver nucleotides into a gene editing system. The gene editing system modifies or affects specific sequences in the cell's endogenous genome. Genome editing systems are useful for targeted genome editing, such as gene disruption, homologous recombination, and gene therapy, which involves inserting therapeutic genes into appropriate chromosomal target sites within the human genome.

[0065] Certain embodiments utilize activator-like effector nucleases (TALENs) as gene editing systems. A TALEN is a fusion protein containing an activator-like effector (TALE) DNA-binding domain and a DNA-cleavage domain. TALENs are used to edit genes and genomes by inducing double-strand breaks (DSBs) in DNA, which trigger repair mechanisms in the cell. Generally, two TALENs must dimerize and bind adjacent to each other on the target DNA site for the DNA-cleavage domain to induce DSBs. DSBs are repaired in the cell by non-homologous end joining (NHEJ) or by homologous recombination (HR) with an exogenous double-stranded donor DNA fragment.

[0066] As shown, TALENs have been engineered, for example, to bind to target sequences in the endogenous genome and cleave DNA at the target sequence site. TALE, a TALEN, is a DNA-binding protein secreted by the bacterium Xanthomonas. The DNA-binding domain of TALE contains 33 or 34 highly conserved amino acid repeats, with diverse residues at positions 12 and 13 of each repeat. These two positions are called Repeat Variable Diresidues (RVDs) and show a strong correlation with the recognition of specific nucleotides. Therefore, targeting specificity can be improved by altering the amino acids in the RVDs and incorporating novel RVD amino acids.

[0067] Examples of DNA cleavage domains usable in TALEN fusions are wild-type and mutant Fokl endonucleases. The Fokl domain functions as a dimer requiring two constructs, each having a distinct DNA-binding domain for a site on the target sequence. The Fokl cleavage domain cleaves within a sequence of five or six base pairs of spacers that separates two inverted half-sites.

[0068] Certain embodiments utilize MegaTAL as a gene editing system. MegaTAL has a single-strand rare-cleavage nuclease structure in which TALE is fused with the DNA cleavage domain of a meganuclease. Meganucleases, also known as homing endonucleases, are single peptide chains that possess both DNA recognition and nuclease function in the same domain. In contrast to TALENs, megaTAL requires only the delivery of a single peptide chain for functional activity.

[0069] Certain embodiments utilize zinc finger nucleases (ZFNs) as a gene editing system. ZFNs are a class of site-specific nucleases engineered to bind to and cleave DNA at specific locations. ZFNs are used to introduce double-strand breaks (DSBs) at specific sites in DNA sequences, enabling ZFNs to target unique sequences within the genome in various different cells. Furthermore, homologous recombination or non-homologous end joining occurs following the double-strand break to repair the DSB, thus enabling genome editing.

[0070] ZFNs are synthesized by fusing a zinc finger DNA-binding domain to a DNA-cleaving domain. The DNA-binding domain contains 3 to 6 zinc finger proteins, which are transcription factors. The DNA-cleaving domain contains, for example, a catalytic domain of a Fokl endonuclease.

[0071] Guide RNA can be used in conjunction with gene editing systems, such as the CRISPR-Cas system. The CRISPR-Cas system includes a set of CRISPR repeats and CRISPR-associated genes (Cas).

[0072] In general, any system capable of bringing about the functional expression of delivered nucleotides can be used within this disclosure. However, in certain embodiments, delivery utilizing viral vectors is excluded.

[0073] (4) Particles. Particles used in the systems and methods disclosed herein can function to condense and protect nucleotides from enzymatic degradation. Materials particularly useful for use in particles for this purpose include polymers containing positively charged lipids and / or poly(β-aminoesters) (PbAEs).

[0074] Examples of positively charged lipids include esters of phosphatidylic acid and amino alcohols, such as esters of dipalmitoylphosphatidylic acid or distealoyliphosphatidylic acid with hydroxyethylenediamine. Further examples of positively charged lipids include 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-chol); N,N'-dimethyl-N,N'-dioctadecylammonium bromide (DDAB); N,N'-dimethyl-N,N'-dioctadecylammonium chloride (DDAC); 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium chloride (DORI); 1,2-dioleoyloxy-3-[trimethylammonio]-propane (DOTAP); N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); dipalmitoylphosphatidylcholine (DPPC); 1,2-dioctadecyloxy-3-[trimethylammonio]-propane (DSTAP); and cationic lipids, for example, those described by Martin et al., Current Pharmaceutical Design 2005, 11, pp. 375-394.

[0075] Examples of positively charged polymers usable in particles of the present disclosure include polyamines; polyorganic amines (e.g., polyethyleneimine (PEI), polyethyleneimine cellulose); poly(amide amines) (PAMAM); polyamino acids (e.g., polylysine (PLL), polyarginine); polysaccharides (e.g., cellulose, dextran, DEAE dextran, starch); spermine, spermidine, poly(vinylbenzyltrialkylammonium), poly(4-vinyl-N-alkylpyridinium), poly(acryloyl-trialkylammonium), and Tat proteins.

[0076] Mixtures of lipids and polymers can be used at any concentration and in any ratio. Mixing different polymer types in different ratios using various grades can result in characteristics borrowed from each contributing polymer. Various end-group chemistry can also be employed.

[0077] In the specific embodiments disclosed herein, porous particles constructed from any material capable of forming a porous network are also available. Exemplary materials include metals, transition metals, and metalloids. Exemplary metals, transition metals, and metalloids include lithium, magnesium, zinc, aluminum, and silica. In specific embodiments, the porous particles include silica. Mesoporous silica has an exceptionally high surface area (1,000 m²). 2 This allows for nucleotide loading at levels exceeding those of conventional DNA carriers such as liposomes (exceeding / g).

[0078] The particles can be formed in a variety of different shapes, including ellipsoidal, cubic, pyramidal, rectangular, cylindrical, donut-shaped, and similar shapes. Nucleotides can be incorporated into the pores of the particles in various ways. For example, nucleotides can be encapsulated in porous particles. In other embodiments, nucleotides can associate with the surface of porous particles or the vicinity just below the surface (e.g., covalently and / or non-covalently). In certain embodiments, nucleotides can be incorporated into porous particles, for example, by being incorporated into the material of the porous particles. For example, nucleotides can be incorporated into the polymer matrix of polymer particles.

[0079] In certain embodiments, the particles disclosed herein include a coating. The coating may function to shield encapsulated nucleotides and / or reduce or prevent off-target binding. Off-target binding is reduced or prevented by lowering the surface charge of the particles to neutral or negative. As further disclosed elsewhere herein, the coating may include a neutral or negatively charged polymer- and / or liposome-based coating. In certain embodiments, the coating is a high-density surface coating of a sufficiently hydrophilic and / or neutrally charged hydrophilic polymer to prevent encapsulated nucleotides from being exposed to the environment before being released into immune cells. In certain embodiments, the coating covers at least 80% or at least 90% of the surface of the particles. In certain embodiments, the coating includes polyglutamic acid (PGA). In certain embodiments, the PGA acts as a linker to bind targeting ligands to the particles. In certain embodiments, the PGA acts as a linker to bind dimannose to the particles. In certain embodiments, the coating includes hyaluronic acid.

[0080] Examples of neutrally charged polymers that can be used as coatings in embodiments of this disclosure include polyethylene glycol (PEG); poly(propylene glycol); and polyalkylene oxide copolymers (PLURONIC®, BASF Corp., Mount Olive, NJ).

[0081] Neutral-charged polymers also include zwitterionic polymers. A zwitterionic polymer is a polymer that possesses both positive and negative charges while maintaining overall charge neutrality. Zwitterionic polymers can behave like regions of a cell membrane that resist cell and protein adhesion.

[0082] Zwitterionic polymers contain zwitterionic structural units that include a pendant group (i.e., a group hanging from the polymer backbone) together with the zwitterionic group. An example of a zwitterionic pendant group is a carboxybetaine group (e.g., -Ra-N+(Rb)(Rc)-Rd-CO2-, where Ra is a linker group that covalently links the polymer backbone to the cation nitrogen center of the carboxybetaine group, Rb and Rc are nitrogen substituents, and Rd is a linker group that covalently links the cation nitrogen center to the carboxyl group of the carboxybetaine group).

[0083] Examples of negatively charged polymers include alginates; carboxylic acid polysaccharides; carboxymethylcellulose; carboxymethylcellulose cysteine; carrageenans (e.g., GELCARIN® 209, GELCARIN® 379, FMC Corporation, Philadelphia, PA); chondroitin sulfate; glycosaminoglycans; mucopolysaccharides; negatively charged polysaccharides (e.g., dextran sulfate); poly(acrylic acid); poly(D-aspartic acid); poly(L-aspartic acid); poly(L-aspartic acid) sodium salt; poly(D-glutamic acid); poly(L-glutamic acid); poly(L-glutamic acid) sodium salt; poly(methacrylic acid); sodium alginate (e.g., PROTANAL® LF 120M, PROTANAL® LF 200M, PROTANAL® LF 200D, FMC Biopolymer) Corp., Drammen, Norway; contains sodium carboxymethylcellulose (CMC); sulfated polysaccharides (heparin, agaropectin); pectin, gelatin, and hyaluronic acid.

[0084] In certain embodiments, the polymers disclosed herein may include “star polymers,” which are branched polymers from which two or more polymer branches extend from a core. The core is an atomic group having two or more functional groups from which branched chains can be extended by polymerization. In certain embodiments, the nanoparticles of the disclosure include star polymers. In certain embodiments, the nanoparticles of the disclosure include star polymers and coatings. In certain embodiments, the nanoparticles of the disclosure include star polymers and coatings containing PGA. In certain embodiments, the nanoparticles of the disclosure include star polymers and coatings containing hyaluronic acid.

[0085] In certain embodiments, the branched chain is a zwitterionic or negatively charged polymer branched chain. The star polymer can be converted from a branched chain precursor to a zwitterionic or negatively charged polymer by hydrolysis, UV irradiation, or heat. The polymer may also be obtained by any polymerization method effective for the polymerization of unsaturated monomers, including atom transfer radical polymerization (ATRP), reversible addition-cleavage-chain transfer polymerization (RAFT), photopolymerization, ring-opening polymerization (ROP), concentration, Michael addition, branch generation / growth reactions, or other reactions.

[0086] Liposomes are microvesicles comprising at least one concentric lipid bilayer. The vesicle-forming lipids are selected to achieve a specified degree of fluidity or rigidity of the final complex. In certain embodiments, liposomes provide a lipid composition that is an outer layer surrounding porous particles. In certain embodiments, the nanoparticles of this disclosure include liposome nanoparticles.

[0087] Liposomes can be neutral (cholesterol) or bipolar and contain phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM), as well as other types of bipolar lipids, including dioleoylphosphatidylethanolamine (DOPE), which has a hydrocarbon chain length in the range of 14 to 22 and is saturated or has one or more double C=C bonds. Lipids that can create stable liposomes alone or in combination with other lipid components include phospholipids such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimide-methyl)cyclohexane-1-carboxylate (DOPE-mal). Lipids that do not contain additional phosphorus and can be incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine lauryl sulfate, alkylaryl sulfate, acetyl palmitate, glycerol ricinolate, hexadecyl stearate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, DDAB, dioctadecyldimethylammonium chloride (DODAC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), DOTAP, DOTMA, DC-Chol, phosphatidic acid (PA), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylglycerol, DOPG, and dicetyl phosphate.In certain embodiments, the lipids used to produce the liposomes disclosed herein include cholesterol, hydrogenated soy phosphatidylcholine (HSPC), and derivatized vesicle-forming lipid PEG-DSPE.

[0088] Methods for forming liposomes are described, for example, in U.S. Patents 4,229,360, 4,224,179, 4,241,046, 4,737,323, 4,078,052, 4,235,871, 4,501,728, and 4,837,028, as well as in Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980) and Hope et al., Chem. Phys. Lip. 40:89 (1986).

[0089] Particle size can vary over a wide range and can be measured in different ways. In a preferred embodiment, the particle size is NP < 130 nm. However, the NP of this disclosure may also have a minimum dimension equal to or less than 500 nm, less than 150 nm, less than 140 nm, less than 120 nm, less than 110 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, or less than 10 nm. In a particular embodiment, the particle size is NP 90 to 130 nm.

[0090] In certain embodiments, the NPs may have minimum dimensions ranging from 5 nm to 500 nm, 10 nm to 100 nm, 20 nm to 90 nm, 30 nm to 80 nm, 40 nm to 70 nm, and 40 nm to 60 nm. In certain embodiments, the dimension is the diameter of the NP or coated NP. In certain embodiments, the group of particles of the Disclosure may also have an average minimum dimension equal to or less than 500 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, or less than 10 nm. In certain embodiments, the group of NPs in the composition of the Disclosure may have an average diameter ranging from 5 nm to 500 nm, 10 nm to 100 nm, 20 nm to 90 nm, 30 nm to 80 nm, 40 nm to 70 nm, and 40 nm to 60 nm, 70 nm to 130 nm, or 75 nm to 125 nm. The particle dimensions can be determined, for example, using conventional techniques such as dynamic light scattering and / or electron microscopy. Although not preferred, in certain embodiments, microparticles may also be used.

[0091] In certain embodiments, the PbAE polymer is mixed with nucleotides (e.g., in vitro transcribed mRNA) in a ratio of 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or more to create a PbAE-nucleotide polyplex. In certain embodiments, the PbAE polymer is mixed with nucleotides (e.g., in vitro transcribed mRNA) in a ratio of 60:1 to create a PbAE-nucleotide polyplex. In certain embodiments, the PbAE-nucleotide polyplex can be combined with PGA / Di-mannose to form a final NP.

[0092] (5) Targeting ligands. Targeting ligands can be used on the surface of particles and can lead to more selective binding of target immune cells within a heterogeneous cell population.

[0093] In certain embodiments, the targeting ligand includes a binding domain derived from a cell marker ligand, receptor ligand, antibody, peptide, peptide aptamer, nucleic acid, nucleic acid aptamer, spiegelmer, or a combination thereof. In certain embodiments, within the context of a cell targeting ligand, the binding domain includes any substance that can bind to another substance to form a complex that mediates endocytosis.

[0094] In certain embodiments, the binding domain is derived from an antibody. The antibody-derived binding domain may include the whole antibody, or it may include antibody binding fragments, such as Fv, Fab, Fab', F(ab')2, Fc, and single-stranded Fv fragments (scFv), or any biologically effective fragment of immunoglobulin that specifically binds to a targeted motif expressed by immune cells. The antibody or antigen-binding fragment may include all or part of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, bispecific antibodies, mini-antibodies, and linear antibodies.

[0095] Antibodies of human origin or humanized antibodies have reduced or no immunogenicity in humans and have fewer non-immunogenic epitopes compared to non-human antibodies. Antibodies and their fragments are generally selected to have reduced or no antigenicity in human subjects.

[0096] Antibodies that specifically bind to motifs expressed by immune cells can be prepared using methods for obtaining monoclonal antibodies, phage display methods, methods for creating human or humanized antibodies, or methods using transgenic animals or plants engineered to produce antibodies known to those skilled in the art (see, for example, U.S. Patents 6,291,161 and 6,291,158). Phage display libraries of partially or completely synthetic antibodies are available and can be screened for antibodies or fragments capable of binding to immune cell motifs. For example, binding domains can be identified by screening Fab phage libraries for Fab fragments that specifically bind to a target of interest (see Hoet et al., Nat. Biotechnol. 23:344, 2005). Phage display libraries of human antibodies are also available. Furthermore, binding domains can be developed by using conventional strategies for hybridoma development that utilize the target of interest as an immunogen in convenient systems (e.g., mice, HuMAb mice®, TC mice®, KM mice®, llamas, chickens, rats, hamsters, rabbits, etc.). In certain embodiments, the antibody specifically binds to a motif expressed by selected immune cells and does not cross-react with nonspecific components or unrelated targets. Once identified, the amino acid or nucleotide sequence encoding the antibody can be isolated and / or determined.

[0097] An intact antibody may contain at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain may have a heavy chain variable region (VH or V in this specification). H It consists of a heavy chain constant region (abbreviated as CH1, CH2, and CH3). Each light chain has a light chain variable region (VL or VL in this specification). LThe antibody consists of a constant region (abbreviated as VH) and a light chain constant region. The constant region of the light chain contains one domain, CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), which are dispersed by more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and host tissues or factors including the first component (Clq) of the classical complement system. In certain embodiments, the antibody includes an antigen-binding moiety from the intact antibody that retains its binding ability. Examples of fragments that retain binding ability include (i) a monovalent fragment containing VL, VH, CL, and CH1 domains, the Fab fragment; (ii) a bivalent fragment containing two Fab fragments linked by disulfide crosslinking at the hinge region, the F(ab')2 fragment; (iii) an Fd fragment containing VH and CH1 domains; (iv) an Fv fragment containing the VL and VH domains of a single arm of the antibody; (v) a dAb fragment containing the VH domain (Ward et al., Nature, 341:544-546 (1989)); and (vi) an isolated complementarity-determining region (CDR).

[0098] The precise amino acid sequence boundaries of a given CDR or FR are described by Kabat et al., (1991) "Sequences of Proteins of Immunological Interest", 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (Kabat numbering scheme); Al-Lazikani et al., (1997) J Mol Biol 273: pp. 927-948 (Chothia numbering scheme); Maccallum et al., (1996) J Mol Biol 262: pp. 732-745 (Contact numbering scheme); Martin et al., (1989) Proc. Natl. Acad. Sci., 86: pp. 9268-9272 (AbM numbering scheme); Lefranc MP et al., (2003) Dev Comp Immunol 27(1): pp. 55-77 (IMGT numbering scheme); and Honegger et al. The numbering scheme can be readily determined using one of several well-known schemes, including the one described by Pluckthun (2001) J Mol Biol 309(3):657-670 ("Aho" numbering scheme). The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, and the Chothia scheme is based on structural information. Numbering in both the Kabat and Chothia schemes is based on the most common antibody region sequence length, with insertions adapted by insertion letters, e.g., "30a", and deletions appearing in some antibodies. The two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of the complex crystal structure and is similar in many ways to the Chothia numbering scheme. In certain embodiments, the antibody CDR sequences disclosed herein follow Kabat numbering.In certain embodiments, the CDR region is found within the Kabat-numbered antibody region, where in the light chain, CDRL1 is amino acids 24-34; CDRL2 is amino acids 50-56; CDRL3 is amino acids 89-97; and in the heavy chain, CDRH1 is amino acids 31-35; CDRH2 is amino acids 50-65; and CDRH3 is amino acids 95-102.

[0099] Peptide aptamers contain peptide loops (specific to the target protein) bound to a protein scaffold at both ends. These dual structural constraints greatly increase the binding affinity of peptide aptamers to levels comparable to antibodies. The variable loop length is typically 8–20 amino acids (e.g., 8–12 amino acids), and the scaffold can be any stable, soluble, small, and non-toxic protein (e.g., thioredoxin-A, stephin A triple mutant, green fluorescent protein, eglin C, and the cell transcription factor Spl). Peptide aptamer selection can be performed using different systems, such as yeast two-hybrid methods (e.g., Gal4 yeast two-hybrid method) or the LexA interaction trap system.

[0100] Nucleic acid aptamers are single-stranded nucleotide sequences (DNA or RNA) that function by folding into specific spherical structures that direct high affinity and specificity to binding to target proteins or other molecules, as described by Osborne et al., Curr. Opin. Chem. Biol. 1: pp. 5-9, 1997; and Cerchia et al., FEBS Letters 528: pp. 12-16, 2002. In certain embodiments, aptamers are small (15 kD; or 15-80 nucleotides or 20-50 nucleotides). Aptamers are generally extracted using a procedure called SELEX (see, for example, Turek et al., Science, 249: pp. 505-510, 1990; Green et al., Methods Enzymology, pp. 75-86, 1991; and Gold et al., Annu. Rev. Biochem., 64: pp. 763-797, 1995).14 ~10 15 They are isolated from a library containing random oligonucleotide sequences. Further methods for creating aptamers are described, for example, in U.S. Patents 6,344,318, 6,331,398, 6,110,900, 5,817,785, 5,756,291, 5,696,249, 5,670,637, 5,637,461, 5,595,877, 5,527,894, 5,496,938, 5,475,096, and 5,270,16. Spiegelmers are similar to nucleic acid aptamers, except that at least one β-ribose unit is replaced by β-D-deoxyribose or a modified sugar unit selected from, for example, β-D-ribose, α-D-ribose, or β-L-ribose.

[0101] In certain embodiments, the targeted cells are TAMs. Targeted cells may also include regulatory T cells (TREGs). TREGs are a subpopulation of T cells that modulate the immune system, maintain tolerance to autoantigens, and suppress autoimmune diseases. TREGs express CD25, CTLA-4, GITR, GARP, and LAP. Selected cell-targeting ligands disclosed herein can bind to CD25, CTLA-4, GITR, GARP, and / or LAP to achieve selective delivery of nucleotides to naive TREGs. Other cell types that can be targeted include myeloid-derived suppressor cells (MDSCs), regulatory dendritic cells (DCregs), neutrophils, helper T17 cells (Th17s), regulatory B cells (Bregs), and / or mesenchymal stromal cells (MSCs). Those skilled in the art can identify appropriate cell markers for targeting these cell types using the targeting ligands disclosed herein.

[0102] M2 binding domain. In certain embodiments, Egr2 is targeted on M2 macrophages. Commercial antibodies against Egr2 are available from Thermo Fisher, Waltham, MA; Abcam, Cambridge, MA; Millipore Sigma, Burlington, MA; Miltenyi Biotec, Bergisch Gladbach, Germany; LifeSpan Biosciences, Inc., Seattle, WA; and Novus Biologicals, Littleton, CO. The production of anti-Egr2 antibodies is discussed, for example, in Murakami K et al., (1993) Oncogene 8(6):1559-1566. Anti-Egr2 antibodies include: rabbit monoclonal anti-Egr2 antibody clone EPR4004; mouse monoclonal anti-Egr2 antibody clone 1G5; mouse monoclonal anti-Egr2 antibody clone OTI1B12; rabbit polyclonal anti-Egr2 antibody that recognizes AA residues 200-300 of human Egr2; rabbit polyclonal anti-Egr2 antibody that recognizes AA residues 340-420 of human Egr2; and rabbit polyclonal anti-Egr2 antibody that recognizes AA residues 370-420 of human Egr2. The binding domain can be derived from these antibodies and other antibodies disclosed herein.

[0103] In certain embodiments, the targeting ligand comprises a human or humanized binding domain (e.g., a nanobody) comprising a variable heavy chain including a CDRH1 sequence containing SGNIFSINAIG (SEQ ID NO: 45), a CDRH2 sequence containing TITLSGSTN (SEQ ID NO: 46), and a CDRH3 sequence containing NTYSDSDVYGY (SEQ ID NO: 47). These reflect CDR sequences that bind to CD206.

[0104] In certain embodiments, the targeting ligand comprises a human or humanized binding domain (e.g., a nanobody) comprising a variable heavy chain including a CDRH1 sequence containing PGFKLDYYAIA (SEQ ID NO: 48), a CDRH2 sequence containing SINSSGGST (SEQ ID NO: 49), and a CDRH3 sequence containing LRRYYGLNLDPGSYDY (SEQ ID NO: 50). These reflect CDR sequences that bind to CD206.

[0105] In certain embodiments, the targeting ligand comprises a human or humanized binding domain (e.g., a nanobody) comprising a variable heavy chain including a CDRH1 sequence containing GFPFNIYPMS (SEQ ID NO: 51), a CDRH2 sequence containing YISHGGTTT (SEQ ID NO: 52), and a CDRH3 sequence containing GYARLMTDSELV (SEQ ID NO: 53). These reflect CDR sequences that bind to CD206.

[0106] Several additional antibodies specific to CD206 are known to those skilled in the art and can be readily characterized in terms of sequence, epitope binding, and affinity. See, for example, International Patent Brochure 2014 / 140376, International Patent Brochure 2013 / 174537, and U.S. Patent No. 7,560,534. Commercial antibodies for CD206 are available from Thermo Fisher, Waltham, MA; Proteintech, Rosemont, IL; BioLegend, San Diego, CA; R&D Systems, Minneapolis, MN; LifeSpan Biosciences, Inc., Seattle, WA; Novus Biologicals, Littleton, CO; and Bio-Rad, Hercules, CA. In certain embodiments, the anti-CD206 antibody comprises the rat monoclonal anti-mouse CD206 monoclonal antibody clone C068C2 (catalog #141732, Biolegend, San Diego, CA).

[0107] In certain embodiments, the targeting ligand comprises a human or humanized binding domain (e.g., scFv) comprising a variable light chain including a CDRL1 sequence containing ASQSVSHDV (SEQ ID NO: 54), a CDRL2 sequence containing YTS, and a CDRL3 sequence containing QDYSSPRT (SEQ ID NO: 56). In certain embodiments, the targeting ligand comprises a human or humanized binding domain (e.g., scFv) comprising a variable heavy chain including a CDRH1 sequence containing GYSITSDY (SEQ ID NO: 57), a CDRH2 sequence containing YSG, and a CDRH3 sequence containing CVSGTYYFDYWG (SEQ ID NO: 59). These reflect the CDR sequences of the Mac2-48 antibody that bind to CD163.

[0108] In certain embodiments, the targeting ligand comprises a human or humanized binding domain (e.g., scFv) comprising a variable light chain including a CDRL1 sequence containing ASQSVSSDV (SEQ ID NO: 60), a CDRL2 sequence containing YAS, and a CDRL3 sequence containing QDYTSPRT (SEQ ID NO: 62). In certain embodiments, the targeting ligand comprises a human or humanized binding domain (e.g., scFv) comprising a variable heavy chain including a CDRH1 sequence containing GYSITSDY (SEQ ID NO: 63), a CDRH2 sequence containing YSG, and a CDRH3 sequence containing CVSGTYYFDYWG (SEQ ID NO: 65). These reflect the CDR sequences of the Mac2-158 antibody that bind to CD163.

[0109] Several additional antibodies or binding domains specific to CD163 are known to those skilled in the art and can be readily characterized in terms of sequence, epitope binding, and affinity. See, for example, International Patent Brochure 2011 / 039510, International Patent Brochure 2002 / 032941, International Patent Brochure 2002 / 076501, and U.S. Patent Application Publication 2005 / 0214871. Commercial antibodies for CD163 are available from Thermo Fisher, Waltham, MA; Enzo Life Sciences, Inc., Farmingdale, NY; BioLegend, San Diego, CA; R&D Systems, Minneapolis, MN; LifeSpan Biosciences, Inc., Seattle, WA; and RDI Research Diagnostics, Flanders, NJ. In certain embodiments, the anti-CD163 antibody may include mouse monoclonal anti-CD163 antibody clone 3D4; mouse monoclonal anti-CD163 antibody clone Ber-Mac3; mouse monoclonal anti-CD163 antibody clone EDHu-1; and mouse monoclonal anti-CD163 antibody clone GHI / 61.

[0110] In certain embodiments, the targeting ligand comprises a human or humanized binding domain (e.g., scFv) comprising a variable light chain including a CDRL1 sequence containing RSSKSLLYKDGKTYLN (SEQ ID NO: 66), a CDRL2 sequence containing LMSTRAS (SEQ ID NO: 67), and a CDRL3 sequence containing QQLVEYPFT (SEQ ID NO: 68). In certain embodiments, the targeting ligand comprises a human or humanized binding domain (e.g., scFv) comprising a variable heavy chain including a CDRH1 sequence containing GYWMS (SEQ ID NO: 69), a CDRH2 sequence containing EIRLKSDNYATHYAESVKG (SEQ ID NO: 70), and a CDRH3 sequence containing FID. These reflect CD23-binding CDR sequences.

[0111] Several additional antibodies or binding domains specific to CD23 are known to those skilled in the art and can be readily characterized in terms of sequence, epitope binding, and affinity. See, for example, U.S. Patent No. 7,008,623, U.S. Patent No. 6,011,138 (antibodies including 5E8, 6G5, 2C8, B3B1, and 3G12), and U.S. Patent Application Publication No. 2009 / 0252725, Rector et al., (1985) J.Immunol. 55: pp. 481-488; Flores-Rumeo et al., (1993) Science 241: pp. 1038-1046; Sherr et al., (1989) J.Immunol. 142: pp. 481-489; and Pene et al., (1988) PNAS 85: pp. 6820-6824. Commercially available antibodies against CD23 are available from Thermo Fisher, Waltham, MA; Abcam, Cambridge, MA; Bioss Antibodies, Inc., Woburn, MA; Bio-Rad, Hercules, CA; LifeSpan Biosciences, Inc., Seattle, WA; and Booster Biological Technology, Pleasanton, CA. In certain embodiments, anti-CD23 antibodies may include mouse monoclonal anti-CD23 antibody clone Tu1; rabbit monoclonal anti-CD23 antibody clone SP23; rabbit monoclonal anti-CD23 antibody clone EPR3617; mouse monoclonal anti-CD23 antibody clone 5B5; mouse monoclonal anti-CD23 antibody clone 1B12; mouse monoclonal anti-CD23 antibody clone M-L23.4; and mouse monoclonal anti-CD23 antibody clone 3A2.

[0112] M1 binding domain. In certain embodiments, the targeting ligand comprises a human or humanized binding domain (e.g., scFv) comprising a variable light chain including a CDRL1 sequence containing SSNIGDNY (SEQ ID NO: 72), a CDRL2 sequence containing RDS, and a CDRL3 sequence containing QSYDSSLSGS (SEQ ID NO: 74). In certain embodiments, the targeting ligand comprises a human or humanized binding domain (e.g., scFv) comprising a variable heavy chain including a CDRH1 sequence containing GFTDDDYG (SEQ ID NO: 75), a CDRH2 sequence containing ISWNGGKT (SEQ ID NO: 76), and a CDRH3 sequence containing ARGSLFHDSSGFYFGH (SEQ ID NO: 77). These reflect the CDR sequences of the Ab79 antibody that bind to CD38.

[0113] In certain embodiments, the targeting ligand comprises a human or humanized binding domain (e.g., scFv) comprising a variable light chain including a CDRL1 sequence containing NSNIGSNT (SEQ ID NO: 78), a CDRL2 sequence containing SDS, and a CDRL3 sequence containing QSYDSSLSGSR (SEQ ID NO: 80). In certain embodiments, the targeting ligand comprises a human or humanized binding domain (e.g., scFv) comprising a variable heavy chain including a CDRH1 sequence containing GFTFNNYG (SEQ ID NO: 81), a CDRH2 sequence containing ISYDGSDK (SEQ ID NO: 82), and a CDRH3 sequence containing ARVYYYGFSGPSMDV (SEQ ID NO: 83). These reflect the CDR sequences of the Ab19 antibody that bind to CD38.

[0114] In certain embodiments, the targeting ligand comprises a human or humanized binding domain (e.g., scFv) comprising a variable light chain including a CDRL1 sequence containing RASQSVSSYLA (SEQ ID NO: 84), a CDRL2 sequence containing DASNRAT (SEQ ID NO: 85), and a CDRL3 sequence containing QQRSNWPPTF (SEQ ID NO: 86). In certain embodiments, the targeting ligand comprises a human or humanized binding domain (e.g., scFv) comprising a variable heavy chain including a CDRH1 sequence containing SFAMS (SEQ ID NO: 87), a CDRH2 sequence containing AISGSGGGTYYADSVKG (SEQ ID NO: 88), and a CDRH3 sequence containing DKILWFGEPVFDY (SEQ ID NO: 89). These reflect the CDR sequences of the daratumumab antibody that binds to CD38 as described in U.S. Patent No. 7,829,693.

[0115] Several antibodies specific to CD38 are known to those skilled in the art and can be readily characterized in terms of sequence, epitope binding, and affinity. See, for example, International Publication Nos. 2005 / 103083, 2006 / 125640, 2007 / 042309, 2008 / 047242, 2012 / 092612, 2006 / 099875, 2011 / 154453, 2015 / 130728, U.S. Patent No. 7,829,693, and U.S. Patent Application Publication No. 2016 / 0200828. Commercially available antibodies against CD38 are available from Thermo Fisher, Waltham, MA; Abcam, Cambridge, MA; and Millipore Sigma, Burlington, MA. In certain embodiments, anti-CD23 antibodies may include rabbit monoclonal anti-CD38 antibody clone GAD-3; mouse monoclonal anti-CD38 antibody clone HIT2; mouse monoclonal anti-CD38 antibody clone AT1; mouse monoclonal anti-CD38 antibody clone AT13 / 5; rat monoclonal anti-CD38 antibody clone NIMR-5; and rat monoclonal IgG2a,κ anti-CD38 antibody clone 90 / CD38 (catalog #BD Biosciences, San Jose, CA).

[0116] In certain embodiments, G protein-coupled receptor 18 (Gpr18) is targeted on M1 macrophages. Commercial antibodies against Gpr18 are available from Assay Biotechnology Company Inc., Sunnyvale, CA; Thermo Fisher, Waltham, MA; Abcam, Cambridge, MA; GeneTex, Inc., Irvine, CA; and Novus Biologicals, Littleton, CO. In certain embodiments, the anti-Gpr18 antibody includes a rabbit polyclonal anti-Gpr18 antibody that recognizes a portion of amino acids 1-50 of human Gpr18; a rabbit polyclonal anti-Gpr18 antibody that recognizes a region containing amino acids 160-240 of human Gpr18; a rabbit polyclonal anti-Gpr18 antibody that recognizes a region containing amino acids 100-180 of human Gpr18; a rabbit monoclonal anti-Gpr18 antibody clone EPR12359; and a rabbit polyclonal anti-Gpr18 antibody that recognizes a region containing amino acids 140-190 of human Gpr18.

[0117] In certain embodiments, formyl peptide receptor 2 (Fpr2) is targeted on M1 macrophages. Commercial antibodies against Fpr2 are available from Atlas Antibodies, Bromma, Sweden; Biorbyt, LLC, San Francisco, CA; Cloud-Clone Corp., Katy, TX; US Biological Life Sciences, Salem, MA; and Novus Biologicals, Littleton, CO. In certain embodiments, anti-fpr2 antibodies include mouse monoclonal anti-fpr2 antibody clone GM1D6; mouse monoclonal anti-fpr2 antibody clone 304405; recombinant anti-fpr2 antibody clone REA663; and rabbit polyclonal anti-fpr2 antibody that recognizes the region of fpr2 containing amino acids 300-350.

[0118] In certain embodiments, the targeting ligand comprises a human or humanized binding domain (e.g., scFv) comprising a variable light chain including a CDRL1 sequence containing RASQSVSSYLA (SEQ ID NO: 90), a CDRL2 sequence containing DASSRAT (SEQ ID NO: 91), and a CDRL3 sequence containing QLRSNWPPYT (SEQ ID NO: 92). In certain embodiments, the targeting ligand comprises a human or humanized binding domain (e.g., scFv) comprising a variable heavy chain including a CDRH1 sequence containing GYGMH (SEQ ID NO: 93), a CDRH2 sequence containing VIWYDGSNKYYADSVKG (SEQ ID NO: 94), and a CDRH3 sequence containing DTGDRFFDY (SEQ ID NO: 95). These reflect CD64-binding CDR sequences.

[0119] Several antibodies specific to CD64 are known to those skilled in the art and can be readily characterized in terms of sequence, epitope binding, and affinity. See, for example, U.S. Patent No. 7,378,504, International Publication No. 2006 / 131953, and International Publication No. 2008 / 074867. Commercial antibodies for CD64 are available from Ancell, Bayport, MN; Thermo Fisher, Waltham, MA; Abcam, Cambridge, MA; LifeSpan Biosciences, Inc., Seattle, WA; and Novus Biologicals, Littleton, CO. In certain embodiments, the anti-CD64 antibody includes mouse monoclonal anti-CD64 antibody clone 32-2; mouse monoclonal anti-CD64 antibody clone UMAB74; rat monoclonal anti-CD64 antibody clone 290322; mouse monoclonal anti-CD64 antibody clone 10.1; and mouse monoclonal anti-CD64 antibody clone 1D3.

[0120] In certain embodiments, CD86 is targeted on M1 macrophages. Several antibodies specific to CD86 are known to those skilled in the art and can be readily characterized for their sequence, epitope binding, and affinity. See, for example, International Publication No. 2004 / 076488, U.S. Patent No. 8,378,082 (mAb 2D4), and U.S. Patent No. 6,346,248 (IG10H6D10). Commercially available antibodies for CD86 are available from Thermo Fisher, Waltham, MA; Miltenyi Biotec, Bergisch Gladbach, Germany; LifeSpan Biosciences, Inc., Seattle, WA; Bio-Rad, Hercules, CA; and Novus Biologicals, Littleton, CO. In certain embodiments, the anti-CD86 antibody includes mouse monoclonal anti-CD86 antibody clone BU63; polyclonal goat anti-CD86 antibody that recognizes the Ala23-His244 region of human CD86; mouse monoclonal anti-CD86 antibody clone IT2.2; rabbit monoclonal anti-CD86 antibody clone BFF-3; and mouse monoclonal anti-CD86 antibody clone C86 / 1146.

[0121] Other agents that can promote the internal migration of lymphocytes and / or lymphocytes by transfection, such as poly(ethyleneimine) / DNA(PEI / DNA) complexes, may also be used.

[0122] (6) Compositions. The particles disclosed herein may be provided as part of a composition formulated for administration to a subject. The composition comprises the particles disclosed herein and a pharmaceutically acceptable carrier.

[0123] Exemplary commonly used pharmaceutically acceptable carriers include all kinds of fillers or packing agents, solvents or co-solvents, dispersions, coatings, surfactants, antioxidants (e.g., ascorbic acid, methionine, vitamin E), preservatives, isotonic agents, absorption retarders, salts, stabilizers, buffers, chelating agents (e.g., EDTA), gels, binders, disintegrants, and / or lubricants.

[0124] Exemplary buffers include citrate buffers, succinate buffers, tartarate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactic acid buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0125] Exemplary preservatives include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halide, hexamethonium chloride, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0126] Examples of isotonic agents include glycerin, erythritol, arabitol, xylitol, sorbitol, or polyhydric sugar alcohols, including trihydric or higher sugar alcohols such as mannitol.

[0127] Examples of stabilizers include organic sugars, polyhydric sugar alcohols, polyethylene glycol, sulfur-containing reducing agents, amino acids, low molecular weight polypeptides, proteins, immunoglobulins, hydrophilic polymers, or polysaccharides.

[0128] In certain embodiments, the compositions are formulated for intraperitoneal, intravenous, or intracranial injection. The compositions disclosed herein may be further formulated for intra-arterial, intra-lymph node, intra-lymphatic, intratumor, intramuscular, oral, and / or subcutaneous administration, and more specifically, by intra-arterial, intra-lymph node, intra-lymphatic, intratumor, intramuscular, and / or subcutaneous injection. The compositions disclosed herein may be formulated for administration by infusion, perfusion, or oral ingestion.

[0129] With regard to injection, the composition can be formulated as an aqueous solution, such as a buffer containing Hanks' solution, Ringer's solution, or physiological saline. The aqueous solution may contain compounding agents such as suspensions, stabilizers, and / or dispersants. Alternatively, the formulation can be freeze-dried and / or in powder form to be composed of a suitable solvent, such as sterile pyrogen-removed water, before use.

[0130] The composition can also be formulated as a depot formulation. The depot formulation can be formulated using a suitable polymer or hydrophobic material (for example, as an emulsion in an acceptable oil) or an ion exchange resin, or as a slightly insoluble derivative, for example, as a slightly insoluble salt.

[0131] Furthermore, the composition can be formulated as a sustained-release formulation using a semipermeable solid polymer matrix containing particles. Various sustained-release materials have been established and are well known to those skilled in the art. Depending on their chemical properties, sustained-release formulations may release particles for several weeks to over 100 days following administration.

[0132] For oral administration, the composition may be formulated as tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, and similar substances.

[0133] When formulated to treat cancer, the disclosed compositions may also include nucleotides carrying one or more anti-oncogenes selected from p53, RB, BRCA1, E1A, bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-I VEGF, angiostatin, oncostatin, endostatin, GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-γ, TNFα and / or HSV-tk.

[0134] Any compositional formulation disclosed herein may favorably include any other pharmaceutically acceptable carrier, including carriers that do not produce significantly adverse, allergic, or other undesirable reactions exceeding the effect of administration, whether for research, prophylactic, and / or therapeutic purposes. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Furthermore, formulations may be prepared to meet the sterility, pyrogenicity, general safety, and purity requirements of the United States FDA Office of Biological Standards and / or other relevant foreign regulatory authorities.

[0135] In a particular embodiment, the particles are provided as part of a composition containing, for example, at least 0.1% w / v or w / w particles; at least 1% w / v or w / w particles; at least 10% w / v or w / w particles; at least 20% w / v or w / w particles; at least 30% w / v or w / w particles; at least 40% w / v or w / w particles; at least 50% w / v or w / w particles; at least 60% w / v or w / w particles; at least 70% w / v or w / w particles; at least 80% w / v or w / w particles; at least 90% w / v or w / w particles; at least 95% w / v or w / w particles; or at least 99% w / v or w / w particles.

[0136] Method of Use. The method disclosed herein involves changing the active state of macrophages from an inactive state to an active state by introducing nanoparticles containing one or more nucleotides encoding IRF and IKKβ into the macrophages. In certain embodiments, the modification reduces the percentage of inactive macrophages (e.g., M2 macrophages) in a population of macrophages treated with nanoparticles containing one or more nucleotides encoding IRF and IKKβ by 5, 10, 15, 20, or more than the percentage of inactive macrophages that have not been treated with nanoparticles containing one or more nucleotides encoding IRF and IKKβ. In certain embodiments, the modification results in a population of macrophages treated with nanoparticles containing one or more IRF and IKKβ encoding nucleotides that is reduced by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more, compared to the number of inactive macrophages that have not been treated with nanoparticles containing one or more IRF and IKKβ encoding nucleotides. In certain embodiments, the modification results in a population of macrophages treated with nanoparticles containing one or more IRF and IKKβ encoding nucleotides that is reduced by 5, 10, 15, 20, or more, compared to the percentage of active macrophages that have not been treated with nanoparticles containing one or more IRF and IKKβ encoding nucleotides. In certain embodiments, the modification results in a population of macrophages treated with nanoparticles containing one or more IRFs and IKKβ encoding nucleotides that are 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more than the number of activated macrophages that have not been treated with nanoparticles containing one or more IRFs and IKKβ encoding nucleotides.

[0137] In certain embodiments, introducing nanoparticles containing one or more nucleotides encoding IRF and IKKβ into macrophages changes the active state of macrophages from an inactive state to an activated state, thereby restoring lymphocyte migration and infiltration into solid tumors; increasing the release of pro-inflammatory (antitumor) cytokines, including IL-1β, IL-12, IFNγ, and / or TNFα, by 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more; decreasing the release of cytokines associated with the M2 macrophage phenotype, including IL-6, by 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more.

[0138] In certain embodiments, changing the active state of a macrophage from an inactive state to an active state involves introducing nanoparticles containing nucleotides encoding IRF5 and IRF8 into the macrophage. In certain embodiments, changing the active state of a macrophage from an inactive state to an active state involves introducing nanoparticles containing nucleotides encoding a constitutively active or more active mutant IRF than its wild-type counterpart IRF into the macrophage.

[0139] The methods disclosed herein include treating subjects (humans, veterinary animals, livestock, and research animals) with the compositions disclosed herein. Treating subjects includes delivering a therapeutically effective dose. A therapeutically effective dose can provide an effective dose, a prophylactic treatment, and / or a therapeutic treatment.

[0140] An "effective dose" is the amount of a compound required to produce a desired physiological change in a subject. Effective doses are often administered for research purposes. The effective doses disclosed herein immunomodulate cells in a subject. In certain embodiments, the immunomodulated cells are immunosuppressed cells. In certain embodiments, the immunomodulated cells are macrophages. In certain embodiments, immunomodulation of macrophages includes switching immunosuppressed macrophages to activated macrophages. In certain embodiments, immunomodulation of macrophages includes switching M2 macrophages to M1 macrophages. In certain embodiments, the immunomodulated cells include immunosuppressed cells including MDSCs, Tregs, DCregs, neutrophils, Th17s, Bregs, and / or MSCs. In certain embodiments, immunomodulation of immunosuppressed cells includes phenotypic and / or functional switching of immunosuppressed cells from protumor to antitumor.

[0141] "Prophylactic treatment" includes treatment administered to subjects who show no signs or symptoms of a disease or condition, or who show only initial signs or symptoms of a disease or condition, for the purpose of reducing, preventing, or decreasing the risk of further development of the disease or condition. Thus, prophylactic treatment functions as a preventive treatment against disease or disorder. In certain embodiments, prophylactic treatment includes administration of compositions disclosed herein to subjects who have had cancer but are in remission, for the purpose of reducing or delaying the occurrence of recurrence.

[0142] "Therapeutic treatment" includes treatments administered to a subject exhibiting symptoms or signs of a disease or condition, for the purpose of reducing or eliminating the symptoms or signs of the disease or condition. In certain embodiments, therapeutic treatment includes administering compositions disclosed herein to a subject with cancer in order to reduce or eliminate tumors and / or metastases.

[0143] In certain embodiments, the therapeutically effective dose provides an anti-cancer effect in the target. Cancer (medical term: malignant neoplasm) is a class of diseases characterized by uncontrolled growth (division beyond normal limits), invasion (invasion and destruction of adjacent tissues), and sometimes metastasis. "Metastasis" refers to the spread of cancer cells from their initial site of growth to another part of the body. The formation of metastasis is a very complex process, depending on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane and entry into body cavities and blood vessels, and then invasion to the target organ after being carried by the blood. Finally, the growth of a new tumor at the target site, i.e., a secondary tumor or metastatic tumor, depends on angiogenesis. Tumor metastasis often occurs even after the primary tumor has been removed, because tumor cells or components may remain and acquire metastatic potential.

[0144] In certain embodiments, a therapeutically effective dose provides an antitumor effect in the target. A “tumor” is a swelling or lesion (called neoplastic cells or tumor cells) formed by the abnormal growth of cells. “Tumor cells” are abnormal cells that divide by rapid, uncontrolled cell proliferation and continue to divide after the stimulus that initiated new division has ceased. Tumors exhibit a partial or complete lack of functional coordination with structural tissues and normal cells and usually form a distinct tissue mass, which may be benign, pre-malignant, or malignant.

[0145] Antitumor effects refer to biological effects, which can be manifested by a reduction in the number of tumor cells, a reduction in the number of metastases, a reduction in tumor volume, an increase in average lifespan, induction of apoptosis in cancer cells, induction of cancer cell death, induction of chemosensibility or radiosensitivity in cancer cells, inhibition of angiogenesis near cancer cells, inhibition of cancer cell proliferation, inhibition of tumor growth, prevention of metastasis, extension of the lifespan of the subject, reduction of cancer-related pain, reduction in the number of metastases, and / or a reduction in cancer relapse or recurrence following treatment. Accordingly, the compositions disclosed herein can be used to treat various cancers, can prevent or significantly delay metastasis, and / or can prevent or significantly delay recurrence. In certain embodiments, the overall survival of subjects whose cancer is treated with the nanoparticle compositions disclosed herein is improved by 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, or more, compared to control subjects whose cancer is not treated with nanoparticles. In certain embodiments, the number of metastases in subjects whose cancer is treated with the nanoparticle compositions disclosed herein is reduced by 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, compared to control subjects whose cancer is not treated with nanoparticles.

[0146] In certain embodiments, therapeutic treatment includes administration to a cancer-bearing subject in combination with other therapies of the compositions disclosed herein to shrink or eliminate tumors. In certain embodiments, therapies used in combination with the compositions disclosed herein include cancer vaccines, CAR immunotherapy (e.g., CAR-T immunotherapy), chemotherapy, radiotherapy, hormone therapy, signaling inhibitors, gene expression regulators, apoptosis inducers, angiogenesis inhibitors, and monoclonal antibodies that deliver toxic molecules. In certain embodiments, administration to a cancer-bearing subject in combination with radiotherapy results in an overall survival improvement of 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, or more, compared to a control subject with the same cancer that was not administered the nanoparticle compositions in combination with radiotherapy.

[0147] Cancers that can be treated with the systems and methods disclosed herein include ovarian cancer, breast cancer, brain cancer, melanoma, lung metastases, seminomas, teratomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, skin cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancers, prostate cancer, uterine cancer, lung cancer, and metastases thereof.

[0148] As shown, the teachings of this disclosure can also be used in methods to inactivate the immune system by modulating the activity state of immune cells in conditions such as autoimmune diseases. In certain embodiments, changing the activity state of macrophages from an activated state to an inactivated state in autoimmune diseases involves introducing nanoparticles containing nucleotides encoding IRFs that induce the M2 phenotype into macrophages. Certain embodiments of the IRFs that induce the M2 phenotype include IRF3 and / or IRF4. In certain embodiments, changing the activity state of macrophages from an activated state to an inactivated state in autoimmune diseases involves introducing nanoparticles containing nucleotides encoding the GILZ (glucocorticoid-inducing leucine zipper) transcription factor, which can mediate glucocorticoid anti-inflammatory effects and induce the M2 phenotype into macrophages. In certain embodiments, changing the activity state of macrophages from an activated state to an inactivated state in autoimmune diseases involves introducing nanoparticles containing nucleotides encoding GILZ and IRF4 into macrophages. Exemplary autoimmune diseases include acute necrotizing hemorrhagic encephalopathy, allergic asthma, alopecia areata, anemia, aphthous ulcers, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, and psoriatic arthritis), asthma, autoimmune thyroiditis, conjunctivitis, Crohn's disease, cutaneous lupus erythematosus, dermatitis (including atopic dermatitis and eczematous dermatitis), diabetes mellitus, diabetes mellitus vera, erythema nodosum leprosy, keratoconjunctivitis, multiple sclerosis, myasthenia gravis, psoriasis, scleroderma, Sjögren's syndrome including keratoconjunctivitis sicca following Sjögren's syndrome, Stevens-Johnson syndrome, systemic lupus erythematosus, ulcerative colitis, vaginitis, and Wegener's granulomatosis.

[0149] Regarding administration, the therapeutically effective dose (also referred to herein as dosage) can be first evaluated based on results from in vitro assays and / or animal model studies. For example, the dose is determined in cell cultures against a specific target. 50 It can be formulated in animal models to achieve a circulating concentration range that includes [specific component]. Using such information, an effective dose for the target subject can be determined more accurately.

[0150] The actual dosage administered to a specific subject can be determined by a physician, veterinarian, or researcher, taking into account parameters such as the target, body weight, severity of the condition, type of disease, previous or concurrent therapeutic interventions, the subject's idiopathic nature, and physical and physiological factors including the route of administration.

[0151] Useful doses often fall within the range of 0.1–5 μg / kg or 0.5–1 μg / kg. In specific embodiments, doses may be 1 μg / kg, 5 μg / kg, 10 μg / kg, 15 μg / kg, 20 μg / kg, 25 μg / kg, 30 μg / kg, 35 μg / kg, 40 μg / kg, 45 μg / kg, 50 μg / kg, 55 μg / kg, 60 μg / kg, 65 μg / kg, 70 μg / kg, 75 μg / kg, 80 μg / kg, 85 μg / kg, 90 μg / kg, 95 μg / kg, 100 μg / kg, 1 50μg / kg, 200μg / kg, 250μg / kg, 350μg / kg, 400μg / kg, 450μg / kg, 500μg / kg, 550μg / kg, 600μg / kg, 650μg / kg, 700 μg / kg, 750 μg / kg, 800 μg / kg, 850 μg / kg, 900 μg / kg, 950 μg / kg, 1000 μg / kg, 0.1-5 mg / kg or 0.5-1 mg / kg. In a particular embodiment, the doses are 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg, 95 mg / kg, 10 It may contain 0 mg / kg, 150 mg / kg, 200 mg / kg, 250 mg / kg, 350 mg / kg, 400 mg / kg, 450 mg / kg, 500 mg / kg, 550 mg / kg, 600 mg / kg, 650 mg / kg, 700 mg / kg, 750 mg / kg, 800 mg / kg, 850 mg / kg, 900 mg / kg, 950 mg / kg, 1000 mg / kg, or more.

[0152] The therapeutically effective dose can be achieved by administering single or multiple doses during the course of the treatment plan (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every year). In certain embodiments, the therapeutically effective dose can be achieved by administering repeated doses during the course of the treatment plan.

[0153] The nanoparticle compositions described herein may be administered by injection, inhalation, infusion, perfusion, lavage, or oral ingestion. The routes of administration may include intravenous, intradermal, intra-arterial, parenteral, intranasal, intra-lymph node, intra-lymphatic, intraperitoneal, intracranial, intrafocal, intraprostatic, intravaginal, intrarectal, topical, subarachnoid, intratumoral, intramuscular, intravesicular, oral, subcutaneous, and / or sublingual administration, and more specifically, intravenous, intratumoral, intraperitoneal, and / or intracranial injection. Topical administration involves administering a therapeutically effective amount of the composition disclosed herein to a specific area, organ, or cavity of the body. For example, intraperitoneal injection may be used to deliver a therapeutic agent for treating ovarian cancer, or intracranial injection may be used to deliver a therapeutic agent for treating glioma. Administration of therapeutic agents at the tumor site may include ligand-mediated targeting of the therapeutic agent (e.g., a nanoparticle composition) to tumor cells and / or tumor-supporting cells using the targeting ligands described above, where the therapeutic agent does not target healthy tissue. Administration of therapeutic agents at the tumor site may also include passive targeting of the therapeutic agent (e.g., a nanoparticle composition) to tumor cells and / or tumor-supporting cells, where the therapeutic agent does not target healthy tissue. Specific embodiments of passive targeting may include a nanoparticle size range and enhanced permeability and retention (EPR) phenomena based on the leaky vascular structure and impaired lymphatic drainage of tumor tissue. In contrast, systemic administration extends throughout the body and is typically achieved by intravenous injection of the composition or therapeutic agent into circulation. Systemic administration of therapeutic agents can be useful for less localized forms of cancer, such as metastatic cancer.

[0154] Figure 5 provides exemplary sequences (SEQ ID NOs: 1-44, 110, and 111) supporting this disclosure. CDR sequences are also described herein. This disclosure includes variants of these sequences. Variants of protein sequences may include sequences having one or more conserved amino acid substitutions or one or more non-conserved substitutions that do not adversely affect the function of the protein. "Conservative substitutions" include substitutions found in one of the following conservative substitution groups: Group 1: alanine (Ala), glycine (Gly), serine (Ser), threonine (Thr); Group 2: aspartic acid (Asp), glutamic acid (Glu); Group 3: asparagine (Asn), glutamine (Gln); Group 4: arginine (Arg), lysine (Lys), histidine (His); Group 5: isoleucine (Ile), leucine (Leu), methionine (Met), valine (Val); and Group 6: phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp).

[0155] Furthermore, amino acids can be classified into conserved substitution groups based on similar functions or chemical structures or compositions (e.g., acidic, basic, aliphatic, aromatic, sulfur-containing). For example, the aliphatic classification may include Gly, Ala, Val, Leu, and Ile for substitution purposes. Other groups containing amino acids considered to be conserved substitutions of each other include: sulfur-containing: Met and cysteine ​​(Cys); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, WH Freeman and Company.

[0156] Protein fragments consist of less than the complete amino acid sequence of the corresponding protein, but retain the function of the full-length protein.

[0157] Nucleotide sequence variants may include one or more degenerate codons, sequence polymorphisms, and mutations, where such changes do not affect the function of the encoded activating regulator, nor do they have a substantial effect on the function of the encoded activating regulator.

[0158] Certain embodiments and sequence variants include sequences having at least 70% sequence identity, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequences described or disclosed herein.

[0159] "% Array identity" refers to the relationship between two or more sequences, when determined by comparing them. In this art, "identity" also means the degree of sequence association between sequences, determined by the match between the strings of such sequences. "Identity" (often called "similarity") can be readily calculated by known methods, including those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J. eds.) Oxford University Press, NY (1992). The preferred method for determining % sequence identity is designed to provide the best fit between the sequences being tested. Methods for determining % sequence identity and similarity are integrated into publicly available computer programs. Sequence alignment and % sequence identity calculation can also be performed using the Megalign program (DNASTAR, Inc., Madison, Wisconsin) in the LASERGENE bioinformatics computing suite. Multiple alignment of sequences can also be performed using the cluster method of alignment with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10) (Higgins and Sharp CABIOS, 5, pp. 151-153 (1989)). Related programs include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul et al., J.Mol.Biol.215: pp. 403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput.Methods Genome Res., [Proc.Int.Symp.] (1994), Meeting Date 1992, pp. 111-120). Editor(s): Suhai, Sandor. Publisher: Plenum, New This includes York, NY. In the context of this disclosure, if sequence analysis software is used for the analysis, it is understood that the results of the analysis will be based on the “default values” of the referenced program. “Default values” means any set of values ​​or parameters that are originally loaded into the software when they are first initialized.

[0160] The exemplary embodiments and examples below are included to demonstrate specific embodiments of the Disclosure. Those skilled in the art should recognize that many modifications can be made to the specific embodiments disclosed herein in light of the Disclosure, and that similar or analogous results can still be obtained without departing from the spirit and scope of the Disclosure.

[0161] Exemplary embodiment. 1. A method for in vivo altering the activity state of immune cells, A method for in vivo altering the activity state of immune cells, comprising administering nanoparticles containing nucleotides encoding one or more interferon regulatory factors (IRFs). 2. The method of Embodiment 1, wherein the immune cells are macrophages, regulatory T cells (TREGs), myeloid-derived suppressor cells (MDSCs), regulatory dendritic cells (DCregs), neutrophils, helper T17 cells (Th17s), regulatory B cells (Bregs), and / or mesenchymal stromal cells (MSCs). 3. The method of Embodiment 1 or 2, wherein the nanoparticles include a positively charged core, a poly(β)aminoester core, a star-shaped polymer, a polyglutamic acid coating, a hyaluronic acid coating, a neutrally charged coating, and / or liposome nanoparticles. 4. Any one of Embodiments 1 to 3, wherein the nanoparticles are <130 nm. 5. Any one of Embodiments 1 to 4, wherein the nucleotide comprises in vitro transcribed mRNA. 6. Any one of embodiments 1 to 5, wherein the nucleotide is encapsulated within the core. 7. Any one of embodiments 1 to 6, wherein one or more encoded IRFs lack functional autoinhibitory domains. 8. Any one of embodiments 1 to 7, wherein one or more encoded IRFs lack a functional nuclear export signal (NES). 9. Any one of Embodiments 1 to 8, wherein the administration is local administration. 10. The method of Embodiment 9, wherein local administration is intraperitoneal or intracranial. 11. Any one of Embodiments 1 to 9, wherein the administration is systemic. 12. Any one of embodiments 1 to 11, wherein the nanoparticles further comprise a targeting ligand. 13. The method of Embodiment 12, wherein a targeting ligand is linked to the coating. 14. Any one of Embodiments 1 to 13, wherein the active state is changed from an inactive state to an active state. 15. Any one of Embodiments 1 to 14, wherein the immune cells include macrophages. 16. The method of embodiment 15, wherein macrophages are present within the tumor. 17. The method of Embodiment 16, wherein the tumor is an ovarian cancer tumor, a glioblastoma tumor, or a metastatic lung cancer tumor. 18. Any one of Embodiments 1 to 17, wherein one or more coded IRFs are selected from IRF1, IRF3, IRF5, IRF7, IRF8, and / or a fusion of IRF7 and IRF3. 19. Any one of Embodiments 1 to 18, wherein one or more coded IRFs are selected from sequences having greater than >90%, >95%, or 98% identity to sequence numbers 1 to 17. 20. Any one of the embodiments 1 to 19, wherein one or more coded IRFs are IRF5 selected from sequence numbers 1 to 7. 21. The method of Embodiment 20, wherein IRF5 is Sequence ID No. 1. 22. The method of Embodiment 20 or 21, wherein IRF5 is Sequence ID 1 or Sequence ID 3 having one or more mutations selected from S156D, S158D and T160D. 23. Any one of the methods of Embodiments 20 to 22, wherein IRF5 is Sequence ID No. 2 having one or more mutations selected from T10D, S158D, S309D, S317D, S451D, and S462D. 24. Any one of the methods of Embodiments 20 to 23, wherein IRF5 is Sequence ID No. 4 having one or more mutations selected from S425D, S427D, S430D, and S436D. 25. Any one of Embodiments 1 to 24, wherein one or more coded IRFs are IRF1 selected from Sequence IDs 8 and 12. 26. Any one of Embodiments 1 to 24, wherein one or more coded IRFs are IRF8 selected from Sequence IDs 11, 16, and 17. 27. The method of Embodiment 26, wherein IRF8 is Sequence ID No. 11 having the K310R mutation. 28. Any one of Embodiments 1 to 27, wherein one or more encoded IRFs comprise an IRF7 / IRF3 fusion protein comprising an N-terminal IRF7 DNA-binding domain (DBD) and a constitutive activity domain (CAD), and a C-terminal IRF3 NES (nuclear export signaling) and an association domain. 29. The method of Embodiment 28, wherein the IRF7 / IRF3 fusion protein includes a mutation that mimics phosphorylation in the IRF3 association domain. 30. The method of Embodiment 28 or 29, wherein the IRF7 / IRF3 fusion protein is shown in Sequence ID No. 15. 31. Any one of Embodiments 1 to 30, wherein the nanoparticles further comprise a nucleotide encoding IKKβ. 32. The method of Embodiment 31, wherein the encoded IKKβ is selected from sequences having greater than >90%, >95%, or 98% identity to sequence numbers 18-22. 33. The method of Embodiment 31 or 32, wherein the coded IKKβ is selected from Sequence IDs 18-22. 34. Any one of Embodiments 1 to 33, wherein the nucleotide comprises a sequence selected from Sequence IDs 23 to 44. 35. Any one of embodiments 12 to 34, wherein the targeting ligand binds to CD206, CD163, or CD23. 36. Any one of embodiments 12 to 35, wherein the targeting ligand is dimannose. 37. Any one of embodiments 31 to 36, wherein one or more nucleotides encoding IRF and IKKβ are encapsulated in the same nanoparticle. 38. Any one of embodiments 31 to 37, wherein one or more nucleotides encoding IRF and IKKβ are encapsulated in different nanoparticles. 39. Any one of Embodiments 1 to 38, comprising altering the activity state of immune cells to reduce the percentage of inactive immune cells in a population of immune cells by 5, 10, 15, 20, or more. 40. Any one of Embodiments 1 to 39, wherein changing the activity state of immune cells reduces the number of inactivated immune cells in a population of immune cells by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more. 41. Any one of Embodiments 1 to 40, wherein changing the activation state of immune cells involves increasing the percentage of activated immune cells in a population of immune cells by 5, 10, 15, 20, or more. 42. Any one of Embodiments 1 to 41, wherein changing the activation state of immune cells involves increasing the number of activated immune cells in a population of immune cells by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more. 43. Any one of Embodiments 1 to 13, wherein the active state is changed from an active state to an inactive state. 44. Any one of embodiments 1 to 13 and 43, wherein the immune cells include macrophages. 45. Any one of embodiments 1 to 13, 43 and 44, wherein one or more coded IRFs are IRF4. 46. ​​Any one of Embodiments 1-13 and 43-45, wherein the nanoparticles further comprise a nucleotide encoding a glucocorticoid-induced leucine zipper (GILZ). 47. Any one of Embodiments 12 and 43-46, wherein the targeting ligand binds to CD38, G protein-coupled receptor 18 (Gpr18), formyl peptide receptor 2 (Fpr2), CD64, or CD68. 48. Any one of Embodiments 1-13 and 43-47, wherein altering the activation state of immune cells reduces the percentage of activated immune cells in a population of immune cells by 5, 10, 15, 20, or more. 49. Any one of Embodiments 1 to 13 and 43 to 48, wherein changing the activation state of immune cells reduces the number of activated immune cells in a population of immune cells by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more. 50. Any one of Embodiments 1-13 and 43-49, wherein changing the activity state of immune cells involves increasing the percentage of inactivated immune cells in a population of immune cells by 5, 10, 15, 20, or more. 51. Any one of Embodiments 1 to 13 and 43 to 50, wherein changing the activity state of immune cells involves increasing the number of inactivated immune cells in a population of immune cells by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more. 52. A method for treating cancer in a subject requiring treatment for cancer, comprising changing the activity state of tumor-associated macrophages in a tumor within the subject from inactivation to activation, thereby treating the cancer in the subject requiring treatment for cancer. 53. The method of Embodiment 52, wherein the tumor is an ovarian cancer tumor, a glioblastoma tumor, or a metastatic lung cancer tumor. 54. The method of Embodiment 52 or 53, wherein the change occurs following the administration of a therapeutically effective dose of nanoparticles containing nucleotides encoding one or more transcription factors that change the active state of tumor-associated macrophages from inactivation to activation. 55. The method of Embodiment 54, wherein the nanoparticles include a positively charged core, a poly(β)aminoester core, a star-shaped polymer, a polyglutamic acid coating, a hyaluronic acid coating, a neutrally charged coating, and / or liposome nanoparticles. 56. The method of Embodiment 54 or 55, wherein the nanoparticles are <130 nm. 57. Any one of embodiments 54 to 56, wherein the nucleotide comprises in vitro transcribed mRNA. 58. Any one of embodiments 54 to 57, wherein the nucleotide is encapsulated within the core. 59. Any one of embodiments 54 to 58, wherein the administration is local administration. 60. The method of Embodiment 59, wherein the local administration is intraperitoneal or intracranial. 61. Any one of embodiments 54 to 59, wherein the administration is systemic. 62. Any one of the embodiments 54 to 61, wherein one or more encoded transcription factors include one or more interferon regulatory factors (IRFs). 63. The method of embodiment 62, wherein one or more encoded IRFs lack functional autoinhibitory domains. 64. The method of embodiment 62 or 63, wherein one or more encoded IRFs lack a functional nuclear export signal (NES). 65. Any one of embodiments 62 to 64, wherein one or more coded IRFs are selected from IRF1, IRF3, IRF5, IRF7, IRF8, and / or a fusion of IRF7 and IRF3. 66. Any one of embodiments 62 to 65, wherein one or more coded IRFs are selected from sequences having greater than >90%, >95%, or 98% identity to sequence numbers 1 to 17. 67. Any one of embodiments 62 to 66, wherein one or more coded IRFs are IRF5 selected from sequence numbers 1 to 7. 68. The method of Embodiment 67, wherein IRF5 is Sequence ID No. 1. 69. The method of Embodiment 67 or 68, wherein IRF5 is Sequence ID 1 or Sequence ID 3 having one or more mutations selected from S156D, S158D and T160D. 70. A method according to any one of embodiments 67 to 69, wherein IRF5 is Sequence ID No. 2 having one or more mutations selected from T10D, S158D, S309D, S317D, S451D, and S462D. 71. Any one of the methods of Embodiments 67 to 70, wherein IRF5 is Sequence ID No. 4 having one or more mutations selected from S425D, S427D, S430D, and S436D. 72. Any one of embodiments 62 to 71, wherein one or more coded IRFs are IRF1 selected from sequence numbers 8 and 12. 73. Any one of the embodiments 62 to 72, wherein one or more coded IRFs are IRF8 selected from sequence numbers 11, 16, and 17. 74. The method of Embodiment 73, wherein IRF8 is Sequence ID No. 11 having the K310R mutation. 75. Any one of Embodiments 62 to 74, wherein one or more encoded IRFs comprise an IRF7 / IRF3 fusion protein comprising an N-terminal IRF7 DNA-binding domain (DBD) and a constitutive activity domain (CAD), as well as a C-terminal IRF3 NES (nuclear export signaling) and an association domain. 76. The method of Embodiment 75, further comprising a mutation in the IRF7 / IRF3 fusion protein that mimics phosphorylation in the IRF3 association domain. 77. The method of Embodiment 75 or 76, wherein the IRF7 / IRF3 fusion protein is shown in Sequence ID No. 15. 78. Any one of embodiments 54 to 77, wherein the nanoparticles further comprise a nucleotide encoding IKKβ. 79. The method of Embodiment 78, wherein the encoded IKKβ is selected from sequences having greater than >90%, >95%, or 98% identity to sequence numbers 18-22. 80. The method of Embodiment 78 or 79, wherein the coded IKKβ is selected from Sequence IDs 18-22. 81. Any one of the embodiments 54 to 79, wherein the nucleotide comprises a sequence selected from sequence numbers 23 to 44. 82. Any one of embodiments 54 to 81, wherein the nanoparticles further comprise a targeting ligand. 83. The method of Embodiment 82, wherein a targeting ligand is linked to the coating. 84. The method of Embodiment 82 or 83, wherein the targeting ligand binds to CD206, CD163, or CD23. 85. Any one of embodiments 82 to 84, wherein the targeting ligand is dimannose. 86. Any one of embodiments 54 to 85, wherein one or more nucleotides encoding IRF and IKKβ are encapsulated in the same nanoparticle. 87. Any one of embodiments 54 to 86, wherein one or more nucleotides encoding IRF and IKKβ are encapsulated in different nanoparticles. 88. Any one of embodiments 54 to 87, comprising altering the activity state of macrophages to reduce the percentage of inactive macrophages in a tumor population by 5, 10, 15, 20, or more. 89. Any one of embodiments 54 to 88, wherein altering the activity state of macrophages reduces the number of inactive macrophages in a tumor population by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more. 90. Any one of Embodiments 54 to 89, wherein altering the activity state of macrophages increases the percentage of activated macrophages in a tumor population by 5, 10, 15, 20, or more. 91. Any one of Embodiments 54 to 90, wherein altering the activity state of macrophages increases the number of active macrophages in a tumor population by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more. 92. Any one of embodiments 54 to 91, further comprising administering a therapy selected from cancer vaccines, chimeric antigen receptor (CAR) immunotherapy, chemotherapy, radiotherapy, hormone therapy, signaling inhibitors, gene expression regulators, apoptosis inducers, angiogenesis inhibitors, and monoclonal antibodies that deliver toxic molecules, in combination with a therapeutically effective amount of nanoparticles. 93. A method for treating an autoimmune disease in a subject requiring treatment for an autoimmune disease, comprising changing the activity state of macrophages in the subject from activation to inactivation, thereby treating the autoimmune disease in the subject requiring treatment for an autoimmune disease. 94. The method of Embodiment 93, wherein the autoimmune disease includes acute necrotizing hemorrhagic encephalopathy, allergic asthma, alopecia areata, anemia, aphthous ulcers, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, and psoriatic arthritis), asthma, autoimmune thyroiditis, conjunctivitis, Crohn's disease, cutaneous lupus erythematosus, dermatitis (including atopic dermatitis and eczematous dermatitis), diabetes mellitus, diabetes mellitus vera, erythema nodosum leprosy, keratoconjunctivitis, multiple sclerosis, myasthenia gravis, psoriasis, scleroderma, Sjögren's syndrome including keratoconjunctivitis sicca following Sjögren's syndrome, Stevens-Johnson syndrome, systemic lupus erythematosus, ulcerative colitis, vaginitis, and Wegener's granulomatosis. 95. The method of Embodiment 93 or 94, wherein the change occurs following the administration of a therapeutically effective dose of nanoparticles containing nucleotides encoding one or more transcription factors that change the active state of macrophages from activated to inactivated. 96. The method of Embodiment 94, wherein the nanoparticles include a positively charged core, a poly(β)aminoester core, a star-shaped polymer, a polyglutamic acid coating, a hyaluronic acid coating, a neutrally charged coating, and / or liposome nanoparticles. 97. The method of Embodiment 94 or 95, wherein the nanoparticles are <130 nm. 98. Any one of the embodiments 95 to 97, wherein the nucleotide comprises in vitro transcribed mRNA. 99. Any one of embodiments 95 to 98, wherein the nucleotide is encapsulated within the core. 100. Any one of the embodiments 95 to 99, wherein the administration is local administration. 101. The method of Embodiment 100, wherein the local administration is intraperitoneal or intracranial. 102. Any one of the embodiments 92 to 99, wherein the administration is systemic. 103. Any one of Embodiments 95 to 102, wherein one or more encoded transcription factors include one or more interferon regulatory factors (IRFs). 104. The method of Embodiment 103, wherein one or more encoded IRFs lack functional autoinhibitory domains. 105. The method of Embodiment 103 or 104, wherein one or more encoded IRFs lack a functional nuclear export signal (NES). 106. Any one of embodiments 103 to 105, wherein one or more coded IRFs are IRF4. 107. Any one of embodiments 95 to 106, wherein the nanoparticles further comprise a nucleotide encoding a glucocorticoid-induced leucine zipper (GILZ). 108. Any one of embodiments 85 to 107, wherein the nanoparticles further comprise a targeting ligand. 109. The method of Embodiment 108, wherein the targeting ligand is linked to the coating. 110. The method of Embodiment 108 or 109, wherein the targeting ligand binds to CD38, G protein-coupled receptor 18 (Gpr18), formyl peptide receptor 2 (Fpr2), CD64, or CD68. 111. Any one of embodiments 93 to 110, wherein altering the activity state of macrophages reduces the percentage of active macrophages in a macrophage population by 5, 10, 15, 20, or more. 112. Any one of embodiments 93 to 111, wherein changing the activity state of macrophages reduces the number of active macrophages in a macrophage population by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more. 113. Any one of embodiments 95 to 112, wherein changing the activity state of macrophages increases the percentage of inactive macrophages in a macrophage population by 5, 10, 15, 20, or more. 114. Any one of embodiments 95 to 113, wherein changing the activity state of macrophages increases the number of inactive macrophages in a macrophage population by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more. 115. A composition comprising nanoparticles containing one or more nucleotides encoding interferon regulatory factors (IRFs). 116. The composition of Embodiment 115, further comprising a pharmaceutically acceptable carrier. 117. A composition of Embodiment 115 or 116, wherein the nanoparticles include a positively charged core, a poly(β)aminoester core, a star-shaped polymer, a polyglutamic acid coating, a hyaluronic acid coating, a neutrally charged coating, and / or liposome nanoparticles. 118. Any one of the compositions of embodiments 115 to 117, wherein the nanoparticles are <130 nm. 119. A composition according to any one of embodiments 115 to 118, wherein the nucleotide comprises in vitro transcribed mRNA. 120. A composition according to any one of embodiments 115 to 119, wherein a nucleotide is encapsulated within a core. 121. Any one of the compositions of embodiments 115 to 120, wherein one or more encoded IRFs lack functional autoinhibitory domains. 122. A composition of Embodiment 115 or 121, wherein one or more encoded IRFs lack a functional nuclear export signal (NES). 123. Any one of the compositions of Embodiments 115 to 122, wherein one or more coded IRFs are selected from IRF1, IRF3, IRF5, IRF7, IRF8, and / or a fusion of IRF7 and IRF3. 124. Any one of the compositions of Embodiments 115 to 123, wherein one or more coded IRFs are selected from sequences having greater than >90%, >95%, or 98% identity to sequence numbers 1 to 17. 125. Any one of the compositions of Embodiments 115 to 124, wherein one or more coded IRFs are IRF5 selected from Sequence IDs 1 to 7. 126. The composition of Embodiment 125, wherein IRF5 is Sequence ID No. 1. 127. The composition of Embodiment 125 or 126, wherein IRF5 is Sequence ID No. 1 or Sequence ID No. 3 having one or more mutations selected from S156D, S158D and T160D. A composition according to any one of embodiments 125 to 127, wherein 128.IRF5 is Sequence ID No. 2 having one or more mutations selected from T10D, S158D, S309D, S317D, S451D, and S462D. 129. A composition of any one of embodiments 125 to 128, wherein IRF5 is Sequence ID No. 4 having one or more mutations selected from S425D, S427D, S430D, and S436D. 130. Any one of the compositions of Embodiments 115 to 129, wherein one or more coded IRFs are IRF1 selected from Sequence IDs 8 and 12. 131. Any one of the compositions of Embodiments 115 to 130, wherein one or more coded IRFs are IRF8 selected from Sequence IDs 11, 16, and 17. 132. The composition of Embodiment 131, wherein IRF8 is Sequence ID No. 11 having the K310R mutation. 133. Any one of the compositions of Embodiments 115 to 132, wherein one or more encoded IRFs comprise an IRF7 / IRF3 fusion protein comprising an N-terminal IRF7 DNA-binding domain (DBD) and a constitutive activity domain (CAD), and a C-terminal IRF3 NES (nuclear export signaling) and an association domain. 134. The composition of Example 133, further comprising a mutation in the IRF7 / IRF3 fusion protein that mimics phosphorylation in the IRF3 association domain. 135. A composition of Embodiment 133 or 134, wherein the IRF7 / IRF3 fusion protein is shown in SEQ ID NO: 15. 136. Any one of embodiments 115 to 135, wherein the nanoparticles further comprise a nucleotide encoding IKKβ. 137. The composition of Embodiment 136, wherein the encoded IKKβ is selected from sequences having greater than >90%, >95%, or 98% identity to sequence numbers 18-22. 138. The composition of Embodiment 136 or 137, wherein the coded IKKβ is selected from Sequence ID Nos. 18-22. 139. A composition according to any one of embodiments 115 to 138, wherein the nucleotide comprises a sequence selected from sequence numbers 23 to 44. 140. Any one composition of Embodiments 115 to 139, further comprising nanoparticles containing nucleotides carrying one or more anti-oncogenes selected from p53, RB, BRCA1, E1A, bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-I VEGF, angiostatin, oncostatin, endostatin, GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-γ, TNFα and / or HSV-tk. 141. Any one of the compositions from Embodiments 115 to 122, wherein one or more coded IRFs are IRF4. 142. Any one of embodiments 115-122 and 141, wherein the nanoparticles further comprise a nucleotide encoding a glucocorticoid-induced leucine zipper (GILZ). 143. Any one of the compositions from Embodiments 115 to 142, further comprising nanoparticles as a targeting ligand. 144. The composition of Embodiment 143, wherein a targeting ligand is linked to the coating. 145. The composition of Embodiment 143 or 144, wherein the targeting ligand is bound to CD206, CD163, or CD23. 146. The composition of Embodiment 145, wherein the targeting ligand is dimannose. 147. The composition of Embodiment 143 or 144, wherein the targeting ligand binds to CD38, G protein-coupled receptor 18 (Gpr18), formyl peptide receptor 2 (Fpr2), CD64, or CD68. 148. Any one of the compositions of Embodiments 115 to 147, wherein one or more nucleotides encoding IRF, IKKβ, and / or GILZ are encapsulated in the same nanoparticle. 149. Any one of the compositions of Embodiments 115 to 148, wherein one or more nucleotides encoding IRF, IKKβ, and / or GILZ are encapsulated in different nanoparticles. [Examples]

[0162] [Example 1] Materials and Method. PbAE Synthesis. The method used to synthesize the polymer had already been described (Mangraviti A et al., (2015) ACS Nano 9: pp. 1236-1249). 1,4-Butanediol diacrylate was combined with 4-amino-1-butanol in a 1:1 molar ratio of diacrylate to amine monomer. The acrylate-terminal poly(4-amino-1-butanol-1,4-butanediol diacrylate copolymer) was formed by heating the mixture to 90°C with stirring for 24 hours. 2.3 g of this polymer was dissolved in 2 mL of tetrahydrofuran (THF). To form the piperazine-capped 447 polymer, 786 g of 1-(3-aminopropyl)-4-methylpiperazine was added to the polymer / THF solution in 13 mL of THF and stirred at room temperature (RT) for 2 hours. The capped polymer was precipitated with 5 volumes of diethyl ether, washed with 2 volumes of fresh ether, and dried under vacuum for 1 day. The pure polymer was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 100 mg / mL and stored at -20°C.

[0163] Conjugation of PGA to Dimannose. α-D-mannopyranosyl-(1→2)-α-D-mannopyranosose (Di-mannose, Omicron Biochemicals Inc.) was modified into a glucosylamine before conjugation with polyglutamic acid (PGA). First, 157 mg of dimannose was dissolved in 10.5 mL of saturated aqueous ammonium carbonate and stirred at room temperature for 24 hours. On the second day, more solid ammonium carbonate was added until dimannose precipitated from the reaction mixture. The mixture was stirred until complete, and then freeze-dried to remove excess ammonium carbonate. Complete removal of volatile salts was achieved by redissolving the solid in methanol. These steps created an amine on the anomeric carbon for future conjugation with PGA.

[0164] To conjugate amination dimannose to PGA, the substrate was dissolved in water to a concentration of 30 mg / mL and then sonicated for 10 minutes. Ethyl-N'-(3-dimethylaminopropyl)carbodiimide·HCl (4 mg / mL, 30 equivalents) was added in water while mixing for 4 minutes at room temperature. N-hydroxysulfosuccinimide (30 mg / mL, 35 equivalents) was incubated with the PGA / EDC solution for 1 minute. Amination dimannose in phosphate-buffered saline (PBS) was combined with the resulting activated PGA in a molar ratio of 44:1 and mixed at room temperature for 6 hours. Excess reagent was removed by dialyzing against water for 24 hours.

[0165] For mRNA synthesis, codon-optimized mRNAs for eGFP, IRF5, and IKK (TriLink Biotechnologies) were capped with the anti-reverse cap analog 3'-O-Me-m7G(5')ppp(5')G(ARCA) and completely substituted with modified ribonucleotides pseudouridine (ψ) and 5-methylcytidine (m5C).

[0166] Nanoparticle preparation. IRF5 and IKKβ mRNA were combined in a 3:1 (w:w) ratio and diluted to 100 μg / mL in 25 mM sodium acetate (NaOAc) buffer (pH=5.2). Poly(β-aminoaster)-447 (PbAE-447) polymer was diluted in DMSO (prepared as above) from 100 μg / μL to 6 μg / μL in NaOAc buffer. To form nanoparticles, PbAE-447 polymer was added to mRNA in a 60:1 (w:w) ratio and immediately vortexed at medium speed for 15 seconds. The mixture was then incubated at room temperature for 5 minutes to form a PbAE-mRNA polyplex. In the next step, 100 μg / mL of PGA / dimannose in NaOAc buffer was added to the polyplex solution, vortexed at medium speed for 15 seconds, and incubated at room temperature for 5 minutes. In this process, PGA / dimannose coated the surface of the PbAE-mRNA polyplex to form the final nanoparticle (NP). For long-term storage, D-sucrose (60 mg / mL) was added to the NP solution as a cryoprotectant. The nanoparticles were flash-frozen with dry ice and then freeze-dried. The dried NPs were stored at -20°C or -80°C until use. In in vivo experiments, the freeze-dried NPs were resuspended in water in a 1:20 (w:v) ratio.

[0167] Characterization of nanoparticle size distribution and zeta potential. The physicochemical properties of NPs (including hydrodynamic radius, polydispersity, zeta potential, and stability) were characterized using a Zetapals instrument (Brookhaven Instrument Corporation) at 25°C. To measure hydrodynamic radius and polydispersity based on dynamic light scattering, NPs were diluted 5-fold in 25 mM NaOAc (pH=5.2). To measure zeta potential, NPs were diluted 10-fold in 10 mM PBS (pH=7.0). To evaluate the stability of NPs, freshly prepared particles were diluted in 10 mM PBS buffer (pH=7.4). The hydrodynamic radius and polydispersity of NPs were measured every 10 minutes for 5 hours, and their size and particle concentration were derived from particle tracking analysis using a Nanosite 300 instrument (Malvern). To characterize the NPs using a transmission electron microscope, we followed a previously described protocol (Smith TT et al., (2017) Nat Nanotechnol 12: pp. 813-820). Newly prepared NPs (25 μL containing 0.83 μg of mRNA) were deposited onto a glow-discharge treated 200-mesh carbon / Formvar coated copper grid. After 30 seconds, the grid was sequentially treated with 50% Karnovsky fixative, 0.1 M cacodylate buffer, dH2O, and then 1% (w / v) uranyl acetate. The samples were imaged using a JEOL JEM-1400 transmission electron microscope (JEOL USA) operating at 120 kV.

[0168] Myelo-derived macrophages (BMDMs) and other cell lines. To prepare BMDMs, myeloprogenitor cells were collected from mouse femurs according to an established protocol (Zhang X et al., (2008) Curr Protoc Immunol Chapter 14: Unit 14 11). These cells were cultured in complete medium [DMEM supplemented with 4.5 g / L D-glucose, L-glutamine, 10% heat-inactivated fetal bovine serum (FBS), 100 U / mL penicillin, and 20 ng / mL M-CSF (Peprotech, catalog #315-02), 100 μg / mL, Glutamax 50 mL / 500 mL] at a seeding density of 0.5–1.0 e6 / ml. The cells were differentiated into BMDMs ex vivo for 7 days under 5% CO2 at 37°C. Next, the cells were conditioned in macrophage conditioning medium [complete macrophage medium supplemented with 20 ng / mL MPLA (Sigma, catalog #L6895) or 20 ng / mL IL4 (eBiosience, catalog #34-8041)]. BMDM was used ex vivo for 7–21 days. Mouse ovarian cancer cell line ID8, donated by Dr. Katherine Roby (University of Kansas Medical Center, Kansas City, KS), was cultured in DMEM supplemented with 10% FBS, 100 U / mL penicillin, 5 μg / mL insulin, 5 μg / mL transferrin, and 5 ng / mL sodium selenite (all Sigma-Aldrich). To create a more invasive vascular endothelial growth factor (VEGF)-expressing ID8 strain, ID8 tumor cells were transfected with the pUNO1 plasmid (Invivogen) encoding mouse VEGF together with a blastosidine resistance gene. To obtain stable transfectants, tumor cells were cultured for 3 weeks in complete medium containing 10 μg / mL of blastosidine (Invivogen).B16F10 melanoma cell line (Cell Culture Lineage Preservation Agency, USA) was cultured in complete RPMI 1640 medium containing 10% FBS, 100 U / mL penicillin, 2 mM / L glutamine, 1.5 g / L sodium bicarbonate, 4.5 g / L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, and 0.05 mM 2-mercaptoethanol. In vivo bioluminescence imaging was performed to retrovirally transduce firefly luciferase into both ID8-VEGF and B16F10 cell lines. DF-1 cell lines carrying RACS-PDGFβ or RCAS-cre retrovirus were cultured at 39°C under 5% CO2 in complete medium supplemented with 10% FBS and 100 U / mL penicillin.

[0169] mRNA transfection of BMDM. One day prior to transfection, BMDM were re-seed in macrophage complete medium at a concentration of 250,000 cells / well on 24-well plates. Before transfection, the complete medium was replaced with 300 μL of unsupplemented DMEM. To transfect these cells, NPs containing 2 μg of mRNA were added to the basal medium and co-cultured with BMDM at 37°C. After 1 hour, the medium containing the NPs was removed, and the cells were cultured for a further 24 hours before evaluating transfection efficiency and cell viability.

[0170] Transfection of BMDM for macrophage signature gene analysis. BMDM cells were re-seed on 24-well plates in conditioned medium 24 hours prior to transfection to transform the cells into their phenotype. Next, M2-like macrophages were exposed to IRF5 / IKKβ NPs carrying 25% eGFP mRNA or eGFP NPs carrying 2 μg of mRNA (control) as reporters, according to the transfection protocol described above. After 24 hours, 10% of the highly transfected BMDM cells (measured by eGFP expression) were selected 24 hours post-transfection and re-challenged in low-dose (10 ng / mL) IL4 medium for a further 48 hours before RNA isolation. RNA extracted from these cells was compared to RNA from standard M1 or M2-like macrophages to identify signature genes associated with IRF5-NP treatment.

[0171] RNA isolation and preparation. To collect RNA, BMDM was dissolved in Trizol reagent (Ambion), and total RNA was extracted and purified using RNeasy® Plus Universal Mini-Kits (QIAGEN) according to the manufacturer's instructions. Sample RNA was quantified using a NanoDrop Microvolume spectrophotometer (Thermo Fisher) and then subjected to quality control performed by the FHCRC Genome Shared Resources using an Agilent 4200 TapeStation analyzer (Agilent).

[0172] Macrophage signature gene analysis using nanostring technology. Gene expression levels from stimulated BMDM cultures were measured using the nCounter® bone marrow innate immune panel (NanoString Technologies, Seattle, WA), which analyzes 770 genes present in 19 different pathways and processes genes across 7 different myeloid cell types. Samples were tested using the nCounter analysis system (NanoString Technologies, Seattle, WA). Raw data were processed for quality checks using the R / Bioconductor NanoStringQCPro software package (Nickles D, Sandmann T, Ziman R and Bourgon R (2018) NanoStringQCPro: Quality metrics and data processing methods for NanoString mRNA gene expression data. R package version 1.10.0). Expression levels were normalized to the geometric mean of housekeeping genes and log2 transformed using nSolver 4.0 software (NanoString Technologies, Seattle, WA). The false positive rate for proportional data was calculated from the p-values ​​returned by a t-test using the Benjamini-Yekutieli method.

[0173] Flow cytometry and cell sorting. Cells obtained from the spleen, blood, peritoneal dialysis, and bronchoalveolar lavage were analyzed by flow cytometry using the anti-mouse antibody probes shown in Figure 9 in a bone marrow and lymphoid immunophenotyping panel. Data were collected using a BD LSRFortessa analyzer running FACSDIVA software (Beckton Dickinson). CD11b+ and F4 / 80+ peritoneal macrophages were sorted using BD FACS ARIA II. All collected data were analyzed using FlowJo 10.0 software.

[0174] Cytokine analysis. Cytokine levels were assessed using the Luminex 200 system (Luminex) at the FHCRC Immunotherapy Shared Resource Center. In ex vivo studies, cell culture supernatants were collected for measurement of IL-6, IL-12p70, INFγ, and TNFα concentrations. In in vivo studies, plasma concentrations of GM-CSF, INFγ, IL-12p70, IL-2, IL-6, and TNFα were measured.

[0175] qRT-PCR analysis. Gene expression levels were determined by qRT-PCR. To measure selected macrophage signature genes (serpin B2, Retnla, Ccl5, Ccl11, codon-optimized IRF5, endogenous IRF5, and housekeeping GAPD gene), total RNA was isolated using RNeasy mini-columns (Qiagen) according to the manufacturer's instructions. cDNA was synthesized using the qScript cDNA synthesis kit (Quanta). For each sample, PerfeCTa qPCR SuperMix Low was used, employing gene-specific probes from Roche's Universal Probe Library (UPL) and PCR primers optimized with ProbeFinder (Roche). Three methods were performed via ROX(Quanta): Serpine B2, UPL-049, F-ACTGGGGCAGTTATGACAGG (SEQ ID NO: 96), R-GATGATCGGCCACAAACTG (SEQ ID NO: 97); Retnla, UPL-078, F-TTGTTCCCTTCTCATCTGCAT (SEQ ID NO: 98), R-CCTTGACCTTATTCTCCACGA (SEQ ID NO: 99); Ccl5, UPL-105, F-CCTACTCCCACTCGGTCCT (SEQ ID NO: 100), R-CTGATTTCTTGGGTTTGCTGT (distribution) Column number 101); Ccl11, UPL-018, F-AGAGCTCCACAGCGCTTC (SEQ ID NO: 102), R-CAGCACCTGGGAGGTGAA (SEQ ID NO: 103); Codon-optimized IRF5, UPL-022, F-TCTTAAAGACCACATGGTAGAACAGT (SEQ ID NO: 104), R-AGCTGCTGTTGGGATTGC (SEQ ID NO: 105); Endogenous IRF5, UPL-011, F-GCTGTGCCCTTAACAAAAGC (SEQ ID NO: 106), R-GGCTGAGGTGGCATGTCT (SEQ ID NO: 107). Signature gene mRNA levels were normalized based on amplification of GAPD, UPL-060, F-AGCCACATCGCTCAGACAC (SEQ ID NO: 108) and R-GCCCAATACGACCAAATCC (SEQ ID NO: 109).All qRT-PCR reactions were performed using a Quant Studio 5 RT-PCR instrument running QuantStudio6 software (Applied Biosystems). If the amplification plot did not cross the threshold and no Ct value was obtained ("undetermined"), a Ct value equal to the maximum number of cycles in the assay (40 cycles) was used for comparison of relative expression.

[0176] Mice and in vivo tumor models. Except for experiments related to brain tumor models, the mice used in these experiments were obtained from the Jackson Laboratory, and the other mice were bred and housed in the FHCRC animal facility. All mice were used in accordance with protocols approved by the center's Animal Experimentation Committee. To model ovarian tumors, 5×10 6 vascular endothelial growth factor (VEGFP)-expressing ID8 cells were injected into female albino B6 (C57BL / 6J-Tyr <c-2j>The drug was injected intraperitoneally (ip) into mice and allowed to establish for two weeks. For survival studies, animals were treated with ip treatment with 50 μg of mRNA-carrying IRF5 NP / eGFP NP (two doses per week for nine weeks, or until a healthy state was reached that required euthanasia). For mechanistic studies, treatment was used for one, two, or three weeks, followed by euthanasia 48 hours after the final dose. Peritoneal dialysis was performed to collect peritoneal cells. To compare the efficiency of IRF5 / IKKβ NPs with current macrophage targeting therapies, one group of mice received treatment with IRF5 / IKKβ NPs carrying 50 μg of mRNA twice a week for three weeks; a second group received oral administration of 15 mg / kg of the PI3Kγ inhibitor IPI-594 (MedKoo Biosciences Inc.) formulated with a solvent (5% 1-methyl-2-pyrrolidinone in polyethylene glycol 400) daily for three weeks; and a third group received intravenous injections of 30 mg / kg of the CSF1R inhibitor pexidartinib (PLX3397, MedKoo Biosciences Inc.) formulated with the same solvent daily for three weeks.

[0177] To model metastatic lung cancer, F-luc was transduced and suspended in 200 μL of RPMI culture medium, resulting in a sample of 2.5 × 10⁶ cells. 4 16F10 cells were taken from a 4-6 week old female albino B6 (C57BL / 6J-Tyr <c-2j>Mice (Jackson Laboratories) were injected and allowed to establish for one week. For survival studies, mice were treated in the posterior orbit with (or without) IRF5 / IKKβ or eGFP NPs carrying 30 μg of mRNA suspended in PBS. Mice were treated for three weeks at three doses per week, or until a healthy state was reached that required euthanasia. For mechanism studies, mice received the same treatment for two weeks. Bronchoalveolar lavage was performed to collect alveolar cells for analysis.

[0178] Mice with gliomas were prepared according to a published protocol (Uhrbom L et al., (2004) Nat Med 10: pp. 1257-1260). Tori DF-1 cells producing RCAS-PDGFβ and RCAS-cre retrovirus were injected intracranially into both brain hemispheres (coordinates: 1 mm caudal from the anterior vertex, 2 mm lateral, 2 mm deep from the dura mater) of Nestin-tv-a / Ink4a-arf- / -;Pten- / - mice (C57BL / 6) at 4-6 weeks postnatal. The tumors were allowed to establish for 2 weeks. On day 15, one hemisphere of the mouse received 10 Gy of radiation, while the unirradiated hemisphere was shielded with lead. The following day, the mice were assigned to either receive posterior orbital injections of IRF5 / IKKβ NPs carrying 30 μg of mRNA (3 doses per week for 3 weeks) or to a PBS control group.

[0179] In vivo bioluminescence imaging. D-luciferin (Xenogen) (15 mg / mL) in PBS was used as a substrate for firefly luciferase imaging. Bioluminescence images were acquired using the Xenogen IVIS Spectrum imaging system (Xenogen). Mice were anesthetized with 2% isoflurane (Forane, Baxter Healthcare) before and during imaging. For ID8-VEGF ovarian tumors, each mouse received an intravenous injection of 300 μg of D-luciferin, and images were acquired 10 minutes later. For B16F10 lung metastatic tumors, mice received an intravenous injection of 3 mg of D-luciferin, and images were acquired 15 minutes later. For brain tumor models, mice received a postorbital injection of 75 mg / kg of D-luciferin, and images were acquired 4 minutes later. Capture times ranged from 10 seconds to 5 minutes.

[0180] In vivo distribution analysis. To determine the in vivo distribution of IRF5 NPs in the ID8-VEGF ovarian tumor model, 7-8 groups of mice received an ip or post-orbital dose of NPs carrying 50 μg of mRNA. 24 hours after injection, whole blood was collected, the mice were euthanized with CO2, and organs (liver, spleen, lungs, kidneys, heart, intestines, pancreas, and diaphragm) were collected. All tissues were stabilized with RNAlater and then frozen on dry ice. Codon-optimized IRF5 mRNA levels in each organ were measured using RT-qPCR.

[0181] Toxicity analysis. To measure the potential in vivo toxicity of repeatedly injected macrophage-targeting NPs, six consecutive doses of IRF5 / IKKβ or eGFP NPs carrying 50 μg of mRNA were intravenously injected into mice (5 / group) over a 3-week course. Controls were untreated. 24 hours after the final injection, mice were anesthetized, blood was collected by post-orbital hemorrhage, and total blood count was determined. Blood was also collected for serological and cytokine profile analysis (performed by Phoenix Central Laboratories, Mukilteo, WA). The animals were then euthanized with CO2, organs were collected, and washed with deionized water before fixation in 4% paraformaldehyde. Tissues were routinely processed, and sections were stained with hematoxylin-eosin. Specimens were interpreted in a blinded manner by committee-accredited staff pathologist Dr. Smitha Pillai MVSc, PhD, DACVP.

[0182] Cytokine assays. Cytokine levels were assessed using the Luminex 200 system at FHCRC Immune Monitoring Shared Resources. For ex vivo studies, cell culture supernatants were collected for measurement of IL-6, IL-12p70, INFγ, and TNFα concentrations. For in vivo studies, plasma concentrations of GM-CSF, INFγ, IL-12p70, IL-2, IL-6, and TNFα were measured.

[0183] Statistical analysis. The statistical significance of observed differences was analyzed using independent two-tailed one-way ANOVA. P-values ​​for each measurement are included in the figures or figure captions. Survival data were characterized using log-rank tests. All statistical analyses were performed using GraphPad Prism software version 6.0 or R software.

[0184] Results. We designed NPs to constitute IVT mRNA transfection of TAM. By utilizing the electrostatic interaction between a cationic poly(β-aminoester) (PbAE) polymer and anionic mRNA, we developed a targeted mRNA delivery system that can introduce robust gene expression in targeted cells (Figure 2A). To improve the stability and translation of mRNA encapsulated on the resulting nanocarrier, we incorporated modified ribonucleotide pseudouridine (ψ) (Kariko K et al., (2008) Mol Ther 16:1833-1840) and 5-methylcytidine (m5C) and used a synthetic version of the message capped with ARCA (anti-reverse cap analog) (Quabius ES et al., (2015) N Biotechnol 32:229-235). mRNA is released from the mRNA-PbAE complex in cells by hydrolysis of the ester bond in the PbAE backbone. Efficient in vivo T cell transfection had been previously demonstrated using this system (Smith TT et al., (2017) Nat Nanotechnol). To direct the nanoparticles to the target TAM and further stabilize the mRNA-PbAE complex contained in the nanoparticles, the dimannose moiety was manipulated on its surface using polyglutamic acid (PGA) as a linker (Figure 2A). The NPs were fabricated using a simple two-step charge-driven self-assembly process. First, the synthetic mRNA was complexed with a positively charged PbAE polymer, thereby condensing the mRNA into a nano-sized complex. Following this step, PGA with dimannose functional groups was added, which shielded the positively charged PbAE-mRNA particles, giving macrophage targeting. The resulting mRNA nanocarriers had a size of 99.8 ± 24.5 nm, a polydispersity of 0.183, and a neutral surface charge (zeta potential of 3.40 ± 2.15 mV, Figures 2B-2C). Transfection efficiency was first tested in mouse bone marrow-derived macrophages (BMDMs) using NPs formulated with green fluorescent protein-coding mRNA (GFP-NPs). Briefly, 50,000 BMDMs were exposed to NPs containing 1 μg of mRNA for 1 hour, followed by flow cytometry measurements of GFP expression the following day.Following single NP application, we routinely transfected 31.9% (±8.5%) of these primary macrophages without reducing their viability (Figures 2E-2F). Surface modification of the particles with dimannose was appropriate because the transfection rate on untargeted (but PGA-coated) nanocarriers decreased to an average of 25% (±2.1%) in this intrinsic phagocytic cell type. The NP selectively targeted the CD11b+, F4 / 80+ macrophage population, with 46% of macrophages transfected and expressing high levels of eGFP (Figure 2D). This high transfection efficiency demonstrates the efficacy of the disclosed system and method in targeted mRNA delivery to TAMs. Based on the results of in vitro screening for candidate transcription factors that induce macrophage polarization, two mRNAs were selected for inclusion in the NP: the first mRNA encodes IRF5, a key member of the IRF family that favors the polarization of macrophages toward the M1 phenotype, and the second mRNA encodes IKKβ, a kinase that phosphorylates and activates IRF5.

[0185] Program immunosuppressive macrophages to a pro-inflammatory phenotype. To induce macrophage polarization, two mRNAs were selected for inclusion in the NPs: the first mRNA encodes IRF5, a key member of the interferon regulatory factor family that favors the polarization of macrophages towards the M1 phenotype (Krausgruber T et al., (2011) Nat Immunol 12:231-238); the second mRNA encodes IKKβ, a kinase that phosphorylates and activates IRF5 (Ren J et al., (2014) Proc Natl Acad Sci USA 111:17438-17443). A ratio of 3 IRF5 mRNAs to 1 IKKβ mRNA was used. Using real-time quantitative PCR specific for NP-delivered (and codon-optimized) IRF5 mRNA, it was found that mRNA expression in macrophages was maximal on day 1, resulting in a 1500-fold increase in IRF5 compared to endogenous factor levels (Figure 2A). As expected, gene expression was transient, but IRF5 levels remained strongly upregulated through day 3 (581-fold increase) to day 5 (87-fold increase) before returning to baseline.

[0186] Nanostring gene expression analysis was used to determine whether IRF5 / IKKβ coding NPs could reprogram M2 macrophages into the therapeutically desirable anti-cancer M1 phenotype. BMDM were first cultured in the presence of interleukin-4 (IL-4) to induce a suppressive M2 phenotype (Figure 2H). Following transfection with control GFP-mRNA nanoparticles or IRF5 / IKKβ mRNA-containing NPs, gene expression profiles were analyzed and compared to inflammatory macrophages. These inflammatory macrophages were separately prepared by exposing BMDM to the TLR4 agonist monophosphorythripide A. Macrophages cultured in suppressive IL-4-containing medium but transfected with IRF5 / IKKβ mRNA NPs showed gene expression profiles similar to those of inflammatory macrophages (Figure 2I). Signature M2 macrophage genes such as serpin b2 and Ccl2 (Jablonski K et al., (2015) Plos One 10:e0145342; Varga T et al., (2016) J Immunol 196:pp. 4771-4782) were strongly downregulated, while key M1 differentiation genes such as Ccl5 (Sica A et al., (2012) J Clin Invest 122:pp. 787-795) were upregulated (Figure 2J, 2K). These data demonstrate that NP-mediated expression of IRF5 and its kinases distorts repressive macrophages toward a pro-inflammatory phenotype.

[0187] [Example 2] Therapeutic effect of the NP-delivering pro-M1 gene on disseminated ovarian cancer. To evaluate this treatment approach in a clinically relevant in vivo testing system, a model reproducing late-stage unresectable ovarian tumors was used in C57BL / 6 mice; these animals were injected with ID8 ovarian cancer cells tagged with luciferase to enable continuous bioluminescence imaging of tumor growth (Liao JB et al., (2015) J Immunother Cancer 3:16; Stephan SB et al., (2015) Nat Biotechnol 33:97-101). The tumors were allowed to establish for two weeks. Up to this stage, the mice had developed small nodules throughout the peritoneal wall and in the mesentery of the intestine. The animals were divided into three groups to receive PBS (control), GFPNP (fake), or IRF5 / IKKβ NP treatment at an ip dose of 100 μg mRNA / mouse / week for nine weeks (Figure 4A). In the IRF5 / IKKβ NP-treated group, disease regression was observed, with eventual elimination in 40% of animals (median overall survival time of 142 days versus 60 days for controls; Figures 4B-4C). To understand the mechanism underlying the IRF5 / IKKβ NP-mediated antitumor effect, we first investigated how mannose receptor targeting exclusively restricts NP interactions to phagocytic cells. Flow cytometry of peritoneal dialysis fluid collected 24 hours after the first administration of dimannose-targeted NPs revealed preferential gene transfection into macrophages and monocytes (mean 37.1% and 15.3%, respectively, Figure 4D), with low or no transfection into off-target cells. Detailed phenotypic and functional analyses of macrophage / monocyte populations in the peritoneum of mice with established ovarian cancer following 3 weeks of treatment with IRF5 / IKKβ nanoparticles or PBS (twice weekly injections) were then performed. Flow cytometry analysis revealed that IRF5 / IKKβ NP reduced the immunosuppressive macrophage population (Ly6C-, F4 / 80+, CD206+) from an average of 2.6% ± 2.1% to 43% ± 15.6% compared to the control group (Figures 4E-4F). Conversely, the proportion of M1-like macrophages increased from 0.5% ± 0.2% to 10.2% ± 4.1% (Figures 4E, 4G).IRF5 gene therapy also affected other immune cell populations. In particular, inflammatory monocytes (CD11b+, Ly6C+, Ly6G−) were more abundant (73.4% ± 3.6% compared to 4.5% ± 1.9% in untreated mice). An interesting finding in all IRF5 NP-treated mice was the presence of multiple focal clusters of lymphocytes within or adjacent to the neoplasm (Figure 4H), indicating that genetic programming of immune-stimulatory macrophages may revive lymphocyte migration and infiltration into solid tumors.

[0188] Peritoneal macrophages were isolated by fluorescence-activated cell sorting and their cytokine secretion was analyzed, detecting a robust increase in the release of pro-inflammatory (anti-tumor) cytokines IL-12 (3.4-fold higher), IFN-γ (8.4-fold higher), and TNF-α (1.5-fold higher), while the level of IL-6, a regulatory cytokine associated with differentiation into selectively activated (M2-like) macrophages, decreased 97-fold; (Figure 4I). Genome expression profiling confirmed a shift towards the M1-like macrophage phenotype in IRF5 / IKKβ nanoparticle-treated mice. Gene expression levels of macrophages cultured ex vivo in MPLA or IL-4 were included to provide reference values for classical M1-like or M2-like macrophages, respectively (Figure 4J).

[0189] Biodistribution and Safety. The distribution of nanoparticles in various organs 24 hours after intraperitoneal injection was then quantified using an RT-qPCR assay designed to detect only nanoparticle-delivered (codon-optimized) IRF5. The highest concentrations of IVT mRNA were found in organs located in the peritoneum, including the liver, spleen, intestine, pancreas, and diaphragm (Figure 5A). Small amounts of particle-delivered mRNA were detected in organs located outside the peritoneum (heart, lungs, kidneys), suggesting that some of the iP-injected nanocarriers entered the systemic circulation. Guided by the distribution data, we then evaluated whether these nanoreagents were biocompatible and safe for repeated administration. Mice were injected with a total of eight doses of IRF5 / IKKβ NP (two 50 μg mRNA doses / week over four weeks, Figure 5B). Mice were euthanized 24 hours after the final dose, their body weight was recorded, blood was collected by post-orbital hemorrhage for serological chemistry, and a complete macroscopic examination was performed. There were no significant differences in body weight between groups. The following tissues—liver, spleen, mesentery, pancreas, stomach, kidney, heart, and lung—were evaluated by a committee-certified staff pathologist. Histopathological evaluation revealed multifocal clusters of lymphocytes within or around tumor lesions in all cases, but no evidence of inflammation or apparent necrosis was observed in tissues without neoplastic cells (Figure 5C). Furthermore, serological chemistry in IRF5 / IKKβ NP-treated mice was comparable to that of PBS-controlled mice, indicating no systemic toxicity (Figure 5D). Since small amounts of IRF5-mRNA were detected systemically in biodistribution studies, parallel experiments were designed to quantify inflammatory cytokines in peripheral blood. Following a single intravenous injection of IRF5 / IKKβ NP, moderate and transient increases in serum levels were measured, with interleukin-6 (IL-6) up to a mean of 26.8 pg / mL (Figure 5E) and tumor necrosis factor-α (TNF-α) up to a mean of 94.7 pg / mL (Figure 5F). Based on previous reports, these levels are 1 / 500th of the levels associated with pathological findings and can therefore be considered safe (Tarrant JM (2010) Toxicol Sci 117: pp. 4-16; Copeland S et al., (2005) Clin Diagn Lab Immunol 12: pp. 60-67).

[0190] Controlling systemic tumor metastasis with intravenous injection of IRF5 / IKKβ nanoparticles. Based on the therapeutic response achieved by direct intraperitoneal administration of IRF5 / IKKβ NPs to treat tumor lesions spread throughout the peritoneum, the next question was whether intravenously injected mRNA nanocarriers could systemically program macrophages to control the transmitted disease. RT-qPCR biodistribution studies revealed that IV-injected nanocarriers preferentially delivered their mRNA cargo to organs with high levels of resident macrophages / phagocytic cells, mostly the spleen, liver, and lungs (Figure 6A). To measure the antitumor response in a clinically relevant in vivo testing system, particles containing IRF5 / IKKβ mRNA were administered to mice with disseminated pulmonary melanoma metastases (Figure 6B). Recent studies have described the fundamental roles of monocytes and macrophages in establishing metastases caused by this disease (Butler KL et al., (2017) Sci Rep 7:45593; Nielsen SR et al., (2017) Mediators Inflamm 2017:9624760), and confocal microscopy confirmed that tumor transplantation is synchronized with phagocytic accumulation in the lungs (Figure 6C). Tumor burden was determined by bioluminescence imaging, and mice with detectable tumors were selected into groups with suitable levels. The groups were then randomly assigned to receive either no treatment (PBS) or intravenous injection of GFP- or IRF5 / IKKβ encapsulated nanoparticles. Only IRF5 / IKKβ nanoparticle therapy substantially reduced tumor burden in the lungs; in fact, IRF5 / IKKβ nanoparticle therapy improved overall survival by an average of 1.3 times (Figures 6D-6E). In parallel experiments, mice were sacrificed 22 days after tumor inoculation, and bioluminescent tumor signals were confirmed by counting lung metastases, while macrophage polarization was evaluated by flow cytometry. The total number of metastases in the lungs of IRF5 / IKKβ NP-treated animals was reduced 8.7 times compared to PBS controls (mean 419 ± 139 metastases; Figures 6F-6G) (mean 40 ± 16 metastases).Flow cytometry of bronchoalveolar lavage fluid cells revealed a strong migration from immunosuppressive (CD206+, MHCII-, CD11c+, CD11blow) macrophages to activated (CD206-, MHCII+, CD11c-, CD11b+) phagocytes (Figures 6H-6I).

[0191] We programmed tumor suppressor phagocytic cells to treat gliomas. Regarding the third in vivo trial system, we investigated gliomas, a difficult-to-manage cancer type in which M2-like macrophages represent the majority of non-neoplastic cells and promote tumor growth and invasion (Hambardzumyan D et al., (2016) Nat Neurosci 19:20-27). Currently, the standard treatment for this disease is radiotherapy, which unfortunately only provides temporary stabilization and reduction of symptoms and extends the median survival by 3 months (Mann J et al., (2017) Front Neurol 8:748). To reproduce the genetic events and subsequent molecular evolution of this disease, we used the RCAS-PDGF-B / Nestin-Tv-a;Ink4a / Arf- / -;Pten- / - transgenic mouse model of PDGFβ-driven glioma (PDG mouse (Hambardzumyan D et al., (2009) Transl Oncol 2:89-95; Quail DF et al., (2016) Science 352:aad3018)). Brain tissue was stereotactically injected with a mixture of DF-1 cells transfected with RCAS-PDGFβ or RCAS-cre retrovirus (1:1 mixture, 2 μL). Overexpression of the PDGFβ oncogene and the absence of the tumor suppressor genes Ink4a-arf and Pten in glioma progenitor cells led to the formation of 4-5 mm diameter tumors with nearly complete penetrant within 21 days (Figure 7A) (as previously established (Hambardzumyan D et al., (2009) Transl Oncol 2:89-95)). Immunofluorescence confirmed the presence of tumor-infiltrating (CD68+) macrophages (Figure 7B, shown in the third panel from the left) in established gliomas (shown in the second panel from the left). Flow cytometry revealed that the F4 / 80+, CD11b+ macrophage population accounted for 32.8% of all cells in the tumor, a figure nine times higher than that seen in healthy control mice of the same age (3.7%) (Figure 7C). PDG mice in the experiment expressed firefly luciferase linked to key oncogene promoters.Bioluminescence derived from this reporter has been shown to have a positive correlation with tumor malignancy (Uhrbom L et al., (2004) Nat Med 10: pp. 1257-1260), and therefore, tumor growth was monitored every four days after the start of treatment using this bioluminescence. IRF5 / IKKβ NP was first tested as monotherapy: PDG mice received intravenous infusions of either NP loaded with IRF5 / IKKβ mRNA or 9 doses of PBS in the control group (3 doses / week for 3 weeks). IRF5 / IKKβ NP treatment slightly suppressed tumor progression (resulting in an average survival advantage of only 5 days compared to untreated controls; Figure 7D). However, combining radiotherapy as standard treatment with IRF5 / IKKβ NP injection substantially reduced tumor growth and more than doubled survival time in treated mice compared to the PBS control group (52 days vs. 25 days, respectively, Figures 7E-7F).

[0192] In conclusion, in vivo results from three preclinical solid tumor models demonstrate that nanoparticles, administered locally or systemically, can deliver genes encoding master regulators of macrophage polarization, thereby reprogramming immunosuppressive macrophages into a tumor-clearing phenotype.

[0193] Mouse-to-human macrophage replacement. To confirm that the data obtained in mice is applicable to treating human diseases, we fabricated nanoparticles (huIRF5 NPs) that deliver IVT mRNA encoding human IRF5 and IKKβ. These nanocarriers were tested using the human monocyte cell line THP-1 as an established M1 and M2 macrophage polarization model (Li C et al., (2016) Sci Rep 6:21044; Surdziel E et al., (2017) Plos One 12:e0183679). M2 type macrophages were created by treating THP-1 cells with PMA and polarizing them with IL-4 and IL-13 (Figure 8A). To confirm that huIRF5 NPs are functional and activate the IRF pathway, THP1-Lucia® ISG cells were transfected with nanoparticles loaded with huIRF5 / IKKβ or GFP control mRNA. THP1-Lucia® ISG cells secrete a fluorescent Lucia reporter under the control of an IRF-inducible promoter. This composite promoter contains five IFN-stimulated response elements (ISREs) fused to the ISG54 minimal promoter, which is unresponsive to NF-κB or AP-1 pathway activators. As a result, THP1-Lucia® ISG cells enable monitoring of the IRF pathway by determining Lucia luciferase activity. The huIRF5 NP was found to strongly upregulate luciferase expression in M2-polarized THP-1 cells, demonstrating the functionality of the mRNA construct in human cells (Figures 8B-8C). To determine whether IRF5 pathway activation can reprogram M2-polarized THP-1 cells to an M1-like phenotype, the secretion of the pro-inflammatory cytokine IL-1β following NP transfection was measured. IL-1β production was significantly increased in huIRF5 NP-transfected THP-1 cells versus untransfected controls (mean 21-fold increase; P<0.0001, Figure 8D), and correlated with robust upregulation of the M1 macrophage cell surface marker CD80 (10.9-fold increase in MFI, P<0.0001) (Figure 8E).

[0194] List of sequence numbers Nucleic acid sequences described herein are shown using standard letter abbreviations for nucleotide bases, as defined in 37 C.FR §1.822. Only one strand of each nucleic acid sequence is shown, but complementary strands are understood to be included in embodiments where appropriate. Sequence IDs 55, 58, 61, 64, 71, 73 and 79 are not used in this sequence listing. The attached sequence listing shows the following sequences:

[0195] [Table 3] TIFF0007844410000004.tif236166TIFF0007844410000005.tif245170TIFF0007844410000006.tif22170

[0196] As those skilled in the art will understand, each embodiment disclosed herein essentially consists of or can consist of, its specific described elements, steps, components, or constituents. Therefore, “include” or “including” should be interpreted as a repetition of “include, consist of, or essentially consist of.” The transitional words “comprise” or “comprises” mean “includes,” but can also include, even in large quantities, elements, steps, components, or constituents not explicitly stated. The transitional phrase “consists of” excludes any elements, steps, components, or constituents not explicitly stated. The transitional phrase “essentially consists of” limits the scope of the embodiment to the explicitly stated elements, steps, components, or constituents, and to elements, steps, components, or constituents that do not physically affect the embodiment. Physical effects are considered to cause a statistically significant reduction in the ability to treat the mouse model of ovarian cancer described in Example 2.

[0197] Unless otherwise indicated, all figures used in the specification and claims to represent the quantities and properties of components such as molecular weight, reaction conditions, and other factors should be understood to be modified in all examples by "approximately." Therefore, unless otherwise indicated, the numerical parameters expressed in the specification and the appended claims are approximations that may vary depending on the desired properties sought by the invention. At the very least, and not as an attempt to limit the applicability of the doctrines of the claims equivalent, each numerical parameter should be interpreted at least in light of the reported number of significant figures and by applying the ordinary method of rounding. Where further clarity is required, “approximately” has the meaning that a person skilled in the art would reasonably expect the term to have when used in conjunction with a given number or range, namely, within the range of ±20%; ±19%; ±18%; ±17%; ±16%; ±15%; ±14%; ±13%; ±12%; ±11%; ±10%; ±9%; ±8%; ±7%; ±6%; ±5%; ±4%; ±3%; ±2%; or ±1% of the given value.

[0198] While the ranges of numbers and parameters representing the broad scope of this invention are approximations, the numerical values ​​shown in specific examples are reported as accurately as possible. However, any numerical value inherently contains a certain degree of error that inevitably arises from the standard deviation found in each test measurement.

[0199] In the context of describing the present invention (particularly in the context of the following claims), “a,” “an,” “the,” and similar reference subjects should be interpreted as including both singular and plural unless otherwise specified herein or clearly inconsistent with the context. The enumeration of value ranges herein is intended solely as a simple way to refer individually to each separate value that falls within that range. Unless otherwise specified herein, each individual value is incorporated into the specification as if it were individually enumerated herein. All methods described herein may be performed in any appropriate order unless otherwise specified herein or clearly inconsistent with the context. The use of any and all embodiments or exemplary language provided herein (e.g., “such as”) is intended solely to better illustrate the present invention and does not limit the scope of the present invention as otherwise claimed. Any language in the specification should be interpreted as referring to any unclaimed elements essential to the execution of the present invention.

[0200] The grouping of other elements or embodiments of the Invention disclosed herein should not be construed as limiting. Members of each group may be referred to and claimed individually or in any combination with other members of that group or other elements found herein. It is anticipated that one or more members of a group may be included in or excluded from a group for convenience and / or patentability reasons. In the event of any such inclusion or exclusion, the specification shall be deemed to contain a group that has been amended and therefore satisfies the written description of all Markush groups used in the appended claims.

[0201] Certain embodiments of the present invention, including the best mode known to the inventors for carrying out the invention, are described herein. Naturally, variations of these described embodiments will be apparent to those skilled in the art by reading the preceding description. The inventors anticipate that those skilled in the art will use such variations as needed, and they intend that the invention will be carried out in ways other than those specifically described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter enumerated in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements in any conceivable variation is incorporated herein unless otherwise indicated herein or otherwise clearly contradicts the context.

[0202] Furthermore, numerous patents, publications, journal articles, and other documents have been referenced throughout this specification (references). Each of these references is individually incorporated into this specification by reference for the teaching it refers to.

[0203] Finally, it should be understood that the embodiments of the present invention disclosed herein illustrate the principles of the present invention. Other modifications that can be used are within the scope of the invention. Therefore, as an example, but not limiting, other configurations of the present invention may be used in accordance with the teachings of this specification. Thus, the present invention is not limited to the invention shown and described herein.

[0204] The features described herein are provided as examples and are intended solely to empirically examine preferred embodiments of the invention, and are presented to provide what is considered to be the most useful and readily understandable description of the principles and concepts of various embodiments of the invention. In this regard, no attempt has been made to describe the structural details of the invention in more detail than is necessary for a basic understanding of the invention, and the description is made in conjunction with drawings and / or examples that will make it clear to those skilled in the art how some forms of the invention may be put into practice.

[0205] In the following embodiments, unless explicitly and clearly modified, it is meant and intended that the definitions and descriptions used herein shall prevail in any future interpretation, or where the application of the meaning renders any interpretation meaningless or essentially meaningless. Where an interpretation of a term is considered meaningless or essentially meaningless, the definition should be taken from a dictionary known to those skilled in the art, such as Webster's Dictionary, Third Edition or the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

Claims

1. A pharmaceutical composition for treating tumors by in vivo altering the activity state of immune cells, The pharmaceutical composition comprises nanoparticles containing a polynucleotide encoding one or more interferon regulatory factors (IRFs) that activate the expression of type I interferon (IFN), and a polynucleotide encoding a molecule that phosphorylates the one or more IRFs. Immune cells include tumor-associated macrophages (TAMs), One or more encoded IRFs that activate IFN expression include IRF8 and / or a fusion protein of IRF7 and IRF3, and The pharmaceutical composition further comprises nanoparticles, dimannose bound to a coating on the nanoparticles, or nanoparticles, a targeting ligand bound to a coating on the nanoparticles.

2. The pharmaceutical composition according to claim 1, further comprising a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 1, wherein the active state is changed from an inactive state to an active state.

4. One or more encoded IRFs activate IFN expression, IRF8 having a sequence represented by sequence number 11, 16, or 17, or a sequence having greater than 90%, 95%, or 98% identity with the sequence represented by sequence number 11, 16, or 17, and / or A fusion protein of IRF7 and IRF3 having the sequence represented by SEQ ID NO: 15, or a sequence having greater than 90%, 95%, or 98% identity to the sequence represented by SEQ ID NO:

15. A pharmaceutical composition according to claim 1, comprising:

5. The pharmaceutical composition according to claim 1, wherein IRF8 has a sequence represented by Sequence ID No. 11 containing the K310R mutation.

6. The pharmaceutical composition according to claim 1, wherein the IRF7 / IRF3 fusion protein comprises an N-terminal IRF7 DNA-binding domain (DBD) and a constitutive activity domain (CAD), and a C-terminal IRF3 NES (nuclear export signaling domain) and an association domain.

7. The pharmaceutical composition according to claim 1, wherein the IRF7 / IRF3 fusion protein further comprises a phosphorylation-mimicking mutation in the IRF3 association domain.

8. The pharmaceutical composition according to claim 1, wherein the encoded molecule that phosphorylates one or more IRFs contains IKKβ.

9. The pharmaceutical composition according to claim 8, wherein the encoded IKKβ has a sequence represented by SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22, or a sequence having greater than 90%, 95%, or 98% identity with the sequence represented by SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO:

22.

10. Polynucleotides The sequence represented by SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44, or The pharmaceutical composition according to claim 3, comprising sequences having greater than 90%, 95%, or 98% identity with sequences represented by sequence numbers 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44.

11. The pharmaceutical composition according to claim 8, wherein one or more polynucleotides encoding IRF and polynucleotides encoding IKKβ that activate IFN expression are encapsulated in the same nanoparticle or in different nanoparticles.

12. The pharmaceutical composition according to claim 1, further comprising a polynucleotide encoding an anti-oncogene including p53, RB, BRCA1, E1A, bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-I VEGF, angiostatin, oncostatin, endostatin, GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-γ, TNFα and / or HSV-tk.

13. The pharmaceutical composition according to claim 1, wherein the tumor is an ovarian cancer tumor, a glioblastoma tumor, or a metastatic lung cancer tumor.

14. The pharmaceutical composition according to claim 1, which is used in combination with a cancer vaccine, chimeric antigen receptor (CAR) immunotherapy, chemotherapy, radiotherapy, hormone therapy, signaling inhibitors, gene expression regulators, apoptosis inducers, angiogenesis inhibitors, or monoclonal antibodies that deliver toxic molecules.

15. The pharmaceutical composition according to claim 1, wherein macrophages are converted from an M2 phenotype to an M1 phenotype.

16. The pharmaceutical composition according to claim 1, wherein the nanoparticles further comprise a polynucleotide encoding a glucocorticoid-induced leucine zipper (GILZ).

17. The pharmaceutical composition according to claim 1, wherein the nanoparticles comprise a positively charged poly(β)aminoester (PBAE) core, a star-shaped polymer, a polyglutamic acid coating, a hyaluronic acid coating, and / or liposomes.

18. The pharmaceutical composition according to claim 17, wherein the nanoparticles comprise a positively charged PBAE core, and the polynucleotides are encapsulated within the positively charged PBAE core.

19. The pharmaceutical composition according to claim 1, wherein the diameter of the nanoparticles is <130 nm.

20. The pharmaceutical composition according to claim 1, wherein the polynucleotide comprises in vitro transcribed mRNA.

21. The pharmaceutical composition according to claim 1, wherein one or more encoded IRFs that activate IFN expression lack a functional autoinhibitory domain.

22. The pharmaceutical composition according to claim 1, wherein one or more encoded IRFs that activate IFN expression lack a functional nuclear export signal (NES).

23. The pharmaceutical composition according to claim 1, which is administered topically.

24. The pharmaceutical composition according to claim 23, which is administered intraperitoneally or intracranially.

25. The pharmaceutical composition according to claim 1, which is administered systemically.

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