Engineered cells and their uses

Abstract

The present disclosure relates to antigen-presenting cells and their use for treating sepsis. In some embodiments, disclosed herein are antigen-presenting cells comprising lipid-based nanoparticles comprising a recombinant polynucleotide comprising a first nucleic acid encoding an antimicrobial peptide, a second nucleic acid encoding cathepsin B, and a third nucleic acid encoding a linker, and a vitamin-lipid.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 898,846, filed September 11, 2019, which is expressly incorporated herein by reference.

[0002] Statement Regarding Federally Sponsored Research This invention was made with government support under Grant No. R35GM119679 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] The present disclosure relates to engineered cells and uses thereof. [Background technology]

[0004] Sepsis was once considered an uncontrolled inflammatory response to pathogens. However, after more than 40 failed clinical trials of anti-inflammatory drugs, researchers are reevaluating sepsis treatment approaches. Recent clinical data reveal that although more than 60% of sepsis patients survive the initial inflammatory storm, they rapidly progress to a longer, immunosuppressed state characterized by immune cell paralysis and death, an inability to eliminate invading pathogens, increased susceptibility to hospital-acquired infections, and high mortality rates. Therefore, what is needed are novel compositions and methods for manipulating cells and methods for treating diseases (e.g., sepsis). Summary of the Invention

[0005] In some embodiments, disclosed herein is an antigen-presenting cell, comprising: lipid-based nanoparticles, the lipid-based nanoparticles comprising: A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid encoding cathepsin B, and a recombinant polynucleotide comprising a third nucleic acid encoding a linker; Vitamin-lipid and antigen-presenting cells.

[0006] In some embodiments, the first nucleic acid and the second nucleic acid are linked by a third nucleic acid.

[0007] In some embodiments, the recombinant polynucleotide is encapsulated by a vitamin-lipid.

[0008] In some embodiments, the recombinant polynucleotide comprises RNA or DNA.

[0009] In some embodiments, the antimicrobial peptide comprises the sequence of SEQ ID NO:1.

[0010] In some embodiments, the first nucleic acid comprises the sequence of SEQ ID NO:2.

[0011] In some embodiments, the second nucleic acid comprises the sequence of SEQ ID NO:4.

[0012] In some embodiments, the linker comprises a cathepsin B-sensitive linker. In some embodiments, the third nucleic acid comprises the sequence of SEQ ID NO:6.

[0013] In some embodiments, the recombinant polynucleotide comprises the sequence of SEQ ID NO:8.

[0014] In some embodiments, the vitamin-lipid comprises a vitamin moiety, and the vitamin moiety comprises vitamin B3, vitamin C, vitamin D, vitamin E, vitamin H, or a derivative thereof. In some embodiments, the vitamin moiety is vitamin C.

[0015] In some embodiments, the vitamin-lipid is selected from the group consisting of: [ka] In the formula, R is [ka] is.

[0016] In some embodiments, the antigen-presenting cells comprise macrophages or dendritic cells. In some embodiments, the macrophages comprise bone marrow-derived macrophages or monocyte-derived macrophages. In some embodiments, the dendritic cells comprise bone marrow-derived dendritic cells, monocyte-derived dendritic cells, conventional dendritic cell-1, or conventional dendritic cell-2.

[0017] In some embodiments, disclosed herein is a method of treating sepsis, comprising administering to a subject one or more antigen-presenting cells, wherein the antigen-presenting cells: nanoparticles, the nanoparticles comprising: A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid encoding cathepsin B, and a recombinant polynucleotide comprising a third nucleic acid encoding a linker; a vitamin-lipid mixture.

[0018] In some embodiments, the antigen-presenting cells are obtained from a subject. In some embodiments, the subject comprises a human. In some embodiments, the human has or is suspected of having sepsis.

[0019] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. [Brief explanation of the drawings]

[0020] [Figure 1A]Schematic diagram of adoptive macrophage transfer and chemical structure of vitamin-derived lipids. a) Construction of MAC for sepsis therapy. MAC represents macrophages loaded with antimicrobial peptides / cathepsin B in lysosomes. AMP-CatB mRNA is encapsulated in vitamin C lipid nanoparticles (VCLNPs) and delivered to macrophages. The mRNA is translated in the reticulum and translocated into lysosomes. Within the lysosome, the cleavable linker is cleaved by lysosomal CatB, releasing AMP-IB367. After phagosomes carrying MDR bacteria fuse with lysosomes, the ingested MDR bacteria are eradicated by the pre-stored AMP-IB367. b) Chemical structures of vitamin-derived lipids, including VB3-lipid, VC-lipid, VD-lipid, VE-lipid, and VH-lipid. [Figure 1B] Schematic diagram of adoptive macrophage transfer and chemical structure of vitamin-derived lipids. a) Construction of MAC for sepsis therapy. MAC represents macrophages loaded with antimicrobial peptides / cathepsin B in lysosomes. AMP-CatB mRNA is encapsulated in vitamin C lipid nanoparticles (VCLNPs) and delivered to macrophages. The mRNA is translated in the reticulum and translocated into lysosomes. Within the lysosome, the cleavable linker is cleaved by lysosomal CatB, releasing AMP-IB367. After phagosomes carrying MDR bacteria fuse with lysosomes, the ingested MDR bacteria are eradicated by the pre-stored AMP-IB367. b) Chemical structures of vitamin-derived lipids, including VB3-lipid, VC-lipid, VD-lipid, VE-lipid, and VH-lipid. [Figure 2]Synthesis of VB3-lipid: Compound 1 (150 mg, 0.23 mmole) was dissolved in a mixture of 2 mL of CHCl and 2 mL of DMF. Vitamin B3 derivative (62 mg, 0.21 mmole), EDC (87 mg, 0.46 mmole), and DMPA (10 mg) were added to the solution. The mixture was stirred overnight at room temperature. The reaction mixture was purified by column chromatography using a CombiFlash RF system with a RediSep Gold Resolution silica column (Teledyne Isco) with a gradient elution (CHCl and ultra) of 100% CHCl to 0% CHCl (ultra: CHCl / MeOH / NHOH = 75 / 22 / 3 by volume) to obtain 80 mg of colorless oil VB3-lipid in 37% yield. 1H NMR (400MHz, CDCl3): δ=10.75(1H,s),9.92(1H,s),9.17-9.16(1H,d,J=4),8 .94(1H,s),8.09-8.06(1H,t,J=4),6.20(1H,s),4.88-4.85(2H,t,J=4),4.06 -4.03(2H,t,J=4),2.47(11H,m),2.31-2.28(2H,t,J=4),2.13(2H,s),1.64- 1.63(7H,m),1.45(12H,m),1.26(56H,s),0.89-0.87(9H,t,J=4).MS(m / z):M+ Calculated for C57H109N4O3: 897.8494; Found: 897.8496. [Figure 3]Synthesis of VC-lipid: Compound 1 (150 mg, 0.23 mmole) was dissolved in 2 mL of CHCl. ​​Vitamin C derivative (77 mg, 0.23 mmole), EDC (87 mg, 0.46 mmole), and DMPA (10 mg) were added to the solution. The mixture was stirred overnight at room temperature. The reaction mixture was purified by column chromatography using a CombiFlash RF system with a RediSep Gold Resolution silica column (Teledyne Isco) with a gradient elution (CHCl and ultra) of 100% CHCl to 80% CHCl (ultra: CHCl / MeOH / NHOH = 75 / 22 / 3 by volume) to obtain 105 mg of colorless oil VC-lipid in 44% yield. 1H NMR (400MHz, CDCl3): δ=7.40-7.25(10H,m),5.26-5.13(4H,m),4.67(1H,s),4.33-4.08(3H,m),2.6 5(1H,s),2.42(12H,m),1.67-1.60(13H,m),1.28(57H,s),0.92-0.89(9H,t,J=4).MS(m / z):[M+H]+ Calculated value of C65H111N2O7: 1031.8391; Actual value: 1031.8379. [Figure 4]Synthesis of VD-lipid: Compound 1 (150 mg, 0.23 mmole) was dissolved in 2 mL of CHCl. ​​Vitamin D (83 mg, 0.23 mmole), EDC (87 mg, 0.46 mmole), and DMPA (10 mg) were added to the solution. The mixture was stirred overnight at room temperature. The reaction mixture was purified by column chromatography using a CombiFlash RF system with a RediSep Gold Resolution silica column (Teledyne Isco) with a gradient elution (CHCl and ultra) of 100% CHCl to 85% CHCl (ultra: CHCl / MeOH / NHOH = 75 / 22 / 3 by volume), yielding 40 mg of colorless oil VD-lipid in 16% yield. 1H NMR (400MHz, CDCl3): δ=6.22-6.19(1H,d,J=12),6.04-6.02(1H,d,J=8),5.06(1H,s),4.94(1H,s),4.84(1H,s),2.82-2.56(12H,m ),2.38-2.28(4H,m),1.99-1.96(5H,m),1.67-1.49(15H,m),1.30-1.26(71H,m),0.93-0.87(21H,m),0.54(2H,s).MS(m / z):[M+H]+ Calculated value of C72H135N2O2: 1060.0524; Actual value: 1060.0529. [Figure 5]Synthesis of VE-lipid: Compound 1 (150 mg, 0.23 mmole) was dissolved in 2 mL of CHCl. ​​Vitamin E (99 mg, 0.23 mmole), EDC (87 mg, 0.46 mmole), and DMPA (10 mg) were added to the solution. The mixture was stirred overnight at room temperature. The reaction mixture was purified by column chromatography using a CombiFlash RF system with a RediSep Gold Resolution silica column (Teledyne Isco) with a gradient elution (CHCl and ultra) of 100% CHCl to 85% CHCl (ultra: CHCl / MeOH / NHOH = 75 / 22 / 3 by volume), yielding 66 mg of colorless oil VE-lipid in 26% yield. 1H NMR(400MHz,CDCl3):δ=2.83-2.57(14H,m),2.08(3H,s),2.00(3H,s),1.96(3H,s),1.81(4H,m),1.6 2(5H,m),1.54-1.52(11H,m),1.28-1.23(67H,m),1.14(7H,m),0.89-0.84(24H,m).MS(m / z):[M+H]+ Calculated value of C74H141N2O3: 1106.0942; Actual value: 1106.0944. [Figure 6]Synthesis of VH-lipid: Compound 1 (100 mg, 0.15 mmole) was dissolved in 3 mL of THF. NHS (50 mg, 0.43 mmole) and DCC (80 mg, 0.39 mmole) were added to the solution, which was stirred overnight. Vitamin H derivative (140 mg, 0.46 mmole) and 200 μL of trimethylamine were added to the solution. The mixture was stirred overnight at room temperature. The reaction mixture was purified by column chromatography using a CombiFlash RF system with a RediSep Gold Resolution silica column (Teledyne Isco) with a gradient elution (CHCl and ultra) of 100% CHCl to 75% CHCl (ultra: CHCl / MeOH / NHOH = 75 / 22 / 3 by volume), to obtain 60 mg of colorless oil VH-lipid in 41% yield. 1H NMR (400MHz, CDCl3): δ=7.11(1H,s),6.70(1H,s),5.98(1H,s),4.52-4.49(1H,t,J=4),4.33-4.30(1H,t,J=4),3.27-3.28(4H,m),2. 75-2.60(11H,m),2.25-2.19(4H,m),1.74-1.64(11H,m),1.51-1.49(11H,m),1.28(59H,s),0.90-0.87(9H,t,J=4).MS(m / z):[M+H]+ Calculated value of C58H115N6O3S: 975.8751; Actual value: 975.8629. [Figure 7A] Screening and characterization of VLNPs. Size. Data are mean ± sd, triplicates. [Figure 7B] Screening and characterization of VLNPs. PDI. Data are mean ± sd, triplicates. [Figure 7C] Screening and characterization of VLNPs. Encapsulation efficiency. Data are mean ± sd, triplicates. [Figure 7D] Screening and characterization of VLNPs. Zeta potential of VLNPs. Data are mean ± sd, triplicates. [Figure 7E] Screening and characterization of VLNPs. Orthogonal array table L16(4)4 and Kn* values. [Figure 7F] Screening and characterization of VLNPs. Cryo-TEM images of optimal VLNP formulations (scale bar = 50 nm). [Figure 8A] Screening and characterization of vitamin-lipid nanoparticles (VLNPs). mRNA delivery efficiency of VLNPs in RAW264.7 cells. Data are mean ± SD, n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant (two-tailed Student's t-test). [Figure 8B] Screening and characterization of vitamin-lipid nanoparticles (VLNPs). Expression kinetics of mRNA delivered by VLNPs in RAW264.7 cells. Data are mean ± SD, n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant (two-tailed Student's t-test). [Figure 8C] Screening and characterization of vitamin-lipid nanoparticles (VLNPs). First round of characterization: four levels of components and effect trends of each VLNP. [Figure 8D] Screening and characterization of vitamin-lipid nanoparticles (VLNPs). Formulation table for validation of predicted formulations and second round of characterization: VC-lipid:mRNA mass ratio. [Figure 8E] Screening and characterization of vitamin-lipid nanoparticles (VLNPs). Fold change in luminescence intensity in two rounds of characterization. Data are mean ± sd, n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant (two-tailed Student's t-test). [Figure 8F] Screening and characterization of vitamin-lipid nanoparticles (VLNPs). Characterization of optimal VLNP formulations including size distribution, polydispersity index (PDI), encapsulation efficiency, zeta potential, and cryo-TEM images (scale bar = 100 nm). Data are mean ± sd, n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant (two-tailed Student's t-test). [Figure 8G] Screening and characterization of vitamin-lipid nanoparticles (VLNPs). Confocal microscopy of RAW264.7 cells incubated with VLNPs encapsulating eGFP-CatB mRNA (scale bar = 10 μm). eGFP-CatB (green) and LysoTracker® Red DND-99 (red) colocalized to lysosomes with a Pearson correlation coefficient of 0.91 ± 0.15. [Figure 8H] Screening and characterization of vitamin-lipid nanoparticles (VLNPs). Intracellular viability of MDR Staphylococcus aureus (MDRSA) in RAW264.7 cells exposed to PBS (PBS-RAW), free AMP-CatB mRNA (Fr-RAW), empty VCLNP (Em-RAW), AMP-CatB mRNA VCLNP / CatB inhibitor II (In-RAW), and AMP-CatB mRNA VCLNP (MAC-RAW). Data are mean ± SD, n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant (two-tailed Student's t-test). [Figure 9A] The therapeutic effect of MAC-RAW in MDRSA induced septic mice with immunosuppression. Bacterial load in the blood 24 hours after cell transplantation. The number of mice in the PBS, PBS-RAW (ip + iv), MAC-RAW (ip), and MAC-RAW (ip + iv) groups was 8, 10, 10, and 12, respectively. Data are mean ± sd. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant (two-tailed Student's t-test). ND, not detected. [Figure 9B] The therapeutic effect of MAC-RAW in MDRSA-induced septic mice with immunosuppression. Survival rate of septic mice. The number of mice in the PBS, PBS-RAW (ip + iv), MAC-RAW (ip), and MAC-RAW (ip + iv) groups was 8, 10, 10, and 12, respectively. [Figure 9C]The therapeutic effect of MAC-RAW in MDRSA induced septic mice with immunosuppression. Body weight (BW), white blood cell (WBC), and lymphocyte (LYM) counts of septic mice were shown. BW. The number of mice in the PBS, PBS-RAW (ip + iv), MAC-RAW (ip), and MAC-RAW (ip + iv) groups was 8, 10, 10, and 12, respectively. Data are mean ± sd. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant (two-tailed Student's t-test). ND, not detected. [Figure 9D] The therapeutic effect of MAC-RAW in MDRSA induced septic mice with immunosuppression. Body weight (BW), white blood cell (WBC), and lymphocyte (LYM) counts of septic mice were measured. WBC. The number of mice in the PBS, PBS-RAW (ip + iv), MAC-RAW (ip), and MAC-RAW (ip + iv) groups was 8, 10, 10, and 12, respectively. Data are mean ± sd. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant (two-tailed Student's t-test). ND, not detected. [Figure 9E] The therapeutic effect of MAC-RAW in MDRSA induced septic mice with immunosuppression. Body weight (BW), white blood cell (WBC), and lymphocyte (LYM) counts of septic mice were compared. LYM. The number of mice in the PBS, PBS-RAW (ip + iv), MAC-RAW (ip), and MAC-RAW (ip + iv) groups was 8, 10, 10, and 12, respectively. Data are mean ± sd. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant (two-tailed Student's t-test). ND, not detected. [Figure 9F] The therapeutic effect of MAC-RAW in MDRSA-induced septic mice with immunosuppression. The bacterial load in the blood of each mouse treated with MAC-RAW (ip + iv) was measured. The number of mice in the PBS, PBS-RAW (ip + iv), MAC-RAW (ip), and MAC-RAW (ip + iv) groups was 8, 10, 10, and 12, respectively. [Figure 9G] The therapeutic effect of MAC-RAW in MDRSA-induced septic mice with immunosuppression. The bacterial load in the blood of each mouse treated with MAC-RAW (ip + iv) was measured. The number of mice in the PBS, PBS-RAW (ip + iv), MAC-RAW (ip), and MAC-RAW (ip + iv) groups was 8, 10, 10, and 12, respectively. [Figure 10A] Screening of VLNPs in BMDMs and intracellular survival of MDR bacteria in BMDMs. F4 / 80, mature macrophage marker, positive cells (83.5 ± 0.7%). *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant (two-tailed Student's t test). Data in this figure are means ± sd, n = 3 independent experiments. [Figure 10B] Screening of VLNPs in BMDMs and intracellular survival of MDR bacteria in BMDMs. mRNA delivery efficiency of VLNPs in BMDMs. *P<0.05, **P<0.01, ***P<0.001, ns, not significant (two-tailed Student's t-test). Data in this figure are means ± sd, n=3 independent experiments. [Figure 10C] Screening of VLNPs in BMDMs and intracellular survival of MDR bacteria in BMDMs. Expression kinetics of mRNA delivered by VLNPs in BMDMs. *P<0.05, **P<0.01, ***P<0.001, ns, not significant (two-tailed Student's t-test). Data in this figure are means ± sd, n=3 independent experiments. [Figure 10D]Screening of VLNPs in BMDM and intracellular survival of MDR bacteria in BMDM. PBS (PBS-BMDM), free AMP-CatB mRNA (Fr-BMDM), empty VCLNP (Em-BMDM), AMP-CatB mRNA VCLNP / CatB inhibitor II (In-BMDM), and AMP-CatB mRNA VCLNP (MAC-BMDM) were used. d) MDRSA, f) MDR E. coli. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). Data in this figure are means ± SD, n=3 independent experiments. [Figure 10E] Screening of VLNPs in BMDMs and intracellular survival of MDR bacteria in BMDMs. Percentage of BMDMs normalized to the PBS-BMDM group at 12 hours. MDRSA. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). Data in this figure are means ± SD, n=3 independent experiments. [Figure 10F] Screening of VLNPs in BMDM and intracellular survival of MDR bacteria in BMDM. PBS (PBS-BMDM), free AMP-CatB mRNA (Fr-BMDM), empty VCLNP (Em-BMDM), AMP-CatB mRNA VCLNP / CatB inhibitor II (In-BMDM), and AMP-CatB mRNA VCLNP (MAC-BMDM) were used. d) MDRSA, f) MDR E. coli. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). Data in this figure are means ± SD, n=3 independent experiments. [Figure 10G] Screening of VLNPs in BMDMs and intracellular survival of MDR bacteria in BMDMs. Percentage of BMDMs normalized to the PBS-BMDM group at 12 hours. MDR E. coli. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). Data in this figure are means ± SD, n=3 independent experiments. [Figure 10H] Screening of VLNPs in BMDMs and intracellular survival of MDR bacteria in BMDMs. The intracellular survival of VLNPs encapsulating AMP-CatB mRNA in BMDMs was measured by MTT assay. *P<0.05, **P<0.01, ***P<0.001, ns, not significant (two-tailed Student's t-test). Data in this figure are mean ± sd, n=3 independent experiments. [Figure 11A] The therapeutic effect of MAC-BMDM in MDRSA-induced septic mice with immunosuppression. Bacterial load in the blood 24 hours after cell transplantation. The number of mice in the PBS, PBS-BMDM, and MAC-BMDM groups was 8, 10, and 12, respectively. Data are mean ± standard deviation (SD). *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). ND, not detected. [Figure 11B] The therapeutic effect of MAC-BMDM in MDRSA-induced septic mice with immunosuppression. Survival rate of mice with sepsis. The number of mice in the PBS, PBS-BMDM, and MAC-BMDM groups was 8, 10, and 12, respectively. [Figure 11C] The therapeutic effect of MAC-BMDM in MDRSA induced sepsis in mice with immunosuppression. BW, WBC, and LYM of mice with sepsis. BW. The number of mice in the PBS, PBS-BMDM, and MAC-BMDM groups was 8, 10, and 12, respectively. Data are mean ± sd. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). ND, not detected. [Figure 11D] The therapeutic effect of MAC-BMDM in MDRSA induced sepsis in mice with immunosuppression. BW, WBC, and LYM in mice with sepsis. WBC. The number of mice in the PBS, PBS-BMDM, and MAC-BMDM groups was 8, 10, and 12, respectively. Data are mean ± sd. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). ND, not detected. [Figure 11E] The therapeutic effect of MAC-BMDM in MDRSA induced sepsis in mice with immunosuppression. BW, WBC, and LYM of mice with sepsis. LYM. The number of mice in the PBS, PBS-BMDM, and MAC-BMDM groups was 8, 10, and 12, respectively. Data are mean ± sd. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). ND, not detected. [Figure 11F] The therapeutic effect of MAC-BMDM on MDRSA induced sepsis in mice with immunosuppression. The bacterial load in the blood of each mouse treated with MAC-BMDM was measured. The number of mice in the PBS, PBS-BMDM, and MAC-BMDM groups was 8, 10, and 12, respectively. [Figure 11G] The therapeutic effect of MAC-BMDM on MDRSA induced sepsis in mice with immunosuppression. The bacterial load in the blood of each mouse treated with MAC-BMDM was measured. The number of mice in the PBS, PBS-BMDM, and MAC-BMDM groups was 8, 10, and 12, respectively. [Figure 12A] The therapeutic effect of MAC-BMDM on mixed MDRSA bacteria (Staphylococcus aureus and Escherichia coli) induced septic mice with immunosuppression. Bacterial load in the blood 24 hours after cell transplantation. The number of mice in the PBS, PBS-BMDM, and MAC-BMDM groups was 8, 10, and 12, respectively. Data are mean ± SD. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). ND, not detected. [Figure 12B] The therapeutic effect of MAC-BMDM in mixed MDRSA bacteria (Staphylococcus aureus and Escherichia coli) induced sepsis in mice with immunosuppression. Survival rate of mice with sepsis. The number of mice in the PBS, PBS-BMDM, and MAC-BMDM groups was 8, 10, and 12, respectively. [Figure 12C]The therapeutic effect of MAC-BMDM on mixed MDRSA bacteria (Staphylococcus aureus and Escherichia coli) induced septic mice with immunosuppression. BW, WBC, and LYM of mice with sepsis. BW. The number of mice in the PBS, PBS-BMDM, and MAC-BMDM groups was 8, 10, and 12, respectively. Data are mean ± SD. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). ND, not detected. [Figure 12D] The therapeutic effect of MAC-BMDM on mixed MDRSA bacteria (Staphylococcus aureus and Escherichia coli) induced septic mice with immunosuppression. BW, WBC, and LYM of mice with sepsis. WBC. The number of mice in the PBS, PBS-BMDM, and MAC-BMDM groups was 8, 10, and 12, respectively. Data are mean ± SD. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). ND, not detected. [Figure 12E] The therapeutic effect of MAC-BMDM on mixed MDRSA bacteria (Staphylococcus aureus and Escherichia coli) induced septic mice with immunosuppression. BW, WBC, and LYM of mice with sepsis. LYM. The number of mice in the PBS, PBS-BMDM, and MAC-BMDM groups was 8, 10, and 12, respectively. Data are mean ± SD. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). ND, not detected. [Figure 12F] The therapeutic effect of MAC-BMDM on mixed MDRSA bacteria (Staphylococcus aureus and Escherichia coli) induced sepsis in mice with immunosuppression. The bacterial load in the blood of each mouse treated with MAC-BMDM was measured. The number of mice in the PBS, PBS-BMDM, and MAC-BMDM groups was 8, 10, and 12, respectively. [Figure 13A] Therapeutic effects of MAC-RAW on MDRSA-induced septic mice involving cellular uptake, endocytic pathway, endosomal escape, and immunosuppression. Cellular uptake after treatment with VCLNP, Lipofectamine 3000, and electroporation. Percentage of Alexa-Fluor 647-positive cells. Data are mean ± SD, triplicate. There were six mice in each group. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). [Figure 13B] Therapeutic effect of MAC-RAW on MDRSA-induced septic mice involving cellular uptake, endocytic pathway, endosomal escape, and immunosuppression. Cellular uptake after treatment with VCLNP, Lipofectamine 3000, and electroporation. Cellular fluorescence intensity. Data are mean ± SD, triplicate. There were six mice in each group. *P<0.05, **P<0.01, ***P<0.001, ns, not significant (two-tailed Student's t-test). [Figure 13C] Therapeutic effects of MAC-RAW in MDRSA-induced septic mice involving cellular uptake, endocytic pathway, endosomal escape, and immunosuppression. Cellular uptake in the presence of endocytosis inhibitors EIPA, MβCD, and CPZ, which inhibit macropinocytosis, caveolae-, and clathrin-mediated endocytosis, respectively. Percentage of Alexa-Fluor 647-positive cells. Data are mean ± SD, triplicate. There were six mice in each group. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). [Figure 13D]Therapeutic effects of MAC-RAW on MDRSA-induced septic mice involving cellular uptake, endocytic pathway, endosomal escape, and immunosuppression. Cellular uptake in the presence of endocytosis inhibitors EIPA, MβCD, and CPZ, which inhibit macropinocytosis, caveolae-, and clathrin-mediated endocytosis, respectively. Cellular fluorescence intensity. Data are mean ± SD, triplicate. There were six mice in each group. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). [Figure 13E] Therapeutic effects of MAC-RAW on MDRSA-induced septic mice involving cellular uptake, endocytic pathway, endosomal escape, and immunosuppression. Confocal microscopy of RAW264.7 cells incubated with calcein alone or calcein and VCLNP containing Alexa-Fluor 647 RNA. Calcein alone. There were six mice in each group. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). [Figure 13F] Therapeutic effects of MAC-RAW on MDRSA-induced septic mice involving cellular uptake, endocytic pathway, endosomal escape, and immunosuppression. Confocal microscopy of RAW264.7 cells incubated with calcein alone or calcein and VCLNP containing Alexa-Fluor 647 RNA. Calcein and VCLNP containing Alexa-Fluor 647 RNA. There were six mice in each group. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). [Figure 13G] Therapeutic effects of MAC-RAW on MDRSA-induced septic mice involving cellular uptake, endocytic pathway, endosomal escape, and immunosuppression. 3D confocal microscopy images of RAW264.7 cells incubated with eGFP-CatB mRNA VCLNP. There were six mice in each group. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). [Figure 13H] Therapeutic effects of MAC-RAW on MDRSA-induced septic mice involving cellular uptake, endocytic pathway, endosomal escape, and immunosuppression. Percentage of RAW264.7 cells normalized to the PBS-RAW group at 12 hours. Data are mean ± SD, triplicate. There were six mice in each group. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). [Figure 13I] Therapeutic effects of MAC-RAW on MDRSA-induced septic mice involving cellular uptake, endocytic pathway, endosomal escape, and immunosuppression. Percentage survival rate of mice with sepsis treated with PBS, MAC-RAW (iv), or MAC-RAW (ip+iv). The number of mice was 6 in each group. *P<0.05, **P<0.01, ***P<0.001; ns, not significant (two-tailed Student's t-test). [Figure 14] Biodistribution of BMDM and MDRSA in mice. a) Distribution of BMDM in peritoneal fluid, blood, and major organs 6 hours after challenge in healthy or septic mice. b) Distribution of bacteria in peritoneal fluid, blood, and major organs 6 hours after infection. [Figure 15] VCLNP mediates luciferase mRNA delivery in bone marrow-derived dendritic cells. VCLNP is 17-fold more effective than Lipofectamine 3000 (Lipo3000) and 8-fold more effective than electroporation at the same mRNA concentration in bone marrow-derived dendritic cells. DETAILED DESCRIPTION OF THE INVENTION

[0021] Sepsis has traditionally been viewed as an uncontrolled inflammatory response to pathogens. However, researchers are reevaluating sepsis treatment approaches after more than 40 failed clinical trials of anti-inflammatory drugs. Clinical data reveal that although more than 60% of sepsis patients survive the initial inflammatory storm, they rapidly progress to a longer, immunosuppressed state characterized by immune cell paralysis and death, which prevents them from eliminating invading pathogens, increases their susceptibility to hospital-acquired infections, and increases their mortality rate. As a result, potential therapeutic targets have been extensively explored to treat sepsis, such as removal of the anaphylatoxin C5a or blockade of the C5a receptor. Meanwhile, approaches aimed at restoring immune function have been developed and are being tested in sepsis patients.

[0022] Macrophages are one of the most efficient pathogen scavengers during infection. In patients with sepsis, impaired macrophages / monocytes may be a major cause of inadequate antibacterial defense. Several small-scale clinical trials of immunostimulants have demonstrated the benefit of reversing inactivated macrophages / monocytes and promoting infection eradication. In contrast, a meta-analysis of large-scale clinical trials showed no significant change in reducing patient mortality. Several reasons may lead to these disparate clinical outcomes. First, immunostimulants cannot restore impaired macrophage / monocyte function to its original level. Second, invading bacteria are typically trapped in macrophage phagosomes, which then fuse with lysosomes to form phagolysosomes. Within the phagolysosome, reactive oxygen species (ROS), reactive nitrogen species (RNS), and lytic enzymes act synergistically to eliminate bacteria. However, many bacteria, such as Staphylococcus aureus and Escherichia coli, have evolved immune evasion mechanisms to prevent phagolysosomal killing, including scavenging reactive oxygen species (ROS) and reactive oxygen species (RNS) and resisting lytic enzymes, resulting in intracellular survival and recurrent infection. Third, although combination antibiotic therapy is the standard treatment in sepsis clinical guidelines, 70%–80% of sepsis deaths are associated with persistent infection, demonstrating the prevalence of antibiotic resistance and the lack of new antimicrobial agents. As an alternative, adoptive cell transfer-based immunotherapy bypasses the need to restore dysfunctional immune cells and thus offers potential benefits to immunocompromised patients.

[0023] Disclosed herein is adoptive transfer using macrophages (MACs) loaded with antimicrobial peptide / cathepsin B in the lysosome. To construct MACs, antimicrobial peptide / cathepsin B (AMP-CatB) mRNA was engineered. Vitamin C lipid nanoparticles (V) C LNP) was identified for its more efficient delivery of mRNA than Lipofectamine 3000 and electroporation in both the RAW264.7 cell line and bone marrow-derived macrophages (BMDMs). CLNPs enable the specific accumulation of AMP-CatB in macrophage lysosomes, a key site for antibacterial activity. Also disclosed herein is that adoptive MAC transfer eliminates MDR bacteria, including Staphylococcus aureus and Escherichia coli, improves the health of mice in an immunodeficiency sepsis model, providing a strategy for overcoming MDR bacteria-induced sepsis and highlighting the development of nanoparticle cell therapy for infectious diseases. Reference will now be made in detail to embodiments of the invention, examples of which are illustrated in the drawings and examples. However, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0025] term Terms used throughout this application should be interpreted with the meaning common and typical to those skilled in the art. However, applicant wishes to give specific definitions as defined below.

[0026] As used herein, the articles "a," "an," and "the" mean "at least one" unless the context in which the article is used clearly dictates otherwise.

[0027] As used herein, the term "comprising" and variations thereof are used synonymously with the term "including" and variations thereof and are open-ended, non-limiting terms. Although the terms "comprising" and "including" have been used herein to describe various embodiments, the terms "consisting essentially of" and "consisting of" may be used in place of "comprising" and "including" to provide more specific embodiments, and are disclosed.

[0028] As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases where the condition occurs and cases where the condition does not occur. Thus, for example, a statement that a formulation "may include an excipient" is meant to include cases where the formulation includes an excipient and cases where the formulation does not include an excipient.

[0029] The terms "about" and "approximately," as understood by one of ordinary skill in the art, are defined as "near." In one non-limiting embodiment, the term is defined as within 10%. In another non-limiting embodiment, the term is defined as within 5%. In yet another non-limiting embodiment, the term is defined as within 1%.

[0030] As used herein, the term "nucleic acid" refers to a polymer composed of nucleotides, such as deoxyribonucleotides (DNA) or ribonucleotides (RNA).

[0031] As used herein, the terms "ribonucleic acid" and "RNA" refer to a polymer composed of ribonucleotides.

[0032] As used herein, the terms "deoxyribonucleic acid" and "DNA" refer to a polymer composed of deoxyribonucleotides.

[0033] The term "oligonucleotide" refers to a single-stranded or double-stranded nucleotide polymer. Suitable oligonucleotides can be prepared by the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett., 22:1859-1862 (1981), or the triester method by Matteucci, et al., J. Am. Chem. Soc., 103:3185 (1981) (both incorporated herein by reference), or by other chemical methods using either a commercial automated oligonucleotide synthesizer or VLSIPS™ technology. When an oligonucleotide is referred to as "double-stranded," those skilled in the art will understand that a pair of oligonucleotides typically exists in a hydrogen-bonded helical arrangement associated with, for example, DNA. As used herein, the term "double-stranded" is meant to refer to 100% complementary forms of double-stranded oligonucleotides as well as forms that include structural features such as bulges and loops, which are more fully described in biochemistry textbooks such as Stryer, Biochemistry, Third Ed., (1988), which is incorporated herein by reference for all purposes.

[0034] The term "polynucleotide" refers to a single- or double-stranded polymer composed of nucleotide monomers.

[0035] The term "polypeptide" refers to a compound composed of a single chain of D- or L-amino acids, or a mixture of D- and L-amino acids linked by peptide bonds.

[0036] The term "promoter" or "regulatory element" refers to a region or sequence determinant located upstream or downstream from the start of transcription and involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. The promoter need not be of bacterial origin; for example, promoters derived from viruses or other organisms can be used in the compositions, systems, or methods described herein.

[0037] The term "recombinant" refers to a human-engineered nucleic acid (e.g., polynucleotide) or a copy or complement of a human-engineered nucleic acid (e.g., polynucleotide), or, with respect to a protein (i.e., a "recombinant protein"), to the protein (e.g., polynucleotide) encoded by a recombinant nucleic acid. In some embodiments, a recombinant expression cassette comprising a promoter operably linked to a second nucleic acid (e.g., polynucleotide) can include a promoter that is heterologous to the second nucleic acid (e.g., polynucleotide) as a result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In another example, a recombinant expression cassette can include nucleic acids (e.g., polynucleotides) that are combined such that the nucleic acids (e.g., polynucleotides) are highly unlikely to be found in nature. For example, a human-engineered restriction site or a plasmid vector sequence may flank or separate a promoter from a second nucleic acid (e.g., polynucleotide). Those skilled in the art will recognize that nucleic acids (e.g., polynucleotides) can be engineered in many ways and are not limited to the above examples.

[0038] The terms "expression cassette" and "vector" refer to nucleic acid constructs that, when introduced into a host cell, result in the transcription and / or translation of an RNA or polypeptide, respectively. In some embodiments, an expression cassette comprising a promoter operably linked to a second nucleic acid (e.g., a polynucleotide) can comprise a promoter that is heterologous to the second nucleic acid (e.g., a polynucleotide) as a result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)).

[0039] The term "identity" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences is determined using the BLAST or BLAST 2.0 sequence comparison algorithms using the default parameters described below, or by manual alignment and visual inspection (e.g., NCBI "Substantially identical" refers to two or more sequences or subsequences that are identical or have a specified percentage of amino acid residues or nucleotides that are identical (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region. Such sequences are said to be "substantially identical." This definition refers to or can be applied to the complement of a test sequence. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. As described below, preferred algorithms account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably, over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent amino acid sequence identity (%) is defined as the percentage of amino acids in a candidate sequence that are identical to those in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for determining percent sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared, can be determined by known methods.

[0040] For sequence comparison, typically, one sequence serves as a reference sequence, and test sequence is compared with it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and subsequence coordinates are designated as needed, and sequence algorithm program parameters are designated.Preferably, default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the sequence identity percentage of test sequence with reference sequence based on program parameters.

[0041] A preferred example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms (described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always greater than 0) and N (penalty score for mismatching residues; always less than 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum performance value, when the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses by default a word length of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989), Proc. Natl. Acad. Sci. USA 89:10915) alignment (B) of 50, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

[0042] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability when comparing the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

[0043] The phrase "codon optimization," when referring to genes or coding regions of a nucleic acid molecule intended for transformation into various hosts, refers to modifying the codons in the genes or coding regions of that polynucleic acid molecule to reflect the typical codon usage of a selected organism, without altering the polypeptide encoded by the DNA. Such optimization includes replacing at least one, more than one, or a significant number of codons with one or more codons that are more frequently used in the genes of the selected organism.

[0044] The term "nucleobase" refers to the portion of a nucleotide that possesses the Watson / Crick base pairing function. The most common naturally occurring nucleobases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T), possess the hydrogen-bonding function to link one nucleic acid strand to another in a sequence-specific manner.

[0045] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA or RNA sequences being linked are contiguous with each other, and, in the case of a secretory leader, contiguous and in reading phase. However, operably linked nucleic acids (e.g., enhancer and coding sequence) need not be contiguous. Linkage is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice. In some embodiments, a promoter is operably linked to a coding sequence if it is capable of affecting expression of a protein from that coding sequence (e.g., regulating relative to the absence of the promoter) (i.e., when the coding sequence is under the transcriptional control of the promoter).

[0046] The term "gene" or "gene sequence" refers to a coding sequence or a control sequence, or a fragment thereof. A gene may include any combination of coding and control sequences, or fragments thereof. Thus, a "gene" referred to herein can be all or a portion of a naturally occurring gene. A polynucleotide sequence referred to herein may be used interchangeably with the term "gene" or may include any coding, non-coding, or control sequence, fragments thereof, and combinations thereof. The term "gene" or "gene sequence" includes, for example, control sequences upstream of the coding sequence (e.g., ribosome binding site).

[0047] The term "nanoparticle," as used herein, refers to a particle or structure that is biocompatible and sufficiently resistant to chemical and / or physical destruction by the environment of such use, such that a sufficient number of nanoparticles remain substantially intact after delivery to the site of application or treatment, and whose size is in the nanometer range. For purposes of the present invention, nanoparticles typically range from about 1 nm to about 1000 nm, about 50 nm to about 500 nm, about 50 nm to about 350 nm, about 100 nm to about 250 nm, or about 110 nm to about 150 nm.

[0048] The phrase "symptoms of sepsis" refers to any symptom characteristic of a subject with sepsis, including, but not limited to, arterial hypotension, metabolic acidosis, fever, decreased systemic vascular resistance, tachypnea, and organ dysfunction. Sepsis can result from sepsis (i.e., organisms, their metabolic end products, or toxins in the bloodstream), such as bacteremia (i.e., bacteria in the blood), and toxemia (i.e., toxins in the blood), such as endotoxemia (i.e., endotoxins in the blood). The term "sepsis" also encompasses fungemia (i.e., fungi in the blood), viremia (i.e., viruses or virus particles in the blood), and parasitosis (i.e., helminth or protozoan parasites in the blood). Thus, phenotypes associated with sepsis and septic shock (acute circulatory failure resulting from sepsis is often associated with multiple organ failure and high mortality) are symptoms of sepsis.

[0049] As used herein, the term "treating" or "treatment" of a subject includes administering a drug to a subject for the purpose of curing, healing, alleviating, relieving, altering, treating, mitigating, improving, stabilizing, or affecting a disease or disorder, or the symptoms of a disease or disorder. The terms "treating" and "treatment" can also refer to reducing the severity and / or frequency of symptoms, eliminating symptoms and / or underlying causes, and ameliorating or repairing damage.

[0050] A "therapeutic agent" refers to any composition that has a beneficial biological effect. A beneficial biological effect includes both therapeutic effects, such as, for example, treating a disorder or other undesirable physiological condition, and prophylactic effects, such as, for example, preventing a disorder or other undesirable physiological condition. These terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term "therapeutic agent" is used, or when a particular agent is specifically identified, the term includes the agent itself, as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, and the like.

[0051] A "therapeutically effective amount" or "therapeutically effective dose" of a composition refers to an amount effective to achieve a desired therapeutic result. In some embodiments, the desired therapeutic result is treatment of sepsis. In some embodiments, the desired therapeutic result is restoring the immune system of a septic patient. In some embodiments, the desired therapeutic result is the reduction or clearance of a pathogen. The therapeutically effective amount of a given therapeutic agent will typically vary with factors such as the type and severity of the disorder or disease being treated and the age, sex, and weight of the subject. The term can also refer to the amount of therapeutic agent effective to promote a desired therapeutic effect, or the rate of delivery of the therapeutic agent (e.g., amount over time). The precise desired therapeutic effect will vary depending on the condition being treated, the subject's tolerance, the drug and / or drug formulation being administered (e.g., the potency of the therapeutic agent, the concentration of the drug in the formulation, etc.), and various other factors understood by those skilled in the art. In some cases, the desired biological or medical response is achieved after administering multiple doses of the composition to the subject over a period of days, weeks, or years.

[0052] A "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") generally refers to a carrier or excipient that is safe and non-toxic and useful in preparing pharmaceutical or therapeutic compositions, and includes carriers that are acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutically acceptable carrier" can include, but is not limited to, phosphate buffered saline, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents.

[0053] As used herein, the term "carrier" includes any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material known in the art for use in pharmaceutical formulations. The choice of carrier for use in a composition depends on the intended route of administration of the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described, for example, in Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005. Examples of physiologically acceptable carriers include buffers such as saline, glycerol, DMSO, phosphate buffers, citrate, and buffers containing other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN™ (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, NJ). To provide for administration of such dosages for the desired therapeutic treatment, the compositions disclosed herein can advantageously contain from about 0.1% to 99% by weight of the total amount of one or more of the subject compounds, based on the weight of the total composition including any carrier or diluent.

[0054] As used throughout, "subject" (or "host") means an individual. Thus, a "subject" can include, for example, domestic animals such as cats, dogs, etc., livestock (e.g., cows, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animals. The subject can be a mammal, such as a primate or a human.

[0055] antigen presenting cells In some embodiments, disclosed herein is an antigen-presenting cell, comprising: lipid-based nanoparticles, the lipid-based nanoparticles comprising: A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid encoding cathepsin B, and a recombinant polynucleotide comprising a third nucleic acid encoding a linker; Vitamin-lipid and antigen-presenting cells.

[0056] In some embodiments, disclosed herein is an antigen-presenting cell, comprising: lipid-based nanoparticles, the lipid-based nanoparticles comprising: A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid encoding a cathepsin B peptide, and a recombinant polynucleotide comprising a third nucleic acid encoding a linker; Vitamin-lipid and antigen-presenting cells.

[0057] In some embodiments, disclosed herein is an antigen-presenting cell, comprising: lipid-based nanoparticles, the lipid-based nanoparticles comprising: A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid encoding a cathepsin peptide, and a recombinant polynucleotide comprising a third nucleic acid encoding a linker; Vitamin-lipid and antigen-presenting cells.

[0058] In some embodiments, the antigen-presenting cells comprise macrophages or dendritic cells.

[0059] As used herein, the term "antigen-presenting cells" or "APCs" should be understood to refer to a heterogeneous group of immune cells that can process and present antigens to stimulate responses in specific lymphocytes (e.g., T cells and B cells). Classical APCs include, for example, dendritic cells, macrophages, B cells, and neutrophils.

[0060] It should be understood herein that macrophages are generally known to phagocytose immune cells (Meszaros et al., 1999). They also secrete factors such as chemokines or cytokines. In addition to phagocytosis and antigen presentation, these cells can also play a supportive role through their diverse repertoire of plasma membrane and secreted molecules (Gordon 1995, BioEssays, Volume 17, Issue 11). This has previously been shown for erythroblasts, hepatocytes, and neurons (Sadahira & Morr, Pathol Int. 1999 Oct;49(10):841-8), (Takeishi, Hirano, et al., Arch Histol Cytol. 1999 Dec;62(5):413-22), (Polazzi, Gianni, et al., Glia. 2001 Dec;36(3):271-80). "Macrophage" generally refers to a cell that exhibits characteristics described for macrophages, including phagocytosis and expression of defined cell surface markers such as CD64, CD14, and HLA-DR antigen expression. Macrophages according to the present invention can be isolated from tissues by ex vivo differentiation from blood monocytes (also referred to herein as "monocyte-derived macrophages"), bone marrow progenitor cells (also referred to herein as "bone marrow-derived macrophages"), or any other possible precursor, or preferentially by using any differentiation method, precursor, and any method known by those skilled in the art. Thus, in some embodiments, macrophages include bone marrow-derived macrophages. In some embodiments, macrophages include monocyte-derived macrophages. In some embodiments, macrophages include iPSC-derived macrophages. In some embodiments, macrophages include macrophage cell lines, including, for example, RAW264.7, THP-1, U937, IC-21, J774A.1, MV-4-11, or KG1.

[0061] It should also be understood that "dendritic cells" or "DCs," as used herein, refer to a type of antigen-presenting cell and are typically distinguished by the expression of one or more of the following markers on their cell surface: CD1a, CD1b, and CD1c, CD4, CD11c, CD33, CD40, CD80, CD86, CD83, and HLA-DR. In some embodiments, dendritic cells are mature DCs. In some embodiments, dendritic cells are immature DCs. DCs according to the present invention can be isolated from tissues by ex vivo differentiation from blood monocytes (also referred to herein as "monocyte-derived dendritic cells"), bone marrow progenitor cells (also referred to herein as "bone marrow-derived dendritic cells"), or any other possible precursor, or preferentially by using any differentiation method, precursor, and method known by those skilled in the art. Thus, in some embodiments, dendritic cells include bone marrow-derived dendritic cells. In some embodiments, dendritic cells are monocyte-derived dendritic cells. In some embodiments, dendritic cells are iPSC-derived dendritic cells. In some embodiments, the dendritic cells are conventional dendritic cells 1 (or cDC1, lymphoid DC), which are typically distinguished by the expression of one or more of the following markers on their cell surface: CD141, CLEC9A, and XCR1. In some embodiments, the dendritic cells are conventional dendritic cells 2 (or cDC2, myeloid DC), which are typically distinguished by the expression of one or more of the following markers on their cell surface: CD1c and CD172a. In some embodiments, the dendritic cells are plasmacytoid DCs (or pDC), which are typically distinguished by the expression of one or more of the following markers on their cell surface: CD123, CD303, and CD304.

[0062] In some embodiments, the antigen-presenting cells are derived from a subject selected from the group consisting of a mouse, a rat, a human, or a non-human primate. In some embodiments, the antigen-presenting cells are derived from a mouse. In some embodiments, the antigen-presenting cells are derived from a rat. In some embodiments, the antigen-presenting cells are derived from a human. In some embodiments, the antigen-presenting cells are derived from a non-human primate.

[0063] nanoparticles Although numerous nanoparticles are described herein, additional nanoparticles known in the art can also be used herein.

[0064] In some embodiments, disclosed herein are lipid-based nanoparticles comprising: A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid encoding cathepsin B, and a recombinant polynucleotide comprising a third nucleic acid encoding a linker; The present invention relates to lipid-based nanoparticles comprising a vitamin-lipid.

[0065] In some embodiments, disclosed herein are lipid-based nanoparticles comprising: A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid encoding a cathepsin B peptide, and a recombinant polynucleotide comprising a third nucleic acid encoding a linker; The present invention relates to lipid-based nanoparticles comprising a vitamin-lipid.

[0066] In some embodiments, disclosed herein are lipid-based nanoparticles comprising: A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid encoding a cathepsin peptide, and a recombinant polynucleotide comprising a third nucleic acid encoding a linker; Lipid-based nanoparticles containing vitamins and lipids.

[0067] As used herein, the term "vitamin-lipid" refers to a compound comprising a vitamin moiety and a lipid, where the lipid may be a lipid-like moiety. The term "vitamin-lipid" is also meant to refer to the forms more fully described in WO2019 / 027999, which is incorporated herein by reference for all purposes. Thus, a vitamin-lipid may be, for example, a compound of formula A: [ka] or a salt thereof, wherein: R 1 is an alkyl or ether linker, where the alkyl or ether linker is substituted with a vitamin moiety; R 2 is alkyl, cycloalkyl, heterocycloalkyl, alkylheterocycloalkyl, amide, alkylamide, ether, alkylether, [ka] In the formula, m is an integer of 1 to 20, n is an integer from 1 to 3, Each R 3 is independently selected from alkyl, alkenyl, alkynyl, ester, or alkyl ester.

[0068] In one embodiment, the vitamin-lipid is a compound of formula I [ka] or a salt thereof, wherein: R 1 is an alkyl or ether linker, the alkyl or ether linker being substituted with a carbohydrate moiety, a phosphate moiety, or a vitamin moiety; Each R 3is independently selected from alkyl, alkenyl, alkynyl, ester, or alkyl ester.

[0069] The vitamin moiety can be, for example, vitamin B3, vitamin C, vitamin D, vitamin E, vitamin H, or a derivative thereof. In some embodiments, the vitamin moiety is vitamin C or a derivative thereof. Vitamin moieties are shown below. [ka]

[0070] In some embodiments, the vitamin-lipid comprises: [ka]

[0071] In some embodiments, the vitamin-lipid [ka] or salts thereof.

[0072] In some embodiments, the vitamin-lipid [ka] or salts thereof.

[0073] In some embodiments, the lipid-based nanoparticles comprise a vitamin-lipid molar ratio of about 10% to about 60%. In some embodiments, the lipid-based nanoparticles comprise a vitamin-lipid molar ratio of about 10% to about 40%. In some embodiments, the lipid-based nanoparticles comprise a vitamin-lipid molar ratio of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, or about 80%. In one embodiment, the lipid-based nanoparticles comprise a vitamin-lipid molar ratio of about 30%.

[0074] In some embodiments, the lipid-based nanoparticles further comprise a non-cationic lipid, and the non-cationic lipid may be 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), DPPC (1 ,2-dipalmitoyl-sn-glycero-3-phosphotidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphotidylcholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-5 / 7-glycero-3-phospho(l'-rac-glycerol) (DOPG), or combinations thereof.

[0075] In some embodiments, the lipid-based nanoparticles further comprise polyethylene glycol lipids (PEG lipids), which can include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. Representative polyethylene glycol lipids include DMG-PEG, DLPE-PEG, DMPE-PEG, DPPC-PEG, and DSPE-PEG.

[0076] In some embodiments, the lipid-based nanoparticles further comprise PEGylated cholesterol, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine, or a combination thereof.

[0077] In some embodiments, the lipid-based nanoparticle further comprises a recombinant polynucleotide, wherein the polynucleotide may be encapsulated by the vitamin-lipid.

[0078] Polynucleotides and Polypeptides In some embodiments, the recombinant polynucleotide comprises RNA or DNA. In some embodiments, the recombinant polynucleotide is RNA. In some embodiments, the recombinant polynucleotide is mRNA. In some embodiments, the recombinant polynucleotide is DNA.

[0079] The term "antimicrobial peptide" disclosed herein includes peptides or derivatives thereof having antimicrobial activity against one or more bacteria, such as Gram-positive bacteria and Gram-negative bacteria, and fungi, such as yeast and mold. In some embodiments, the antimicrobial peptide comprises SEQ ID NO: 1, or a fragment or functionally active variant thereof. In some embodiments, the antimicrobial peptide is selected from the group comprising a polypeptide sequence at least 60% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 1. In some embodiments, the antimicrobial peptide is selected from protegrin 1, C16G2, omiganan, β-defensins, hLF1-11, LL37, or MSI-78, or a fragment or functionally active variant thereof. These antimicrobial peptide sequences and additional antimicrobial peptides are known in the art, see, for example, US Pat. No. 8,754,039, which is incorporated herein by reference in its entirety.

[0080] The antimicrobial peptide of any of the aforementioned aspects can be encoded by a first nucleic acid of a recombinant polynucleotide. In some embodiments, the first nucleic acid comprises SEQ ID NO:2, or a fragment or functionally active variant thereof. In some embodiments, the first nucleic acid is selected from the group comprising polynucleotide sequences at least 60% identical (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) to SEQ ID NO:2.

[0081] Cathepsin B disclosed herein comprises the polypeptide sequence set forth in SEQ ID NO: 3, or a fragment or functionally active variant thereof. In some embodiments, the cathepsin B is selected from the group comprising a polypeptide sequence at least 60% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 3.

[0082] The cathepsin B of any of the aforementioned aspects can be encoded by a second nucleic acid of the recombinant polynucleotide. In some embodiments, the second nucleic acid comprises SEQ ID NO:4, or a fragment or functionally active variant thereof. In some embodiments, the second nucleic acid is selected from the group comprising polynucleotide sequences at least 60% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO:4.

[0083] In some embodiments, disclosed herein is an antigen-presenting cell, comprising: lipid-based nanoparticles, the lipid-based nanoparticles comprising: A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid, and a recombinant polynucleotide comprising a third nucleic acid encoding a linker; Vitamin-lipid and antigen-presenting cells.

[0084] In some embodiments, disclosed herein is an antigen-presenting cell, comprising: lipid-based nanoparticles, the lipid-based nanoparticles comprising: A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid encoding a cathepsin peptide, and a recombinant polynucleotide comprising a third nucleic acid encoding a linker; Vitamins and lipids, and antigen presenting cells.

[0085] In some embodiments, the second nucleic acid encoding a cathepsin peptide comprises cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, or cathepsin G, or a fragment or functionally active variant thereof. These sequences are known in the art and can be found on the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov). In some embodiments, the sequence is derived from a mammal. In some embodiments, the sequence is derived from a mouse. In some embodiments, the sequence is derived from a primate. In some embodiments, the sequence is derived from a human.

[0086] In some embodiments, the linker comprises a cathepsin B-sensitive linker. In some embodiments, the linker comprises a polypeptide sequence set forth in SEQ ID NO: 5, or a fragment or functionally active variant thereof. In some embodiments, the linker is selected from the group comprising a polypeptide sequence at least 60% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 5.

[0087] The linker of any of the foregoing aspects can be encoded by a third nucleic acid. In some embodiments, the third nucleic acid comprises SEQ ID NO:6, or a fragment or functionally active variant thereof. In some embodiments, the third nucleic acid is selected from the group comprising a polynucleotide sequence at least 60% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO:6.

[0088] In some embodiments, the linker comprises a polypeptide sequence set forth in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, or a fragment or functionally active variant thereof. In some embodiments, the linker is selected from the group comprising a polynucleotide sequence that is at least 60% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some embodiments, the linker can also be Phe-Lys.

[0089] In some embodiments, the recombinant polynucleotide of any of the foregoing aspects comprises SEQ ID NO: 7, or a fragment or functionally active variant thereof. In some embodiments, the recombinant polynucleotide is selected from the group comprising a polynucleotide sequence that is at least 60% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 7.

[0090] In some embodiments, the recombinant polynucleotide encodes a recombinant polypeptide comprising the sequence set forth in SEQ ID NO: 8, or a fragment or functionally active variant thereof. In some embodiments, the recombinant polynucleotide is selected from the group comprising a polynucleotide sequence that is at least 60% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 8.

[0091] In some embodiments, the first nucleic acid and the second nucleic acid are linked by a third nucleic acid.

[0092] Treatment methods In some embodiments, disclosed herein is a method of treating sepsis, comprising administering to a subject one or more antigen-presenting cells, wherein the antigen-presenting cells:

[0093] nanoparticles, the nanoparticles comprising: A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid encoding cathepsin B, and a recombinant polynucleotide comprising a third nucleic acid encoding a linker; a vitamin-lipid mixture.

[0094] In some embodiments, disclosed herein is a method of treating sepsis, comprising administering to a subject one or more antigen-presenting cells, wherein the antigen-presenting cells: nanoparticles, the nanoparticles comprising: A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid, and a recombinant polynucleotide comprising a third nucleic acid encoding a linker; a vitamin-lipid mixture.

[0095] In some embodiments, disclosed herein is a method of treating sepsis, comprising administering to a subject one or more antigen-presenting cells, wherein the antigen-presenting cells: nanoparticles, the nanoparticles comprising: A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid encoding a cathepsin peptide, and a recombinant polynucleotide comprising a third nucleic acid encoding a linker; a vitamin-lipid mixture.

[0096] In some embodiments, disclosed herein is a method of treating sepsis, comprising administering to a subject one or more antigen-presenting cells, wherein the antigen-presenting cells: nanoparticles, the nanoparticles comprising: A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid encoding a cathepsin B peptide, and a recombinant polynucleotide comprising a third nucleic acid encoding a linker; a vitamin-lipid mixture.

[0097] As disclosed above, the term "sepsis" also encompasses bacteremia (i.e., bacteria in the blood), toxemia (i.e., toxins in the blood), fungemia (i.e., fungi in the blood), viremia (i.e., viruses or virus particles in the blood), and parasitosis (i.e., helminthic or protozoan parasites in the blood). Thus, phenotypes associated with sepsis and septic shock (acute circulatory failure resulting from sepsis is often associated with multiple organ failure and high mortality) are symptoms of sepsis. Accordingly, disclosed herein are methods of treating, inhibiting, or reducing sepsis or symptoms of sepsis (e.g., reducing the bacterial, toxin, fungal, viral, or parasitic load in the blood and / or restoring, maintaining, or improving the immune system of an affected subject).

[0098] In some embodiments, the antigen-presenting cells of any preceding aspect are derived from a subject. In some embodiments, the antigen-presenting cells are derived from a subject. In some embodiments, the antigen-presenting cells are derived from a different subject. In some embodiments, the subject is a human. In some embodiments, the human has or is suspected of having sepsis.

[0099] In some embodiments, a method for treating sepsis comprises administering to a subject one or more antigen-presenting cells of any of the foregoing aspects, wherein the one or more antigen-presenting cells are prepared and administered together with a pharmaceutically acceptable carrier.

[0100] Because the timing of sepsis is often unpredictable, it is understood that the disclosed methods of treating, preventing, reducing, and / or inhibiting sepsis can be used to treat, prevent, inhibit, and / or reduce sepsis before or after the onset of sepsis symptoms. Here, the disclosed methods can be performed any time before sepsis. In one aspect, the disclosed methods can be performed 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 month, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 days, 60, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 19, 20, 21, 22, 23, 24, 25, 26, 27, 27, 28, 29, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 8, 36, 30, 24, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2 hours, 60, 45, 30, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute, or after sepsis, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 60, 75, 90, 105, 120 minutes, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 24, 30, 36, 48, 60 hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 Can be used for 9, 30, 45, 60, 90 or more days, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, and 1 year.

[0101] The antigen-presenting cells of the present invention can be administered to suitable subjects by any method known in the art, for example, orally, intramuscularly, intravenously, sublingually, intraarterially, intrathecally, intradermally, intraperitoneally, intranasally, intrapulmonary, intraocularly, intravaginally, intrarectally, or subcutaneously.They can be introduced into the digestive tract or respiratory tract.When used, parenteral administration of the composition is generally characterized by injection.

[0102] In some embodiments, disclosed herein is a method of treating a disease, comprising administering to a subject one or more antigen-presenting cells, wherein the antigen-presenting cells: nanoparticles, the nanoparticles comprising: A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid (e.g., encoding a cathepsin peptide, such as a cathepsin B peptide), and a recombinant polynucleotide comprising a third nucleic acid encoding a linker; a vitamin-lipid mixture.

[0103] In some embodiments, disclosed herein is a method of treating cancer, comprising administering to a subject one or more antigen-presenting cells, wherein the antigen-presenting cells: nanoparticles, the nanoparticles comprising: A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid (e.g., encoding a cathepsin peptide, such as a cathepsin B peptide), and a recombinant polynucleotide comprising a third nucleic acid encoding a linker; a vitamin-lipid mixture.

[0104] In some embodiments, the disease is selected from a lysosomal storage disorder, aspartylglucosaminuria, Gaucher disease, GM1 gangliosidosis, or mucopolysaccharidosis.

[0105] In some embodiments, disclosed herein is a method of treating a neurodegenerative disease, comprising administering to a subject one or more antigen-presenting cells, wherein the antigen-presenting cells: nanoparticles, the nanoparticles comprising: A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid (e.g., encoding a cathepsin peptide, such as a cathepsin B peptide), and a recombinant polynucleotide comprising a third nucleic acid encoding a linker; a vitamin-lipid mixture.

[0106] In some embodiments, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, or Huntington's disease.

[0107] In yet other embodiments, disclosed herein are methods for treating a disease, comprising administering a therapeutically effective amount of an immune cell of any preceding aspect to a subject. In some embodiments, the disease is selected from lysosomal storage disorders, aspartylglucosaminuria, Gaucher disease, GM1 gangliosidosis, or mucopolysaccharidosis.

[0108] Additional Compositions and Methods In some embodiments, disclosed herein is an immune cell comprising: lipid-based nanoparticles, the lipid-based nanoparticles comprising: a recombinant polynucleotide encoding an immunity protein; Vitamins, lipids, and The immune cells are immune cells, including T cells.

[0109] In some embodiments, the recombinant polynucleotide is encapsulated by a vitamin-lipid.

[0110] In some embodiments, the recombinant polynucleotide comprises RNA or DNA. In some embodiments, the recombinant polynucleotide is RNA. In some embodiments, the recombinant polynucleotide is mRNA. In some embodiments, the recombinant polynucleotide is DNA.

[0111] In some embodiments, the immunity protein comprises a chimeric antigen receptor or a cytotoxic cytokine.

[0112] In some embodiments, the immunity protein is a chimeric antigen receptor, which in some embodiments comprises an antigen binding domain, a transmembrane domain, a costimulatory signaling region, or a CD3 zeta signaling domain.

[0113] In some embodiments, the antigen binding domain binds to a tumor antigen.

[0114] In some embodiments, the immunity protein is a cytotoxic cytokine, including, for example, interferon gamma, tumor necrosis factor, granzyme A, granzyme B, or perforin.

[0115] In some embodiments, disclosed herein is a method of treating cancer, comprising administering to a subject a therapeutically effective amount of an immune cell of any of the foregoing aspects.

[0116] In yet other embodiments, disclosed herein are methods for treating a disease, comprising administering a therapeutically effective amount of an immune cell of any preceding aspect to a subject. In some embodiments, the disease is selected from lysosomal storage disorders, aspartylglucosaminuria, Gaucher disease, GM1 gangliosidosis, or mucopolysaccharidosis.

[0117] In some embodiments, disclosed herein are methods for treating a neurodegenerative disease, comprising administering to a subject a therapeutically effective amount of an immune cell of any preceding aspect, in some embodiments, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, or Huntington's disease.

[0118] In some embodiments, the subject is a human, hi some embodiments, the human has or is suspected of having cancer. [Example]

[0119] The following examples are set forth below to illustrate compositions, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention that would be apparent to one skilled in the art.

[0120] Example 1. Vitamin lipid nanoparticles provide therapeutic adoptive macrophage transfer Multidrug-resistant bacterial sepsis The data disclosed herein demonstrate that adoptive transfer of macrophages (MACs) loaded with lysosomal antimicrobial peptides / cathepsin B provides immunocompromised septic hosts with enhanced innate immunity, preventing bacterial immune evasion, and eliminating multidrug-resistant (MDR) bacteria. As shown in Figure 1a, mRNA (AMP-CatB) was designed and constructed with multiple components encoding the antimicrobial peptide IB367 (AMP-IB367), cathepsin B (CatB), and a cleavable linker. AMP-IB367 is a broad-spectrum AMP with rapid bactericidal activity, which has been confirmed in clinical trials. The endogenous protein CatB is first translated as an inactive precursor in the cytoplasm and translocated into lysosomes. These precursors are then processed into mature CatB. The functions of the CatB components are integrated to transport AMP-IB367 into lysosomes, which then fuse with bacteria-containing phagosomes. To eradicate bacteria, free AMP-IB367 must be released from the AMP-CatB protein. Therefore, a CatB-sensitive linker was added to the mRNA sequence. Lysosomes contain large amounts of CatB protein, facilitating the release of AMP-IB367. To deliver the designed mRNA to macrophages, an efficient mRNA delivery system for macrophages is required, as macrophages are one of the most difficult cells to transfect. Macrophages incorporate different vitamins to achieve their biological functions.

[0121] Through initial screening and two rounds of characterization, vitamin C lipid nanoparticles (V C The formulation of AMP-CatB (LNP) effectively delivered AMP-CatB mRNA to macrophages. The mRNA was translated into functional protein in the cytoplasm, and the protein was further translocated to lysosomes. In the lysosome, the CatB-sensitive linker was cleaved by the CatB protein, thus releasing APM-IB367. When the phagosome encapsulating the bacterium fuses with the lysosome, the ingested bacterium is exposed to both pre-stored AMP-IB367 and lysosomal antibacterial components. While immune evasion strategies can protect MDR bacteria from the phagolysosomal killing mechanism, AMP-IB367 can kill these bacteria because it has high antibacterial activity against MDR bacteria in animal models and humans. Therefore, adoptive transfer of MAC rescues MDR bacteria that induce sepsis accompanied by immunosuppression by restoring innate immunity, overcoming bacterial immune evasion, and eradicating the infection.

[0122] Five vitamins were initially selected: vitamin B3, vitamin C, vitamin D, vitamin E, and vitamin H (also known as vitamin B7). Then, lipid tails were incorporated using a previously reported method. Amino acid lipids were attached to these vitamins via ester or amide bonds (Figure 1b). These five vitamin-derived lipids were then synthesized as V B3 -Lipids, V C- lipid, V D -Lipids, V E -lipids, and V H The tertiary amines in the lipid chains are ionized under acidic conditions and can interact with mRNA. The structure of these vitamin-derived lipids is 1 This was confirmed by 1 H NMR and mass spectrometry (MS) (Figures 2 to 6).

[0123] These vitamin-derived lipids were then formulated into vitamin-lipid nanoparticles (VLNPs) accordingly. The particle sizes of the VLNPs ranged from 127±1 to 174±1 nm, and the polydispersity index (PDI) was 1.0.H The VLNPs were <0.3 except for VLNPs (Figures 7a and 7b). The mRNA entrapment efficiency ranged from 52% to 99%, and all VLNPs were positively charged (Figures 7c and 7d). Initial screening of RAW264.7 cells using mRNA encoding firefly luciferase demonstrated that VLNPs C VLNPs were 20 times more effective for mRNA delivery than the other four VLNPs (Figure 8a). C LNP was 10-fold better than Lipofectamine 3000 and 50-fold better than electroporation at the same mRNA concentration (Figure 8a). C The highest luminescence intensity of the LNP group was observed at 12 hours between 6 and 24 hours (Figure 8b). C To further explore the formulation of LNPs, an orthogonal array design was performed to fine-tune the component ratios. 16 (4) 4 Based on the orthogonal array design table (Figure 7e), 16 different formulations were prepared. From this study, formulation B2 (lipid:DOPE:cholesterol = 30:30:40) was obtained (Figures 8c and 8e). The delivery efficiency was then verified by comparing it with the top-ranked formulation A10 (lipid:DOPE:cholesterol = 30:30:50) in the orthogonal array. The predicted luminescence intensity of formulation B2 was significantly higher than that of formulation A10 (P<0.05, Figures 8d and 8e). To further improve mRNA delivery efficiency, the V of formulation B2 was adjusted from 5:1 to 20:1. C- The lipid:mRNA mass ratio was investigated (Figure 8d). In the second round of characterization, V C- As the lipid:mRNA mass ratio increased up to 15:1, the luminescence intensity increased (formulation C5, Figure 8e). C LNP formulation C5 was positively charged with a spherical morphology as seen in cryo-TEM images (Figures 8f and 7f). Based on these results, this V formulation showed a more than 7-fold improvement in mRNA delivery efficiency compared to its initial formulation. C The LNP formulation was selected for further study.

[0124] Using a fluorescent probe, Alexa-Fluor 647-labeled RNA, 99.2% of Alexa-Fluor 647-positive cells were VC 21.5% in the LNP group, 21.5% in the Lipofectamine 3000 group, and 2.4% in the electroporation group (Figure 13a). Furthermore, the fluorescence intensity was significantly higher than that of the V C In cells treated with LNP, the V was approximately 4-fold and 16-fold higher than in cells treated with Lipofectamine 3000 or electroporation, respectively (Figure 13b). C Efficient cellular uptake of LNPs was demonstrated. Cells were then incubated in the presence of different endocytosis inhibitors, 5-(N-methyl-N-isopropyl) amiloride (EIPA), methyl-beta-cyclodextrin (MβCD), and chlorpromazine hydrochloride (CPZ), which inhibit macropinocytosis, caveolae-, and clathrin-mediated endocytosis, respectively. C Incubated with LNP. C The cellular uptake of LNPs was dramatically reduced by about 96% in the MβCD group (Figs. 13c and 13d), which indicates that these V C This demonstrated the key role of caveolae-mediated endocytosis for LNPs. To explore the endosomal escape mechanism, a calcein assay was performed. Calcein, a membrane-impermeable dye, is normally trapped in cellular endosomes. Cells were incubated with calcein and V. C Treated with both LNPs, diffuse green fluorescence was observed in the cytoplasm, resulting in endosomal membrane rupture and V C It was shown that LNPs were released into the cytoplasm (Figs. 13e and 13f).

[0125] To test whether cathepsin B (CatB) could transport payloads to lysosomes, eGFP-CatB mRNA was constructed and expressed as V CAMP-IB367 was delivered to RAW264.7 cells using LNPs. Live-cell confocal microscopy showed that eGFP-CatB colocalized with LysoTracker® Red DND-99 in the lysosomes, with a Pearson correlation coefficient of 0.91 ± 0.15 (Figures 8g and 13g), indicating that CatB delivers its payload into the lysosomes. Next, to assess the bactericidal activity of macrophages (MACs) loaded with AMP-IB367 in lysosomes, we used PBS (PBS-RAW), free AMP-CatB mRNA (Fr-RAW), empty V-AMP, and AMP-IB367. C LNP(Em-RAW), AMP-CatB mRNA V C LNP / CatB inhibitor II (In-RAW) and AMP-CatB mRNA V C The intracellular viability of multidrug-resistant Staphylococcus aureus (MDRSA) was quantified in RAW264.7 cells treated with LNP (MAC-RAW). Compared with the other four treatments, MAC-RAW demonstrated the strongest bactericidal activity at all time points tested, with inhibition rates ranging from 33% to 87% (Figure 8h). Inhibiting CatB function using CatB inhibitor II dramatically reduced bactericidal activity (Figure 8h), demonstrating the importance of AMP-IB367 release. No significant differences in cell counts were observed in all five groups (Figure 13h). Therefore, these results demonstrated that pre-stored AMP-IB367 prevented immune evasion by bacteria and eliminated them within the phagolysosome.

[0126] Given the potent in vitro bactericidal activity of MAC-RAW, we conducted a study to evaluate the therapeutic efficacy of immunosuppression in mice with MDRSA-induced sepsis. After three consecutive days of cyclophosphamide (CY) treatment, decreases in body weight (BW), white blood cells (WBC), and lymphocytes (LYM) resembled the immunocompromised state in septic patients. After infection with MDRSA, mice were treated with PBS, PBS-RAW, or MAC-RAW. PBS-RAW was injected both intraperitoneally (ip) and intravenously (iv) to treat local and blood bacteria. MAC-RAW was administered in three different ways with the same total cell count: ip injection alone, iv injection alone, and ip + iv injection. Because lethality in immunosuppressed sepsis is associated with nonradical pathogens, bacterial colony-forming units (CFU) in mouse blood were measured 24 h after cell transfer. Similar to PBS treatment, PBS-RAW did not reduce the bacterial load in the blood. However, MAC-RAW administered via ip injection only (MAC-RAW(ip)) and MAC-RAW administered via both ip and iv injections (MAC-RAW(ip+iv)) significantly reduced bacterial CFU in the blood (P<0.01 and P<0.001, respectively) (Fig. 9a). These results indicated in vivo bactericidal activity. Interestingly, MAC-RAW(ip+iv) demonstrated a much stronger bacterial elimination ability than MAC-RAW(ip) (P<0.001, Fig. 3a). Furthermore, on day 30, the survival rate of the MAC-RAW(ip+iv) group was 58%, a significant improvement compared with the MAC-RAW(ip) group (P<0.01, Fig. 9b). Similarly, MAC-RAW(ip+iv) demonstrated a superior therapeutic effect on survival rate (P<0.05) compared with MAC-RAW(iv) (Fig. 13i).

[0127] In the MAC-RAW (ip + iv) group, three of the seven mice that survived at 480 hours had undetectable bacteria in the blood (Figure 9f). Four mice with persistent infection were then treated repeatedly, resulting in the elimination of residual bacteria in these mice (Figure 9g). One month later, the BW, WBC, and LYM levels of the seven mice were completely recovered (Figures 9c-9e). Furthermore, bacteria were undetectable in the blood and major organs (heart, liver, spleen, lungs, and kidneys) of these mice.

[0128] Next, we evaluated the bactericidal activity using primary bone marrow-derived macrophages (BMDMs), which are translatable for clinical use. BMDMs were generated from mouse bone marrow as reported in the literature, and approximately 83.5% of the cells were confirmed to be F4 / 80 positive (Figure 10a). VLNPs were then also screened and tested for their expression profile in BMDMs. Similar to the results in RAW264.7 cells, VLNPs were also screened and tested for their expression profile in BMDMs. C VLNPs were 5-fold more effective in mRNA delivery than the other four VLNPs, 6-fold more effective than Lipofectamine 3000, and 150-fold more effective than electroporation (Fig. 10b). C LNP followed a consistent expression profile with maximum luminescence intensity at 12 hours (Fig. 10c). Next, AMP-CatB mRNA V C MAC-BMDMs were prepared using LNPs and evaluated for in vitro bactericidal activity against MDRSA and multidrug-resistant Escherichia coli (MDR E. coli). PBS (PBS-BMDM), free AMP-CatB mRNA (Fr-BMDM), empty V C LNP(Em-BMDM) and AMP-CatB mRNA V C Compared with BMDMs treated with LNP / CatB inhibitor II (In-BMDM), AMP-CatB mRNA V CLNP(MAC-BMDM) showed the strongest bactericidal activity against both MDRSA and MDR E. coli with maximum percentage inhibition of 85% and 74%, respectively (Figures 10d and 10f). In addition, all five groups showed comparable cell numbers (Figures 10e and 10g), and AMP-CatB mRNA V C LNPs did not induce obvious cytotoxicity in BMDMs (Fig. 10h).

[0129] After verifying the results in vitro, MAC-BMDMs were administered to immunosuppressed mice to treat MDRSA-induced sepsis. Based on the data from RAW264.7 cells, MAC-BMDMs were administered to mice by both i.p. and i.v. injection. Analysis of blood samples at 24 h showed that MAC-BMDMs were more effective at eliminating bacteria than PBS-BMDMs (P<0.001, Figure 11a). Furthermore, MAC-BMDMs rescued 58% of mice from immunosuppressed sepsis, compared with only 10% rescued by PBS-BMDMs (P<0.01, Figure 11b). Except for one mouse in the MAC-BMDM group that showed persistent infection, no bacteria were found in the blood of the other six surviving mice at 480 h (Figures 11f and 11g). Similarly, after repeated treatment of the mice, all mice showed normal levels of BW, WBC, and LYM (Figures 11c-11e) and undetectable levels of MDRSA in their blood and major organs (heart, liver, spleen, lungs, and kidneys).

[0130] Bacteria distribute to multiple organs within hours of infection. To profile the biodistribution of these macrophages, mRNA encoding firefly luciferase was delivered to BMDMs (FLuc-BMDMs). Six hours after ip+iv administration of FLuc-BMDMs, luminescence intensity was measured in the following major organs commonly infected by bacteria in this mouse model: peritoneal cavity, spleen, liver, lungs, kidneys, heart, and blood. The results showed similar biodistribution in healthy and septic mice, except that higher luminescence intensity was detected in the lungs of septic mice than in healthy mice (Figure 14a). In septic mice, the order of luminescence intensity was as follows: peritoneal cavity (52.7%), spleen (21.1%), lungs (12.9%), liver (9.1%), and blood (3.0%). The biodistribution of BMDMs was relatively consistent with the bacterial distribution in these septic mice (Figure 14b).

[0131] Because septic hosts are usually exposed to mixed bacterial infections, which is a difficult challenge for treating sepsis, a murine sepsis model was established with infection by both MDRSA and MDR E. coli. Because mixed infections result in more severe symptoms compared with single bacterial infections, mice were infected with a total of 2 × 10 8 The mice were infected with a bacterial CUF containing 2.5-fold less bacteria than the single-infection model. Treatment with MAC-BMDM significantly reduced the bacterial burden in the blood by 43% (P < 0.01) and 39% (P < 0.05) compared with treatment with PBS and PBS-BMDM (Figure 12a), demonstrating the enhanced ability of MAC-BMDM to eliminate mixed MDR bacteria. The therapeutic effect of MAC-BMDM was also reflected in a much higher survival rate (83%) than the PBS group (P < 0.01) and PBS-BMDM group (P < 0.05) (Figure 12b). In contrast, PBS-BMDM did not significantly alter survival compared with PBS (Figure 12b). Finally, normal levels of BW, WBC, and LYM were observed in all surviving mice (Figures 12c–12f), and no persistent infection was detected in the blood or major organs (heart, liver, spleen, lungs, and kidneys) at 480 h.

[0132] Presented herein are vitamin C lipid nanoparticles (V C Macrophages (MACs) loaded with antimicrobial peptides / cathepsin B in their lysosomes were delivered via mRNA encapsulated in LNPs. The data show that adoptive transfer of MACs beneficially reduced bacterial burden and improved survival in immunosuppressed mice with sepsis induced by multidrug-resistant (MDR) bacteria by restoring innate immune defense, preventing bacterial immune evasion, and killing MDR bacteria. MACs were effective against sepsis induced by mixed strains of MDR bacteria. The superior therapeutic effect of MACs administered via IP + IV injection compared with IP or IV injection alone may be attributed to the infectious process of sepsis. In septic mice, bacteria are transported to the blood by the lymphatic system within a short time and then distributed to other organs via the blood circulation. IV + IP administration contributes to the elimination of bacteria that have invaded the blood or colonized the peritoneal cavity, reducing bacterial CFU and improving survival. When sepsis is diagnosed in the early stages, autologous macrophages can be prepared and manipulated in approximately 7 days. These autologous MACs can then be infused back into patients with immunosuppression, which represents the majority of sepsis patients under current treatment guidelines. Furthermore, with advances in induced pluripotent stem cell (iPSC) technology, allogeneic "universal" macrophages can be produced, enabling iPSC-derived MACs to become an off-the-shelf therapy for a wide range of clinical applications, including sepsis. Overall, adoptive transfer of MACs offers a potentially curative strategy for patients with sepsis and MDR bacterial infections in the future.

[0133] Example 2. Materials and Methods Chemicals and Reagents. The following reagents were purchased from Sigma-Aldrich, including cholesterol, gentamicin, cathepsin B (CatB) inhibitor II, cyclophosphamide (CY), and Accutase cell detachment solution. The following reagents were obtained from Thermo Fisher Scientific, including F4 / 80 monoclonal antibody, recombinant mouse macrophage colony-stimulating factor (M-CSF), and LysoTracker® DND-99. 2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was purchased from Avanti Polar Lipids. Bright-Glo luciferase substrate was purchased from Promega.

[0134] Cells and bacteria. The RAW264.7 cell line was obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle's medium (DMEM, ATCC) with 10% fetal bovine serum (Gibco, Invitrogen). Murine bone marrow-derived macrophages (BMDMs) were cultured in DMEM containing 10% fetal bovine serum and 100 ng / mL M-CSF and obtained by adaptation of a previous procedure. MDR Staphylococcus aureus (MDRSA, ATCC BAA-44) was grown in trypticase soy agar or broth at 37°C with aeration. According to data from ATCC, MDRSA is resistant to the following antibiotics: amoxicillin / clavulanic acid, penicillin, ciprofloxacin, cephalothin, doxycycline, gentamicin, erythromycin, imipenem, methicillin, tetracycline, oxacillin, azithromycin, clindamycin, ceftriaxone, rifampin, amikacin, and tobramycin. Multidrug-resistant Escherichia coli (MDR E. coli, ATCC BAA-2340) was grown in nutrient agar or nutrient broth (BD Biosciences) at 37°C with aeration. According to ATCC data, MDR E. coli is resistant to the following: amoxicillin / clavulanic acid, ticarcillin, piperacillin, ampicillin / sulbactam, cephalothin, cefuroxime, cefotetan, cefpodoxime, cefotaxime, ceftizoxime, cefazolin, cefoxitin, ceftazidime, ceftriaxone, cefepime, doripenem, meropenem, ertapenem, imipenem, nalidixic acid, moxifloxacin, norfloxacin, ciprofloxacin, levofloxacin, tobramycin, aztreonam, and trimethoprim / sulfamethoxazole.

[0135] Synthesis of Vitamin-Derived Lipids. Compound 1, a vitamin B3 derivative, a vitamin C derivative, and a vitamin H (also called vitamin B7) derivative were synthesized according to previously reported methods.

[0136] The preparation and characterization of VLNP.mRNA used herein was constructed using an mRNA platform based on reported methods. The preparation of mRNA LNPs was previously reported. Briefly, newly synthesized vitamin-derived lipids were formulated with DOPE, cholesterol, and firefly luciferase (FLuc) mRNA by pipetting for in vivo screening or by a microfluidic mixing device for ex vivo studies. Size and zeta potential were measured using a NanoZS Zetasizer (Malvern). Entrapment efficiency was measured by Ribogreen assay. V C LNP morphology was examined using a Thermo Scientific™ Glacios™ CryoTEM using previously described methods. Initial screening was performed using five vitamin-lipid nanoparticles (VLNPs), Lipofectamine 3000, and electroporation to effectively deliver mRNA to macrophages. In the initial screening, newly synthesized vitamin-derived lipids were formulated with DOPE, cholesterol (lipid:DOPE:cholesterol = 20:30:40, molar ratio), and FLuc mRNA (lipid:mRNA = 10:1, mass ratio). The mRNA delivery efficiency was determined by luciferase expression assay. Next, the kinetics of FLuc expression, followed by two rounds of characterization, were performed after the initial screening. Briefly, VcLNPs A-1 to A-16 were designed using the orthogonal array design table L. 16 (4) 4 The top formulations were prepared based on the FLuc expression data and predicted by the FLuc expression data. After the top formulations were validated, a second round of characterization was performed. C -Focused on fine tuning the lipid:mRNA mass ratio. Electroporation for macrophages was performed using the Nucleofector kit (Lonza) and the protocol suggested by the Nucleofector 2b Device.

[0137] Cellular uptake and endosomal escape. RAW264.7 cells were cultured in 6-well plates at 10 5Cells were seeded at 1000 x 1000 cells / well and cultured for 24 hours. Cells were then treated with FLuc mRNA and Alexa-Fluor 647-labeled RNA (1:1, weight ratio) using Lipofectamine 3000, VcLNP, or electroporation. After 3 hours of incubation, cellular uptake was quantified using a flow cytometer (LSRII, BD). To study the endocytic pathway of VcLNP, cellular uptake assays were performed in the presence of different endocytic inhibitors, including 5-(N-methyl-N-isopropyl) amiloride (EIPA), methyl-beta-cyclodextrin (MβCD), and chlorpromazine hydrochloride (CPZ). For endosomal escape assays, 2 x 10 cells were cultured at 1000 x 1000 cells / well. 4 Cells were seeded in imaging dishes (ibidi) for 24 h, and then 150 μg / mL of calcein was added to the cells with or without VcLNPs containing Alexa-Fluor 647-labeled RNA for 6 h at 37 °C. After washing with PBS to remove extracellular calcein and nanoparticles, the cells were actively imaged under a Nikon A1R Live Cell Imaging Confocal Microscope via 487 nm and 647 nm lasers.

[0138] Analysis of lysosomal colocalization. To test whether AMP-CatB can specifically accumulate in lysosomes, eGFP-CatB mRNA was prepared and expressed in V. C The eGFP-CatB was delivered into RAW264.7 cells using LNPs. Lysosomes were then stained with LysoTracker® Red DND-99, a well-established lysosomal probe. Colocalization of eGFP-CatB and LysoTracker® Red DND-99 was analyzed under a Nikon A1R Live Cell Imaging Confocal Microscope using 487 nm and 561 nm lasers.

[0139] Cytotoxicity of vitamin C lipid nanoparticles in BMDM. C The cytotoxicity of LNPs was examined by MTT assay.4 BMDMs were seeded into each well of a 96-well plate in 100 μL of growth medium. C LNP, AMP-CatB mRNA V C LNP / CatB inhibitor II and AMP-CatB mRNA V C After 12 hours of incubation with LNP, MTT solution was added. After an additional 4 hours of incubation, 100 μL of 10% SDS-HCl was added to each well. The purple formazan was allowed to dissolve overnight, and the absorbance was measured at 570 nm using a plate reader.

[0140] In vitro antibacterial assay. Intracellular antibacterial assay was performed according to the reported method. Briefly, PBS, free AMP-CatB mRNA, empty V C LNP, AMP-CatB mRNA V C LNP / CatB inhibitor II and AMP-CatB mRNA V C After treatment with LNP, RAW264.7 cells or BMDMs were incubated with MDRSA or MDR E. coli at a multiplicity of infection (MOI) of 25 for 120 min. After washing with PBS, the medium was replenished with gentamicin (100 μg / mL), and the cells were incubated for an additional 1 h to remove extracellular bacteria. At different time points, the cells were washed with PBS and lysed with 0.1% Triton-X100. Finally, the lysates were plated on nutrient agar or trypticase soy agar for counting bacterial colony-forming units (CFU).

[0141] In vivo treatment of MDR-induced sepsis mice with immunosuppression. All mouse experiments were performed under protocols approved by the Ohio State University Institutional Animal Care and Use Committee (IACUC). C57BL / 6 mice (6-7 weeks old) were purchased from Jackson Lab. The immunosuppressed sepsis model was performed as previously described. Briefly, C57BL / 6 mice were intraperitoneally injected with cyclophosphamide (CY) at a dosage of 100 mg / kg for three consecutive days of bacterial infection. Their immunocompromised state was assessed by monitoring body weight (BW), counting white blood cells (WBC) with a hemocytometer, and counting lymphocytes (LYM) with Kwik-Diff staining. Mice were then inoculated with 0.1 mL of bacterial suspension (5 × 10 for MDRSA infection). 8 CFU / mouse, 2 × 10 for MDRSA and MDR E. coli infections 8 Mice were intraperitoneally inoculated with 10 ...

[0142] Macrophage and bacterial biodistribution. Macrophage biodistribution was performed in both healthy and septic C57BL / 6 mice (6–7 weeks of age). In this experiment, mRNA encoding firefly luciferase was first delivered to BMDM (FLuc-BMDM) for 12 hours. Each mouse then received 0.2 mL of PBS or 0.2 mL of cell suspension (4 million cells total) via intraperitoneal (ip) and intravenous (iv) injection. Six hours later, the mice were intravenously injected with 150 μL of D-luciferin substrate (30 mg / mL), and then euthanized by CO2 8 minutes after the injection. Bioluminescence signals in the blood, peritoneal fluid, and major organs were immediately measured using a Xenogen IVIS imaging system (Caliper, Alameda, CA). Bacterial CFU in the blood, peritoneal fluid, and major organs of septic mice were quantified 6 hours postinfection.

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[0144] array SEQ ID NO: 1, amino acid sequence of antimicrobial peptide RGGLCYCRGRFCVGR SEQ ID NO: 2, nucleotide sequence encoding antimicrobial peptide AGGGGCGGCUUGUGCUACUGUCGCGGAAGGUUUUGUGUAGGCAGA SEQ ID NO: 3, amino acid sequence of cathepsin B MWWSLILLSCLLALTSAHDKPSFHPLSDDLINYINKQNTTWQAGRNFYNVDISYLKKLCGTVLGGPKLPGRVAFGEDIDLPETFDAREQWSNCPTIGQIRDQGSCGSCWAFGAVEAISDRTCIHTNGRVNVEVSAEDLLTCCGIQCGDGCNGGYPSGAWSFWTKKGLVS GGVYNSHVGCLPYTIPPCEHHVNGSRPPCTGEGDTPRCNKSCEAGYSPSYKEDKHFGYTSYSVSNSVKEIMAEIYKNGPVEGAFTVFSDFLTYKSGVYKHEAGDMMGGHAIRILGWGVENGVPYWLAANSWNLDWGDNGFFKILRGENHCGIESEIVAGIPRTDQYWGR SEQ ID NO: 4, nucleotide sequence encoding cathepsin B SEQ ID NO: 5, amino acid sequence of the linker FGFLG SEQ ID NO: 6, nucleotide sequence encoding the linker UUCGGAUUUCUGGGC SEQ ID NO:7 MWWSLILLSCLLALTSAHDKPSFHPLSDDLINYINKQNTTWQAGRNFYNVDISYLKKLCGTVLGGPKLPGRVAFGEDIDLPETFDAREQWSNCPTIGQIRDQGSCGSCWAFGAVEAISDRTCIHTNGRVNVEVSAEDLLTCCGIQCGDGCNGGYPSGAWSFWTKKGLVSGGVYNSHVGC LPYTIPPCEHHVNGSRPPCTGEGDTPRCNKSCEAGYPSYKEDKHFGYTSYSVSNSVKEIMAEIYKNGPVEGAFTVFSDFLTYKSGVYKHEAGDMMGGHAIRILGWGVENGVPYWLAANSWNLDWGDNGFFKILRGENHCGIESEIVAGIPRTDQYWGRFGFLGRGGLCYCRGRFCVGR SEQ ID NO:8 SEQ ID NO: 9 Amino acid sequence of linker Gly Phe Leu Gly SEQ ID NO: 10 Amino acid sequence of the linker Ala Leu Ala Leu SEQ ID NO: 11 Amino acid sequence of linker Ala Gly Val Phe SEQ ID NO: 12 Amino acid sequence of the linker Val Lys Lys Arg

[0145] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. Publications and the materials for which they are cited herein are specifically incorporated by reference.

[0146] Those skilled in the art will understand that many changes and modifications may be made to the preferred embodiment of the present invention and that such changes and modifications may be made without departing from the spirit of the present invention. It is, therefore, intended by the appended claims to cover all such equivalent variations which fall within the true spirit and scope of the invention. For example, the present disclosure provides the following embodiments. [1] An antigen-presenting cell, lipid-based nanoparticles, the lipid-based nanoparticles comprising: 1. A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid encoding cathepsin B, and the recombinant polynucleotide comprising a third nucleic acid encoding a linker; and a vitamin-lipid. [2] Item 1, wherein the first nucleic acid and the second nucleic acid are linked by the third nucleic acid. [3] Item 3. The antigen-presenting cell according to Item 1 or 2, wherein the recombinant polynucleotide is encapsulated by the vitamin-lipid. [4] Item 4. The antigen-presenting cell according to any one of Items 1 to 3, wherein the recombinant polynucleotide comprises RNA or DNA. [5] Item 5. The antigen-presenting cell according to any one of Items 1 to 4, wherein the antimicrobial peptide comprises the sequence of SEQ ID NO:1. [6] Item 6. The antigen-presenting cell according to any one of Items 1 to 5, wherein the first nucleic acid comprises the sequence of SEQ ID NO:2. [7] Item 7. The antigen-presenting cell according to any one of Items 1 to 6, wherein the second nucleic acid comprises the sequence of SEQ ID NO:4. [8] Item 8. The antigen-presenting cell according to any one of Items 1 to 7, wherein the linker is a cathepsin B-sensitive linker. [9] Item 9. The antigen-presenting cell according to any one of Items 1 to 8, wherein the third nucleic acid comprises the sequence of SEQ ID NO:6.

[10] Item 10. The antigen-presenting cell according to any one of Items 1 to 9, wherein the recombinant polynucleotide comprises the sequence of SEQ ID NO:8.

[11] Item 11. The antigen-presenting cell according to any one of Items 1 to 10, wherein the vitamin-lipid comprises a vitamin moiety, and the vitamin moiety comprises vitamin B3, vitamin C, vitamin D, vitamin E, vitamin H, or a derivative thereof.

[12] Item 12. The antigen-presenting cell of Item 11, wherein the vitamin moiety is vitamin C.

[13] The vitamin-lipid compound of formula A: [ka] or a salt thereof, wherein R 1 is an alkyl or ether linker, said alkyl or ether linker being substituted with a vitamin moiety; R 2Item 13. The antigen-presenting cell according to any one of Items 1 to 12, wherein is alkyl, cycloalkyl, heterocycloalkyl, alkylheterocycloalkyl, amide, alkylamide, ether, or alkyl ether.

[14] The vitamin-lipid is selected from the group consisting of: [ka] In the formula, R is [ka] Item 14. The antigen-presenting cell according to any one of Items 1 to 13,

[15] Item 15. The antigen-presenting cells according to any one of Items 1 to 14, wherein the antigen-presenting cells include macrophages or dendritic cells.

[16] Item 16. The antigen-presenting cell of Item 15, wherein the macrophage comprises a bone marrow-derived macrophage or a monocyte-derived macrophage.

[17] Item 17. The antigen-presenting cell of Item 16, wherein the dendritic cells comprise bone marrow-derived dendritic cells, monocyte-derived dendritic cells, conventional dendritic cells-1, or conventional dendritic cells-2.

[18] 1. A method for treating sepsis, comprising: administering to a subject one or more antigen-presenting cells comprising nanoparticles, wherein the nanoparticles comprise: 1. A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid encoding cathepsin B, and the recombinant polynucleotide comprising a third nucleic acid encoding a linker; a vitamin-lipid.

[19] 20. The method of claim 18, wherein the first nucleic acid and the second nucleic acid are linked by the third nucleic acid.

[20] 20. The method of claim 18 or 19, wherein the recombinant polynucleotide is encapsulated by the vitamin-lipid. [twenty one] 21. The method according to any one of items 18 to 20, wherein the recombinant polynucleotide comprises RNA or DNA. [twenty two] Item 22. The method according to any one of Items 18 to 21, wherein the antimicrobial peptide comprises the sequence of SEQ ID NO:1. [twenty three] 23. The method according to any one of items 18 to 22, wherein the first nucleic acid comprises the sequence of SEQ ID NO:2. [twenty four] 24. The method according to any one of items 18 to 23, wherein the second nucleic acid comprises the sequence of SEQ ID NO:4. [twenty five] Item 25. The method according to any one of Items 18 to 24, wherein the linker comprises a cathepsin B-sensitive linker.

[26] Item 26. The method according to any one of Items 18 to 25, wherein the third nucleic acid comprises the sequence of SEQ ID NO:6.

[27] Item 27. The method according to any one of Items 18 to 26, wherein the recombinant polynucleotide comprises the sequence of SEQ ID NO:8.

[28] Item 28. The method according to any one of Items 18 to 27, wherein the vitamin-lipid comprises a vitamin moiety, and the vitamin moiety comprises vitamin B3, vitamin C, vitamin D, vitamin E, vitamin H, or a derivative thereof.

[29] 29. The method of claim 28, wherein the vitamin moiety is vitamin C.

[30] The vitamin-lipid compound of formula A: [ka] or a salt thereof, wherein R 1 is an alkyl or ether linker, said alkyl or ether linker being substituted with a vitamin moiety; R 2Item 30. The method according to any one of Items 18 to 29, wherein is alkyl, cycloalkyl, heterocycloalkyl, alkylheterocycloalkyl, amide, alkylamide, ether, or alkyl ether.

[31] The vitamin-lipid is selected from the group consisting of: [ka] In the formula, R is [ka] Item 31. The method according to any one of Items 18 to 30, wherein

[32] Item 32. The method according to any one of Items 18 to 31, wherein the antigen-presenting cells are macrophages or dendritic cells.

[33] 33. The method of claim 32, wherein the macrophages comprise bone marrow-derived macrophages or monocyte-derived macrophages.

[34] 33. The method of paragraph 32, wherein the dendritic cells comprise bone marrow-derived dendritic cells, monocyte-derived dendritic cells, conventional dendritic cells-1, or conventional dendritic cells-2.

[35] Item 35. The method according to any one of Items 18 to 34, wherein the antigen-presenting cells are derived from the subject.

[36] Item 36. The method according to any one of Items 18 to 35, wherein the subject includes a human.

[37] 37. The method of paragraph 36, wherein the human has or is suspected of having sepsis.

Claims

1. An antigen-presenting cell, lipid-based nanoparticles, the lipid-based nanoparticles comprising: A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid encoding cathepsin B, and a third nucleic acid encoding a cleavable linker; the recombinant polynucleotide, wherein the first nucleic acid and the second nucleic acid are linked by the third nucleic acid; and A vitamin-lipid selected from the group consisting of: 【Chemistry 1】 In the formula, R is 【Chemistry 2】 Vitamin-lipid The antigen-presenting cell.

2. The antigen-presenting cell of claim 1, wherein the recombinant polynucleotide is encapsulated by the vitamin-lipid.

3. The antigen-presenting cell of claim 1 or 2, wherein the recombinant polynucleotide comprises RNA or DNA.

4. The antigen-presenting cell according to any one of claims 1 to 3, wherein the antimicrobial peptide comprises the sequence of SEQ ID NO:

1.

5. The antigen-presenting cell according to any one of claims 1 to 4, wherein the first nucleic acid comprises the sequence of SEQ ID NO:

2.

6. The antigen-presenting cell according to any one of claims 1 to 5, wherein the second nucleic acid comprises the sequence of SEQ ID NO:

4.

7. The antigen-presenting cell according to any one of claims 1 to 6, wherein the linker is a cathepsin B-sensitive linker.

8. The antigen-presenting cell according to any one of claims 1 to 7, wherein the third nucleic acid comprises the sequence of SEQ ID NO:

6.

9. The antigen-presenting cell according to any one of claims 1 to 8, wherein the recombinant polynucleotide comprises the sequence of SEQ ID NO:

8.

10. The antigen-presenting cell of claim 1 , wherein the vitamin moiety is vitamin C.

11. The antigen-presenting cell according to any one of claims 1 to 10, wherein the antigen-presenting cell comprises a macrophage or a dendritic cell.

12. The antigen-presenting cell of claim 11 , wherein the macrophage comprises a bone marrow-derived macrophage or a monocyte-derived macrophage.

13. The antigen-presenting cell of claim 11, wherein the dendritic cells comprise bone marrow-derived dendritic cells, monocyte-derived dendritic cells, conventional dendritic cells-1, or conventional dendritic cells-2.

14. 1. A pharmaceutical composition for the treatment of sepsis comprising one or more antigen-presenting cells, the pharmaceutical composition is for administration to a subject, and the one or more antigen-presenting cells comprise nanoparticles, A recombinant polynucleotide comprising: a first nucleic acid encoding an antimicrobial peptide; a second nucleic acid encoding cathepsin B, and a third nucleic acid encoding a cleavable linker; the recombinant polynucleotide, wherein the first nucleic acid and the second nucleic acid are linked by the third nucleic acid; and A vitamin-lipid selected from the group consisting of: 【Transformation 3】 In the formula, R is 【Chemistry 4】 Vitamin-lipid The pharmaceutical composition comprising:

15. 15. The pharmaceutical composition of claim 14, wherein the recombinant polynucleotide is encapsulated by the vitamin-lipid.

16. 16. The pharmaceutical composition of claim 14 or 15, wherein the recombinant polynucleotide comprises RNA or DNA.

17. The pharmaceutical composition according to any one of claims 14 to 16, wherein the antimicrobial peptide comprises the sequence of SEQ ID NO:

1.

18. The pharmaceutical composition according to any one of claims 14 to 17, wherein the first nucleic acid comprises the sequence of SEQ ID NO:

2.

19. The pharmaceutical composition of any one of claims 14 to 18, wherein the second nucleic acid comprises the sequence of SEQ ID NO:

4.

20. The pharmaceutical composition of any one of claims 14 to 19, wherein the linker comprises a cathepsin B-sensitive linker.

21. The pharmaceutical composition of any one of claims 14 to 20, wherein the third nucleic acid comprises the sequence of SEQ ID NO:

6.

22. The pharmaceutical composition according to any one of claims 14 to 21, wherein the recombinant polynucleotide comprises the sequence of SEQ ID NO:

8.

23. 15. The pharmaceutical composition of claim 14, wherein the vitamin moiety is vitamin C.

24. The pharmaceutical composition according to any one of claims 14 to 23, wherein the antigen-presenting cells are macrophages or dendritic cells.

25. 25. The pharmaceutical composition of claim 24, wherein the macrophages comprise bone marrow-derived macrophages or monocyte-derived macrophages.

26. 25. The pharmaceutical composition of claim 24, wherein the dendritic cells comprise bone marrow-derived dendritic cells, monocyte-derived dendritic cells, conventional dendritic cells-1, or conventional dendritic cells-2.

27. The pharmaceutical composition according to any one of claims 14 to 26, wherein the antigen-presenting cells are derived from the subject.

28. The pharmaceutical composition of any one of claims 14 to 27, wherein the subject comprises a human.

29. 29. The pharmaceutical composition of claim 28, wherein the human has or is suspected of having sepsis.

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