Novel secretory signal peptides

Abstract

Provided herein are novel engineered and isolated signal peptide sequences and compositions comprising these signal peptides. In addition, compositions and methods of using these signal peptides to secrete heterologous polypeptides of therapeutic, diagnostic and commercial value are also provided.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 376,696, filed on September 22, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted in the WIPO ST.26.xml format via the Patent Center, which is incorporated herein by reference in its entirety. The.xml copy is named "106546 - 757419UTSD3964.xml" and is 66 KB in size. Background of the Invention

[0005] 1. Field

[0006] The present invention relates to the therapeutic applications of novel secretory signal peptides.

[0007] 2. Background

[0008] Recent scientific discoveries have highlighted numerous therapeutic applications of mRNA due to its modular nature and ability to provide customized "instructions" to create functional proteins. Clinical studies have continuously confirmed its good efficacy, further highlighting its potential as a next - generation gene medicine, while scientists' research on its potential applications has just begun. Along with the progress in mRNA biology, significant progress has also been made in the fields of lipid nanoparticles (LNPs), polymer nanoparticles, and other methods for mediating the safe and effective delivery of nucleic acids. To date, most research has focused on expanding the intracellular expression of encoded polypeptides. However, less attention has been paid to the post - translational control of encoded proteins and the targeted localization and secretion of proteins in vivo.

[0009] Inside the cell, there are multiple unique pathways and processes that can transport proteins to various organelles such as the nucleus and mitochondria, and export proteins to the extracellular space through the secretory pathway. However, to utilize these transport systems, the mRNA encoding each protein also contains a metaphorical transport tag, the signal peptide (SP), upstream of the protein sequence. This tag then notifies the appropriate cellular machinery to transport the protein to a specific location within the cell or package it for secretion into the extracellular space. Designing novel signal sequences for therapeutic mRNA can significantly facilitate the delivery, localization, and clearance of encoded therapeutic proteins. Summary of the Invention

[0010] In some aspects, the present disclosure encompasses an engineered signal peptide comprising any one of the amino acid sequences of SEQ ID NOs: 9-29 and variants or derivatives thereof. In some aspects, the signal peptide is fused to a heterologous polypeptide. In some aspects, the heterologous polypeptide is a therapeutic or diagnostic polypeptide, non-limiting examples of which include anti-cancer, anti-inflammatory, immunomodulatory, antiviral, antimicrobial, antifungal, anti-helminth, hypocholestrolemic, anti-diabetic, anti-fibrotic, analgesic, anesthetic, anti-aging, anti-depressant, neuromodulatory, anti-dermatitis, anti-edema, anti-allergy, anti-hyperkeratolyte, antimycotic, anti-itch, cardiovascular therapeutic, chemotherapy, hormone, protein replacement therapeutic, polypeptide therapeutic, vaccine, viral vaccine, theranostic, diagnostic or secretory therapeutic polypeptide.

[0011] In some aspects, the heterologous polypeptide is an enzyme, nutrient, food additive, flavor enhancer and / or cosmetic. In some aspects, the heterologous polypeptide is a reporter polypeptide, non-limiting examples of which include fluorescent proteins, LacZ (β-galactosidase), CAT (chloramphenicol acetyltransferase) or luciferase. In some aspects, the fluorescent protein is any one of GFP (green fluorescent protein), BFP (blue fluorescent protein), YFP (yellow fluorescent protein), RFP (red fluorescent protein), mCherry or luciferase.

[0012] In some aspects, the present disclosure further encompasses a recombinant polynucleotide sequence comprising a nucleic acid sequence encoding any one of the signal peptides of SEQ ID NOs: 9-29. In some aspects, the nucleic acid sequence is a DNA sequence. In some aspects, the nucleic acid sequence is an RNA sequence. In some aspects, the recombinant polynucleotide comprises any one of the nucleic acid sequences of SEQ ID NOs: 30-50 or variants or derivatives thereof. In some aspects, the polynucleotide sequence comprises a ribonucleic acid sequence corresponding to any one of the sequences of SEQ ID NOs: 30-50 or variants or derivatives thereof.

[0013] In some aspects, the recombinant polynucleotide encodes a heterologous polypeptide in-frame with the signal peptide. In some aspects, the heterologous polypeptide is a therapeutic or diagnostic polypeptide, non-limiting examples of which include anti-cancer, anti-inflammatory, immunomodulatory, antiviral, antimicrobial, antifungal, anti-helminth, hypocholestrolemic, anti-diabetic, anti-fibrotic, analgesic, anesthetic, anti-aging, anti-depressant, neuromodulatory, anti-dermatitis, anti-edema, anti-allergy, anti-hyperkeratolyte, antimycotic, anti-itch, cardiovascular therapeutic, chemotherapy, hormone, protein replacement therapeutic, polypeptide therapeutic, vaccine, viral vaccine, theranostic, diagnostic or secretory therapeutic polypeptide.

[0014] In some aspects, the heterologous polypeptide is an enzyme, a nutrient, a food additive, a flavor enhancer, and / or a cosmetic. In some aspects, the heterologous polypeptide is a reporter polypeptide, non-limiting examples of which include fluorescent proteins, LacZ (β-galactosidase), CAT (chloramphenicol acetyltransferase), or luciferase. In some aspects, the fluorescent protein is any one of GFP (green fluorescent protein), BFP (blue fluorescent protein), YFP (yellow fluorescent protein), RFP (red fluorescent protein), mCherry, or luciferase.

[0015] In some exemplary aspects, the recombinant polynucleotide encodes a heterologous polypeptide having any one of the sequences of SEQ ID NOs: 55 - 58 or a functional fragment, derivative, or variant thereof. In some aspects, the heterologous polypeptide is an anti-PD-L1 antibody, Enbrel, mCherry, or hEPO, or a functional fragment, derivative, or variant thereof.

[0016] In some aspects, the present disclosure also encompasses a therapeutic composition comprising a delivery system and a polynucleotide sequence comprising a nucleic acid sequence encoding a signal peptide and a therapeutic polypeptide. In some aspects, the signal peptide comprises any one of the amino acid sequences of SEQ ID NOs: 9 - 29 or a variant or derivative thereof. In some aspects, the polynucleotide sequence encoding the signal peptide comprises a nucleic acid sequence corresponding to any one of the sequences of SEQ ID NOs: 30 - 50 or a variant or derivative thereof.

[0017] In some aspects, the delivery system is any one of a polymer, polymer complexes (polyplexes), lipids, lipid analogs (lipidoids), lipid complexes (lipoplexes), liposomes, polymer nanoparticles, nanoparticles, lipid nanoparticles (LNP), core-shell nanoparticles, solid lipid nanoparticles, metal nanoparticles, self-assembling nucleic acid nanoparticles, hyaluronidase, nanoparticle mimics, ribonucleoproteins, positively charged peptides, small molecule RNA conjugates, aptamer RNA chimeras, RNA fusion protein complexes, and any combination thereof. In some aspects, the delivery system is a lipid nanoparticle comprising an ionizable amino lipid. In some aspects, the lipid nanoparticle further comprises one or more of phospholipids, cholesterol, or polymer lipids. In some aspects, the delivery system comprises any one of iPhos LNP, mDLNP, liver SORT LNP, lung SORT LNP, or spleen SORT LNP. In some aspects, the delivery system is a controlled system selected from a synthetic material depot, a polymer depot, a lipid depot, a controlled release hydrogel depot, or a controlled release polymer depot.

[0018] In some aspects, the therapeutic composition further comprises one or more pharmaceutically acceptable excipients. In some aspects, the therapeutic polypeptide in the therapeutic composition is an anti-cancer, anti-inflammatory, immunomodulatory, antiviral, anti-microbial, anti-fungal, anti-helminthic, cholesterol-lowering, anti-diabetic, anti-fibrotic, analgesic, anesthetic, anti-aging, anti-depressant, neuromodulatory, anti-dermatitis, anti-edema, anti-allergic, anti-hyperkeratotic, anti-mildew, anti-itch, cardiovascular therapeutic, chemotherapy, hormone, protein replacement therapeutic, polypeptide therapeutic, vaccine, viral vaccine, theranostic, diagnostic or secretory therapeutic polypeptide.

[0019] In some aspects, the present disclosure also encompasses a method of diagnosis, prevention or treatment, comprising administering to a subject in need thereof an effective amount of the composition disclosed herein.

[0020] In some aspects, the method of diagnosis, prevention or treatment comprises administering the composition disclosed herein by one or more of the following routes: parenteral, oral, intra-adipose, intra-arterial, intra-articular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrarectal, intracapsular, intratracheal, intratumoral, intraumbilical, intravaginal, intravenous, intravascular, intravitreal, liposomal, topical, mucosal, parenteral, rectal, subconjunctival, subcutaneous, sublingual, topical, buccal, or transdermal route. In some aspects, the administration is carried out by a controlled system selected from an implant, a synthetic material depot, a polymer depot, a lipid depot, a controlled release hydrogel depot, or a controlled release polymer depot.

[0021] In some aspects, the subject in need of diagnosis, prevention or treatment is suspected of having or has been diagnosed with any one of an autoimmune disorder, cancer, diabetes, cardiovascular disease, neurological disease, bacterial infection, fungal infection, viral infection, or fibrosis. In some aspects, the subject is in need of prevention. In some aspects, the therapeutic polypeptide disclosed herein is systemically secreted in a subject in need thereof. In some aspects, the therapeutic polypeptide is suitable for expression in any one of the lung, liver or spleen. In some aspects, the subject is a mammal. In some aspects, the subject is a human.

[0022] In some aspects, the present disclosure also encompasses a recombinant polypeptide comprising: a signal peptide corresponding to any one of the sequences of SEQ ID NOs: 1-4 or a variant or derivative thereof; and a heterologous polypeptide.

[0023] In some aspects, the present disclosure also encompasses a recombinant polynucleotide comprising a nucleic acid sequence encoding a signal peptide corresponding to any one of the sequences of SEQ ID NO.1-4 or a variant or derivative thereof and a heterologous polypeptide in-frame with the signal peptide. In some aspects, the nucleic acid sequence can be a DNA or RNA sequence corresponding to SEQ ID NOs: 5-8 or a variant or derivative thereof.

[0024] In some aspects, the heterologous polypeptide is a therapeutic or diagnostic polypeptide, such as an anti-cancer, anti-inflammatory, immunomodulatory, antiviral, antimicrobial, antifungal, anti-helminth, cholesterol-lowering, anti-diabetic, anti-fibrotic, analgesic, anesthetic, anti-aging, antidepressant, neuromodulatory, anti-dermatitis, anti-edema, anti-allergy, anti-hyperkeratosis, fungistatic, anti-itch, cardiovascular therapeutic, chemotherapy, hormone, protein replacement therapeutic, polypeptide therapeutic, vaccine, viral vaccine, theranostic, diagnostic or secretory therapeutic polypeptide. In some aspects, the heterologous polypeptide is an enzyme, a nutrient, a food additive, a flavor enhancer and / or a cosmetic. In some aspects, the heterologous polypeptide is a reporter polypeptide selected from a fluorescent protein, LacZ (β-galactosidase), CAT (chloramphenicol acetyltransferase) or luciferase. In some aspects, the heterologous polypeptide is, for example, an anti-PD-L1 antibody, Enbrel, hEPO or a functional fragment, derivative or variant thereof.

[0025] In some aspects, the present disclosure also encompasses a therapeutic composition comprising a delivery system and a polynucleotide sequence comprising a nucleic acid sequence encoding a signal peptide corresponding to any one of SEQ ID NOs: 1-4 or a variant or derivative thereof and a therapeutic polypeptide. In some aspects, the polynucleotide sequence encoding the signal peptide comprises any one of the nucleic acid sequences of SEQ ID NOs: 5-8 or a variant or derivative thereof. In some aspects, the polynucleotide sequence encoding the signal peptide comprises a ribonucleic acid sequence corresponding to any one of SEQ ID NOs: 5-8 or a variant or derivative thereof. In some aspects, non-limiting examples of the delivery system of the therapeutic composition disclosed herein include polymers, polymer complexes, lipids, lipid analogs, lipid complexes, liposomes, polymer nanoparticles, nanoparticles, lipid nanoparticles (LNP), core-shell nanoparticles, solid lipid nanoparticles, metal nanoparticles, self-assembling nucleic acid nanoparticles, hyaluronidase, nanoparticle mimetics, ribonucleoproteins, positively charged peptides, small molecule RNA conjugates, aptamer RNA chimeras, RNA fusion protein complexes and any combination thereof. In some aspects, the delivery system is a lipid nanoparticle comprising an ionizable amino lipid. In some aspects, the lipid nanoparticle further comprises one or more of phospholipids, cholesterol, or polymeric lipids. In some aspects, the delivery system includes any one of iPhos LNP, mDLNP, liver SORT LNP, lung SORT LNP, or spleen SORT LNP. In some aspects, the delivery system is a controlled system selected from a synthetic material depot, a polymer depot, a lipid depot, a controlled release hydrogel depot, or a controlled release polymer depot.

[0026] In some aspects, a therapeutic composition comprising a signal peptide corresponding to SEQ ID NO: 1-4 or a variant or derivative thereof further comprises one or more pharmaceutically acceptable excipients. In some aspects, the therapeutic composition comprises a therapeutic polypeptide, such as an anti-cancer, anti-inflammatory, immunomodulatory, antiviral, antimicrobial, antifungal, anti-helminthic, cholesterol-lowering, anti-diabetic, anti-fibrotic, analgesic, anesthetic, anti-aging, antidepressant, neuromodulatory, anti-dermatitis, anti-edema, anti-allergic, anti-hyperkeratotic, anti-mildew, anti-itch, cardiovascular therapeutic, chemotherapy, hormone, protein replacement therapeutic, polypeptide therapeutic, vaccine, viral vaccine, theranostic, diagnostic or secretory therapeutic polypeptide.

[0027] In some aspects, the present disclosure also encompasses a method of diagnosis, prevention or treatment, comprising administering to a subject in need thereof an effective amount of a composition comprising a signal peptide corresponding to SEQ ID NO: 1-4 or a variant or derivative thereof. These therapeutic compositions can be administered, for example, by one or more of the following routes: parenteral, oral, intra-adipose, intra-arterial, intra-articular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrarectal, intracapsular, intratracheal, intratumoral, intraumbilical, intravaginal, intravenous, intravascular, intravitreal, liposomal, topical, mucosal, parenteral, rectal, subconjunctival, subcutaneous, sublingual, transdermal, transmucosal, or transdermal route. In some aspects, the administration is carried out by a controlled system selected from an implant, a synthetic material depot, a polymer depot, a lipid depot, a controlled release hydrogel depot, or a controlled release polymer depot. In some aspects, the subject is suspected of having or has been diagnosed with any one of an autoimmune disorder, cancer, diabetes, cardiovascular disease, neurological disease, bacterial infection, fungal infection, viral infection, or fibrosis. In some aspects, the subject is a mammal in need of prevention, such as a human. In some aspects, the composition is systemically secreted in a subject in need thereof or is specifically expressed in an organ-specific manner in any one of the lungs, liver, or spleen. In some aspects, the subject is suspected of having or has been diagnosed with any one of an autoimmune disorder, cancer, diabetes, or fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Aspects of the inventive concept are illustrated by examples, in which like reference numerals denote like elements, wherein:

[0029] Figure 1A A schematic diagram is provided of a construct comprising mCherry having a signal peptide at the N-terminus and its use for screening signal peptides by in vitro transfection with pDNA.

[0030] Figure 1BFluorescence microscopy images are provided, which show time-dependent mCherry secretion in Hela cells (exposure time, 1 / 30 s). The mCherry signal was clearly visible in the culture medium after two days of treatment with gLuc-mCherry. PBS, phosphate buffer solution; WT, wild type; gLuc, Gaussia luciferase.

[0031] Figure 1C Quantification of mCherry fluorescence in cell lysates and culture media at different time points is provided. mCherry without a signal peptide was used as a control ("WT-mCherry"). PBS, phosphate buffer solution; WT, wild type; gLuc, Gaussia luciferase.

[0032] Figure 1D Fluorescence microscopy images are provided, which show the secretion of mCherry in Hela cells at 72 h (exposure time, 1 / 70 s). PBS, phosphate buffer solution; NC, negative control; hAlb, human albumin; hApoB, human apolipoprotein B; gLuc, Gaussia luciferase; hFVII, human coagulation factor VII.

[0033] Figure 1E Photographs of cell lysates and culture media imaged at 72 h are provided. PBS, phosphate buffer solution; NC, negative control; hAlb, human albumin; hApoB, human apolipoprotein B; gLuc, Gaussia luciferase; hFVII, human coagulation factor VII.

[0034] Figure 1F Quantification of mCherry fluorescence and cell lysates and culture media after 72 h is provided, and the data are presented as mean ± s.e.m. (n = 5 biologically independent samples). Hela cells in 96-well plates (50 ng per well) were treated with Lipofectamine 2000 / pDNA, and the cells were imaged by microscopy at the given time points. The mCherry signal was quantified by a plate reader or captured by IVIS. A two-tailed unpaired t-test was used to determine the significance of data comparison (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). PBS, phosphate buffer solution; NC, negative control; hAlb, human albumin; hApoB, human apolipoprotein B; gLuc, Gaussia luciferase; hFVII, human coagulation factor VII.

[0035] Figure 1GBar graph showing mCherry signal in cell lysates and media at 72 h, confirming the function of the signal peptide in Huh7 cells. Data are represented as mean ± s.e.m. (n = 4 biologically independent samples). Two-tailed unpaired t-test was used to determine significance of data comparison (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). PBS, phosphate buffered saline; NC, negative control; hAlb, human albumin; hApoB, human apolipoprotein B; gLuc, Gauss luciferase; hFVII, human coagulation factor VII. PBS, phosphate buffered saline; NC, negative control; hAlb, human albumin; hApoB, human apolipoprotein B; gLuc, Gauss luciferase; hFVII, human coagulation factor VII.

[0036] Figure 1H Confocal images are shown, which show the "circle-like" signal (indicated by yellow arrows) observed in Huh7 cells when the signal peptide works well (scale bar, 50 μm). PBS, phosphate buffered saline; NC, negative control; hAlb, human albumin; hApoB, human apolipoprotein B; gLuc, Gauss luciferase; hFVII, human coagulation factor VII.

[0037] Figure 1I Quantification of mCherry fluorescence in cell lysates and media at 72 h post transfection is provided. Data are represented as mean ± s.e.m. (n = 4 biologically independent samples). Two-tailed unpaired t-test was used to determine significance (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). PBS, phosphate buffered saline; NC, negative control; hAlb, human albumin; hApoB, human apolipoprotein B; gLuc, Gauss luciferase; hFVII, human coagulation factor VII.

[0038] Figure 1J Images of cell lysates and media from IVIS imaging are shown. Huh7 cells in 96-well plates (50 ng per well) were treated with Lipofectamine 2000 / pDNA, and at 72 h, mCherry signal was quantified by plate reader or captured by IVIS. PBS, phosphate buffered saline; NC, negative control; hAlb, human albumin; hApoB, human apolipoprotein B; gLuc, Gauss luciferase; hFVII, human coagulation factor VII.

[0039] Figure 2A Schematic diagram showing in vitro transcription (IVT) synthesis of hFVII-mCherry mRNA, in vivo delivery encapsulated by LNP, and imaging using microscope and plate reader.

[0040] Figure 2B Shows time-dependent (100 ng mRNA per well) and dose-dependent (72 hours) mCherry secretion in Huh7 cells, culture medium, and cell lysates.

[0041] Figure 2C Shows quantification of mCherry signal in different cell lines. Cells in 96-well plates were treated with mDLNP-mRNA, and at given time points, the mCherry signal was quantified using a plate reader.

[0042] Figure 2D Shows fluorescence and bright-field images of HEK293T cells treated with hFVII-mCherry mRNA formulation. At day 3 after treatment with hFVII-mCherry mRNA formulation, distinct mCherry signal was observed in the culture medium. HEK293T cells in 96-well plates were treated with different doses of mDLNP-mRNA and imaged using a microscope (exposure time 1 / 6 s for 24 hours and 1 / 15 s for 72 hours). A close-up reproduction of the image showing HEK293T cells treated with mRNA formulation for 24 hours (exposure time 1 / 6 s) is presented. The reproduced image shows a clear "circular-like" mCherry distribution (indicated by yellow arrows) in the hFVII-mCherry mRNA formulation group, which is similar to that Figure 1H shown for pDNA delivery.

[0043] Figure 2E Shows efficient liver-targeted Luc mRNA delivery via mDLNP. Mice were intravenously injected with a dose of 0.1 mg / kg of Luc mRNA and imaged at hour 3.

[0044] Figure 2F Shows successful in vivo secretion of mCherry via liver-targeted delivery mediated by mDLNP / hFVII-mCherry. Mice were intravenously injected with a dose of 0.5 mg / kg of mRNA, and sera were collected from 2 hours to 72 hours after treatment and the mCherry signal was detected using a plate reader. Mice were sacrificed at 55 hours and 72 hours, and tissues were imaged by IVIS.

[0045] Figure 2G Shows mCherry signal in the kidney, indicating mCherry secretion in the blood.

[0046] Figure 2HIt shows that liver SORT LNP, lung SORT LNP, or spleen SORT LNP successfully mediated the delivery of Luc mRNA and hFVII-mCherry in vivo. SORT LNP showed tissue-selective Luc mRNA delivery and all successfully secreted mCherry into the blood. For the Luc assay, mice were intravenously injected with mRNA at a dose of 0.1 mg / kg and imaged at 3 hours. For the mCherry assay, mice were intravenously injected with mRNA at a dose of 0.5 mg / kg and imaged at 24 hours.

[0047] Figure 3A It shows a schematic diagram of Enbrel protein production, TNF-α binding, and disease treatment in a dermatitis model.

[0048] Figure 3B It is a graph showing the dose-dependent cytotoxicity of human TNF-α (hTNF-α) and murine TNF-α (mTNF-α) in L929 cells. Cells were incubated with 1 μg / ml of actinomycin and different concentrations of TNF-α for 24 hours, and then cytotoxicity was measured.

[0049] Figure 3C It is a graph showing cell viability in the presence of TNF-α. L929 cells pretreated with hFVII-Enbrel mRNA were resistant to both murine and human TNF-α. Cells were pretreated with mDLNP-hFVII-Enbrel mRNA (80 ng mRNA per well), and two days later, cells were stimulated with TNF-α at concentrations of 0 - 5 ng / ml and a fixed 1 μg / ml of actinomycin. After another 24 hours, cell viability was detected.

[0050] Figure 3D It is a graph quantifying the dose-dependent viability rescue through LNP after pretreatment. Cells were pretreated with mDLNP-hFVII-Enbrel mRNA at mRNA doses of 0 - 1.25 ng / ml, and two days later, cells were stimulated with TNF-α at a dose of 0.1 ng / ml and a fixed 1 μg / ml of actinomycin. After 24 hours, cell viability was detected.

[0051] Figure 3EIt is a graph showing the dose-dependent rescue of the LNP formulation for display. L929 cells were pretreated with mDLNP-hFVII-Enbrel mRNA at an mRNA dose of 0 - 1.25 ng / ml. Two days later, the cells were stimulated with TNF-α at a dose of 0.02 ng / ml and actinomycin at a fixed 1 μg / ml (upper left figure). After another 24 hours, cell viability was detected. No obvious cytotoxicity was observed with the mRNA formulation alone (upper right figure). The dose-dependent rescue of cell viability in the medium of cells treated with the LNP formulation is shown. Cells were pretreated with mDLNP-hFVII-Enbrel mRNA at an mRNA dose of 0 - 1.25 ng / ml. Two days later, the medium containing secreted Enbrel was transferred to new L929 cells, and at the same time, the cells were stimulated with TNF-α at a dose of 0.1 ng / ml and actinomycin at 1 μg / ml (lower left figure). After another 24 hours, cell viability was detected (lower right figure).

[0052] Figure 3F The workflow for creating and treating the imiquimod-induced psoriasis model is shown.

[0053] Figure 3G The pharmacokinetic comparison after single administration of Enbrel protein and mRNA is shown. Mice were intravenously injected with a protein or mRNA formulation at a dose of 0.5 mg / kg. Serum was collected at different time points, and Enbrel was detected using an ELISA kit.

[0054] Figure 3H Images of the dorsal skin of vehicle control mice or imiquimod-treated mice injected with mCherry mRNA or hFVII-Enbrel mRNA formulations are provided. Images of H&E staining sections, Ki-67 staining, and Gr-1 staining of the dorsal skin of vehicle control mice and imiquimod-treated mice are shown in the figure.

[0055] Figure 3I Quantitative measurements of the epidermal thickness of three groups are provided, as well as the percentage of Ki-67 + cells in epidermal basal cells (per 50 cells) quantified starting from h (****P < 0.0001).

[0056] Figure 4A The workflow of tumor immunotherapy with hFVII-anti-PDL1 mRNA is shown. The protocol for anti-PDL1 antibody production and tumor immunotherapy is shown in the figure.

[0057] Figure 4BShows the experimental design of hFVII-anti-PDL1 mRNA tumor immunotherapy using MC38, MC38-Luc, and B16F10-Luc xenograft mouse models.

[0058] Figure 4C Provides pharmacokinetic study data of anti-PDL1 antibody after single-dose administration of LNP-mRNA formulation. Mice were intravenously injected with the mRNA formulation at a dose of 0.5 mg / kg. Serum was collected at different time points and anti-PDL1 was detected using an ELISA kit.

[0059] Figure 4D Shows the PDL1 expression on the cell membrane surface of MC-38 determined by flow cytometry.

[0060] Figure 4E Shows the luminescence images of MC38-Luc tumors captured by IVIS at different time points.

[0061] Figure 4F Shows the luminescence image of the isolated MC38-Luc tumor on day 32. Tumor images on day 32 (1 / 4 clearance) are also provided. (*P<0.05).

[0062] Figure 4G Shows the quantitative luminescence signals in the tumor region at different time points after tumor immunotherapy with hFVII-anti-PDL1 mRNA. (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0063] Figure 4H Shows the tumor growth of the MC38 model. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0064] Figure 4I Shows the survival data of mice during mRNA formulation treatment. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0065] Figure 4J Shows the luminescence images of B16F10-Luc tumors on day 3 and day 16 treated with different formulations.

[0066] Figure 4K Shows the representative PDL1 expression on the cell membrane surface of B16F10-Luc determined by flow cytometry.

[0067] Figure 4LShows the tumor growth of the B16F10-Luc model. (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0068] Figure 4M Shows the survival of mice during mRNA formulation treatment. (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0069] Figure 4N Shows better pharmacokinetic curves of the mRNA formulation compared to the protein. The pharmacokinetic comparison of PD1 protein and mRNA formulation after single-dose administration is shown. Mice were intravenously injected with the mRNA formulation or protein at a dose of 0.5 mg / kg. Serum was collected at different time points and PD1 was detected using an ELISA kit.

[0070] Figure 5 Shows the schematic diagram of matrix generation for constructing a novel signal peptide sequence.

[0071] Figure 6 Is an overview of the signal peptide structure, mRNA sequence, and generation of SP-mRNA by in vitro transcription.

[0072] Figure 7 Is a graph showing the serum hEPO concentration after injection. The mRNA with SP(1-21)-hEPO was encapsulated into iPhosLNP and intravenously injected into mice at a dose of 0.5 mg / kg. For each construct, blood was collected from mice at 6, 24, 48, and 72 hours, and the serum hEPO concentration in mIU / mL was determined by hEPO enzyme-linked immunosorbent assay (ELISA).

[0073] The drawings do not limit the inventive concept to the specific aspects disclosed and described herein. The drawings are not necessarily drawn to scale, and the emphasis is on clearly illustrating the principles of certain aspects of the inventive concept. Detailed Description

[0074] The following detailed description refers to the drawings that illustrate various aspects of the inventive concept. The drawings and the detailed description are intended to fully and detailedly illustrate various aspects of the inventive concept so that those skilled in the art can practice the inventive concept. Other components may be used and changes may be made without departing from the scope of the inventive concept. Therefore, the following description should not be construed as restrictive. The scope of the inventive concept is only defined by the full scope of the appended claims and their equivalents.

[0075] This disclosure is the result of intensive research by the inventors to identify and / or develop novel secretory signal peptides for therapeutic and non-therapeutic applications. While much work has been done to maximize the expression of therapeutic proteins after nucleic acid delivery to cells, post-translational control of protein localization by elucidating and exploiting endogenous protein translocation pathways has been largely overlooked. Systemic delivery or targeted delivery to specific organs of nucleic acid-encoded therapeutic-related polypeptides within cells has important benefits for the field of nucleic acid therapeutics. Current research has used carefully designed experiments and bioinformatics methods to identify and isolate naturally occurring or develop synthetic secretory signal peptides that can control the systemic and organ-specific secretion of polypeptides. Extensive experimental validation has subsequently been performed, and these signal peptide sequences have been used to develop therapeutic and non-therapeutic compositions. These naturally occurring and synthetic signal peptides can be fused to any heterologous protein of interest to control its trafficking and localization. This work is a pathway for the development of novel protein therapeutics that may impact the treatment efficacy of patients with a variety of different disease conditions. In addition, this work can also be used to develop non-therapeutic applications such as diagnostics, industrial production of heterologous proteins, and various laboratory uses.

[0076] I. Terms

[0077] The language and terminology used herein are for descriptive purposes only and should not be regarded as limiting. For example, the use of singular terms, such as "a" or "an", is not intended to limit the number of items. Additionally, the use of relational terms in the specification, such as but not limited to "top", "bottom", "left", "right", "above", "below", "downward", "upward", and "side", is for clarity when specifically referring to the figures and is not intended to limit the scope of the inventive concept or the appended claims.

[0078] In addition, since the inventive concept can be applied to various different forms of aspects, the present disclosure should be regarded as an example of the principles of the inventive concept, rather than intending to limit the inventive concept to the specific aspects shown and described. Any feature of the inventive concept can be used alone or in combination with any other feature. The terms "aspect", "aspects", and / or similar terms mentioned in the specification refer to the feature and / or features mentioned being included in at least one of the described aspects. The terms "aspect", "aspects", and / or similar terms separately mentioned in the description do not necessarily refer to the same aspect and are not mutually exclusive, unless otherwise stated and / or it is readily apparent to those skilled in the art from the description. For example, the features, structures, processes, steps, actions, or the like described in one aspect may also be included in other aspects, but not necessarily. Therefore, the inventive concept may include various combinations and / or integrations of the aspects described herein. In addition, all aspects of the present disclosure described herein are not essential for its practice. Similarly, other systems, methods, features, and advantages of the inventive concept will also be apparent to those skilled in the art after reading the drawings and the specification. All such additional systems, methods, features, and advantages should be included in this specification, fall within the scope of the inventive concept, and be covered by the claims.

[0079] Any degree terms used in the specification and the appended claims, such as but not limited to "substantially", should be understood to include an exact or approximate but not exact configuration. For example, a "substantially flat surface" means an exactly flat surface or an approximate but not exact flat surface. Similarly, the terms "about" or "approximately" used in the specification and the appended claims should be understood to include the stated value or a value three times or one-third of the stated value. For example, about 3 millimeters includes all values from 1 millimeter to 9 millimeters, and approximately 50 degrees includes all values from 16.6 degrees to 150 degrees. For example, they can refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%.

[0080] The terms "comprising", "including", and "having" can be used interchangeably in the present disclosure. The terms "comprising", "including", and "having" mean including, but not necessarily limited to, what is described.

[0081] Finally, the terms "or" and "and / or" as used herein shall be construed to include and mean any one or any combination. Thus, "A, B, or C" or "A, B, and / or C" means any of the following: "A", "B", or "C"; "A and B"; "A and C"; "B and C"; "A, B, and C". This definition is only an exception when the combination of elements, functions, steps, or acts is mutually exclusive to some extent.

[0082] The terms "nucleic acid", "nucleic acid molecule", and "polynucleotide" are used interchangeably herein. The term "nucleic acid encoding..." or "nucleic acid molecule encoding..." shall be understood to mean a nucleotide sequence encoding a polypeptide.

[0083] The polynucleotides described herein may comprise one or more nucleic acids, each nucleic acid encoding a polypeptide and operably linked (i.e., in a functional relationship) to one or more regulatory sequences such as a promoter. Such polynucleotides may also be referred to herein as "nucleic acid constructs" or "constructs". The term "operably linked" as used herein refers to a functional linkage between a promoter or other regulatory element and the coding sequence of a relevant transcribable DNA sequence or gene (or transgene) such that the promoter etc. operates to initiate, assist, affect, cause, and / or facilitate the transcription and expression of the relevant transcribable DNA sequence or coding sequence at least in certain tissues, developmental stages, and / or conditions.

[0084] The polynucleotide sequences "corresponding" to the provided sequences as used herein encompass DNA, RNA, and cDNA sequences with reference to that sequence. For example, in the case of a provided sequence encoding a heterologous polypeptide, the term encompasses DNA and RNA sequences encoding the heterologous polypeptide. Thus, the polynucleotide may be the same as the reference sequence or a complementary RNA sequence of the reference sequence.

[0085] As used herein, the term "regulatory element" refers to any sequence element that positively or negatively regulates the expression of an operably linked sequence. "Regulatory elements" include, but are not limited to, promoters, enhancers, leaders, transcription start sites (TSSs), linkers, 5' and 3' untranslated regions (UTRs), introns, polyadenylation signals, and termination regions or sequences, etc., which are suitable, necessary, or preferred for regulating or allowing the expression of a gene or transcriptionally active DNA sequence in a cell. These additional regulatory elements may be optional and are used to enhance or optimize the expression of a gene or transcriptionally active DNA sequence. For example, the regulatory sequence may be inducible, non-inducible, constitutive, cell cycle-regulated, metabolism-regulated, etc. The regulatory sequence may be a promoter. As used herein, the term "promoter" refers to a DNA sequence that contains an RNA polymerase binding site, a transcription start site, and / or a TATA box and aids or promotes the transcription and expression of an associated transcriptionally active polynucleotide sequence and / or gene (or transgene). A promoter may be synthetically produced, altered, or derived from a known or naturally occurring promoter sequence or other promoter sequences. A promoter may also include a chimeric promoter that contains a combination of two or more heterologous sequences. Thus, the promoters of the present application may include variants of promoter sequences that are similar but not identical to other promoter sequences known or provided herein. As used herein, the term "enhancer" refers to a DNA sequence region that initiates, aids, affects, causes, and / or promotes the transcription and expression of an associated transcriptionally active DNA sequence or coding sequence, at least in certain tissues, developmental stages, and / or conditions. In one aspect, the enhancer is a cis enhancer. In one aspect, the enhancer is a trans enhancer.

[0086] As used herein, the term "operably linked" refers to a functional linkage between a promoter or other regulatory element and the coding sequence of an associated transcriptionally active DNA sequence or gene (or transgene) such that the promoter, etc., functions to initiate, aid, affect, cause, and / or promote the transcription and expression of the associated transcriptionally active DNA sequence or coding sequence, at least in certain tissues, developmental stages, and / or conditions.

[0087] The terms "polypeptide" and "protein" are used interchangeably herein and refer to amino acid polymers of any length. The polymers may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified either naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. For example, the definition also includes polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. The terms "polypeptide" and "protein" as used herein specifically encompass antibodies.

[0088] An amino acid sequence "derived" from the amino acid sequences disclosed herein can refer to an amino acid sequence that differs from a reference amino acid sequence by one or more amino acids, for example, containing one or more amino acid insertions, deletions, or substitutions disclosed herein. When the terms "derivative", "variant", and "fragment" are used herein to refer to a polypeptide, they refer to a polypeptide related to a wild-type polypeptide, for example, related in any way by amino acid sequence, structure (such as secondary and / or tertiary), activity (such as enzyme activity), and / or function. Compared with the wild-type polypeptide, the derivatives, variants, and fragments of a polypeptide may include one or more amino acid variations (such as mutations, insertions, and deletions), truncations, modifications, or combinations thereof. A portion or fragment of a polypeptide may correspond to at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40% of the length of the polypeptide, for example, a polypeptide having the amino acid sequence identified by a specific SEQ ID NO., or a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the length of the polypeptide (in amino acids).

[0089] In this application, proteins are represented by amino acid sequences, and the corresponding nucleic acid molecules or polynucleotides are represented by nucleic acid sequences. Identity and similarity between sequences: Throughout this application, each time a specific amino acid sequence SEQ ID NO (taking SEQ ID NO:Y as an example) is mentioned, it can be replaced by: a polypeptide represented by the amino acid sequence, which contains a sequence having at least 60% sequence identity or similarity with the amino acid sequence SEQ ID NO:Y. Another preferred level of sequence identity or similarity is 65%. Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 75%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 85%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 98%. Another preferred level of sequence identity or similarity is 99%.

[0090] Each amino acid sequence described herein, by virtue of its percentage identity or similarity, respectively, with a given amino acid sequence, in another preferred aspect, has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity or similarity with the given nucleotide or amino acid sequence. The terms "homology", "sequence identity", etc. are used interchangeably herein. As used herein, sequence identity refers to the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences determined by comparing the sequences. In a preferred aspect, sequence identity is calculated based on the full length or a partial length of two given SEQ ID NOs. The partial length preferably refers to at least 50%, 60%, 70%, 80%, 90%, or 100% of the two SEQ ID NOs. In the art, "identity" also refers to the degree of sequence relatedness between amino acids or nucleic acid sequences, as the case may be, determined by the degree of match between such sequence strings. For example, the degree of sequence identity between two sequences can be determined by comparing the two sequences using computer programs commonly used for this purpose, such as global or local alignment algorithms. Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, or other suitable methods or algorithms. The Needleman and Wunsch global alignment algorithm can be used to align the full length or a partial length (the partial length can refer to at least 50%, 60%, 70%, 80%, 90% of the sequence length) of two sequences to maximize the number of matches and minimize the number of gaps.Default settings can be used. Preferred programs are Needle for pairwise sequence alignment (in one aspect, EMBOSS Needle 6.6.0.0, gap open penalty 10, gap extent penalty 0.5, end gap penalty: false, end gap open penalty: 10, end gap extent penalty: 0.5) and MAFFT for multiple sequence alignment (in one aspect, the default values for MAFFT v7 are: BLOSUM62 [bl62], gap open: 1.53, gap extent: 0.123, order: by alignment, number of tree reconstructions: 2, bootstrap tree output: ON [true], maximum number of iterations: 2, execute FFTS: not used).

[0091] "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conservative amino acid substitutions with the sequence of a second polypeptide. Similar algorithms for determining sequence identity can also be used to determine sequence similarity. Optionally, when determining the degree of amino acid similarity, one of ordinary skill in the art can also consider so-called conservative amino acid substitutions. As used herein, "conservative" amino acid substitutions refer to the interchangeability between residues having similar side chains.

[0092] For example, a group of amino acids having aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxy side chains are serine and threonine; a group of amino acids having amide-containing side chains are asparagine and glutamine; a group of amino acids having aromatic side chains are phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains are lysine, arginine, and histidine; a group of amino acids having sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acid substitutions are valine - leucine - isoleucine, phenylalanine - tyrosine, lysine - arginine, alanine - valine, and asparagine - glutamine. Substitution variants of the amino acid sequences disclosed herein refer to those in which at least one residue in the disclosed sequence is removed and a different residue is inserted in its place. Preferably, the change in amino acid is conservative. The preferred conservative substitutions for each naturally occurring amino acid are as follows: Ala to Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser or Ala; Gln to Asn; Glu to Asp; Gly to Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg; Gln or Glu; Met to Leu to Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and Val to Ile or Leu.

[0093] The term "heterologous", when referring to a nucleic acid molecule (such as a coding sequence) or a polypeptide (such as an enzyme), means a nucleic acid molecule or protein that is not naturally present in a host organism or cell. "Heterologous" also includes a native coding region or a portion thereof that has been removed from a source organism and subsequently reintroduced into the source organism in a form different from the corresponding native gene (e.g., not at its natural location in the organism's genome). A heterologous nucleic acid molecule is deliberately introduced into a host cell. A "heterologous" nucleic acid molecule or protein can be from any source, such as eukaryotes, prokaryotes, viruses, etc. In one aspect, a heterologous nucleic acid molecule can be derived from a eukaryote (such as, for example, another yeast, etc.) or a prokaryote (such as, for example, a bacterium, etc.). The term "heterologous" as used herein also refers to an element (nucleic acid or protein) derived from a source other than endogenous. Thus, for example, a heterologous element can be derived from a different strain of a host cell, or from an organism of a different taxonomic group (e.g., different kingdom, phylum, class, order, family, genus, or species, or any subgroup within these classifications). The term "heterologous" is also synonymous with the term "exogenous" herein.

[0094] As used herein, certain aspects of the present invention are "engineered" when they have characteristics or properties (either structurally or chemically) different from a starting point, wild type, or native molecule. As used herein, for a signal peptide, the term "engineered" or "synthetic" means that one or more amino acids in its amino acid sequence are different from a naturally occurring signal peptide. In some aspects, these signal peptides are designed by performing bioinformatics analysis on a naturally occurring signal peptide. In some aspects, a signal peptide may be derived from a natural signal peptide but is not identical thereto. As used herein, for a polypeptide, the term "engineered" or "recombinant" means a polypeptide whose amino acid sequence has been altered after applying genetic engineering techniques to the nucleic acid encoding the polypeptide and to the cell or organism expressing the polypeptide. The alterations include, but are not limited to, insertions, deletions, or substitutions. For a nucleic acid, the term "recombinant" or "engineered" means a nucleic acid whose nucleic acid sequence has been altered by applying genetic engineering techniques. The alterations include, but are not limited to, insertions, deletions, or substitutions. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning techniques; transfection, transformation, and other gene transfer techniques; homologous recombination; site-directed mutagenesis; and gene fusion.

[0095] The terms "fusion protein" or "fusion polypeptide" are used interchangeably and refer to a polypeptide encoded by a nucleic acid sequence that contains a coding sequence from one nucleic acid molecule and a coding sequence from another nucleic acid molecule, wherein the coding sequences are in the same reading frame such that when the fusion construct is transcribed and translated in a host cell, a protein is produced that contains both proteins. The two molecules can be adjacent in the construct or can be separated by a linker polypeptide containing 1, 2, 3, or more amino acids. The protein product encoded by the fusion construct is called a fusion polypeptide.

[0096] The terms “host cell,” “host cell line,” “host cell culture,” “genetic modification,” or “engineered host” cell, which are used interchangeably herein, refer to a cell into which an exogenous nucleic acid has been introduced, including progeny of such cells. Host cells include “transformants” and “transformed cells,” including primary transformed cells and progeny derived therefrom, regardless of the number of passages. The nucleic acid content of the progeny may not be identical to that of the parental cell, but may contain mutations. This includes mutant progeny having the same function or biological activity that are selected or screened for in the originally transformed cell. In some aspects, the host cell is a genetically modified cell. As defined herein, in such a cell, the nuclear, organellar, or extrachromosomal nucleic acid may have been transformed, modified, or transduced by recombinant DNA techniques to contain a heterologous nucleic acid molecule, and is used interchangeably with “engineered cell,” “transformed cell,” and “transduced cell.” An engineered cell can be a “transduced cell,” in which the cell has been infected with a modified virus, for example, a retrovirus can be used, but other suitable viruses, such as lentiviruses, can also be considered. Non-viral methods, such as transfection, can also be used. Thus, an engineered cell can also be a “stably transfected cell” or a “transiently transfected cell.” Transfection refers to a non-viral method of introducing DNA (or RNA) into a cell to effect gene expression. Transfection methods are well known in the art, such as calcium phosphate transfection, PEG transfection, liposome or lipid complex transfection of nucleic acids. Such transfection can be transient, but can also be stable transfection, in which cells can be selected that have integrated the gene construct into their genome. In some cases, gene engineering systems, such as CRISPR or Argonaute, can be utilized to engineer cells that express the polypeptides described herein.

[0097] A variety of enzymes can catalyze the insertion of foreign DNA into the host genome. Non-limiting examples of gene editing tools and techniques include CRISPR, TALEN, zinc finger nucleases (ZFNs), meganucleases, Mega-TAL, and transposon-based systems. The CRISPR system can be used to insert a polynucleotide sequence encoding a membrane protein or a component thereof into the cell genome. For example, the CRISPR system can introduce a double-strand break at a target site in the genome. There are at least five types of CRISPR systems, all of which contain RNA and CRISPR-associated proteins (Cas). Types I, III, and IV assemble a multi-Cas protein complex capable of cleaving nucleic acids complementary to the crRNA. Both types I and III require pre-crRNA processing before assembling the processed crRNA into the multi-Cas protein complex. Type II and V CRISPR systems include a single Cas protein complexed with at least one guide RNA.

[0098] As used in the specification and / or claims, the term "effective" means sufficient to achieve a desired, expected, or intended result. When used in the context of treating a patient or subject with a compound, an "effective amount", "therapeutically effective amount", or "pharmaceutically effective amount" refers to the amount of the compound that, when administered to a subject or patient to treat a disease, is sufficient to achieve a therapeutic effect against the disease.

[0099] As used herein, the terms "patient" or "subject" refer to a living mammalian organism such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. In certain aspects, the patient or subject is a primate. Non-limiting examples of human subjects include adults, adolescents, infants, and fetuses.

[0100] As used herein, "pharmaceutically acceptable" generally refers to a compound, material, composition, and / or dosage form that, within the scope of sound medical judgment, is suitable for contact with the tissues, organs, and / or bodily fluids of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, and having a reasonable benefit / risk ratio.

[0101] "Prevention / preventing" includes (1) inhibiting the onset of a disease in a subject or patient who is at risk of and / or predisposed to the disease but has not yet experienced or manifested any or all of the pathology or symptoms of the disease, and / or (2) slowing the onset of the disease pathology or symptoms in a subject or patient who is at risk of and / or predisposed to the disease but has not yet experienced or manifested any or all of the pathology or symptoms of the disease.

[0102] "Treatment" refers to therapeutic treatment and prophylactic or preventive measures that are intended to prevent or slow down (mitigate) a target pathological condition or disorder. Persons in need of treatment include those who already have the disorder, as well as those who are predisposed to having the disorder or in need of preventing the disorder.

[0103] A "therapeutic polypeptide" is a polypeptide that can alleviate or reduce symptoms caused by a protein deficiency or defect in a cell or in a subject. Alternatively, a "therapeutic polypeptide" refers to a polypeptide that confers other benefits to a subject, such as an anti-cancer effect or an improvement in transplant survival rate. As used herein, the term "therapeutic polypeptide" also includes proteins that can be used as vaccines, therapeutic agents, and diagnostic agents.

[0104] II. Compositions

[0105] In certain aspects, the present disclosure encompasses compositions and methods comprising novel naturally occurring and synthetic or engineered signal peptides, and their use in controlling protein trafficking and localization. In certain aspects, the present disclosure also encompasses the use of novel delivery formulations for introducing the disclosed compositions into cells or into a subject in need thereof and methods for introducing the disclosed compositions into cells or into a subject in need thereof. These compositions can be used for therapeutic (treatment and diagnosis) and non-therapeutic applications, such as industrial and laboratory applications, to drive the extracellular secretion and distribution of proteins of interest.

[0106] In certain aspects, the present disclosure encompasses engineered signal peptides. These signal peptides are developed through extensive bioinformatics analysis followed by validation studies in cell culture and in vivo. In certain aspects, the signal peptide comprises an amino acid sequence having at least 60% identity to one or more of SEQ ID NOs: 9-29. In certain aspects, the signal peptide can have at least about 60% to about 75%, or about 75% to about 80%, or about 80% to about 85%, or about 85% to about 90%, or about 90% to about 95%, or about 95% to about 100% identity to at least one of SEQ ID NOs: 9-30. In certain aspects, the signal peptide can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 9-29. In certain aspects, the length of the engineered signal peptide can vary from 5 amino acid residues to about 100 amino acid residues. In certain aspects, the signal peptide can have a length of 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, 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, 70, 71, 72, 73, 74, 75 amino acids. In some exemplary aspects, the length of the signal peptide is about 10-50 residues. In certain aspects, the engineered signal peptide can include a stretch of 1-50 leucine amino acid residues. In certain aspects, the stretch of leucine amino acid residues can be divided into one or two or more groups of residues separated by one or two or more amino acid residues other than leucine.

[0107] In some aspects, the signal peptide is derived from a naturally occurring signal sequence, such as a signal peptide from any one of albumin (hAlb), apolipoprotein B (hApoB), Gaussia luciferase (gLuc), or coagulation factor VII (hFVII) sequences. In some aspects, the signal peptide comprises an amino acid sequence having at least 60% identity to one or more of SEQ ID NOs: 1-4. In some aspects, the signal peptide can have at least about 60% to about 75%, or about 75% to about 80%, or about 80% to about 85%, or about 85% to about 90%, or about 90% to about 95%, or about 95% to about 100% identity to any one of SEQ ID NOs: 1-4. In some aspects, the signal peptide can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NO:1-4.

[0108] In some aspects, the disclosure also encompasses recombinant polypeptides that comprise one or more of the signal peptides provided herein and a heterologous polypeptide. The two molecules can be adjacent in the construct or separated by a linker polypeptide containing 1, 2, 3, or more amino acids. For example, linker molecules are described in Huston, J.S. et al., PNAS (Proceedings of the National Academy of Sciences of the United States of America) 85:5879-5883 (1988), Whitlow, M. et al., Protein Engineering 6:989-995 (1993), and Newton, D.L. et al., Biochemistry 35:545-553 (1996). In some aspects, the recombinant polypeptide can further comprise one or more linker sequences, degrons, degradation tags, protease cleavage sites, and / or purification tags. In some aspects, the heterologous polypeptide can be any polypeptide of interest. In some aspects, the heterologous polypeptide can comprise the same amino acid sequence as a naturally occurring protein or a variant, derivative, or fragment thereof. In some aspects, the heterologous polypeptide can comprise a synthetic amino acid sequence. In some aspects, the heterologous polypeptide can comprise the same amino acid sequence as a prokaryotic protein or a variant, derivative, or fragment thereof. In some aspects, the heterologous polypeptide can comprise the same amino acid sequence as a eukaryotic protein or a variant, derivative, or fragment thereof. In some aspects, the heterologous polypeptide is a variant, derivative, or fragment of a non-human mammalian protein (such as mouse, rat, rabbit, dog, monkey, gibbon, chimpanzee, ape, baboon, cow, pig, horse, sheep, cat, and other species). In some aspects, the heterologous polypeptide is a variant, derivative, or fragment of a human protein.

[0109] In some aspects, the heterologous polypeptide is a therapeutic polypeptide. In some aspects, the heterologous polypeptide is a bioactive polypeptide. In some exemplary aspects, the heterologous polypeptide has anti-cancer, anti-inflammatory, immunomodulatory, antiviral, anti-microbial, anti-fungal, anti-helminth, cholesterol-lowering, anti-diabetic, anti-fibrotic, analgesic, anesthetic, anti-aging, anti-depressant, neuromodulatory, anti-dermatitis, anti-edema, anti-allergic, anti-hyperkeratotic, anti-mycotic, anti-itch, cardiovascular, chemotherapeutic and / or hormonal activity.

[0110] In some aspects, the heterologous polypeptide is a protein replacement therapeutic, polypeptide therapeutic, vaccine, viral vaccine, secreted therapeutic, anti-cancer, anti-inflammatory, antiviral, anti-microbial, cholesterol-lowering, anti-diabetic, or anti-fibrotic polypeptide.

[0111] In some aspects, the heterologous polypeptide is a negative checkpoint regulator, non-limiting examples of which include cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), B and T lymphocyte attenuator (BTLA), T cell immunoreceptor with Ig and ITIM domains (TIGIT), T cell activation V domain Ig suppressor (VISTA), adenosine A2a receptor (A2aR), killer cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), CD20, CD39, and CD73.

[0112] In some aspects, the heterologous polypeptide is a tumor antigen, non-limiting examples of which include alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), epithelial tumor antigen (ETA), mucin 1 (MUC1), Tn-MUC1, mucin 16 (MUC16), tyrosinase, melanoma-associated antigen (MAGE), tumor protein p53 (p53), CD4, CD8, CD45, CD80, CD86, programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), NY-ESO-1, PSMA, TAG-72, HER2, GD2, cMET, EGFR, mesothelin, VEGFR, alpha-folate receptor, CE7R, IL-3, cancer-testis antigen, MART-1 gp100, and TNF-related apoptosis-inducing ligand.

[0113] In some aspects, the heterologous polypeptide is an antibody.

[0114] In some aspects, the heterologous polypeptide is a chimeric antigen receptor (CAR).

[0115] In some aspects, the heterologous polypeptide is active as a vaccine.

[0116] In some aspects, the heterologous polypeptide is a diagnostic polypeptide. In some aspects, the heterologous polypeptide is a theranostic polypeptide. In some aspects, the heterologous polypeptide is an antibody-based diagnostic polypeptide. Generally, the heterologous polypeptide is labeled with a radio nucleotide (such as 111In, 99Tc, 14C, 131I, 3H, 32P, or 35S, etc.) and specifically binds to a tumor antigen, so that tumors can be localized using immunoscintiography. In one aspect, the heterologous polypeptide or its fragment can bind to the extracellular domain of a specific cancer biomarker. The heterologous polypeptide for diagnosis can be labeled with a probe suitable for detection by various imaging methods. Detection methods for the probe include but are not limited to fluorescence, light, confocal, and electron microscopy; magnetic resonance imaging and spectroscopy; fluoroscopy, computed tomography, and positron emission tomography. Suitable probes include but are not limited to fluorescein, rhodamine, eosin, and other fluorophores, radioisotopes, gold, gadolinium, and other lanthanide elements, paramagnetic iron, fluorine-18, and other positron-emitting radionuclides. In addition, the probe can have dual or multiple functions and can be detected by more than one of the listed methods.

[0117] In some aspects, the heterologous polypeptide is not a therapeutic polypeptide. In some aspects, the heterologous polypeptide is a reporter polypeptide, such as a fluorescent protein, such as GFP (green fluorescent protein), BFP (blue fluorescent protein), YFP (yellow fluorescent protein), RFP (red fluorescent protein), mCherry, LacZ (β-galactosidase), CAT (chloramphenicol acetyltransferase), luciferase. In some aspects, the heterologous polypeptide is fused with another detectable probe for in vitro or ex vivo applications.

[0118] In some aspects, the heterologous polypeptide has industrial or commercial applications. In some aspects, the polypeptide can be used, for example, as an enzyme, a nutrient, a food additive, a flavor enhancer, and / or a cosmetic. In some aspects, the secretion of the heterologous polypeptide disclosed herein can be beneficial for the production, isolation, or use of commercially relevant polypeptides.

[0119] In some exemplary aspects, the heterologous polypeptide comprises a sequence having at least 60% identity with SEQ ID NO: 55 - 54 or a variant, derivative, or fragment thereof. In some exemplary aspects, the heterologous polypeptide has at least about 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 100% identity with one or more of SEQ ID.NO: 55 - 58 or a functional fragment thereof.

[0120] In some aspects, the present disclosure encompasses a recombinant polypeptide comprising a signal peptide sequence and a heterologous polypeptide, wherein the signal peptide sequence has at least 60% identity with one or more of SEQ ID NOs: 1-4 or 9-29. In some aspects, the signal peptide sequence comprises a sequence having at least about 60% to about 75%, or about 75% to about 80%, or about 80% to about 85%, or about 85% to about 90%, or about 90% to about 95%, or about 95% to about 100% identity with any one of the sequences of SEQ ID NO: 1-4 or 9-29, and a heterologous polypeptide. In some aspects, the recombinant polypeptide may comprise a signal peptide sequence and a heterologous polypeptide, wherein the signal peptide sequence has 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 100% sequence identity with one or more of SEQ ID NOs: 1-4 or 9-29. In some exemplary aspects, the recombinant polypeptide comprises a signal peptide sequence having at least 60% identity with SEQ ID NOs: 1-4 or 9-29 and a heterologous polypeptide sequence having at least 60% identity with SEQ.ID.NO.55-58 or a functionalized fragment thereof.

[0121] In some aspects, the present disclosure further encompasses a polynucleotide comprising a nucleic acid sequence encoding a signal peptide disclosed herein. In some aspects, the polynucleotide comprises a nucleic acid sequence encoding an amino acid sequence having at least 60% identity with any one of the sequences of SEQ ID NO: 1-4 or 9-29. In some aspects, the polynucleotide comprises a nucleic acid sequence encoding an amino acid sequence having at least about 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 100% identity with one or more of SEQ ID NOs: 1-4 or 9-29. In some aspects, the polynucleotide further comprises a nucleic acid sequence encoding a heterologous polypeptide provided herein. In some aspects, the polynucleotide further comprises a nucleic acid sequence encoding a heterologous polypeptide having at least 60% identity with SEQ ID NO: 55-58.

[0122] In certain aspects, the polynucleotide sequence may comprise a nucleic acid sequence encoding a signal peptide having a nucleic acid sequence that is at least 60% identical or complementary to SEQ ID NO: 5-8 or 30-50. In certain aspects, the polynucleotide sequence may comprise a nucleic acid sequence encoding a signal peptide having a nucleic acid sequence that is at least about 60% to about 75%, or about 75% to about 80%, or about 80% to about 85%, or about 85% to about 90%, or about 90% to about 95%, or about 95% to about 100% identical or complementary to any one of SEQ ID NO: 5-8 or 30-50. In certain aspects, the polynucleotide sequence encoding the signal peptide may comprise a nucleic acid sequence having at least about 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 100% identity or corresponding nucleic acid sequence to one or more of SEQ ID NO: 5-8 or 30-50. In certain aspects, the polynucleotide may be a deoxynucleotide sequence (DNA). In certain aspects, the polynucleotide may be a ribonucleic acid sequence (RNA).

[0123] In certain aspects, the polynucleotide sequence may further comprise a nucleic acid sequence corresponding to a sequence encoding a heterologous polypeptide in-frame with the nucleic acid sequence encoding the signal peptide. In certain aspects, the polynucleotide sequence may further comprise a nucleic acid sequence corresponding to a sequence having at least 60% identity to SEQ ID NO: 51-54. In certain aspects, the polynucleotide may be a deoxynucleotide sequence (DNA). In certain aspects, the polynucleotide may be a ribonucleic acid sequence (RNA).

[0124] The nucleic acid encoding the heterologous polypeptide is expected to include sequences based on naturally occurring sequences. Given the degeneracy of the genetic code, the sequence has at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% nucleotide identity to the nucleic acid sequence of the naturally occurring sequence. In another aspect, the nucleic acid is a complementary sequence of the naturally occurring sequence, or is complementary to 75%, 80%, 85%, 90%, 95% and 100%. Polynucleotides encoding 50, 100, 250, 500, 1000, 1500, 2000, 2500, 3000 or longer and intermediate sizes are contemplated herein. In certain aspects, the nucleic acid encoding the heterologous polypeptide may be derived from genomic DNA cloned directly from the genome of a particular organism. But in certain aspects, the nucleic acid will include complementary DNA (cDNA). In certain aspects, the nucleic acid will include mRNA encoding the heterologous polypeptide and the signal peptide in-frame.

[0125] In some aspects, the polynucleotides disclosed herein may further comprise, but are not limited to, one or more of a promoter, enhancer, leader sequence, transcription start site (TSS), linker, 5′ and 3′ untranslated regions (UTRs), Kozak sequence, intron, polyadenylation signal, cap sequence, enhancer, viral sequence, IRES sequence, and termination region or sequence that are suitable, necessary, or preferred for regulating or allowing the expression of a heterologous polypeptide in a cell. The polynucleotide may include one or more regions or portions that function as or have the function of an untranslated region. When the polynucleotide is designed to encode at least one polypeptide of interest, the polynucleotide may include one or more such untranslated regions. By definition, the wild-type untranslated regions (UTRs) of a gene are transcribed but not translated. In an mRNA, the 5′ UTR starts at the transcription start site and extends to the start codon, but does not include the start codon; while the 3′ UTR starts immediately after the stop codon and extends to the transcription termination signal. Regulatory features of the UTR can be incorporated into the polynucleotides of the present invention to, for example, increase the stability of the molecule. Specific features can also be added to ensure controlled downregulation of the transcript when the transcript is misdirected to an undesired organ site. In some aspects, any suitable naturally occurring or synthetic UTR sequence can be incorporated into the polynucleotides disclosed herein. Other non-UTR sequences can also be used as regions or sub-regions in the polynucleotide. For example, an intron or a portion of an intron sequence can be integrated into a region of the polynucleotides of the present invention. The addition of an intron sequence can increase protein production and polynucleotide levels. Combinations of features can be included in the flanking regions or in other features. For example, an ORF may be flanked by a 5′ UTR (which may contain a strong Kozak translation initiation signal) and / or a 3′ UTR (which may contain an oligo(dT) sequence for templating the addition of a poly-A tail). The 5′ UTR may include a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different genes.

[0126] In certain embodiments, the 5′ UTR sequence can be SEQ ID NO:59. In certain embodiments, the 3′ UTR sequence can be SEQ ID NO:60.

[0127] In some aspects, the polynucleotides disclosed herein can be assembled intracellularly. In some aspects, the polynucleotides can be synthesized in vivo. In some aspects, the polynucleotides can be synthesized in vitro using methods known in the art (such as in vitro transcription, DNA, RNA, and cDNA synthesis methods). In some aspects, the polynucleotides disclosed herein can be incorporated into a suitable viral vector, expression cassette, expression vector, transposon, episomal element, integrated into a chromosome, host cell, delivery system.

[0128] In some aspects, the polynucleotide is a chemically modified polynucleotide. In some aspects, the polynucleotide may comprise one or more modified nucleosides, and the nucleoside comprises a modified sugar moiety. Such a compound comprising one or more sugar-modified nucleosides may have desirable properties, such as greater nuclease stability relative to an oligonucleotide comprising only nucleosides with natural sugar molecules. In some aspects, the modified sugar moiety is a substituted sugar moiety. In some aspects, the modified sugar moiety is a sugar surrogate. Such a sugar surrogate may include one or more substitutions corresponding to the substituted sugar moiety. In some aspects, the modified polynucleotide may comprise a modified backbone, such as a phosphorothioate, phosphotriester, morpholino, methylphosphonate, short-chain alkyl or cycloalkyl sugar-linkage, or short-chain heteroatom or heterocyclic sugar-linkage.

[0129] In some aspects, the modified sugar moiety is a substituted sugar moiety comprising one or more non-bridging sugar substituents (including but not limited to substituents at the 2′ and / or 5′ positions). Examples of sugar substituents applicable to the 2′ position include but are not limited to 2′-F, 2′-OCH 3 (“OMe” or “O-methyl”) and 2′-O(CH 2 ) 2 OCH 3 (“MOE”). In some aspects, the sugar substituent at the 2′-position is selected from allyl, amino, azido, thio, O-allyl, O-C1-C10 alkyl, O-C1-C10 substituted alkyl; OCF 3 , O(CH 2 ) 2 SCH 3 , O(CH 2 ) 2 -O-N(Rm)(Rn), and O-CH 2 -C(═O)-N(Rm)(Rn), wherein each Rm and Rn is independently H or substituted or unsubstituted C1-C10 alkyl. Examples of sugar substituents at the 5′-position include but are not limited to 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. In some aspects, the substituted sugar comprises more than one non-bridging sugar substituent, e.g., a T-F-5′-methyl sugar moiety (see, e.g., PCT International Application WO2008 / 101157, additional 5′,2′-b is a substituted sugar moiety and nucleoside).

[0130] A nucleoside comprising a 2′-substituted sugar moiety is called a 2′-substituted nucleoside. In some aspects, the 2′-substituted nucleoside comprises a 2′-substituent group selected from the following: halogen, allyl, amino, azido, SH, CN, OCN, CF 3 , OCF 3, O, S or N(Rm)-alkyl; O, S or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; O-alkenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH 2 ) 2 SCH 3 , O(CH 2 ) 2 -O-N(Rm)(Rn) or O-CH 2 -C(=O)-N(Rm)(Rn), where each Rm and Rn is independently H, an amino protecting group, or a substituted or unsubstituted C1-C10 alkyl. These 2'-substituents may be further substituted by one or more substituents independently selected from hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, nitro (NO 2 ), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

[0131] In certain aspects, the 2'-substituted nucleosides include 2'-substituent groups selected from: F, NH 2 , N 3 , OCF 3 , O-CH 3 , O(CH 2 ) 3 NH 2 , CH 2 -CH=CH 2 , O-CH 2 -CH=CH 2 , OCH 2 CH 2 OCH 3 , O(CH 2 ) 2 SCH 3 , O-(CH 2 ) 2 -O-N(Rm)(Rn), O(CH 2 ) 2 , O(CH 2 )2N(CH 3 ) 2 , and N-substituted acetamide (O-CH 2 -C(=O)-N(Rm)(Rn), where each Rm and R is independently H, an amino protecting group, or a substituted or unsubstituted C1-C10 alkyl. In certain aspects, the 2'-substituted nucleosides include a sugar moiety that includes 2'-substituent groups selected from: F, OCF 3 , O-CH 3 , O 2 CH 2 OCH3 , O(CH 2 ) 2 SCH 3 , O(CH 2 ) 2 -O-N(CH 3 ) 2 , -O(CH 2 ) 2 O(CH 2 ) 2 N(CH 3 ) 2 , and O-CH 2 -C(=O)-N(H)CH 3 . In certain aspects, the 2'-substituted nucleosides include a sugar moiety that contains a 2'-substituent selected from F, O-CH 3 , and OCH 2 CH 2 OCH 3 .

[0132] Certain modified sugar moieties contain a bridging sugar substituent that forms a second ring, thereby forming a bicyclic sugar moiety. In certain such aspects, the bicyclic sugar moiety includes a bridge between the 4' and 2' furanose ring atoms. Examples of such 4'-to-2' sugar substituents include, but are not limited to: -[C(Ra)(Rb)]-, -[C(Ra)(Rb)]n-O-, -C(RaRb)-N(R)-O-, or -C(RaRb)-O-N(R)-; 4'-CH 2 -2', 4'-(CH 2 ) 2 -2', 4'-(CH 2 )-O-2'(LNA); 4'-(CH 2 )-S-2'; 4'-(CH 2 ) 2 -O-2'(ENA); 4'-CH(CH 3 )-O-2'(cEt) and 4'-CH(CH 2 OCH 3 )-O-2', and analogs thereof (see, e.g., U.S. Patent No. 7,399,845); 4'-C(CH 3 )(CH 3 )-O-2' and analogs thereof (see, e.g., WO2009 / 006478); 4'-CH 2 -N(OCH 3 )-2' and analogs thereof (see, e.g., WO2008 / 150729); 4'-CH 2 -O-N(CH 3)-2′(see, e.g., US2004 / 0171570, published on September 2, 2004); 4′-CH 2 -O-N(R)-2′, and 4′-CH 2 -N(R)-O-2′-, where each R is independently H, a protecting group, or a C1-C12 alkyl group; 4′-CH 2 -N(R)-O-2′, where R is H, a C1-C12 alkyl group, or a protecting group (see U.S. Patent No. 7,427,672); 4′-CH 2 -C(H)(CH 3 )-2′(see, e.g., Chattopadhyaya et al., J. Org. Chem. (Journal of Organic Chemistry), 2009, 74, 118-134); and 4′-CH 2 -C(=CH 2 )-2′ and its analogs (see PCT International Application WO2008 / 154401).

[0133] In certain aspects, this 4′ to 2′ bridge independently comprises 1 to 4 linking groups independently selected from -[C(Ra)(Rb)]n-, -C(Ra)═C(Rb)-, -C(Ra)═N-, -C(═NRa)-, -C(═O)-, -C(═S)-, -O-, -Si(Ra) 2 -S(═O)x- and -N(Ra)-; where x is 0, 1, or 2; n is 1, 2, 3, or 4; each Ra and Rb is independently H, a protecting group, a hydroxyl group, a C1-C12 alkyl group, a substituted C1-C12 alkyl group, a C2-C12 alkenyl group, a substituted C2-C12 alkenyl group, a C2-C12 alkynyl group, a substituted C2-C12 alkynyl group, a C5-C20 aryl group, a substituted C5-C20 aryl group, a heterocyclic radical, a substituted heterocyclic radical, a heteroaryl group, a substituted heteroaryl group, a C5-C7 cycloaliphatic radical, a substituted C5-C7 cycloaliphatic radical, a halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, an acyl group (C(═O)-H), a substituted acyl group, CN, a sulfonyl group (S(═O)2-J1), or a sulfinyl group (S(═O)-J1); each J1 and J2 is independently H, a C1-C12 alkyl group, a substituted C1-C12 alkyl group, a C2-C12 alkenyl group, a substituted C2-C12 alkenyl group, a C2-C12 alkynyl group, a substituted C2-C12 alkynyl group, a C5-C20 aryl group, a substituted C5-C20 aryl group, an acyl group (C(═O)-H), a substituted acyl group, a heterocyclic radical, a substituted heterocyclic radical, a C1-C12 aminoalkyl group, a substituted C1-C12 aminoalkyl group, or a protecting group.

[0134] Nucleosides containing a bicyclic sugar moiety are called bicyclic nucleosides or BNA. Bicyclic nucleosides include, but are not limited to: (A) α-L-methylenoxy (4′-CH 2 -O-2′) BNA, (B) β-D-methylenoxy (4′-CH 2 -O-2′) BNA (also known as locked nucleic acid or LNA), (C) ethenoxy (4′-(CH 2 ) 2 -O-2′) BNA, (D) aminooxy (4′-CH 2 -O-N(R)-2′) BNA, (E) oxyamino (4′-CH 2 -N(R)-O-2′) BNA, (F) methyl (methylenoxy) (4′-CH(CH 3 )-O-2′) BNA (also known as constrained ethyl or cEt), (G) methylenesulfide (4′-CH 2 -S-2′) BNA, (H) methyleneamino (4′-CH 2 -N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH 2 -CH(CH 3 )-2′) BNA, (J) propene carbocyclic (4′-(CH 2 ) 3 -2′) BNA, and (K) methoxy (ethenoxy) (4′-CH(CH 2 OMe)-O-2′) BNA (also known as constrained MOE or cMOE).

[0135] Other bicyclic sugar moieties are known in the art, e.g., Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379 (Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 5561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845; WO2004 / 106356, WO1994 / 14226, WO2005 / 021570, and WO2007 / 134181; U.S. Patent Publications US2004 / 0171570, US2007 / 0287831, and US2008 / 0039618; U.S. Patent Application Serial Nos. 12 / 129,154, 60 / 989,574, 61 / 026,995, 61 / 026,998, 61 / 056,564, 61 / 086,231, 61 / 097,787 and 61 / 099,844; and PCT International Applications PCT / US2008 / 064591, PCT / US2008 / 066154 and PCT / US2008 / 068922.

[0136] In certain aspects, the bicyclic sugar moiety and the nucleoside containing such bicyclic sugar moiety can be further defined by the isomeric configuration. For example, a nucleoside containing a 4′-2′ methylene-oxy bridge can be in the α-L configuration or in the β-D configuration. Previously, α-L-methyloxy (4′-CH 2-O-2′) bicyclic nucleosides have been incorporated into antisense polynucleotides that exhibit antisense activity (Frieden et al., Nucleic Acids).

[0137] In certain aspects, the substituted sugar moiety includes one or more non-bridging sugar substituents and one or more bridging sugar substituents (e.g., 5′-substitution and 4′-2′ bridging sugars; PCT International Application WO2007 / 134181, in which LNA is substituted, for example, with 5′-methyl or 5′-vinyl).

[0138] In certain aspects, the modified sugar moiety is a sugar surrogate. In certain aspects, the oxygen atoms of the naturally occurring sugar are substituted, for example, with sulfur, carbon, or nitrogen atoms. In certain aspects, such modified sugar moieties also include the above-mentioned bridging and / or non-bridging substituents. For example, certain sugar surrogates include a 4′-sulfur atom and a substituent at the 2′-position (see published U.S. Patent Application US2005 / 0130923) and / or a substituent at the 5′-position. See, for example, Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740).

[0139] In certain aspects, the sugar surrogate includes a ring having other than 5 atoms. For example, in certain aspects, the sugar surrogate includes tetrahydropyran (THP) of a six-membered ring. Such tetrahydropyran can be further modified or substituted. Nucleosides containing such modified tetrahydropyran include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), mannitol nucleic acid (MNA) (see Leumann, C J. Bioorg. & Med. Chem. (Bioorganic & Medicinal Chemistry) (2002) 10:841-854) and fluoro-HNA (F-HNA).

[0140] Many other bicyclic and tricyclic sugar surrogate ring systems are also known in the art and can be used to modify nucleosides for incorporation into antisense compounds (see, for example, the review article: Leumann, J.C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

[0141] Combinations of various modifications may also be provided, without limitation, such as 2′-F-5′-methyl-substituted nucleosides (for other disclosed 5′,2′-b-substituted nucleosides, see PCT International Application WO2008 / 101157), and substitution of the ribosyl epoxide atom with S and further substitution at the 2′-position (see US Patent Publication US2005 / 0130923), or 5′-substitution of bicyclic nucleic acids (see PCT International Application WO2007 / 134181, where the 4′-CH 2 -O-2′ bicyclic nucleoside is further substituted at the 5′-position with 5′-methyl or 5′-vinyl). The synthesis, preparation, oligomerization, and biochemical studies of carbocyclic bicyclic nucleosides have also been described (see Srivastava et al., 2007).

[0142] In certain aspects, the present invention provides polynucleotides comprising modified nucleosides. These modified nucleotides may include modified sugars, modified nucleobases, and / or modified linkages. Specific modifications are selected to impart desired properties to the resulting polynucleotide. In certain aspects, the polynucleotide includes one or more RNA-like nucleosides. In certain aspects, the polynucleotide includes one or more DNA-like nucleotides.

[0143] In certain aspects, the nucleosides of the present invention include one or more unmodified nucleobases. In certain aspects, the nucleosides of the present invention include one or more modified nucleobases.

[0144] In certain aspects, the modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-Substituted pyrimidines, 6-aza pyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyl adenine, 5-propynyl uracil; 5-propynyl cytosine; 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-amino adenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-fluorouracil and cytosine, 5-propyl (CH3) uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azauracil, cytosine, and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy, and other 8-substituted adenines and guanines, 5-halo especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methyl guanine and 7-methyl adenine, 2-F-adenine, 2-amino adenine, 8-azaguanine and 8-aza adenine, 7-deazaguanine and 7-deaza adenine, 3-deazaguanine and 3-deaza adenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidine (such as 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases also include nucleobases in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza adenine, 7-deaza guanosine, 2-aminopyridine, and 2-pyridone. Other nucleobases include those disclosed in U.S. Patent 3,687,808; those disclosed in the Concise Encyclopedia of Polymer Science and Engineering (Kroschwitz, J.I, editor, John Wiley & Sons, 1990, 858-859); those disclosed by Englisch et al. in 1991; and those disclosed by Sanghvi, Y.S. in 1993.

[0145] Representative U.S. patents teaching the preparation of some of the above-described modified nucleobases, as well as other modified nucleobases, include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, each of which is incorporated herein by reference in its entirety.

[0146] In certain aspects, the present invention provides polynucleotides comprising linked nucleosides. In these aspects, the nucleosides can be linked together using any internucleoside linkage. Two broad classes of internucleoside linking groups are defined based on the presence or absence of a phosphorus atom. Representative phosphorus-containing nucleoside linkages include, but are not limited to, phosphodiester (P=O), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing internucleoside linking groups include, but are not limited to, methylene methylene imino (-CH 2 -N(CH 3 )-O-CH 2 -), thiodiester (-O-C(O)-S-), thiocarbamate (-O-C(O)(NH)-S-), siloxane (-O-Si(H) 2 -O-), and N,N'-dimethylhydrazine (-CH 2 -N(CH 3 )-N(CH 3 )-). Compared to the native phosphodiester linkage, modified linkages can be used to alter (usually increase) the nuclease resistance of the polynucleotide. In certain aspects, internucleoside linkages having chiral atoms can be prepared as racemic mixtures or as individual enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those skilled in the art.

[0147] The polynucleotides described herein may contain one or more asymmetric centers and thus can give rise to enantiomers, diastereomers, and other stereoisomeric configurations, which can be defined as (R) or (S), a or R (such as sugar isomers), or (D) or (L) (such as amino acids, etc.) according to absolute stereochemistry. The antisense compounds provided herein include all such possible isomers, as well as their racemic and optically pure forms.

[0148] Natural internucleoside linkages include, but are not limited to: phosphotriesters, methylphosphonates, MMI (3′-CH 2 -N(CH 3 )-O-5′), amide-3 (3′-CH 2 -C(=O)-N(H)-5′), amide-4 (3′-CH 2 -N(H)-C(=O)-5′), formacetal (3′-O-CH 2 -O-5′), and thioformacetal (3′-S-CH 2 -O-5′). Other neutral internucleoside linkages include nonionic linkages of siloxanes (dialkylsiloxanes), carboxylates, carboxamides, sulfides, sulfonates, and amides (see, e.g., carbohydrate modifications in antisense research; edited by Y.S. Sanghvi and P.D. Cook, ACS Symposium Series 580; Chapters 3 and 4, 40-65). More neutral intermolecular nucleoside linkages include nonionic linkages containing mixed N, O, S, and CH 2 constituents.

[0149] Additional modifications can also be made at other positions of the polynucleotide, particularly at the 3'-position of the sugar of the 3'-terminal nucleotide and the 5'-position of the 5'-terminal nucleotide. For example, an additional modification of the polynucleotide of the present invention involves chemically linking one or more additional moieties or conjugates to the polynucleotide to enhance the activity, cellular distribution, or cellular uptake of the polynucleotide. These moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., 1989), bile acids (Manoharan et al., 1994), thioethers (such as hexyl-5-tritylthiol) (Manoharan et al., 1992; Manoharan et al., 1993), thiolcholesterol (Oberhauser et al., 1992), aliphatic chains (such as dodecanediol or undecyl residues) (Saison-Behmoaras et al., 1991; Kabanov et al., 1990; Svinarchuk et al., 1993), phospholipids (such as bis-hexadecyl-rac-glycerol or 1,2-bis-O-hexadecyl-rac-glycerol-3-H-phosphonate triethylamine) (Manoharan et al., 1995; Shea et al., 1990), polyamine or polyethylene glycol chains (Manoharan et al., 1995), or adamantylacetic acid (Manoharan et al., 1995), adamantylacetic acid (Manoharan et al., 1995), palmitoyl moieties (Mishra et al., 1995), or octadecylamine or hexylamino-carbonyl-oxy-cholesterol moieties (Crooke et al., 1996).

[0150] Representative U.S. patents teaching the preparation of such polynucleotide conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is incorporated herein by reference.

[0151] In some aspects, the present disclosure also encompasses host cells comprising the polynucleotides disclosed herein. In some aspects, the host cell or population of host cells comprises a polynucleotide comprising a nucleic acid sequence encoding a signal peptide. In some aspects, the host cell or population of host cells comprises a polynucleotide comprising a nucleic acid sequence encoding a heterologous polypeptide in-frame with the signal peptide. In some aspects, the host cell is a eukaryotic cell. In some aspects, the host cell is a mammalian cell. In some exemplary aspects, the host cell is a human cell. In some aspects, the host cell is an in vitro cell line or an isolated cell. In some aspects, the host cell is present in vivo. In some aspects, the host cell is a somatic cell. In some aspects, the host cell is a differentiated cell. In some aspects, the host cell is a stem cell. In some aspects, the host cell is a tumor cell. In some aspects, the host cell is selected from: CHO-K1 cells; HEK293 cells; Hela cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells, U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT116 cells; Hu-h7 cells; Huvec cells; Molt4 cells. In some exemplary aspects, the host cell is selected from Hela A549 cells, Huh7 cells, or IGROV1.

[0152] Compositions for cell culture, tissue culture, in vivo and / or in vitro delivery

[0153] In some aspects, the present disclosure encompasses a composition comprising a polypeptide or polynucleotide provided herein in combination with a suitable delivery system for cell culture, tissue culture, in vivo, and / or in vitro delivery. In some aspects, the suitable delivery system is a system for introducing the polypeptide or polynucleotide disclosed herein into a cell.

[0154] In some aspects, the present disclosure encompasses a composition comprising a polynucleotide comprising a nucleic acid sequence encoding a signal peptide and a recombinant polypeptide in combination with a suitable delivery system. In some aspects, the present disclosure encompasses the use of any suitable delivery system known in the art.

[0155] In some aspects, the suitable delivery system can be a viral vector. In some aspects, the viral vector is an RNA viral vector. In some aspects, the viral vector is a DNA viral vector. Non-limiting examples of suitable viral vectors include adenovirus, adeno-associated virus (AAV), retrovirus, herpesvirus, lentivirus, poxvirus, or papillomavirus vectors.

[0156] In some aspects, the delivery system is a non-viral delivery system. Non-limiting examples of non-viral delivery systems include polymers, polymer complexes, lipids, lipid analogs, lipid complexes, liposomes, lipid fusion constructs, polymer nanoparticles, nanoparticles, lipid nanoparticles (LNPs), core-shell nanoparticles, solid lipid nanoparticles, metal nanoparticles, self-assembling nucleic acid nanoparticles, hyaluronidase, nanoparticle mimics, ribonucleoproteins, positively charged peptides, small molecule RNA conjugates, aptamer RNA chimeras, RNA fusion protein complexes, and any combination thereof.

[0157] In some aspects, the polynucleotides of the present disclosure can be formulated using natural and / or synthetic polymers. The polymers can include one or more polymers such as, but not limited to: polyethylene, polyethylene glycol (PEG), poly(L-lysine) (PLL), PEG grafted to PLL, cationic lipid polymers, biodegradable cationic lipid polymers, polyethyleneimine (PEI), crosslinked branched poly(alkyleneimine), polyamine derivatives, modified poloxamers, biodegradable polymers, biodegradable block copolymers, biodegradable random copolymers, biodegradable polyester copolymers, biodegradable polyester block copolymers, biodegradable polyester block random copolymers, linear biodegradable copolymers, poly[a-(4-aminobutyl)-L-glycolic acid] (PAGA), biodegradable crosslinked cationic multi-block copolymers, polycarbonates, polyanhydrides, polyhydroxy acids, polypropyl fumerate, polycaprolactone, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohol, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysines, poly(ethyleneimine), poly(serine esters), poly(L-lactic acid-co-L-lysine), poly(4-hydroxy-L-proline esters), acrylic polymers, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates, amine-containing polymers, or combinations thereof. Non-limiting examples of polymers useful for delivery include, but are not limited to: Dynamic POLYCONJUGATE from Bio, Inc. (Madison, Wisconsin) and Roche Madison, Inc. (Madison, Wisconsin) TM formulations, PHASERX TM polymer formulations including, but not limited to SMARTT POLYMER TECHNOLOGY TM (Seattle, Washington), DMRI / DOPE, poloxamers, from Vical, Inc. (San Diego, California) Adjuvants, chitosan, cyclodextrins, dendrimers, and poly(lactic-co-glycolic acid) (PLGA) polymers from Calando Pharmaceuticals (Pasadena, California). RONDEL TM (RNAi / Oligonucleotide Nanoparticle Delivery) Polymers (Arrowhead Research, Pasadena, California) and pH-responsive co-block polymers such as, but not limited to, PHASERX TM (Seattle, Washington).

[0158] In some aspects, the delivery system includes liposomes. Non-limiting examples include, but are not limited to, N-[1-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), dioleoyl dimethyl ammonium propane (DODAP), dipalmitoyl phosphatidylethanolamine (DOPE), or dioleoyl phosphatidylethanolamine (DPPE), distearoyl phosphatidylcholine (DSPC), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (1,2-dioleoyl-sn-glycero-3-phosphate(1'-rac-glycerol)), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol), 2,3,-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide, and dimethyl dioctadecyl ammonium bromide (DDAB) and any combination thereof.

[0159] In some aspects, the delivery system includes one or more nanoparticles. Nanoparticles can be solid in nature, including polysaccharides, lipids, proteins, polymers, biodegradable polymers, metal oxides, and any combination thereof. Other nanoparticles are in liquid form, mainly liposomes, micelles, or emulsion systems composed of amphiphilic molecules or polymers. Lipid nanoparticles (LNP) are one of the most promising types of nanoparticles because they can effectively encapsulate nucleic acids, have high stability, and compatibility with the biological environment.

[0160] In some aspects, the LNPs can be made of cationic, anionic, zwitterionic, or neutral lipids or any combination thereof. The LNPs can also be composed of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Any lipid or combination of lipids known in the art can be used to produce LNPs. Non-limiting examples of lipids for producing LNPs are DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Non-limiting examples of cationic lipids that are often used are: polyethyleneimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINE TM (e.g., LIPOFECTAMINE TM(2000), DOPE, Cytofectin, Eufectins, 98N12-5, C12-200, DDAB, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Non-limiting examples of commonly used neutral lipids include: DPSC, DPPC, POPC, DOPE, and SM. Non-limiting examples of commonly used PEG-modified lipids include: PEG-DMG, PEG-DSG, PEG-CerC14, and PEG-CerC20. Neutral lipids, such as the fusogenic phospholipid DOPE or the membrane component cholesterol, can be included in the LNP to enhance transfection activity and the stability of the nanoparticles.In some aspects, the lipid nanoparticles comprise ionizable amino lipids (such as 4-dimethylaminobutyric acid heptatriaconta-6,9,28,31-tetraen-19-yl ester, DLin-MC3-DMA), phospholipids, such as phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidic acid (EPA), egg phosphatidylinositol (EPI), soy phosphatidylcholine (SPC), soy phosphatidylglycerol (SPG), soy phosphatidylethanolamine (SPE), soy phosphatidylserine (SPS), soy phosphatidic acid (SPA), soy phosphatidylinositol (SPI), dipalmitoyl phosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylglycerol (DOPG), dimyristoyl phosphatidylglycerol (DMPG), hexadecylphosphocholine (HEPC), hydrogenated soy phosphatidylcholine (HSPC), distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl stearoyl phosphatidylcholine (PSPC), palmitoyl stearoyl phosphatidylglycerol (PSPG), monooleoyl phosphatidylethanolamine (MOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), polyethylene glycol distearoyl phosphatidylethanolamine (PEG-DSPE), dipalmitoyl phosphatidylserine (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), dimyristoyl phosphatidylserine (DMPS), distearoyl phosphatidylserine (DSPS), dipalmitoyl phosphatidic acid (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), dimyristoyl phosphatidic acid (DMPA), distearoyl phosphatidic acid (DSPA), dipalmitoyl phosphatidylinositol (DPPI), 1,2-dioleoyl-sn-glycero-3-phosphatidylinositol (DOPI), dimyristoyl phosphatidylinositol (DMPI), distearoyl phosphatidylinositol (DSPI), and mixtures thereof., cholesterol, and an outer lipid (polyethylene glycol-dimyristoyl glycerol, PEG-DMG), as disclosed, for example, in: Tam et al. (2013); Advances in Lipid Nanoparticles for siRNA delivery, Pharmaceuticals 5(3):498-507.Multiple such LNP systems are known, such as, but not limited to, those disclosed in: Hou, X. et al., Lipid nanoparticles for mRNA delivery, Nat Rev Mater 6, 1078-1094 (2021), U.S. Pat. Nos. 7,166,745; 7,173,154; 7,323,594; 7,470,817; 7,479,573; 7,601,872; 7,915,450; 8,158,827; 8,785,200; 9,358,300, International Patent Publication WO2016 / 011203, and U.S. Patent Publication 2017 / 0107539, all of which are hereby expressly incorporated by reference in their entirety as if fully set forth herein. In certain aspects, the lipid nanoparticles can comprise a combination of lipids, such as cationic lipids, phospholipids (e.g., PEGylated lipids, such as iPhos LNP (9A1-P9 / cholesterol / DODAP / DMG-PEG, 25:30:30:1 mol / mol; 18:1 9A1-P9:nucleic acid, wt / wt)). In certain aspects, the lipid nanoparticles are organ-targeting (SORT) lipid nanoparticles provided in US11304911B2, which is incorporated herein by reference in its entirety. In certain aspects, the LNP can be selected from any one of iPhos LNP, mDLNP, liver SORT LNP, lung SORT LNP, or spleen SORT LNP and any combination thereof.

[0161] In some exemplary aspects, the delivery system comprises one or more lipid nanoparticles (LNP). In certain aspects, the average diameter of the lipid nanoparticles is between about 10 nanometers and about 1000 nanometers. In certain aspects, the diameter of the lipid nanoparticles is less than 300 nanometers. In certain aspects, the diameter of the lipid nanoparticles is between about 10 nanometers and about 300 nanometers. In certain aspects, the diameter of the lipid nanoparticles is less than 200 nanometers. In certain aspects, the diameter of the lipid nanoparticles is between about 25 nanometers and about 200 nanometers. In certain aspects, the lipid nanoparticle formulation (e.g., a composition comprising multiple lipid nanoparticles) has a size distribution with an average size (e.g., diameter) of about 70 nanometers to about 200 nanometers, more typically an average size of about 100 nanometers or less.

[0162] In some aspects, in the composition comprising the LNP, the molar ratio of LNP to nucleic acid ranges from about 5:1 to about 1000:1. In some aspects, the molar ratio of LNP to nucleic acid ranges from about 100:1 to about 1000:1. In other aspects, the molar ratio ranges from about 250:1 to about 750:1. In some aspects, the molar ratio is 5:1, 10:1, 50:1, 100:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1, 1000:1, or any intermediate ratio.

[0163] In some aspects, the compositions disclosed herein can be a therapeutic composition and can further comprise one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients for manufacturing pharmaceutical compositions include, but are not limited to, inert diluents, dispersing agents and / or granulating agents, suspending aids, isotonic agents, thickening agents, surfactants and / or emulsifying agents, disintegrating agents, binding agents, preservatives, buffering agents, lubricants, preservatives, and / or oils. These excipients can be selectively incorporated into the pharmaceutical formulation. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening agents, flavoring agents, and / or aromatic agents can also be present in the composition, at the discretion of the formulation designer. The various excipients for formulating pharmaceutical compositions and the techniques for formulating the compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Maryland, 2006). The use of conventional excipient media can be contemplated within the scope of the present disclosure.

[0164] In some aspects, the delivery system can be a therapeutic delivery system for sustained-release or controlled-release formulations, such as synthetic material depots, polymer depots, lipid depots, controlled-release hydrogel depots, liquid crystal depots, liposome depots, oil-based depots, and controlled-release polymer depots. Depot formulations are a means of administering drugs that can reduce the frequency of drug administration while improving efficacy and patient compliance. In some aspects, these formulations are characterized by a slower rate of release of the therapeutic drug compared to conventional release dosage forms administered by the same route. In some aspects, these formulations are adjustable and release the drug at a predetermined rate for a period of time within the therapeutic range.

[0165] In some aspects, the compositions disclosed herein are formulated for administration to a subject in need thereof by one or more routes, such as orally, intralipally, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, rectally, intracapsularly, intratracheally, intratumorally, intraumbilically, vaginally, intravenously, intravascularly, intravitreally, liposomally, topically, mucosally, parenterally, rectally, subconjunctivally, subcutaneously, sublingually, topically, buccally, transdermally, vaginally, as an ointment, as a lipid composition, via a catheter, via lavage, via continuous infusion, via infusion, via inhalation, via injection, via topical delivery, or via topical perfusion. In some aspects, the pharmaceutical composition is formulated for administration by injection. In some aspects, the pharmaceutical composition is formulated as a unit dose. In some aspects, the formulation may further comprise excipients suitable for administration by the routes provided herein.

[0166] In some aspects, the compositions disclosed herein may further comprise excipients suitable for one or more suitable modes of administration. In some aspects, the composition can be formulated as an injection, a liquid, an emulsion, a suspension, a syrup, a pill, a caplet, a cream, an ointment, a lotion, a patch, a solution, a suppository, a lyophilized product, a gel, and a capsule. Methods of making pharmaceutical compositions are well known in the art (e.g., see Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (ed.), Lippincott, Williams & Wilkins). The pharmaceutical composition can also be formulated as a timed, sustained, pulsed, or continuous release dosage form. The pharmaceutical composition can also be administered by a device, such as a timed, sustained, pulsed, or continuous release device.

[0167] In some aspects, the present disclosure also encompasses compositions comprising a polypeptide disclosed herein and a suitable delivery system. Any delivery system suitable for polypeptides known in the art can be used herein. Examples of suitable delivery systems include, but are not limited to, polymers, polymer complexes, microspheres, lipids, lipid analogs, lipid complexes, liposomes, microparticles, polymer nanoparticles, nanoparticles, lipid nanoparticles (LNP), core-shell nanoparticles, solid lipid nanoparticles, metal nanoparticles, nanoparticle mimics, and any combination thereof. Details of some common delivery systems for delivering polynucleotides are provided in the present disclosure, but can also be suitably used for delivering polypeptides.

[0168] III. Methods

[0169] In some aspects, the present disclosure also encompasses methods and applications of using the compositions disclosed herein. In some aspects, these compositions comprising the signal peptides or polynucleotides encoding the signal peptides provided herein can be used for any suitable application. In some aspects, secreting a protein of interest out of a cell may be beneficial for such applications. In some aspects, the present disclosure provides a number of signal peptide sequences, each of which can be suitable for one or more applications. In some aspects, these compositions can be used for non-therapeutic purposes. In some aspects, these compositions can be used for therapeutic applications.

[0170] In some aspects, the present disclosure provides various engineered polynucleotides, engineered polypeptides, expression cassettes, viral vectors, expression vectors, host cells, and suitable formulations that can secrete a heterologous protein of interest out of a cell. Post-translational secretion of a polypeptide of interest from a cell may be desired, for example, for systemic or organ-specific delivery of therapeutic, diagnostic, theranostic, and / or reporter polypeptides to a subject in need. In some aspects, post-translational secretion of a polypeptide of interest from a cell may be desired in non-therapeutic applications, such as in laboratory experiments. In some aspects, post-translational secretion of a polypeptide of interest from a cell may be desired for industrial applications (for the production and isolation of protein products). The present disclosure contemplates all such applications of the disclosed secretion signal peptides.

[0171] In some exemplary aspects, the present disclosure encompasses diagnostic, prophylactic, and / or therapeutic methods, including administering an effective amount of the compositions disclosed herein. In certain aspects, the compositions include a therapeutic polypeptide fused to a signal peptide disclosed herein, or a polynucleotide composition encoding the same. In certain aspects, the compositions (polypeptides or polynucleotides) corresponding to any therapeutic polypeptide that needs to be secreted can be used in the disclosed methods. For example, these compositions can be used in methods for improving disease effects, preventing diseases, treating diseases, or inhibiting disease progression in a subject in need thereof. These methods include inhibiting cell rolling, inflammation, autoimmune diseases, metastasis, the growth and / or replication of tumor cells or leukemia cells, or inhibiting the increase in the number of tumor cells in a tumor patient or leukemia cells in a leukemia patient. In addition, such methods also include increasing the mortality rate of tumor cells or leukemia cells, altering the susceptibility of diseased cells to damage by an agent against the disease, the susceptibility of tumor cells to damage by an anti-cancer agent, or the susceptibility of leukemia cells to damage by an anti-cancer agent. These methods also include inhibiting or reducing virus entry into cells. Such a method also includes preventing or inhibiting cardiovascular diseases. In certain aspects, the therapeutic polypeptide used in the method can have anti-cancer, anti-inflammatory, immunomodulatory, antiviral, antimicrobial, antifungal, anti-helminthic, cholesterol-lowering, anti-diabetic, anti-fibrotic, analgesic, anti-tumor, anti-aging, anti-depressant, neuromodulatory, anti-dermatitis, anti-edema, anti-allergic, anti-hyperkeratosis, anti-fungal, anti-itch, cardiovascular, chemotherapeutic, hormone activity. In certain aspects, the therapeutic polypeptide is a protein replacement therapeutic, polypeptide therapeutic, vaccine, viral vaccine, secreted therapeutic, anti-cancer, anti-inflammatory, antiviral, antimicrobial, cholesterol-lowering, anti-diabetic, or anti-fibrotic polypeptide. In certain aspects, the therapeutic polypeptide is an antibody. In certain aspects, the polypeptide is a negative checkpoint regulator, non-limiting examples of which include cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin-containing protein 3 (TIM-3), B and T lymphocyte attenuator (BTLA), T cell immunoreceptor with Ig and ITIM domains (TIGIT), T cell activation V domain Ig suppressor (VISTA), adenosine A2a receptor (A2aR), killer cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), CD20, CD39, and CD73.In certain aspects, the polypeptide is a tumor antigen, non-limiting examples of which include alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), epithelial tumor antigen (ETA), mucin 1 (MUC1), Tn-MUC1, mucin 16 (MUC16), tyrosinase, melanoma-associated antigen (MAGE), tumor protein p53 (p53), CD4, CD8, CD45, CD80, CD86, programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), NY-ESO-1, PSMA, TAG-72, HER2, GD2, cMET, EGFR, mesothelin, VEGFR, alpha-folate receptor, CE7R, IL-3, cancer-testis antigen, MART-1 gp100, and tumor necrosis factor-related apoptosis-inducing ligand. In certain aspects, the heterologous polypeptide is an antibody. In certain aspects, the polypeptide is a chimeric antigen receptor (CAR). In certain aspects, the polypeptide is active as a vaccine. In certain aspects, the method also encompasses the use of diagnostic polypeptides. In certain aspects, the polypeptide is a theranostic polypeptide. In certain aspects, the polypeptide is an antibody-based diagnostic polypeptide. Generally, the polypeptide is labeled with a radio nucleotide (such as 111In, 99Tc, 14C, 131I, 3H, 32P, or 35S) and specifically binds to the tumor antigen so that the tumor can be localized by immunoscintigraphy. In one aspect, the polypeptide or its fragment can bind to the extracellular domain of a specific cancer biomarker. The polypeptide for diagnosis can be labeled with a probe detectable by various imaging methods. Detection methods for the probe include but are not limited to fluorescence, light, confocal, and electron microscopy; magnetic resonance imaging and spectroscopy; fluoroscopy, computed tomography, and positron emission tomography. Suitable probes include but are not limited to fluorescein, rhodamine, eosin, and other fluorophores, radioisotopes, gold, gadolinium, and other lanthanide elements, paramagnetic iron, fluorine-18, and other positron-emitting radionuclides.

[0172] The effective dose / amount and schedule of administration of the composition can be determined empirically and making such a decision is within the purview of one of ordinary skill in the art. One of ordinary skill in the art will understand that the dose at which the compositions disclosed herein must be administered will vary depending on, for example, the subject receiving the composition, the route of administration, the particular type of composition used, and other drugs being administered. For example, for anti-cancer treatment, a therapeutically administered composition amount that can prevent tumor growth, shrink the tumor, and / or prevent the occurrence of new tumors compared to the course of the disease without administration is an effective dose. The composition can be administered in a single dose or repeated as needed. In certain aspects, the treatment method can further include the administration of additional treatments, including additional drugs, such as anti-inflammatory drugs, analgesics, antibacterial drugs, or therapies, such as radiotherapy.

[0173] In certain aspects, the subject in the treatment method can include an animal (human or non-human) to which the method according to the present disclosure is provided. The present disclosure contemplates human and veterinary applications. The term includes, but is not limited to, birds, reptiles, amphibians, and mammals such as humans, other primates, pigs, rodents (such as mice and rats), rabbits, guinea pigs, hamsters, horses, cows, cats, dogs, sheep, chickens, and goats. In certain aspects, the subject is a human. Both children and adults are included.

[0174] In certain aspects, the present disclosure also encompasses methods of using the compositions provided herein for in vivo diagnosis. In certain aspects, the composition comprises or encodes a diagnostic antibody. In some exemplary aspects, the methods disclosed herein are used for tumor detection.

[0175] In certain aspects, the present disclosure also encompasses methods of using the compositions disclosed herein to secrete a heterologous polypeptide in cell culture and tissue culture. In certain aspects, the method encompasses contacting the cell with the composition disclosed herein. In one exemplary aspect, the cell can be transfected with the polynucleotide composition provided herein. In certain aspects, the heterologous polypeptide can be a reporter polypeptide, such as a fluorescent polypeptide or an antibody, and the secreted polypeptide can be used for visualization by microscopy or other suitable techniques.

[0176] In certain aspects, the present disclosure also encompasses methods of using the compositions disclosed herein for industrial applications. The secreted protein provides various advantages for the industrial production of products. In certain exemplary aspects, the purification of the secreted protein may be easier and more desirable than extracting heterologous proteins from cells or tissues. In certain exemplary aspects, the systemic secretion of the protein can be used in the food industry to add flavor to meat products. The applications disclosed herein are only exemplary and should not be considered restrictive.

[0177] IV. Kits

[0178] In certain aspects, the compositions and methods provided herein can also be provided in the form of a kit, accompanied by instructions for use. In certain aspects, the kit comprises at least one composition that comprises a polynucleotide encoding a signal peptide provided herein, and optionally a suitable substrate, reagent, buffer, diluent, cell, standard, container, and instructions for use. In certain aspects, the kit can comprise at least one cell comprising the polynucleotide or polypeptide disclosed herein, and optionally a suitable substrate, reagent, buffer, diluent, cell, standard, container, and instructions for use.

[0179] In some aspects, a manufactured article or kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, microcentrifuge tubes, bottles, vials, assay plates, strips, substrates, and the like. The container can be made of a variety of materials such as glass, plastic, paper, and the like. The kit can further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0180] A "package insert" is used to refer to the instructions that are typically included in the commercial packaging of a product and that contain usage information and the like.

[0181] The instructions in the kit can be affixed to the packaging material or can be in the form of a package insert. While the instructions are typically written or printed materials, they are not limited thereto. Any medium capable of storing such instructions and communicating them to the end user is within the contemplation of the present disclosure. Such media include, but are not limited to, electronic storage media (such as magnetic disks, tapes, cartridges, chips), optical media (such as CD ROMs), and the like. The term "instructions" as used herein can include the Internet website address providing the instructions.

[0182] Examples

[0183] The following examples are intended to illustrate the preferred aspects of the present disclosure. Those skilled in the art will appreciate that the techniques disclosed in the examples represent techniques discovered by the inventors to function well in the practice of the present disclosure, and thus can be considered to constitute a preferred mode for practicing the present disclosure. However, those skilled in the art will appreciate that, in light of the present disclosure, many changes can be made to the specific aspects disclosed and still obtain the same or similar results without departing from the spirit and scope of the present disclosure.

[0184] Methods

[0185] Construct the SP-mCherry plasmid (pDNA)

[0186] To determine the optimal signal peptide (SP), an SP-modified mCherry plasmid was constructed. Briefly, the SP-mCherry coding region was directly obtained by PCR using carefully designed primers. Several SPs screened included hAlb (human albumin, SEQ ID NO: 1), hApoB (human apolipoprotein, SEQ ID NO: 2), gLuc (Gauss luciferase, SEQ ID NO: 3), and hFVII (human coagulation factor VII, SEQ ID NO: 4). The polypeptide sequences of the signal sequences fused to mcherry are shown in SEQ ID NOs: 62 - 66 and are described in Table 1. The enzymatically digested SP-mCherry product was cloned into the pCS2-MT vector according to the standard protocol. After sequencing verification, the SP-mCherry plasmid can be used for in vitro screening.

[0187] Table 1

[0188]

[0189]

[0190]

[0191] In vitro SP screening by pDNA transfection

[0192] To screen for SP, pDNA transfection was performed in cells. Hela and Huh7 cells were seeded in 96-well plates at 1 x 10 4 cells per well. After 24 hours, the cells were treated with the Lipo2k-pDNA preparation containing 50 ng of pDNA per well. On day 1, day 2, and / or day 3 after treatment, the cells were immediately imaged with a Keyence Scope. Meanwhile, cell lysates and media were further collected and then the mCherry signal was quantified using a plate reader. To clearly observe the mCherry signal, the cell lysates and media were transferred to EP tubes and imaged using an IVIS Lumina system. Subcellular signal distribution was detected using a confocal microscope. Huh7 cells were treated as described above, rinsed three times with 1xPBS 3 days later, stained with Hoechst 33342, and imaged using a confocal microscope.

[0193] mRNA synthesis

[0194] All mRNAs used in this work were generated by in vitro transcription (IVT) as previously described (Cheng et al., 2020). Briefly, linear pDNAs with optimized 5'(3')-untranslated regions (UTRs) and poly A sequences were first obtained by enzymatic digestion, then the IVT reaction was carried out with a standard protocol and N1-methylpseudouridine-5'-triphosphate modification was performed. Finally, the mRNAs were capped (Cap-1) with vaccinia virus capping enzyme and 2'-O-methyltransferase (NEB).

[0195] Formation of mRNA nanoparticles

[0196] mRNA-loaded LNP formulations were formed by the ethanol dilution method previously described (Cheng et al., 2020). Liver-targeted mRNA formulations (mDLNP) and tissue-selective SORT LNPs have been developed and reported (Cheng et al., 2018, 2020). Briefly, all lipids with a specific molar ratio were first dissolved in ethanol, and the RNA was dissolved in 10 mM citrate buffer (pH 4.0). Then the two solutions were rapidly mixed at a volume ratio of aqueous solution to ethanol of 3:1 (3:1, aqueous solution:ethanol, volume:volume) to meet the final weight ratio of 40:1 (total lipid:mRNA). After incubation at room temperature for 10 minutes, the mRNA LNP formulations were immediately added to cells or dialyzed with PBS for 2 hours for in vivo experiments.

[0197] Optimal hFVII-SP-driven in vitro mCherry secretion verified by mRNA formulations

[0198] hFVII-mCherry mRNA was transfected into several cell lines, including Huh7, 293T, Hela, A549 and IGROV1. Cells were seeded at 1x10 4 per well into 96-well plates and cultured for 24 hours. The mRNA mDLNP formulations were prepared as described above and then cells were treated with different mRNA doses (0 to 750 ng per well) and time points (24 hours to 72 hours). At a given time, cells were directly imaged with a Keyence Scope, and then the mCherry signals in the culture medium and cell lysates were quantified with a plate reader. The WT-mCherry mRNA formulation was used as a control.

[0199] Optimal hFVII-SP-driven in vivo mCherry secretion verified by mRNA formulations

[0200] All animal experiments have been approved by the Animal Care and Use Committee of the University of Texas Southwestern Medical Center and comply with relevant local, state, and federal regulations. C57BL / 6 mice were obtained from the UTSW Mouse Breeding Core Facility. In the luciferase mRNA assay, mDLNP and SORT LNP (liver, lung, and spleen) formulations were injected intravenously into mice at a dose of 0.1 mg / kg mRNA. At 3 hours, D-Luciferin (150 mg / kg, intraperitoneal injection (IP)) was injected into the mice, and imaging was performed using an IVIS Lumina system (Perkin Elmer). To test the secretion of mCherry, hFVII-mCherry mRNA was encapsulated into mDLNP and then injected intravenously into mice at a dose of 0.5 mg / kg mRNA. At different time points (2h, 6h, 24h, 30h, 48h, 55h, and 72h), sera were isolated and the mCherry signal was quantified using a plate reader. Meanwhile, tissues were imaged using IVIS to confirm the secretion of mCherry in the blood. PBS and WT-mCherry formulation-treated groups were used as controls. To further examine the secretion of mCherry, hFVII-mCherry mRNA (0.5 mg / kg) was delivered to the liver, lung, and spleen using liver-, lung-, and spleen-targeted SORT LNP, respectively. Tissues were imaged using IVIS at 24 hours.

[0201] Cytotoxicity rescue of hFVII-Enbrel mRNA

[0202] L929 cells were used to evaluate TNF-α-mediated cytotoxicity. Mouse TNF-α (mTNF-α) and human TNF-α (hTNF-α) were selected. Cells were seeded in 96-well plates at a density of 1×10 4 cells per well and cultured for 24 hours. The medium was replaced with 180 μl of fresh medium containing actinomycin and TNF-α, such that the final concentration of actinomycin was 1 μg / ml and the concentration of TNF-α was 0 to 0.1 ng / ml. After incubation for another 24 hours, cell viability was detected using the CellTiter-Glo kit according to the standard protocol.

[0203] To evaluate the cytotoxicity rescue of the hFVII-Enbrel mRNA formulation, cells were pretreated with hFVII-Enbrel mRNA mDLNP for two days before stimulation with TNF-α. Dose-dependent rescue of both mRNA and TNF-α was tested. For dose-dependent rescue of the mRNA formulation, mRNA doses from 0 ng / ml to 1.25 ng / ml per well were tested, followed by 24-hour stimulation with 0.1 ng / ml of TNF-α. For dose-dependent rescue of TNF-α, the transfected mRNA concentration was fixed at 0.4 ng / ml, and then stimulated with TNF-α from 0 ng / ml to 5 ng / ml.

[0204] To further verify the rescue effect of Enbrel secreted in the medium, cytotoxicity rescue pretreated with functional medium was measured. As described above, cells were seeded and treated with mRNA at concentrations from 0 ng / ml to 1.25 ng / ml, then the medium was collected and transferred to a new 96-well plate with attached L929 cells. At the same time, the new well plate was stimulated with TNF-α (0.1 ng / ml) and actinomycin (1 μg / ml). After another 24 hours, cell viability was detected.

[0205] Pharmacokinetic study

[0206] Male C57BL / 6 mice weighing 20 g were randomly grouped. Enbrel protein and hFVII-Enbrel mRNA mDLNP formulation were intravenously injected at a dose of 0.5 mg / kg. Serum was collected at time points from 2 hours to 216 hours, and Enbrel in the serum was quantified using an ELISA kit (MyBioSource).

[0207] Psoriasis treatment

[0208] In the imiquimod-induced psoriasis-like hyperplasia model, 8-week-old female C57BL / 6 mice were shaved and chemically depilated with Nair (recorded as day 1). On day 3, the mice were intravenously injected with the hFVII-Enbrel formulation at a dose of 0.5 mg / kg. Then, the shaved dorsal skin samples were topically treated with 60 mg of Aldara cream (5% imiquimod) (Aldara, 3M Pharmaceuticals) once a day for 5 days. On day 9, the whole body was photographed to show the differences between groups, with mice treated with lanolin + PBS and imiquimod + mCherry mDLNP as controls. Finally, the dorsal skin of the mice was collected, stained with H&E to measure the thickness, and cell proliferation was analyzed by Ki-67 and Gr-1 immunohistochemistry.

[0209] Evaluation of PDL1 expression

[0210] The MC38 and B16F10 cell lines were used to study tumor immunotherapy in vivo. Flow cytometry was used to evaluate the expression of PDL1 on the cell membrane. Cells were seeded at a density of 3×10 5 cells per well in 6-well plates and cultured for 24 hours. After incubation with IFN-γ (100 ng / ml) for another 24 hours, cells were stained with anti-PDL1 primary antibody and Alexa Fluor 647-labeled secondary antibody, and the expression of PDL1 was analyzed by flow cytometry. Cells stained with isotype antibody were used as gating controls.

[0211] Tumor immunotherapy

[0212] MC38 or MC38-Luc (stably expressing luciferase) cells were grown in DMEM medium containing 10% FBS. On day 0, a total of 1×10 6 cells in 100 μl PBS were subcutaneously injected into the right abdomen of C57BL / j mice. The hFVII-anti-PDL1 mRNA mDLNP formulation was intravenously injected continuously at a dose of 0.5 mg / kg mRNA on day 3, three times every 4 days. Tumor measurements were taken for the MC38 model and survival curves were monitored. For the MC38-Luc model, luciferase expression was continuously captured by IVIS on days 3, 10, 24, and 32, and luciferase signals were quantified using IVIS software. For the B16F10-Luc model, a total of 4×10 5 cells were subcutaneously injected, and the mRNA formulation was intravenously injected as described above. Luciferase signals, tumor size, and survival were monitored from day 0 to day 32. For both tumor models, the mCherry mDLNP formulation was used as a control. Tumors were measured using digital calipers, and the tumor size was calculated using the formula: volume = 0.5 × length × width. Mice were sacrificed and recorded as dead when the tumor volume reached 1500 cm 3 or greater.

[0213] Example 1: Selection of signal peptides for therapeutic applications

[0214] Initially, a variety of naturally occurring signal peptides (SPs) were screened to test their ability to drive the secretion of a reporter protein in cell culture. For this purpose, several different signal peptides from three known endogenous secreted proteins (albumin, hAlb; apolipoprotein B, hApoB; coagulation factor VII, hFVII) and one known synthetic secreted protein (Gauss luciferase, gLuc), as well as a negative control (NC) SP leader sequence, were cloned into the pCS2-MT plasmid backbone directly upstream of the reporter gene mCherry mRNA sequence.

[0215] The front of the construct is the SP6 promoter and the optimized 5'UTR, and the back is the optimized 3'UTR and polyA tail( Figure 1A ). Initially, HeLa cells were transfected with wild-type (WT) mCherry pDNA without SP and gLuc-mCherry pDNA using Lipofectamine2000. Intracellular and extracellular fluorescence were quantified by fluorescence microscopy at 24, 48, and 72 hours after transfection. Among them, gLuc SP induced a high level of mCherry secretion into the culture medium( Figure 1B ). In addition, at 24, 48, and 72 hours, the mCherry protein content in the cell culture medium and cell lysates was quantified by a fluorescence microplate reader. The results showed that the mCherry secretion in the culture medium increased over time, and at the same time, the mCherry fluorescence ratio in the culture medium and cell lysates also increased in the gLuc SP group( Figure 1C ). Then the SP group was expanded to include a negative control (disruptive sequence), hAlb, hApoB, and hFVII in addition to gLuc. HeLa cells were transfected again with the pDNA construct using Lipofectamine2000. Images taken by fluorescence microscopy and IVIS 72 hours after transfection showed that SPs such as hApoB, gLuc, and hFVII all produced a high level of mCherry protein secretion, while the NC and hAlb constructs effectively mediated the intracellular expression of mCherry but did not significantly promote extracellular secretion( Figure 1D - Figure 1F ). The same set of SPs was evaluated in the hepatoma cell line Huh7, and the mCherry secretion trend observed in HeLa cells persisted( Figure 1I - Figure 1J ); however, the transfected cells were also analyzed using a confocal microscope, and morphological differences in the mCherry signal were found between the SPs that promoted extracellular mCherry secretion and the SPs that only promoted intracellular mCherry expression( Figure 1G - Figure 1H ). Overall, it was determined that hFVII SP could cultivate the highest level of protein secretion in both cell lines.

[0216] Example 2: Organ delivery of mRNA encoding signal peptide and secretion of encoded polypeptide

[0217] Based on the above observations, there was interest in understanding whether mRNA containing an integrated SP sequence would produce similar observations to pDNA. To explore this question, hFVII-mCherry mRNA was generated from the FVII-mCherry-pCS2-MT plasmid by in vitro transcription (IVT) Figure 2A)。It was tested whether mDLNP lipid nanoparticles could be used as the initial carrier of RNA. The results of transfecting multiple different cell lines with mDLNP containing FVII-mCherry mRNA showed that the protein output into the culture medium was positively correlated with the time and dose after transfection, and greater fluorescence signal intensity was observed in the cell lines at longer time intervals and higher doses( Figure 2B - Figure 2D )。

[0218] Next, the liver-targeted mDLNP test delivered FVII-mRNA to the mouse liver. First, the liver-targeting ability of mDLNP was verified by intravenous injection of mDLNP loaded with luciferase mRNA, and IVIS analysis showed bright fluorescence emitted 6 h after injection( Figure 2E )。On this basis, hFVII-mCherry mRNA and WT-mCherry mRNA were encapsulated into mDLNP and injected into mice intravenously. To determine whether mDLNP containing hFVII mCherry mRNA could secrete mCherry into the systemic circulation, thus enabling the liver to act as a protein factory, blood was collected at 2, 6, 24, 30, 48, 55, and 72 h after injection.

[0219] Fluorescence analysis of mouse serum revealed that there was no secretion in the WT-mCherry group, but mCherry signals were present in the serum of the FVII-mCherry group at all time points, with the peak concentration occurring 6 h after injection( Figure 2F )。IVIS images were also taken of the WT-mCherry group and hFVII-mCherry group mice at 55 h and 72 h after injection. Interestingly, mCherry fluorescence was visible in the livers of WT group mice at 55 h after injection, and the signal completely disappeared after 72 h. However, bright mCherry fluorescence could be seen in the kidneys of mice injected with mDLNP containing hFVII mCherry mRNA at both time points, indicating that mCherry protein was being cleared from the systemic circulation through renal filtration( Figure 2G )。

[0220] Next, it was tested whether the liver and extrahepatic organs including the lungs and spleen could promote the secretion of mCherry after targeting and transfecting the liver, lung, and spleen SORT LNP preparations loaded with hFVII mCherry mRNA, respectively. To further investigate this hypothesis, the targeting ability of the SORT technology was first confirmed by intravenous injection of liver, lung, and spleen SORT LNP loaded with luciferase.

[0221] In fact, after injection of the corresponding SORT LNPs, bright fluorescence was emitted from each organ. Then each SORT LNP formulation was loaded with hFVII mCherry mRNA and injected intravenously into mice, and imaging was performed by IVIS 24 hours after injection. Due to the addition of hFVII SP, all SORT LNPs were able to secrete mCherry protein from their respective tissues. Signals were observed in the kidneys of all groups, indicating that the mCherry protein was systemically cleared through the kidneys. The signal in the liver SORT group was significantly higher. In summary, it was determined that the liver, lungs, and spleen could all mediate intracellular mCherry protein production and extracellular protein secretion into the systemic circulation after transfection with liver, lung, and spleen SORT LNPs loaded with hFVII mCherry mRNA( Figure 2H ).

[0222] Example 3: Treatment of psoriasis with hFVII-Enbrel mRNA formulation

[0223] To test the application of signal peptides in therapy, mRNA encoding hFVII SP and the therapeutic synthetic dimer fusion protein Enbrel (etanercept), which was encapsulated into mDLNPs, was tested in L929 cells and an in vivo model of imiquimod-induced psoriasis( Figure 3A ). L929 cells were first treated with murine and human TNF-α at doses ranging from 0.001 to 0.1 ng / mL. At a concentration of only 0.02 ng / mL, less than 20% of the cells survived( Figure 3B ). Next, the cells were pretreated with 80 ng of LNP loaded with hFVII-Enbrel mRNA, and then 48 hours after treatment with hFVII-Enbrel mRNA LNP, the cells were stimulated by administering murine or human TNF-α at doses ranging from 0.002 ng / mL to 5 ng / mL. In both treatment groups, pretreatment with hFVII-Enbrel mRNA mDLNP significantly increased cell viability across the dose range of TNF-α compared to the PBS and mCherry mRNA control groups( Figure 3C ). In addition, L929 cells were pretreated with hFVII Enbrel mRNA mDLNP at doses ranging from 0.05 ng / mL to 1.25 ng / mL for 48 hours and then 0.1 ng / mL of murine or human TNF-α was administered. As expected, cell viability increased in a dose-dependent manner with increasing concentration of the pretreated hFVII Enbrel mRNA( Figure 3D)。Finally, L929 cells treated with 0.1 ng / mL murine or human TNF-α were able to be rescued in a dose-dependent manner after treatment with the medium from cells pretreated with hFVII Enbrel mRNA mDLNP (at doses from 0.05 ng / mL to 1.25 ng / mL). Figure 3E , upper panel), with the viability in the human TNF-α group restored to nearly 100% at an mRNA dose of only 0.4 ng / mL. Figure 3E , lower panel).

[0224] To evaluate the therapeutic potential of mDLNP-mediated hFVII Enbrel mRNA, an in vivo imiquimod-induced psoriasis model was designed. Mice were first shaved and depilated, and then divided into three groups: a negative control group without LNP and treated with a low-dose lanolin cream on days 4 - 8; and two experimental groups, one of which was intravenously injected with hFVII Enbrel mRNA mDLNP 3 days after shaving and depilation, and the other was intravenously injected with mCherry mDLNP 3 days after shaving and depilation. In both experimental groups, mice were administered imiquimod on days 4 - 8 to induce a psoriasis-like phenotype. After intravenous injection of 0.5 mg / kg of hFVII Enbrel mDLNP or Enbrel protein, the serum pharmacokinetics of Enbrel were first evaluated by blood sampling and Enbrel ELISA. The serum concentration of mice injected with Enbrel protein reached a peak at 2 hours after injection and then decreased rapidly. However, in the group injected with hFVII Enbrel mRNA mDLNP, the serum concentration continued to rise within 48 hours after injection and was still detectable until 168 hours after injection. Compared with Enbrel protein, the AUC of hFVII Enbrel mRNA mDLNP increased by more than 10-fold and the Tmax (hours) increased by more than 20-fold (107548.73, + / - 4321.8 vs. 8919.09 + / - 4325.51; 2 hours vs. 40 hours), respectively. Figure 3G ). H&E staining, Ki-67 and Gr-1 IHC staining were performed on skin tissue sections of all mice. In addition to the obvious inflammation shown in the mouse images, the epidermal thickness and Ki-67 positive cells were also objectively elevated in the mCherry imiquimod-treated group compared with the lanolin control group and the hFVII Enbrel mRNA imiquimod group, thus indicating that hFVII Enbrel mRNA mDLNP was able to provide significant therapeutic benefits in an in vivo mouse psoriasis model. Figure 3H - Figure 3I )。

[0225] Example 4: Tumor Immunotherapy with hFVII-anti-PDL1 mRNA

[0226] Next, the use of the disclosed signal peptides was tested with anti-cancer polypeptides. For this purpose, two xenograft tumor models were used. MC38-Luc cells (an aggressive murine adenocarcinoma cell line containing a luciferase reporter gene) or B16F10-Luc cells (a murine melanoma cell line with a luciferase reporter gene construct) were subcutaneously injected into the right hind leg of C57BL6 mice to allow tumor growth. To determine the anti-cancer therapeutic potential of SP-mRNA LNPs, mice were first inoculated with tumor cells and then intravenously injected with mDLNP containing mCherry mRNA or mDLNP encapsulating mRNA encoding the upstream hFVIISP of anti-PDL1 antibody on days 3, 7, and 11 after inoculation ( Figure 4A - Figure 4B ).

[0227] The pharmacokinetic profile of serum anti-PDL1 levels was established, with peak serum concentration reached 48 hours after intravenous injection - essentially reflecting the curve of the previous hFVII Enbrel mRNA mDLNP, and the PDL1 expression on the surface of MC38 cells was determined using flow cytometry ( Figure 4C - Figure 4D ). On days 3, 10, 24, and 32, tumor growth was evaluated by IVIS luminescence imaging. In the group treated with mCherry mDLNP, tumor development was rapid, with significant increases in luminescence and volume at each time point. By day 32, the tumor volume had filled the entire hind leg of each mouse. However, in the group treated with hFVII anti-PDL1 mRNA mDLNP, tumor growth significantly decreased at day 24, indicating that treatment with hFVII anti-PDL1 mRNA mDLNP could effectively inhibit tumor progression. As expected, after tumor resection on day 32, this reduction was paralleled by a decrease in luminescence, a significant slowdown in tumor development, and an overall reduction in tumor volume ( Figure 4E - Figure 4H ). Most notably, there was an extension of survival mediated by hFVII anti-PDL1 mRNA mDLNP. The survival time of mice in this treatment group was almost twice that of the control group ( Figure 4I ). Similarly, mice inoculated with B16F10-Luc tumors were intravenously injected with mDLNP containing mCherry mRNA or hFVII anti-PDL1 mRNA. IVIS imaging on days 3 and 16 showed that compared with the control group, the tumor volume and luminescence in the hFVII anti-PDL1 mRNA group decreased, the growth rate significantly slowed down, and the overall survival period was extended ( Figure 4J - Figure 4N ).

[0228] Example 5: Design of a novel signal peptide sequence

[0229] Attempts have been made to engineer novel signal peptide (SP) sequences encoding secretion into specific mRNA sequences, enabling proteins that are normally confined to the intracellular space to be secreted into the circulation. To further investigate this issue, an exhaustive search for known SPs was conducted in multiple databases, and the search results were merged with the current findings into a master database. Based on this, the known SPs were classified, and a new list was generated that contains the amino acid sequences of 643 previously identified naturally occurring SPs associated with secreted proteins. Due to the heterogeneity of the amino acid sequence lengths, the list of 643 sequences was sorted by length, and sequences of the same length were grouped accordingly (e.g., all SP sequences containing 18 amino acids were grouped into one group, those of 19 amino acids were grouped into another group, and so on). After stratifying the signal sequences by length, a matrix could be created to identify the frequency of each amino acid at the corresponding position in the peptide segment among all sequences of a specific length (see Figure 5 ). Then, the amino acids with the highest frequency of occurrence at each position were selected and concatenated to form novel amino acid sequences of a specific length. Through this process, 21 new SP sequences (shown in Table 2) were generated, with lengths ranging from 15 to 35 amino acids, which do not exist in nature (referred to as SP1 - SP21).

[0230] Table 2: List of Engineered Sequences

[0231]

[0232]

[0233] To determine whether these 21 novel SP sequences could induce secretion, these sequences were integrated upstream of the amino acid sequence encoding human erythropoietin (SEQ ID NO: 58, hEPO without a native signal peptide). However, it is known that hEPO itself contains its own signal peptide sequence, so a pDNA backbone (NF - NSP - hEPO) containing hEPO lacking the endogenous signal peptide was created. Using a reverse codon generation tool, the nucleic acid sequences of the 21 peptides were synthesized, and the corresponding oligonucleotides were subsequently ordered. Through a series of cloning and PCR reactions, pDNA vectors containing each of the 21 SPs were constructed, with the SP directly upstream of NF - NSP - hEPO ( Figure 6) To ensure the response of the novel SP to any possible secretion, a pDNA backbone containing functional hEPO (without its SP) was also constructed as a control. However, the Kozak sequence of hEPO is located upstream of the endogenous SP of hEPO, and the previous endogenous SP has been removed. Therefore, a kozak sequence (GCCACCATG) was inserted upstream of the truncated SP hEPO sequence, which allows the translation of hEPO mRNA but removes its secretion ability (NSP-hEPO). After obtaining the pDNA backbone, in vitro transcription (IVT) reactions were performed on each novel SP-hEPO sequence and NSP-hEPO sequence to generate a product containing an optimized 5'UTR (SEQ ID NO:59), Kozak sequence, novel signal peptide sequence, hEPO protein sequence, 3'UTR (SEQ ID NO:60), and finally an optimized poly-A tail (SEQ ID NO:61).

[0234] The newly synthesized mRNA was then encapsulated into iPhos LNPs (9A1-P9 / cholesterol / DODAP / DMG-PEG, 25:30:30:1 mol / mol; 18:1, 9A1-P9:nucleic acid, weight / weight) and intravenously injected into mice at a dose of 0.5 mg / kg. For each construct, mouse blood was collected at 6, 24, 48, and 72 hours and analyzed by hEPO enzyme-linked immunosorbent assay (ELISA) to determine the serum hEPO concentration in mIU / mL ( Figure 7 ). The successful secretion of SP-hEPO had stronger differential kinetic characteristics (C max , t 1 / 2 and area under the curve (AUC)) compared to the NSP-hEPO control group.

[0235] Sequence:

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244] References

[0245] Hou, X., Zaks, T., Langer, R., et al., Lipid nanoparticles for mRNA delivery, Nature Reviews Materials, Vol. 6, pp. 1078 - 1094 (2021).

[0246] Cheng, Q., Wei, T., Farbiak, L., Johnson, L. T., Dilliard, S. A. & Siegwart, D. J., Selective organ targeting (SORT) nanoparticles for tissue - specific mRNA delivery and CRISPR - Cas gene editing, Nature Nanotechnology, Vol. 15, pp. 313 - 320 (2020).

[0247] Cheng, Q., Wei, T., Jia, Y., Farbiak, L., Zhou, K., Zhang, S., Wei, Y., Zhu, H. & Siegwart, D. J., Dendrimer - based lipid nanoparticles deliver therapeutic FAH mRNA to normalize liver function and extend survival in a mouse model of hepatorenal tyrosinemia type I, Advanced Materials, Vol. 30, No. e1805308 (2018).

[0248] Liu, S.; Cheng, Q.; Wei, T.; Yu, X.; Johnson, L. T.; Farbiak, L. & Siegwart, D. J., Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing (for organ-selective mRNA delivery and CRISPR-Cas gene editing of membrane-destabilizing ionizable phospholipids), Nature Materials (Nature Materials), Volume 20, 701-710 (2021).

Claims

1. An engineered signal peptide comprising any amino acid sequence of SEQ ID NO: 9 - 29, its variants or derivatives.

2. The engineered signal peptide according to claim 1, which is fused with a heterologous polypeptide.

3. The engineered signal peptide according to claim 2, wherein the heterologous polypeptide is a therapeutic or diagnostic polypeptide.

4. The engineered signal peptide according to claim 3, wherein the heterologous polypeptide is any one of an anti - cancer, anti - inflammatory, immunomodulatory, antiviral, antimicrobial, antifungal, anti - worm, cholesterol - lowering, anti - diabetic, anti - fibrotic, analgesic, anesthetic, anti - aging, anti - depressive, neuromodulatory, anti - dermatitis, anti - edema, anti - allergic, anti - hyperkeratotic, fungistatic, anti - pruritic, cardiovascular therapeutic, chemotherapy, hormone, protein replacement therapeutic, polypeptide therapeutic, vaccine, viral vaccine, theranostic, diagnostic or secretory therapeutic polypeptide.

5. The engineered signal peptide according to claim 2, wherein the heterologous polypeptide is an enzyme, a nutrient, a food additive, a flavor enhancer, and / or a cosmetic.

6. The engineered signal peptide according to claim 2, wherein the heterologous polypeptide is a reporter polypeptide selected from a fluorescent protein, LacZ (β - galactosidase), CAT (chloramphenicol acetyltransferase), or luciferase.

7. The engineered signal peptide according to claim 6, wherein the fluorescent protein is any one of GFP (green fluorescent protein), BFP (blue fluorescent protein), YFP (yellow fluorescent protein), RFP (red fluorescent protein), mCherry, or luciferase.

8. A recombinant polynucleotide sequence comprising a nucleic acid sequence encoding the signal peptide of SEQ ID NO: 9 - 29.

9. The recombinant polynucleotide sequence according to claim 8, wherein the nucleic acid sequence is a DNA sequence.

10. The recombinant polynucleotide sequence according to claim 8, wherein the nucleic acid sequence is an RNA sequence.

11. The recombinant polynucleotide sequence according to claim 9, comprising any nucleic acid sequence of SEQ ID NO: 30 - 50 or its variants or derivatives.

12. The recombinant polynucleotide sequence according to claim 10, comprising a ribonucleic acid sequence corresponding to any sequence of SEQ ID NO: 30 - 50 or its variants or derivatives.

13. The recombinant polynucleotide according to claim 8, further encoding a heterologous polypeptide in - frame with the signal peptide.

14. The recombinant polynucleotide according to claim 13, wherein the heterologous polypeptide is a therapeutic or diagnostic polypeptide.

15. The recombinant polynucleotide according to claim 14, wherein the heterologous polypeptide is any one of an anti - cancer, anti - inflammatory, immunomodulatory, antiviral, antimicrobial, antifungal, anti - worm, cholesterol - lowering, anti - diabetic, anti - fibrotic, analgesic, anesthetic, anti - aging, anti - depressive, neuromodulatory, anti - dermatitis, anti - edema, anti - allergic, anti - hyperkeratotic, fungistatic, anti - pruritic, cardiovascular therapeutic, chemotherapy, hormone, protein replacement therapeutic, polypeptide therapeutic, vaccine, viral vaccine, theranostic, diagnostic or secretory therapeutic polypeptide.

16. The recombinant polynucleotide according to claim 13, wherein the heterologous polypeptide is an enzyme, a nutrient, a food additive, a flavor enhancer, and / or a cosmetic.

17. The recombinant polynucleotide according to claim 13, wherein the heterologous polypeptide is a reporter polypeptide selected from a fluorescent protein, LacZ (β-galactosidase), CAT (chloramphenicol acetyltransferase), or luciferase.

18. The recombinant polynucleotide according to claim 13, wherein the heterologous polypeptide is any one of the sequences of SEQ ID NOs: 55 - 58 or a functional fragment, derivative, or variant thereof.

19. The recombinant polynucleotide according to claim 13, wherein the heterologous polypeptide is an anti-PD-L1 antibody, Enbrel, mCherry, or hEPO or a functional fragment, derivative, or variant thereof.

20. A therapeutic composition, which comprises: a. A delivery system; and b. A polynucleotide sequence comprising a nucleic acid sequence encoding the following peptides: i. A signal peptide; and ii. A therapeutic polypeptide.

21. The therapeutic composition according to claim 20, wherein the signal peptide comprises any one of the amino acid sequences of SEQ ID NOs: 9 - 29 or a variant or derivative thereof.

22. The therapeutic composition according to claim 20, wherein the polynucleotide sequence encoding the signal peptide comprises a nucleic acid sequence corresponding to any one of the sequences of SEQ ID NOs: 30 - 50 or a variant or derivative thereof.

23. The therapeutic composition according to claim 20, wherein the signal peptide has a length of 10 - 50 amino acids.

24. The therapeutic composition according to any one of claims 20 - 23, wherein the delivery system is any one of a polymer, a polymer complex, a lipid, a lipid analog, a lipid complex, a liposome, a polymer nanoparticle, a nanoparticle, a lipid nanoparticle (LNP), a core-shell nanoparticle, a solid lipid nanoparticle, a metal nanoparticle, a self-assembled nucleic acid nanoparticle, hyaluronidase, a nanoparticle mimic, a ribonucleoprotein, a positively charged peptide, a small molecule RNA conjugate, an aptamer RNA chimera, an RNA fusion protein complex, and any combination thereof.

25. The therapeutic composition according to claim 24, wherein the delivery system is a lipid nanoparticle comprising an ionizable amino lipid.

26. The therapeutic composition according to claim 25, wherein the lipid nanoparticle further comprises one or more of phospholipid, cholesterol, or a polymer lipid.

27. The therapeutic composition according to any one of claims 24 - 26, wherein the delivery system comprises any one of iPhosLNP, mDLNP, liver SORT LNP, lung SORT LNP, or spleen SORT LNP.

28. The therapeutic composition according to any one of claims 20 - 24, wherein the delivery system is a controlled system selected from a synthetic material depot, a polymer depot, a lipid depot, a controlled release hydrogel depot, or a controlled release polymer depot.

29. The therapeutic composition according to any one of claims 20-28, further comprising one or more pharmaceutically acceptable excipients.

30. The therapeutic composition according to claim 20, wherein the therapeutic polypeptide is any one of an anti-cancer, anti-inflammatory, immunomodulatory, antiviral, anti-microbial, anti-fungal, anti-helminthic, anti-cholesterol, anti-diabetic, anti-fibrotic, analgesic, anesthetic, anti-aging, anti-depressant, neuromodulatory, anti-dermatitis, anti-edema, anti-allergic, anti-hyperkeratotic, anti-fungal, anti-itch, cardiovascular therapeutic, chemotherapy, hormone, protein replacement therapeutic, polypeptide therapeutic, vaccine, viral vaccine, theranostic, diagnostic, or secretory therapeutic polypeptide.

31. A method for diagnosis, prevention or treatment, comprising administering to a subject in need thereof an effective amount of the composition according to any one of claims 20-30.

32. The method for diagnosis, prevention or treatment according to claim 31, wherein the administration is carried out by one or more of the following routes: parenteral, oral, intra-adipose, intra-arterial, intra-articular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrarectal, intracapsular, intratracheal, intratumoral, intraumbilical, intravaginal, intravenous, intravascular, intravitreal, liposomal, topical, mucosal, parenteral, rectal, subconjunctival, subcutaneous, sublingual, topical, transbuccal, or transdermal routes.

33. The method for diagnosis, prevention or treatment according to claim 31, wherein the administration is carried out by a controlled system selected from an implant, a synthetic material depot, a polymer depot, a lipid depot, a controlled release hydrogel depot, or a controlled release polymer depot.

34. The method for diagnosis, prevention or treatment according to claim 31, wherein the subject is suspected of having or has been diagnosed with any one of an autoimmune disorder, cancer, diabetes, cardiovascular disease, neurological disease, bacterial infection, fungal infection, viral infection, or fibrosis.

35. The method for diagnosis, prevention or treatment according to claim 31, wherein the subject is in need of prevention.

36. The method for diagnosis, prevention or treatment according to claim 31, wherein the therapeutic polypeptide according to claim 15 is systemically secreted in a subject in need thereof.

37. The method for diagnosis, prevention or treatment according to claim 31, wherein the therapeutic polypeptide according to claim 21 is directionally expressed in any one of the lung, liver, or spleen.

38. The method according to claim 31, wherein the subject is a mammal.

39. The method according to claim 38, wherein the subject is a human.

40. Use of the composition according to any one of claims 20-30 in treating a subject in need thereof.

41. A recombinant polypeptide, comprising: i. a signal peptide corresponding to any one of the sequences of SEQ ID NO: 1-4 or a variant or derivative thereof; and ii. a heterologous polypeptide.

42. A recombinant polynucleotide, comprising a nucleic acid sequence encoding the following information: iii. a signal peptide corresponding to any one of SEQ ID NOs: 1-4, or a variant or derivative thereof; and iv. a heterologous polypeptide in-frame with the signal peptide.

43. The recombinant polynucleotide according to claim 42, wherein the nucleic acid sequence is a DNA sequence.

44. The recombinant polynucleotide according to claim 42, wherein the nucleic acid sequence is an RNA sequence.

45. The recombinant polynucleotide sequence according to claim 43, which comprises a nucleic acid sequence corresponding to any one of SEQ ID NOs: 5-8 or a variant or derivative thereof.

46. The recombinant polynucleotide sequence according to claim 44, which comprises a ribonucleic acid sequence corresponding to SEQ ID NOs: 5-8 or a variant or derivative thereof.

47. The recombinant polynucleotide according to claim 42, wherein the heterologous polypeptide is a therapeutic or diagnostic polypeptide.

48. The recombinant polynucleotide according to claim 47, wherein the heterologous polypeptide is any one of an anti-cancer, anti-inflammatory, immunomodulatory, antiviral, antimicrobial, antifungal, anti-helminth, cholesterol-lowering, anti-diabetic, anti-fibrotic, analgesic, anesthetic, anti-aging, anti-depressant, neuromodulatory, anti-dermatitis, anti-edema, anti-allergy, anti-hyperkeratosis, anti-fungal, anti-itching, cardiovascular therapeutic, chemotherapy, hormone, protein replacement therapeutic, polypeptide therapeutic, vaccine, viral vaccine, theranostic, diagnostic or secretory therapeutic polypeptide.

49. The recombinant polynucleotide according to claim 42, wherein the heterologous polypeptide is an enzyme, a nutrient, a food additive, a flavor enhancer, and / or a cosmetic.

50. The recombinant polynucleotide according to claim 42, wherein the heterologous polypeptide is a reporter polypeptide selected from a fluorescent protein, LacZ (β-galactosidase), CAT (chloramphenicol acetyltransferase), or luciferase.

51. The recombinant polynucleotide according to claim 42, wherein the heterologous polypeptide is an anti-PD-L1 antibody, Enbrel, hEPO or a functional fragment, derivative or variant thereof.

52. A therapeutic composition, which comprises: a. a delivery system; and b. a polynucleotide sequence comprising a nucleic acid sequence encoding the following: v. a signal peptide corresponding to any one of SEQ ID NOs: 1-4, or a variant or derivative thereof; and vi. a therapeutic polypeptide.

53. The therapeutic composition according to claim 52, wherein the polynucleotide sequence encoding the signal peptide comprises any nucleic acid sequence of SEQ ID NOs: 5-8 or a variant or derivative thereof.

54. The therapeutic composition according to claim 52, wherein the polynucleotide sequence encoding the signal peptide comprises a ribonucleic acid sequence corresponding to any one of SEQ ID NOs: 5-8 or a variant or derivative thereof.

55. The therapeutic composition according to any one of claims 52 - 54, wherein the delivery system is any one of a polymer, a polymer complex, a lipid, a lipid analogue, a lipid complex, a liposome, a polymer nanoparticle, a nanoparticle, a lipid nanoparticle (LNP), a core - shell nanoparticle, a solid lipid nanoparticle, a metal nanoparticle, a self - assembled nucleic acid nanoparticle, hyaluronidase, a nanoparticle mimetic, a ribonucleoprotein, a positively charged peptide, a small - molecule RNA conjugate, an aptamer - RNA chimera, an RNA fusion protein complex, and any combination thereof.

56. The therapeutic composition according to claim 55, wherein the delivery system is a lipid nanoparticle comprising an ionizable amino lipid.

57. The therapeutic composition according to claim 56, wherein the lipid nanoparticle further comprises one or more of phospholipid, cholesterol, or a polymer lipid.

58. The therapeutic composition according to any one of claims 55 - 57, wherein the delivery system comprises any one of iPhos LNP, mDLNP, liver SORT LNP, lung SORT LNP, or spleen SORT LNP.

59. The therapeutic composition according to any one of claims 52 - 54, wherein the delivery system is a controlled system selected from a synthetic material depot, a polymer depot, a lipid depot, a controlled - release hydrogel depot, or a controlled - release polymer depot.

60. The therapeutic composition according to any one of claims 52 - 59, further comprising one or more pharmaceutically acceptable excipients.

61. The therapeutic composition according to claim 52, wherein the therapeutic polypeptide is any one of an anti - cancer, anti - inflammatory, immunomodulatory, anti - viral, anti - microbial, anti - fungal, anti - helminthic, cholesterol - lowering, anti - diabetic, anti - fibrotic, analgesic, anesthetic, anti - aging, anti - depressive, neuromodulatory, anti - dermatitis, anti - edema, anti - allergic, anti - hyperkeratotic, anti - fungal, anti - pruritic, cardiovascular therapeutic, chemotherapeutic, hormonal, protein replacement therapeutic, polypeptide therapeutic, vaccine, viral vaccine, theranostic, diagnostic, or secretory therapeutic polypeptide.

62. A method of diagnosis, prevention, or treatment, comprising administering to a subject in need thereof an effective amount of the composition according to any one of claims 52 - 61.

63. The method of diagnosis, prevention, or treatment according to claim 62, wherein the administration is carried out by one or more of the following routes: parenteral, oral, intra - adipose, intra - arterial, intra - articular, intracranial, intradermal, intralesional, intramuscular, intranasal, intra - ocular, intra - pericardial, intra - peritoneal, intra - pleural, intra - prostatic, intra - rectal, intra - capsular, intra - tracheal, intratumoral, intra - umbilical, intra - vaginal, intravenous, intravascular, intravitreal, liposomal, topical, mucosal, parenteral, rectal, subconjunctival, subcutaneous, sublingual, topical, trans - buccal, or trans - dermal route.

64. The diagnostic, prophylactic or therapeutic method according to claim 63, wherein said administration is effected by a controlled system selected from an implant, a synthetic material depot, a polymer depot, a lipid depot, a controlled release hydrogel depot, or a controlled release polymer depot.

65. The diagnostic, prophylactic or therapeutic method according to claim 63, wherein the subject is suspected of having or has been diagnosed with any one of an autoimmune disorder, cancer, diabetes, cardiovascular disease, neurological disease, bacterial infection, fungal infection, viral infection, or fibrosis.

66. The diagnostic, prophylactic or therapeutic method according to claim 63, wherein the subject is in need of prophylaxis.

67. The therapeutic method according to claim 63, wherein the therapeutic polypeptide according to claim 52 is systemically secreted in a subject in need thereof.

68. The diagnostic, prophylactic or therapeutic method according to claim 63, wherein the therapeutic polypeptide of claim 52 is expressed specifically in any one of the lung, liver, or spleen.

69. The method according to claim 63, wherein the subject is a mammal.

70. The method according to claim 63, wherein the subject is a human.

71. Use of the composition according to any one of claims 52 - 61 in treating a subject in need thereof.

72. The use according to claim 71, wherein the subject is suspected of having or has been diagnosed with any one of an autoimmune disorder, cancer, diabetes, or fibrosis.

73. The use according to any one of claims 71 or 72, wherein the subject is a mammal.

74. The use according to any one of claims 71 or 72, wherein the subject is a human.

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