Novel secretory signal peptide

Abstract

Provided herein are novel, engineered, isolated signal peptide sequences and compositions comprising them, as well as compositions and methods that use these signal peptides to enable secretion of heterologous polypeptides for therapeutic, diagnostic, and commercial value.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Application No. 63 / 376,696, filed September 22, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] [Sequence table] This application contains a Sequence Listing that has been submitted through the Patent Center in WIPO ST.26xml format, and is incorporated herein by reference in its entirety. This .xml copy is named "106546-757419 UTSD 3964.xml" and is 66KB in size.

[0003] [background] 1. Field The present invention relates to the use of novel secretory signal peptides for therapeutic applications. [Background technology]

[0004] 2.Background Recent scientific discoveries have highlighted the myriad therapeutic applications of mRNA due to its modularity and ability to provide customizable "instructions" for creating functional proteins. Clinical studies continue to confirm its favorable efficacy, further highlighting its potential as a next-generation genetic medicine, for which scientists are only beginning to scratch the surface of potential applications. In parallel with advances in mRNA biology, significant progress has also been made in the fields of lipid nanoparticles (LNPs), polymeric nanoparticles, and other approaches mediating safe and effective nucleic acid delivery. Much of the focus of research to date has been on amplifying intracellular expression of encoded polypeptides. However, less attention has been paid to post-translational regulation of encoded proteins and their targeted localization and secretion within the body.

[0005] Within cells, there are a wide variety of unique pathways and processes that enable the shuttling of proteins into various organelles, such as the nucleus and mitochondria, and the export of proteins to the extracellular space via the secretory pathway. However, to utilize these transport systems, the mRNA encoding each protein also contains a figurative transport label known as a signal peptide (SP) upstream of the protein sequence. This label then signals the appropriate cellular machinery to transport the protein to a specific location within the cell or package it for secretion into the extracellular space. The design of novel signal sequences for therapeutic mRNAs can greatly benefit the delivery, localization, and clearance of the encoded therapeutic proteins. Summary of the Invention

[0006] In some embodiments, the present disclosure encompasses an engineered signal peptide comprising the amino acid sequence of any of SEQ ID NOS: 9-29, and variants or derivatives thereof. In some embodiments, the signal peptide is fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide is a therapeutic or diagnostic polypeptide, non-limiting examples of which include anti-cancer, anti-inflammatory, immunomodulatory, antiviral, antibacterial, antifungal, antihelminthic, cholesterol-lowering, antidiabetic, antifibrotic, analgesic, anesthetic, anti-aging, antidepressant, neuromodulatory, antidermatitis, antiedema, antiallergic, antikeratolytic, antifungal, antipruritic, cardiovascular therapeutic, chemotherapeutic, hormone, protein replacement therapy, polypeptide therapeutic, vaccine, viral vaccine, theragnostic, diagnostic, or secreted therapeutic polypeptide.

[0007] In some embodiments, the heterologous polypeptide is an enzyme, a nutraceutical, a food additive, a flavor enhancer, and / or a cosmetic. In some embodiments, the heterologous polypeptide is a reporter polypeptide, non-limiting examples of which include a fluorescent protein, LacZ (β-galactosidase), CAT (chloramphenicol acetyltransferase), or luciferase. In some embodiments, the fluorescent protein is any of GFP (green fluorescent protein), BFP (blue fluorescent protein), YFP (yellow fluorescent protein), RFP (red fluorescent protein), mCherry, or luciferase.

[0008] In some embodiments, the present disclosure also encompasses a recombinant polynucleotide sequence comprising a nucleic acid sequence encoding a signal peptide of any of SEQ ID NOS: 9-29. In some embodiments, the nucleic acid sequence is a DNA sequence. In some embodiments, the nucleic acid sequence is an RNA sequence. In some embodiments, the recombinant polynucleotide comprises a nucleic acid sequence of any of SEQ ID NOS: 30-50, or a variant or derivative thereof. In some embodiments, the polynucleotide sequence comprises a ribonucleic acid sequence corresponding to any of SEQ ID NOS: 30-50, or a variant or derivative thereof.

[0009] In some embodiments, the recombinant polynucleotide encodes a heterologous polypeptide in frame with a signal peptide. In some embodiments, the heterologous polypeptide is a therapeutic or diagnostic polypeptide, non-limiting examples of which include anti-cancer, anti-inflammatory, immunomodulatory, antiviral, antibacterial, antifungal, antihelminthic, cholesterol-lowering, antidiabetic, antifibrotic, analgesic, anesthetic, anti-aging, antidepressant, neuromodulatory, antidermatitis, antiedema, antiallergic, antikeratolytic, antifungal, antipruritic, cardiovascular therapeutic, chemotherapeutic, hormone, protein replacement therapy, polypeptide therapeutic, vaccine, viral vaccine, theragnostic, diagnostic, or secreted therapeutic polypeptide.

[0010] In some embodiments, the heterologous polypeptide is an enzyme, a nutraceutical, a food additive, a flavor enhancer, and / or a cosmetic. In some embodiments, the heterologous polypeptide is a reporter polypeptide, non-limiting examples of which include a fluorescent protein, LacZ (β-galactosidase), CAT (chloramphenicol acetyltransferase), or luciferase. In some embodiments, the fluorescent protein is any of GFP (green fluorescent protein), BFP (blue fluorescent protein), YFP (yellow fluorescent protein), RFP (red fluorescent protein), mCherry, or luciferase.

[0011] In some exemplary embodiments, the recombinant polynucleotide encodes a heterologous polypeptide having any of SEQ ID NOs: 55-58, or a functional fragment, derivative, or variant thereof. In some embodiments, the heterologous polypeptide is an anti-PD-L1 antibody, Enbrel, mCherry, or hEPO, or a functional fragment, derivative, or variant thereof.

[0012] In some embodiments, the disclosure also encompasses therapeutic compositions comprising a delivery system and a polynucleotide sequence comprising a nucleic acid sequence encoding a signal peptide and a therapeutic polypeptide. In some embodiments, the signal peptide comprises the amino acid sequence of any of SEQ ID NOS: 9-29, or a variant or derivative thereof. In some embodiments, the polynucleotide sequence encoding the signal peptide comprises the nucleic acid sequence corresponding to any of SEQ ID NOS: 30-50, or a variant or derivative thereof.

[0013] In some embodiments, the delivery system is any of polymers, polyplexes, lipids, lipidoids, lipoplexes, liposomes, polymeric nanoparticles, nanoparticles, lipid nanoparticles (LNPs), core-shell nanoparticles, solid lipid nanoparticles, metal nanoparticles, self-assembled nucleic acid nanoparticles, hyaluronidase, nanoparticle mimics, ribonucleoproteins, positively charged peptides, small RNA conjugates, aptamer-RNA chimeras, RNA fusion protein complexes, and any combination thereof. In some embodiments, the delivery system is a lipid nanoparticle containing an ionizable amino lipid. In some embodiments, the lipid nanoparticle further comprises one or more of phospholipids, cholesterol, or polymeric lipids. In some embodiments, the delivery system comprises iPhos LNP, mDLNP, liver SORT LNP, lung SORT LNP, or spleen SORT LNP. In some embodiments, the delivery system is a controlled system selected from a synthetic material depot, a polymer depot, a lipid depot, a sustained-release hydrogel depot, or a sustained-release polymer depot.

[0014] In some embodiments, the therapeutic composition further comprises one or more pharmaceutically acceptable excipients. In some embodiments, the therapeutic polypeptide in the therapeutic composition is an anti-cancer, anti-inflammatory, immunomodulatory, antiviral, antibacterial, antifungal, antihelminthic, cholesterol-lowering, antidiabetic, antifibrotic, analgesic, anesthetic, anti-aging, antidepressant, neuromodulatory, antidermatitis, antiedema, antiallergic, antikeratolytic, antifungal, antipruritic, cardiovascular therapeutic, chemotherapeutic, hormone, protein replacement therapy, polypeptide therapeutic, vaccine, viral vaccine, theragnostic, diagnostic, or secreted therapeutic polypeptide.

[0015] In some embodiments, the present disclosure also encompasses diagnostic, prophylactic, or therapeutic methods comprising administering to a subject in need thereof an effective amount of a composition disclosed herein.

[0016] In some embodiments, the diagnostic, prophylactic, or therapeutic method comprises administering a composition disclosed herein via one or more of the following routes: parenteral, oral, intraadipose, intraarterial, intraarticular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrarectal, intrathecal, intratracheal, intratumoral, intraumbilical, intravaginal, intravenous, intravascular, intravitreal, liposomal, topical, mucosal, parenteral, rectal, subconjunctival, subcutaneous, sublingual, topical, transbuccal, or transdermal. In some embodiments, the administration is via a controlled system selected from an implant, a synthetic material depot, a polymer depot, a lipid depot, a sustained-release hydrogel depot, or a sustained-release polymer depot.

[0017] In some embodiments, the subject in need of diagnosis, prevention, or treatment is suspected of or diagnosed with an autoimmune disease, cancer, diabetes, cardiovascular disease, neurological disease, bacterial infection, fungal infection, viral infection, or fibrosis. In some embodiments, the subject is in need of prevention. In some embodiments, the therapeutic polypeptides disclosed herein are secreted systemically in the subject in need thereof. In some embodiments, the therapeutic polypeptides are adapted for expression in the lung, liver, or spleen. In some embodiments, the subject is a mammal. In some embodiments, the subject is human.

[0018] In some embodiments, the present disclosure also encompasses recombinant polypeptides comprising a signal peptide corresponding to any of SEQ ID NOs: 1-4, or a variant or derivative thereof; and a heterologous polypeptide.

[0019] In some embodiments, the present disclosure also encompasses a recombinant polynucleotide comprising a nucleic acid sequence encoding a signal peptide corresponding to any of SEQ ID NOS: 1-4, or a variant or derivative thereof, and a heterologous polypeptide in-frame with the signal peptide. In some embodiments, the nucleic acid sequence can be a DNA or RNA sequence corresponding to SEQ ID NOS: 5-8, or a variant or derivative thereof.

[0020] In some embodiments, the heterologous polypeptide is, for example, an anti-cancer, anti-inflammatory, immunomodulatory, antiviral, antibacterial, antifungal, antihelminthic, cholesterol-lowering, antidiabetic, antifibrotic, analgesic, anesthetic, anti-aging, antidepressant, neuromodulatory, antidermatitis, antiedema, antiallergic, antikeratolytic, antifungal, antipruritic, cardiovascular therapeutic, chemotherapeutic, hormone, protein replacement therapy, polypeptide therapeutic, vaccine, viral vaccine, theragnostic, diagnostic, or secreted therapeutic polypeptide. In some embodiments, the heterologous polypeptide is an enzyme, nutraceutical, food additive, flavor enhancer, and / or cosmetic. In some embodiments, the heterologous polypeptide is a reporter polypeptide selected from a fluorescent protein, LacZ (β-galactosidase), CAT (chloramphenicol acetyltransferase), or luciferase. In some embodiments, the heterologous polypeptide is, for example, an anti-PD-L1 antibody, Enbrel, hEPO, or a functional fragment, derivative, or variant thereof.

[0021] In some embodiments, the present disclosure also encompasses therapeutic compositions comprising a delivery system and a polynucleotide sequence comprising a nucleic acid sequence encoding a signal peptide corresponding to any of SEQ ID NOS: 1-4, or a variant or derivative thereof, and a therapeutic polypeptide. In some embodiments, the polynucleotide sequence encoding the signal peptide comprises a nucleic acid sequence corresponding to any of SEQ ID NOS: 5-8, or a variant or derivative thereof. In some embodiments, the polynucleotide sequence encoding the signal peptide comprises a ribonucleic acid sequence corresponding to any of SEQ ID NOS: 5-8, or a variant or derivative thereof. In some embodiments, non-limiting examples of delivery systems for the therapeutic compositions disclosed herein are polymers, polyplexes, lipids, lipidoids, lipoplexes, liposomes, polymeric nanoparticles, nanoparticles, lipid nanoparticles (LNPs), core-shell nanoparticles, solid lipid nanoparticles, metal nanoparticles, self-assembled nucleic acid nanoparticles, hyaluronidase, nanoparticle mimics, ribonucleoproteins, positively charged peptides, small RNA conjugates, aptamer-RNA chimeras, RNA fusion protein complexes, and any combination thereof. In some embodiments, the delivery system is a lipid nanoparticle comprising an ionizable amino lipid. In some embodiments, the lipid nanoparticles further comprise one or more of phospholipids, cholesterol, or polymeric lipids. In some embodiments, the delivery system comprises iPhos LNP, mDLNP, liver SORT LNP, lung SORT LNP, or spleen SORT LNP. In some embodiments, the delivery system is a controlled system selected from a synthetic material depot, a polymer depot, a lipid depot, a sustained-release hydrogel depot, or a sustained-release polymer depot.

[0022] In some embodiments, a therapeutic composition comprising a signal peptide corresponding to SEQ ID NOs: 1-4, or a variant or derivative thereof, further comprises one or more pharmaceutically acceptable excipients. In some embodiments, a therapeutic composition comprises, for example, an anti-cancer, anti-inflammatory, immunomodulatory, antiviral, antibacterial, antifungal, antihelminthic, cholesterol-lowering, antidiabetic, antifibrotic, analgesic, anesthetic, anti-aging, antidepressant, neuromodulatory, antidermatitis, anti-edema, anti-allergic, antikeratolytic, antifungal, antipruritic, cardiovascular therapeutic, chemotherapeutic, hormone, protein replacement therapy, polypeptide therapeutic, vaccine, viral vaccine, theragnostic, diagnostic, or secreted therapeutic polypeptide.

[0023] In some embodiments, the present disclosure also encompasses diagnostic, prophylactic, or therapeutic methods comprising administering to a subject in need thereof an effective amount of a composition comprising a signal peptide corresponding to SEQ ID NOs: 1-4, or a variant or derivative thereof. These therapeutic compositions may be administered, for example, via one or more of the following routes: parenteral, oral, intraadipose, intra-arterial, intra-articular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrarectal, intrathecal, intratracheal, intratumoral, intraumbilical, intravaginal, intravenous, intravascular, intravitreal, liposomal, topical, mucosal, parenteral, rectal, subconjunctival, subcutaneous, sublingual, topical, transbuccal, or transdermal. In some embodiments, administration is via a controlled system selected from an implant, a synthetic material depot, a polymer depot, a lipid depot, a sustained-release hydrogel depot, or a sustained-release polymer depot. In some embodiments, the subject is suspected of having or has been diagnosed with an autoimmune disease, cancer, diabetes, cardiovascular disease, neurological disease, bacterial infection, fungal infection, viral infection, or fibrosis. In some embodiments, the subject is a mammal, such as a human, in need of prevention. In some embodiments, the composition is secreted systemically in the subject in need thereof, or directed for organ-specific expression in the lung, liver, or spleen. In some embodiments, the subject is suspected of having or has been diagnosed with an autoimmune disease, cancer, diabetes, or fibrosis.

[0024] Aspects of the inventive concepts are illustrated by the following examples, in which like reference numerals refer to like elements. [Brief explanation of the drawings]

[0025] [Figure 1A] Figure 1A provides a schematic diagram of a construct containing mCherry with a signal peptide at the N-terminus and its use for signal peptide screening by pDNA transfection in vitro. [Figure 1B] Figure 1B provides fluorescence microscopy images (exposure time, 1 / 30 s) showing time-dependent mCherry secretion in HeLa cells. mCherry signals were clearly observed in the medium 2 days after treatment with gLuc-mCherry. PBS, phosphate-buffered saline; WT, wild-type; gLuc, Gaussia luciferase. [Figure 1C] Figure 1C provides quantification of mCherry fluorescence in cell lysates and medium at different time points. mCherry without the signal peptide was used as a control ("WT-mCherry"). PBS, phosphate-buffered saline; WT, wild-type; gLuc, Gaussia luciferase. [Figure 1D] Figure 1D provides a fluorescence microscopy image (exposure time, 1 / 70 s) showing mCherry secretion in HeLa cells after 72 h. PBS, phosphate-buffered saline; NC, negative control; hAlb, human albumin; hApoB, human apolipoprotein B; gLuc, Gaussia luciferase; hFVII, human factor VII. [Figure 1E] Figure 1E provides photographs of cell lysates and medium taken after 72 hours. PBS, phosphate-buffered saline; NC, negative control; hAlb, human albumin; hApoB, human apolipoprotein B; gLuc, Gaussia luciferase; hFVII, human factor VII. [Figure 1F]Figure 1F provides quantification of mCherry fluorescence in cell lysates and medium after 72 hours. Data are shown as mean ± standard error (sem) (n = 5, biologically independent samples). HeLa cells in 96-well plates were treated with Lipofectamine 2000 / pDNA (50 ng per well). At the indicated time points, cells were photographed under a microscope, and mCherry signals were quantified using a plate reader or captured using IVIS. A two-tailed unpaired t-test was used to determine the significance of data comparisons (*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, Gaussia luciferase; hFVII, human factor VII. [Figure 1G] Figure 1G shows a bar graph quantifying the mCherry signal in cell lysates and medium after 72 hours, confirming the functionality of the signal peptide in Huh7 cells. Data are shown as mean ± sem (n = 4, biologically independent samples). A two-tailed unpaired t-test was used to determine the significance of data comparisons (*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, Gaussia luciferase; hFVII, human factor VII. PBS, phosphate-buffered saline; NC, negative control; hAlb, human albumin; hApoB, human apolipoprotein B; gLuc, Gaussia luciferase; hFVII, human factor VII. [Figure 1H] Figure 1H shows a confocal image demonstrating the "circular" signal (indicated by the yellow arrow) observed in Huh7 cells when the signal peptide functioned well (scale bar, 50 µm). PBS, phosphate-buffered saline; NC, negative control; hAlb, human albumin; hApoB, human apolipoprotein B; gLuc, Gaussia luciferase; hFVII, human factor VII. [Figure 1I]Figure 1I provides quantification of mCherry fluorescence in cell lysates and medium at 72 hours post-transfection. Data are shown as mean ± sem (n = 4, biologically independent samples). Significance was determined using a two-tailed unpaired t-test (*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, Gaussia luciferase; hFVII, human factor VII. [Figure 1J] Figure 1J shows images of cell lysates and media captured with IVIS. Huh7 cells in a 96-well plate were treated with Lipofectamine 2000 / pDNA (50 ng per well), and after 72 hours, mCherry signals were quantified with a plate reader or captured with IVIS. PBS, phosphate-buffered saline; NC, negative control; hAlb, human albumin; hApoB, human apolipoprotein B; gLuc, Gaussia luciferase; hFVII, human factor VII. [Figure 2A] Figure 2A shows a schematic diagram of the synthesis of hFVII-mCherry mRNA by in vitro transcription (IVT), in vivo delivery encapsulated by LNPs, and imaging using a microscope and plate reader. [Figure 2B] Figure 2B provides a graph showing time-dependent (100 ng of mRNA per well) and dose-dependent (72 hour time point) mCherry secretion in Huh7 cells, medium, and cell lysates. [Figure 2C] Figure 2C shows the quantification of mCherry signals in different cell lines. Cells in a 96-well plate were treated with mDLNPs-mRNA, and at predetermined time points, mCherry signals were quantified using a plate reader. [Figure 2D]Figure 2D shows fluorescent and bright-field images of HEK293T cells treated with the hFVII-mCherry mRNA formulation. A clear mCherry signal was observed in the medium 3 days after treatment with the hFVII-mCherry mRNA formulation. HEK293T cells in a 96-well plate were treated with different doses of mDLNPs-mRNA and photographed under a microscope (exposure time: 1 / 6 sec for 24 hours, 1 / 15 sec for 72 hours). A close-up reproduction of the image (exposure time: 1 / 6 sec) showing HEK293T cells treated with the mRNA formulation for 24 hours is shown. The reproduced image shows a clear "circular" distribution of mCherry (indicated by yellow arrows) in the hFVII-mCherry mRNA formulation group, which was similar to the pDNA delivery shown in Figure 1H. [Figure 2E] Figure 2E shows effective liver-targeted Luc mRNA delivery by mDLNPs. Mice were intravenously injected with Luc mRNA at a dose of 0.1 mg / kg and imaged 3 hours later. [Figure 2F] Figure 2F shows successful in vivo mCherry secretion via liver-targeted delivery via mDLNP / hFVII-mCherry. Mice were intravenously injected with mRNA at a dose of 0.5 mg / kg. Serum was collected 2 to 72 hours after treatment, and mCherry signals were detected using a plate reader. After 55 and 72 hours, the mice were sacrificed, and tissues were imaged using IVIS. [Figure 2G] Figure 2G shows mCherry signal in the kidney, indicating mCherry secretion in the blood. [Figure 2H] Figure 2H shows successful in vivo Luc mRNA and hFVII-mCherry delivery via SORT LNPs in the liver, lung, and spleen. SORT LNPs demonstrated tissue-selective Luc mRNA delivery, and all successfully achieved mCherry secretion into the blood. For the Luc assay, mice were intravenously injected with mRNA at a dose of 0.1 mg / kg and imaged 3 hours later. For the mCherry assay, mice were intravenously injected with mRNA at a dose of 0.5 mg / kg and imaged 24 hours later. [Figure 3A] FIG. 3A shows a schematic diagram of Enbrel protein production, TNF-α binding, and disease treatment in a dermatitis model. [Figure 3B] Figure 3B is a graph showing the dose-dependent cytotoxicity of human-derived TNF-α (hTNF-α) and mouse-derived TNF-α (mTNF-α) in L929 cells. Cells were incubated in 1 μg / ml actinomycin and various concentrations of TNF-α for 24 hours before determining cytotoxicity. [Figure 3C] Figure 3C is a graph showing cell viability in the presence of TNF-α. L929 cells pretreated with hFVII-Enbrel mRNA were resistant to both mouse and human TNF-α. Cells were pretreated with 80 ng of mDLNP-hFVII-Enbrel mRNA per well. Two days later, the cells were challenged with TNF-α at concentrations ranging from 0 to 5 ng / ml and a fixed concentration of 1 μg / ml actinomycin. Cell viability was measured after an additional 24 hours. [Figure 3D] Figure 3D shows a graph quantifying the dose-dependent viability rescue by LNPs after pretreatment. Cells were pretreated with mDLNP-hFVII-Enbrel mRNA at mRNA doses ranging from 0 to 1.25 ng / ml. Two days later, the cells were challenged with TNF-α at a dose of 0.1 ng / ml and a fixed concentration of 1 μg / ml actinomycin. After an additional 24 hours, cell viability was detected. [Figure 3E]Figure 3E is a graph showing dose-dependent rescue by LNP formulations. L929 cells were pretreated with mDLNP-hFVII-Enbrel mRNA at mRNA doses ranging from 0 to 1.25 ng / ml. Two days later, the cells were challenged with TNF-α at a dose of 0.02 ng / ml and a fixed concentration of 1 μg / ml actinomycin (top left). Cell viability was detected after an additional 24 hours. No significant cytotoxicity was observed with the mRNA formulation alone (top right). Dose-dependent viability rescue by medium from LNP formulation-treated cells. Cells were pretreated with mDLNP-hFVII-Enbrel mRNA at mRNA doses ranging from 0 to 1.25 ng / ml. Two days later, the medium containing secreted Enbrel was transferred to fresh L929 cells, and the cells were simultaneously challenged with TNF-α at a dose of 0.1 ng / ml and 1 μg / ml actinomycin (bottom left). Cell viability was detected after an additional 24 hours (bottom right). [Figure 3F] Figure 3F shows a scheme of the workflow for the generation and treatment of the imiquimod-induced psoriasis model. [Figure 3G] Figure 3G shows the pharmacokinetic comparison between Enbrel protein and mRNA after a single dose. Mice were intravenously injected with a 0.5 mg / kg dose of either protein or mRNA formulation. Serum samples were collected at different time points, and Enbrel was detected using an ELISA kit. [Figure 3H] Figure 3H provides images of the dorsal skin of lanolin control or imiquimod-treated mice injected with mCherry mRNA or hFVII-Enbrel mRNA formulations. Images of H&E-stained sections, Ki-67 staining, and Gr-1 staining of the dorsal skin of lanolin control or imiquimod-treated mice. [Figure 3I] Figure 3I provides quantitative measurements of epidermal thickness in the three groups and the percentage of Ki-67+ cells (per 50 cells) among epidermal basal cells quantified from h (****P<0.0001). [Figure 4A] Figure 4A shows the workflow of tumor immunotherapy using hFVII-anti-PDL1 mRNA. The scheme of anti-PDL1 antibody production and tumor immunotherapy is shown. [Figure 4B] Figure 4B shows the scheme of the experimental design of tumor immunotherapy with hFVII-anti-PDL1 mRNA using MC38, MC38-Luc, and B16F10-Luc xenograft mouse models. [Figure 4C] Figure 4C shows the pharmacokinetics of anti-PDL1 antibodies after a single administration of the LNP-mRNA formulation. Mice were intravenously injected with a 0.5 mg / kg dose of the mRNA formulation. Serum samples were collected at different time points, and anti-PDL1 antibodies were detected using an ELISA kit. [Figure 4D] FIG. 4D shows PDL1 expression on the membrane surface of MC-38 cells as determined by flow cytometry. [Figure 4E] FIG. 4E shows luminescence images of MC38-Luc tumors captured by IVIS at different time points. [Figure 4F] Figure 4F shows a luminescence image of an isolated MC38-Luc tumor at day 32. A tumor image at day 32 is also provided (1 / 4 disappeared). (*P<0.05) [Figure 4G] Figure 4G shows the quantified luminescence signals in the tumor area at different time points after tumor immunotherapy with hFVII-anti-PDL1 mRNA (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). [Figure 4H] Figure 4H shows tumor growth in the MC38 model. (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001) [Figure 4I] Figure 4I shows mouse survival data during treatment with mRNA formulations. (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001) [Figure 4J] FIG. 4J shows luminescence images of B16F10-Luc tumors at days 3 and 16 treated with different formulations. [Figure 4K] FIG. 4K shows representative PDL1 expression on the membrane surface of B16F10-Luc cells as determined by flow cytometry. [Figure 4L]Figure 4L is a graph showing tumor growth in the B16F10-Luc model. (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001) [Figure 4M] Figure 4M shows the survival time of mice treated with mRNA formulations. (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001) [Figure 4N] Figure 4N shows that the mRNA formulation provided a better pharmacokinetic curve than the protein. Pharmacokinetic comparison between PD1 protein and mRNA formulations after a single dose. Mice were intravenously injected with either the mRNA formulation or the protein at a dose of 0.5 mg / kg. Serum was collected at different time points, and PD1 was detected using an ELISA kit. [Figure 5] FIG. 5 shows a schematic diagram of matrix generation for constructing novel signal peptide sequences. [Figure 6] FIG. 6 is a summary of the signal peptide structure along with the mRNA sequence and the generation of SP-mRNA using in-vitro transcription. [Figure 7] Figure 7 shows serum hEPO concentrations after injection. SP(1-21)-hEPO mRNA was encapsulated in iPhos LNPs and administered intravenously to mice at a dose of 0.5 mg / kg. Blood was collected from mice at 6, 24, 48, and 72 hours after injection for each construct and analyzed by hEPO enzyme-linked immunosorbent assay (ELISA) to determine serum hEPO concentrations in mIU / mL. DETAILED DESCRIPTION OF THE INVENTION

[0026] The drawings are not intended to limit the inventive concepts to the specific embodiments disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of certain embodiments of the inventive concepts.

[0027] [Detailed explanation] The following detailed description refers to the accompanying drawings that illustrate various aspects of the concepts of the present invention. The drawings and the description are intended to describe aspects of the concepts of the present invention in sufficient detail to enable those skilled in the art to implement the concepts of the present invention. Other components can be utilized and modifications can be made without departing from the scope of the concepts of the present invention. Accordingly, the following description should not be construed in a limiting sense. The scope of the concepts of the present invention is defined only by the appended claims and the full scope of equivalents to which such claims are entitled.

[0028] This disclosure is the result of intensive investigations by the inventors to identify and / or develop novel secretory signal peptides for therapeutic and non-therapeutic applications. Although many studies have been conducted on maximizing the expression of therapeutic proteins after nucleic acid delivery into cells, post-translational control of protein localization through elucidation and utilization of the trafficking pathways of endogenous proteins has been largely overlooked. Delivery of therapeutically important polypeptides encoded by nucleic acids into cells, the body, or specific organs can be highly beneficial for the field of nucleic acid pharmaceuticals. In this investigation, using sophisticated experimental and bioinformatics approaches, naturally occurring secretory signal peptides that can control the systemic and organ-specific secretion of polypeptides were identified and isolated, or synthetic secretory signal peptides were developed. Subsequently, extensive experimental validation and development of therapeutic and non-therapeutic compositions using these signal peptide sequences were conducted. These naturally occurring signal peptides and synthetic signal peptides can be fused to any heterologous protein of interest to control its transport and localization. This research provides an entry point for the development of novel protein therapeutics that are likely to impact patient outcomes for multiple different disease states. In addition, this research can also be used for the development of non-therapeutic applications such as diagnostic agents, industrial production of heterologous proteins, and various laboratory purposes.

[0029] <I. Terms> The phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. For example, the use of singular terms such as "a" or "an" is not intended as a limitation on the number of items. Additionally, the use of relational terms such as, but not limited to, "top," "bottom," "left," "right," "upper," "lower," "downward," "upward," and "side" are used in the description for clarity with particular reference to the drawings and are not intended to limit the concepts of the invention or the scope of the appended claims.

[0030] Furthermore, because the inventive concept is susceptible to embodiment in many different forms, the present disclosure should be considered as an example of the principles of the inventive concept and is not intended to limit the inventive concept to the specific embodiments shown and described. Any feature of the inventive concept may be used separately or in combination with any other feature. Reference to "an embodiment," "embodiments," and / or similar terms in the description means that the referenced feature and / or features are included in at least one embodiment of the description. Separate references to "an embodiment," "embodiments," and / or similar terms in the description do not necessarily refer to the same embodiment, nor are they mutually exclusive, unless so stated and / or readily apparent to one of ordinary skill in the art from the description. For example, features, structures, processes, steps, actions, etc. described in one embodiment may, but are not necessarily, included in other embodiments. Thus, the inventive concept may include various combinations and / or integrations of the embodiments described herein. Additionally, not all aspects of the present disclosure described herein are required for its practice. Similarly, other systems, methods, features, and advantages of the inventive concepts will be or become apparent to one with skill in the art upon examination of the figures and description, and it is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the inventive concepts, and be covered by the claims.

[0031] As used in the description and the appended claims, terms of degree, such as, but not limited to, "substantially," should be understood to include exact or similar but not exact configurations. For example, a "substantially flat surface" means having an exactly flat surface or a similar but not exactly flat surface. Similarly, as used in the description and the appended claims, the terms "about" or "approximately" should be understood to include the stated value, or values ​​three times greater or one-third of the stated value. For example, about 3 mm includes all values ​​from 1 mm to 9 mm, and approximately 50 degrees includes all values ​​from 16.6 degrees to 150 degrees. For example, they may refer to ±5% or less, e.g., ±2% or less, e.g., ±1% or less, e.g., ±0.5% or less, e.g., ±0.2% or less, e.g., ±0.1% or less, e.g., ±0.05% or less.

[0032] In this disclosure, the terms "comprising," "including," and "having" are used interchangeably. The terms "comprising," "including," and "having" mean including, but not necessarily limited to, what is stated.

[0033] Finally, the terms "or" and "and / or" as used herein should be construed as being inclusive or meaning either or any combination. Thus, "A, B or C" or "A, B and / or C" means any of "A," "B," or "C"; "A and B"; "A and C"; "B and C"; "A, B and C." Exceptions to this definition occur only when combinations of elements, features, steps, or actions are inherently mutually exclusive in some way.

[0034] The terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably herein. The term "nucleic acid encoding" or "nucleic acid molecule encoding" should be understood to refer to a sequence of nucleotides that encodes a polypeptide.

[0035] The polynucleotides described herein may comprise one or more nucleic acids, each encoding a polypeptide, which may be operably linked (i.e., in a functional relationship) to one or more regulatory sequences, such as a promoter. Such polynucleotides may alternatively be referred to herein as "nucleic acid constructs" or "constructs." As used herein, the term "operably linked" refers to the functional linkage between a promoter or other regulatory element and an associated transcribable DNA sequence or coding sequence of a gene (or transgene), such that the promoter or the like acts to initiate, assist, influence, induce, and / or promote the transcription and expression of the associated transcribable DNA sequence or coding sequence, at least in particular tissues, developmental stages, and / or conditions.

[0036] As used herein, the polynucleotide sequence "corresponding" to the provided sequence encompasses the DNA, RNA and cDNA sequences related to that sequence.For example, with respect to the provided sequence encoding a heterologous polypeptide, this term includes both the DNA sequence and the RNA sequence encoding the heterologous polypeptide.Therefore, the polynucleotide can be identical to the reference sequence or its complementary RNA sequence.

[0037] As used herein, "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., that are appropriate, necessary, or preferable for regulating or enabling the expression of a gene or transcribable DNA sequence in a cell. Such additional regulatory elements are optional and can be used to enhance or optimize the expression of a gene or transcribable DNA sequence. Regulatory sequences can be, for example, inducible, non-inducible, constitutive, cell cycle-regulated, metabolically-regulated, etc. A regulatory sequence may be a promoter. As used herein, the term "promoter" refers to a DNA sequence that comprises an RNA polymerase binding site, a transcription initiation site, and / or a TATA box, and that assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and / or gene (or transgene). Promoters can be synthetically produced, modified, or derived from known or naturally occurring promoter sequences or other promoter sequences. Promoters can also include chimeric promoters, which contain a combination of two or more heterologous sequences. Thus, promoters of the present application can include variants of promoter sequences that are similar in composition but not identical to other promoter sequences known or provided herein. As used herein, the term "enhancer" refers to a region of a DNA sequence that acts to initiate, assist, influence, induce, and / or promote the transcription and expression of an associated transcribable DNA sequence or coding sequence, at least under specific tissues, developmental stages, and / or conditions. In one embodiment, an enhancer is a cis enhancer. In one embodiment, an enhancer is a trans enhancer.

[0038] As used herein, the term "operably linked" refers to a functional linkage between a promoter or other regulatory element and an associated transcribable DNA sequence or coding sequence of a gene (or transgene), such that the promoter etc. acts to initiate, assist, influence, induce, and / or promote the transcription and expression of the associated transcribable DNA sequence or coding sequence, at least in particular tissues, developmental stages, and / or conditions.

[0039] The terms "polypeptide" and "protein," used interchangeably herein, refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers modified 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. Also included within the definition are polypeptides containing, for example, one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. As used herein, the terms "polypeptide" and "protein" specifically encompass antibodies.

[0040] An amino acid sequence "derived from" an amino acid sequence disclosed herein can refer to an amino acid sequence that differs by one or more amino acids compared to a reference amino acid sequence, e.g., an amino acid sequence that includes an insertion, deletion, or substitution of one or more amino acids as disclosed herein. The terms "derivative," "variant," and "fragment," as used herein with respect to a polypeptide, refer to a polypeptide related to the wild-type polypeptide, e.g., a polypeptide related by any of amino acid sequence, structure (e.g., secondary and / or tertiary structure), activity (e.g., enzymatic activity), and / or function. Derivatives, variants, and fragments of a polypeptide can include one or more amino acid changes (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof, compared to the wild-type polypeptide. 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, such as a polypeptide having an amino acid sequence identified by a particular SEQ ID NO, or a polypeptide having at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the length (number of amino acids) of that polypeptide.

[0041] In the context of this application, proteins are represented by amino acid sequences, and correspondingly, nucleic acid molecules or polynucleotides are represented by nucleic acid sequences. Identity and similarity between sequences: Throughout this application, whenever a specific amino acid sequence SEQ ID NO:Y is mentioned, it may be replaced by: a polypeptide represented by the amino acid sequence comprising a sequence having at least 60% sequence identity or similarity with the amino acid sequence of 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%.

[0042] Each amino acid sequence described herein by a percentage of identity or similarity to a given amino acid sequence, respectively, is in further preferred embodiments 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%, or at least 76% identity or similarity to the given nucleotide or amino acid sequence, respectively. , 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. The terms "homology," "sequence identity," and the like are used interchangeably herein. Sequence identity is described herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred embodiment, sequence identity is calculated based on the entire length of two given SEQ ID NOs, or based on a portion thereof. A portion thereof preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NOs. In the art, "identity" also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between the strings of such sequences. The degree of sequence identity between two sequences can be determined, for example, by comparing the two sequences using a computer program commonly used for this purpose, such as a global or local alignment algorithm.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 two sequences over their entire length or over a portion thereof (where a portion may mean at least 50%, 60%, 70%, 80%, 90% of the length of the sequences), maximizing the number of matches and minimizing the number of gaps. Default settings can be used, with preferred programs being Needle for pairwise alignments (in one embodiment EMBOSS Needle 6.6.0.0, gap opening penalty 10, gap extension penalty: 0.5, end gap penalty: false, end gap opening penalty: 10, end gap extension penalty: 0.5 are used) and MAFFT for multiple sequence alignments (in one embodiment MAFFT v7 default values ​​are used: BLOSUM62 [bl62], gap opening: 1.53, gap extension: 0.123, order: aligned, number of tree rebuilds: 2, guide tree output: ON [true], maximum iterations: 2, run FFTS: none).

[0043] The "similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutions with the sequence of a second polypeptide. The same algorithm used to determine sequence identity can be used to determine sequence similarity. Optionally, when determining the degree of amino acid similarity, those skilled in the art may also take into account so-called conservative amino acid substitutions. As used herein, "conservative" amino acid substitution refers to the interchangeability of residues with similar side chains.

[0044] For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitution variants of the amino acid sequences disclosed herein are those in which at least one residue in the disclosed sequence is removed and a different residue is inserted in its place. Preferably, the amino acid changes are conservative. Preferred conservative substitutions for each natural 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 or 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.

[0045] The term "heterologous," when used with reference to a nucleic acid molecule (e.g., a coding sequence) or a polypeptide (e.g., an enzyme), refers to a nucleic acid molecule or protein that is not naturally found in the host organism or cell. "Heterologous" also includes a native coding region or portion thereof that has been removed from a source organism and then reintroduced into the source organism in a form that differs from the corresponding native gene, e.g., not in its natural location in the organism's genome. A heterologous nucleic acid molecule is intentionally introduced into a host cell. A "heterologous" nucleic acid molecule or protein can be derived from any source, e.g., a eukaryote, a prokaryote, a virus, etc. In one aspect, a heterologous nucleic acid molecule can be derived from a eukaryote (e.g., another yeast, etc.) or a prokaryote (e.g., a bacterium, etc.). As used herein, the term "heterologous" also refers to an element (nucleic acid or protein) derived from a source other than the endogenous source. Thus, for example, a heterologous element can be derived from a different strain of the host cell, or from an organism of a different taxonomic group (e.g., a different kingdom, phylum, class, order, family, genus, or species, or any subgroup within these taxa). The term "heterologous" is also used interchangeably with the term "exogenous" herein.

[0046] As used herein, an embodiment of the invention is "engineered" if it has characteristics or properties, whether structural or chemical, that differ from the starting, wild-type, or naturally occurring molecule. As used herein with respect to signal peptides, the terms "engineered" or "synthetic" refer to having an amino acid sequence that has one or more amino acids in the sequence that differ from a naturally occurring signal peptide. In some embodiments, these signal peptides are designed using bioinformatics analysis of naturally occurring signal peptides. In some embodiments, the signal peptide is derived from, but may not be identical to, a naturally occurring signal peptide. As used herein with respect to a polypeptide, the terms "engineered" or "recombinant" refer to having an altered amino acid sequence as a result of the application of genetic engineering techniques to a nucleic acid encoding the polypeptide and to a cell or organism that expresses the polypeptide. Alterations include, but are not limited to, insertions, deletions, or substitutions. With respect to nucleic acids, the terms "recombinant" or "engineered" refer to having an altered nucleic acid sequence as a result of the application of genetic engineering techniques. 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.

[0047] The terms "fusion protein" or "fusion polypeptide" are used interchangeably and refer to a polypeptide encoded by a nucleic acid sequence that includes a coding sequence from one nucleic acid molecule and a coding sequence from another nucleic acid molecule, where 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 containing the two proteins is produced. The two molecules can be adjacent in the construct or separated by a linker polypeptide containing one, two, three, or more amino acids. The protein product encoded by a fusion construct is referred to as a fusion polypeptide.

[0048] The terms "host cell," "host cell line," "host cell culture," "genetically modified," or "genetically engineered host" cells, used interchangeably herein, refer to cells into which exogenous nucleic acid has been introduced and include the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. In some embodiments, the host cell is a genetically engineered cell. As defined herein, in such cells, the nucleus, organelles, or extrachromosomal nucleic acid may be transformed, modified, or transduced using recombinant DNA technology to contain a heterologous nucleic acid molecule, and the terms "genetically engineered cell," "transformed cell," and "transduced cell" are used interchangeably. Genetically engineered cells may also be "transduced cells," in which the cells are infected, for example, with a modified virus; for example, a retrovirus may be used, although other suitable viruses, such as lentiviruses, are also contemplated. Non-viral methods, such as transfection, can also be used. Thus, genetically engineered cells can also be "stably transfected cells" or "transiently transfected cells." Transfection refers to a non-viral method for transferring DNA (or RNA) into cells so that the gene is expressed. Transfection methods are widely known in the art, such as calcium phosphate transfection, PEG transfection, and liposome or lipoplex transfection of nucleic acids. Such transfection is transient and Although it may be possible to obtain a stable transfection, it may also be possible to select cells that have integrated the gene construct into their genome. In some cases, genetic engineering systems such as CRISPR or Argonaute may be used to design genetically engineered cells that express the polypeptides described herein.

[0049] Various enzymes can catalyze the insertion of foreign DNA into a host genome. Non-limiting examples of gene editing tools and technologies include CRISPR, TALEN, zinc finger nucleases (ZFNs), meganucleases, Mega-TALs, and transposon-based systems. CRISPR systems can be used to facilitate the insertion of polynucleotide sequences encoding membrane proteins or their components into a cellular genome. For example, CRISPR systems can introduce double-strand breaks at target sites within the genome. There are at least five types of CRISPR systems, all of which incorporate RNA and CRISPR-associated proteins (Cas). Types I, III, and IV assemble a multi-Cas protein complex that can cleave nucleic acids complementary to the crRNA. Both Types I and III require pre-crRNA processing before the processed crRNA can assemble into a multi-Cas protein complex. Types II and V CRISPR systems contain a single Cas protein complexed with at least one guide RNA.

[0050] The term "effective," as used herein and / or in the claims, 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" means the amount of a compound that, when administered to a patient or subject for treating a disease, is sufficient to effect such treatment for the disease.

[0051] As used herein, the term "patient" or "subject" refers to a living mammal, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human subjects are adults, juveniles, infants, and fetuses.

[0052] As generally used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues, organs and / or body fluids within the scope of sound medical judgment, without undue toxicity, irritation, allergic reaction, or other problems or complications, and in a reasonable benefit / risk ratio.

[0053] "Prevention" or "preventing" includes the following: (1) inhibiting the onset of a disease in a subject or patient who has a risk of and / or is potentially predisposed to the disease, but has not yet experienced or exhibited any or all of the pathological conditions or symptoms of the disease, and / or (2) delaying the onset of the pathological conditions or symptoms of a disease in a subject or patient who has a risk of and / or is potentially predisposed to the disease, but has not yet experienced or exhibited any or all of the pathological conditions or symptoms of the disease.

[0054] "Treatment" refers to both therapeutic treatment and prophylactic or preventive treatment, the purpose of which is to prevent or delay (alleviate) the targeted pathological condition or disorder. Those who require treatment include those who already have a disorder, as well as those who tend to have a disorder or those in whom the disorder should be prevented.

[0055] A "therapeutic polypeptide" is a polypeptide that can alleviate or reduce the symptoms resulting from the lack or deficiency of a protein in a cell or subject. Alternatively, a "therapeutic polypeptide" provides other benefits to the subject, such as, for example, an anti-cancer effect or an improvement in transplant survival rate. As used herein, the term "therapeutic polypeptide" also encompasses proteins useful as vaccines, therapeutic agents, and diagnostic agents.

[0056] <II. Compositions> In some embodiments, the present disclosure encompasses compositions and methods comprising novel naturally occurring and synthetic or engineered signal peptides, and their use for controlled protein transport and localization. In some embodiments, the present disclosure also encompasses the use of novel delivery formulations and methods for introducing the disclosed compositions into cells or subjects in need thereof. These compositions can be used in both therapeutic (treatment and diagnostic) and non-therapeutic applications, such as industrial and laboratory applications, to drive extracellular secretion and distribution of target proteins.

[0057] In some embodiments, the present disclosure encompasses engineered signal peptides. These signal peptides were developed using extensive bioinformatics analysis followed by cell culture and in vivo validation studies. In some embodiments, the signal peptide comprises an amino acid sequence at least about 60% identical to one or more of SEQ ID NOs: 9-29. In some embodiments, the signal peptide can be 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 to any of SEQ ID NOs: 9-30. In some embodiments, 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 some embodiments, the engineered signal peptide can vary in length from 5 amino acid residues to about 100 amino acid residues. In some embodiments, the signal peptide can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 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, or 75 amino acids in length. In some exemplary embodiments, the signal peptide is about 10-50 residues in length. In some embodiments, the engineered signal peptide may comprise a stretch of leucine amino acid residues ranging in number from 1 to 50. In some embodiments, the stretch of leucine amino acid residues may be divided into sets of one, two, or more residues separated by one, two, or more non-leucine amino acid residues.

[0058] In some embodiments, the signal peptide is derived from a naturally occurring signal sequence, such as the signal peptide from any of the following: albumin, hAlb; apolipoprotein B, hApoB; Gaussia luciferase, gLuc; or Factor VII, hFVII sequences. In some embodiments, the signal peptide comprises an amino acid sequence at least about 60% identical to one or more of SEQ ID NOs: 1-4. In some embodiments, the signal peptide can be 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 to any of SEQ ID NOs: 1-4. In some embodiments, the signal peptide may 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: 1-4.

[0059] In some embodiments, the present disclosure also encompasses recombinant polypeptides comprising one or more of the signal peptides provided herein and a heterologous polypeptide. The two molecules can be adjacent within the construct or separated by a linker polypeptide comprising one, two, three, or more amino acids. Linker molecules are described, for example, in Huston, J.S., et al., PNAS 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 embodiments, the recombinant polypeptide can further comprise one or more of a linker sequence, a degron, a degradation tag, a protease cleavage site, and / or a purification tag. In some embodiments, the heterologous polypeptide can be any polypeptide of interest. In some embodiments, the heterologous polypeptide can comprise an amino acid sequence identical to a naturally occurring protein or a variant, derivative, or fragment thereof. In some embodiments, a heterologous polypeptide may comprise a synthetic amino acid sequence. In some embodiments, a heterologous polypeptide may comprise an amino acid sequence that is identical to, or a variant, derivative, or fragment of, a prokaryotic protein. In some embodiments, a heterologous polypeptide may comprise an amino acid sequence that is identical to, or a variant, derivative, or fragment of, a eukaryotic protein. In some embodiments, a heterologous polypeptide is a variant, derivative, or fragment of a non-human mammalian protein (e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimpanzee, ape, baboon, cow, pig, horse, sheep, cat, and other species). In some embodiments, a heterologous polypeptide is a variant, derivative, or fragment of a human protein.

[0060] In some embodiments, the heterologous polypeptide is a therapeutic polypeptide. In some embodiments, the heterologous polypeptide is a biologically active polypeptide. In some exemplary embodiments, the heterologous polypeptide has one or more of anti-cancer, anti-inflammatory, immunomodulatory, antiviral, antibacterial, antifungal, antihelminthic, cholesterol-lowering, antidiabetic, antifibrotic, analgesic, anesthetic, anti-aging, antidepressant, neuromodulatory, antidermatitis, anti-edema, antiallergic, antikeratolytic, antifungal, antipruritic, cardiovascular, chemotherapeutic, and / or hormonal activities.

[0061] In some embodiments, the heterologous polypeptide is a protein replacement therapy, polypeptide therapy, vaccine, viral vaccine, secreted therapy, anti-cancer, anti-inflammatory, anti-viral, anti-bacterial, cholesterol-lowering, anti-diabetic, or anti-fibrotic polypeptide.

[0062] In some embodiments, 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 mucin-containing protein 3 (TIM-3), B- and T-lymphocyte attenuator (BTLA), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), V-domain Ig suppressor of T-cell activation (VISTA), adenosine A2a receptor (A2aR), killer cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), CD20, CD39, and CD73.

[0063] In some embodiments, 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 cell death ligand 1 (PD-L1), programmed cell 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.

[0064] In some embodiments, the heterologous polypeptide is an antibody.

[0065] In some embodiments, the heterologous polypeptide is a chimeric antigen receptor (CAR).

[0066] In some embodiments, the heterologous polypeptide is active as a vaccine.

[0067] In some embodiments, the heterologous polypeptide is a diagnostic polypeptide. In some embodiments, the heterologous polypeptide is theragnostic. In some embodiments, the heterologous polypeptide is an antibody-based diagnostic agent. Generally, the heterologous polypeptide is labeled with a radionuclide (e.g., In, Tc, C, I, H, P, or S) and specifically binds to tumor antigens, allowing tumor localization using immunoscintigraphy. In one embodiment, the heterologous polypeptide or a fragment thereof binds to the extracellular domain of a specific cancer biomarker. Diagnostic heterologous polypeptides can be labeled with probes suitable for detection by various imaging methods. Probe detection methods include, but are not limited to, fluorescence, optical, 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 lanthanides, paramagnetic iron, fluorine-18 and other positron-emitting radionuclides. In addition, probes may be bifunctional or multifunctional and may be detectable by one or more of the methods listed.

[0068] In some embodiments, the heterologous polypeptide is not a therapeutic polypeptide. In some embodiments, the heterologous polypeptide is a reporter polypeptide, for example, 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), or luciferase. In some embodiments, the heterologous polypeptide is fused to one or more detectable probes for in vitro or ex vivo use.

[0069] In some embodiments, the heterologous polypeptide has industrial or commercial applications. In some embodiments, the polypeptide may be, or may be useful in, for example, an enzyme, a nutraceutical, a food additive, a flavor enhancer, and / or a cosmetic. In some embodiments, the secretion of the heterologous polypeptides disclosed herein may provide benefits in the production, isolation, or use of commercially relevant polypeptides.

[0070] In some exemplary embodiments, the heterologous polypeptide comprises a sequence at least about 60% identical to SEQ ID NOs: 55-54 or variants, derivatives, or fragments thereof. In some exemplary embodiments, the heterologous polypeptide is 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% identical to one or more of SEQ ID NOs: 55-58 or functional fragments thereof.

[0071] In some embodiments, the disclosure encompasses a recombinant polypeptide comprising a signal peptide sequence that is at least 60% identical to one or more of SEQ ID NOs: 1-4 or 9-29, and a heterologous polypeptide. In some embodiments, the signal peptide sequence comprises a 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 to any of SEQ ID NOs: 1-4, 9-29, and the heterologous polypeptide. In some embodiments, the recombinant polypeptide comprises a signal peptide sequence that can be 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% identical to one or more of SEQ ID NOs: 1-4 or 9-29, and a heterologous polypeptide. In some exemplary embodiments, the recombinant polypeptide comprises a signal peptide sequence that is at least 60% identical to SEQ ID NOs: 1-4 or 9-29, and a heterologous polypeptide sequence that is at least 60% identical to SEQ ID NOs: 55-58, or a functional fragment thereof.

[0072] In some embodiments, the present disclosure also encompasses polynucleotides comprising nucleic acid sequences encoding signal peptides disclosed herein. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding an amino acid sequence at least about 60% identical to any of SEQ ID NOS: 1-4 or 9-29. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding an amino acid sequence 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% identical to one or more of SEQ ID NOS: 1-4 or 9-29. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a heterologous polypeptide as provided herein. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a heterologous polypeptide that is at least about 60% identical to SEQ ID NOs:55-58.

[0073] In some embodiments, the polynucleotide sequence may comprise a nucleic acid sequence encoding a signal peptide having a nucleic acid sequence that is at least about 60% identical to, or complementary to, SEQ ID NOs: 5-8 or 30-50. In some embodiments, 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 to, or complementary to, any of SEQ ID NOs: 5-8 or 30-50. In some embodiments, the polynucleotide sequence encoding the signal peptide may comprise a nucleic acid sequence that is 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% identical to or corresponds to one or more of SEQ ID NOs: 5-8 or 30-50. In some embodiments, the polynucleotide may be a deoxyribonucleotide sequence (DNA). In some embodiments, the polynucleotide may be a ribonucleic acid sequence (RNA).

[0074] In some embodiments, the polynucleotide sequence may further comprise a nucleic acid sequence corresponding to a sequence encoding a heterologous polypeptide in frame with a nucleic acid sequence encoding a signal peptide. In some embodiments, the polynucleotide sequence may further comprise a nucleic acid sequence corresponding to a sequence at least about 60% identical to SEQ ID NOs: 51-54. In some embodiments, the polynucleotide may be a deoxyribonucleotide sequence (DNA). In some embodiments, the polynucleotide may be a ribonucleic acid sequence (RNA).

[0075] Nucleic acids encoding heterologous polypeptides are contemplated to include sequences based on naturally occurring sequences. Taking into account the degeneracy of the genetic code, sequences having at least about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 100% identical nucleotides to the nucleic acid sequence of a naturally occurring sequence. In another embodiment, the nucleic acid is a sequence complementary to a naturally occurring sequence, or a sequence that is 75%, 80%, 85%, 90%, 95%, or 100% complementary. Polynucleotides encoding 50, 100, 250, 500, 1000, 1500, 2000, 2500, 3000, or more, and intermediate sizes are contemplated herein. In some embodiments, nucleic acids encoding heterologous polypeptides can be derived from genomic DNA cloned directly from the genome of a specific organism. However, in some embodiments, the nucleic acid will comprise complementary DNA (cDNA). In some embodiments, the nucleic acid will comprise an mRNA encoding a signal peptide in frame with the heterologous polypeptide.

[0076] In some embodiments, the polynucleotides disclosed herein may further comprise one or more of the following, including but not limited to, promoters, enhancers, leaders, transcription start sites (TSSs), linkers, 5' and 3' untranslated regions (UTRs), Kozak sequences, introns, polyadenylation signals, capping sequences, enhancers, viral sequences, IRES sequences, and termination regions or sequences, which are suitable, necessary, or preferred for regulating or enabling the expression of heterologous proteins in cells, and may include one or more regions or portions that act or function as untranslated regions. When a polynucleotide is designed to encode at least one polypeptide of interest, the polynucleotide may comprise one or more of these untranslated regions. By definition, the wild-type untranslated region (UTR) of a gene is transcribed but not translated. In mRNA, the 5'UTR begins at the transcription start site and continues up to, but not including, the start codon; while the 3'UTR begins immediately after the stop codon and continues until the transcription termination signal. For example, regulatory features of UTRs can be incorporated into the polynucleotides of the present invention to increase the stability of the molecule. Specific features can also be incorporated to ensure controlled downregulation of transcripts if they are misdirected to undesirable organ sites. In some embodiments, 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 subregions within the polynucleotide. For example, introns or portions of intron sequences can be incorporated into regions of the polynucleotides of the present invention. The incorporation of intron sequences can increase protein production and polynucleotide levels. A combination of features can be included in the flanking regions, or within other features. For example, an ORF can be flanked by a 5' UTR, which can contain a strong Kozak translation initiation signal, and / or a 3' UTR, which can contain an oligo(dT) sequence for templated addition of a polyA tail. The 5' UTR can contain a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different genes.

[0077] In some embodiments, the 5'UTR sequence can be SEQ ID NO: 59. In some embodiments, the 3'UTR sequence can be SEQ ID NO: 60.

[0078] In some embodiments, the polynucleotides disclosed herein can be assembled in cells. In some embodiments, the polynucleotides can be synthesized in vivo. In some embodiments, the polynucleotides can be synthesized in vitro using methods known in the art, such as in vitro transcription, DNA, RNA, and cDNA synthesis. In some embodiments, the polynucleotides disclosed herein can be incorporated into suitable viral vectors, expression cassettes, expression vectors, transposons, extrachromosomal elements, chromosomally integrated, or incorporated into host cells or delivery systems.

[0079] In some embodiments, the polynucleotide is a chemically modified polynucleotide. In some embodiments, the polynucleotide may contain one or more modified nucleosides containing modified sugar moieties. Such compounds containing one or more sugar-modified nucleosides may have desirable properties, such as improved nuclease stability, compared to oligonucleotides containing only nucleosides containing naturally occurring sugar moieties. In some embodiments, the modified sugar moiety is a substituted sugar moiety. In some embodiments, the modified sugar moiety is a sugar surrogate. Such sugar surrogates may contain one or more substitutions corresponding to those of the substituted sugar moiety. In some embodiments, the modified polynucleotide may contain a modified backbone, such as, for example, a phosphorothioate, a phosphotriester, a morpholino, a methylphosphonate, a short alkyl or cycloalkyl intersugar linkage, or a short heteroatom or heterocyclic intersugar linkage.

[0080] In some embodiments, the modified sugar moiety is a substituted sugar moiety containing one or more non-bridging sugar substituents, including, but not limited to, substituents at the 2' and / or 5' positions. Examples of suitable sugar substituents at the 2' position include, but are not limited to, 2'-F, 2'-OCH ("OMe" or "O-methyl"), and 2'-O(CH)OCH ("MOE"). In certain embodiments, 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, O(CH)SCH, O(CH)-ON(Rm)(Rn), and O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn is independently H or a 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 embodiments, the substituted sugar comprises multiple non-bridging sugar substituents, such as a TF-5'-methyl sugar moiety (see, e.g., PCT International Application WO2008 / 101157 for additional 5',2'-disubstituted sugar moieties and nucleosides).

[0081] Nucleosides containing a 2'-substituted sugar moiety are referred to as 2'-substituted nucleosides. In some embodiments, the 2'-substituted nucleoside contains a 2'-substituent selected from halo, allyl, amino, azido, SH, CN, OCN, CF, OCF, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S, or N(Rm)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2-ON(Rm)(Rn), or O-CH2-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 with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

[0082] In some embodiments, the 2' substituted nucleoside comprises a 2' substituent selected from F, NH, N, OCF, O-CH, O(CH)NH, CH-CH=CH, O-CH-CH=CH, OCHCHOCH, O(CH)SCH, O-(CH)-ON(R)(R), O(CH)O(CH)N(CH), and N-substituted acetamide (O-CH-C(=O)-N(R)(R), where each R and R is independently H, an amino protecting group, or a substituted or unsubstituted acetamide. or unsubstituted C1-C10 alkyl. In some embodiments, 2'-substituted nucleosides comprise a sugar moiety comprising a 2'-substituent selected from F, OCF3, O-CH3, O2CHOCH3, O(CH2)2SCH3, O(CH2)2-ON(CH3)2, -O(CH2)2O(CH2)2N(CH3)2, and O-CH2-C(=O)-N(H)CH3. In some embodiments, 2'-substituted nucleosides comprise a sugar moiety comprising a 2'-substituent selected from F, O-CH3, and OCH2CHOCH3.

[0083] Certain modified sugar moieties include a bridging sugar substituent that forms a second ring, resulting in a bicyclic sugar moiety. In some such embodiments, the bicyclic sugar moiety includes a bridge between the 4' and 2' furanose ring atoms. Examples of such 4' to 2' sugar substituents include -[C(Ra)(Rb)]-, -[C(Ra)(Rb)]nO-, -C(RaRb)-N(R)-O-, or -C(RaRb)-ON(R)-; 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (cEt) and 4'-CH(CHOCH3)-O-2', and analogs thereof (see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH3)(CH3)-O-2' and analogs thereof (see, e.g., WO 2009 / 0064 78); 4'-CH2-N(OCH3)-2' and analogs thereof (see, e.g., WO2008 / 150729); 4'-CH2-ON(CH3)-2' (see, e.g., US2004 / 0171570 published September 2, 2004); 4'-CH2-ON(R)-2', and 4'-CH2-N(R)-O-2'-, where each R is independently H, a protecting group, or a C1-C12 alkyl; 4'-CH2-N(R)-O-2', where R is H, a C1-C12 alkyl, or a protecting group (see U.S. Pat. No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' and analogs thereof (see PCT International Application WO2008 / 154401).

[0084] In some embodiments, such 4' to 2' bridges independently comprise 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; and each Ra and Rb is independently H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocyclic radical, substituted heterocyclic radical. , heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); and each J1 and J2 is independently H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.

[0085] Nucleosides containing a bicyclic sugar moiety are called bicyclic nucleosides or BNAs. Bicyclic nucleosides include: (A) α-L-methyleneoxy (4'-CH2-O-2') BNAs, (B) β-D-methyleneoxy (4'-CH2-O-2') BNAs (also called locked nucleic acids or LNAs), (C) ethyleneoxy (4'-(CH2)2-O-2') BNAs, (D) aminooxy (4'-CH2-ON(R)-2') BNAs, (E) oxyamino (4'-CH2-N(R)-O-2') BNAs, and (F) methyl(methyleneoxy) (4'-CH(CH3)-O-2') BNAs (constrained ethyleneoxy) BNAs. (G) methylenethio (4'-CH2-S-2') BNA, (H) methyleneamino (4'-CH2-N(R)-2') BNA, (I) methylcarbocyclic (4'-CH2-CH(CH3)-2') BNA, (J) propylenecarbocyclic (4'-(CH2)3-2') BNA, and (K) methoxy(ethyleneoxy) (4'-CH(CHOMe)-O-2') BNA (also called constrained MOE or cMOE).

[0086] Additional 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. USA, 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. 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. Patent 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 Publication Nos. US2004 / 0171570, US2007 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 Application Nos. PCT / US2008 / 064591, PCT / US2008 / 066154, and PCT / US2008 / 068922.

[0087] In some embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by their isomeric configuration. For example, nucleosides comprising a 4'-2' methylene-oxy bridge can be in the α-L or β-D configuration. Previously, α-L-methyleneoxy (4'-CH2-O-2') bicyclic nucleosides have been incorporated into antisense polynucleotides that have demonstrated antisense activity (Frieden et al., Nucleic Acids).

[0088] In some embodiments, the substituted sugar moiety comprises one or more non-bridging sugar substituents and one or more bridging sugar substituents (e.g., 5'-substituted and 4'-2'-bridging sugars; PCT International Application WO2007 / 134181, where LNA is substituted with, e.g., 5'-methyl or 5'-vinyl groups).

[0089] In some embodiments, the modified sugar moiety is a sugar surrogate. In some such embodiments, the oxygen atom of a naturally occurring sugar is replaced with, for example, a sulfur, carbon, or nitrogen atom. In some such embodiments, such modified sugar moieties also contain bridging and / or non-bridging substituents, as described above. For example, certain sugar surrogates contain substitutions at the 4' sulfur atom and the 2' position (see, e.g., published U.S. patent application US2005 / 0130923) and / or the 5' position. As an additional example, carbocyclic bicyclic nucleosides with a 4'-2' bridge have been described (see, e.g., Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740).

[0090] In some embodiments, the sugar surrogate contains a ring with more than five atoms. For example, in some embodiments, the sugar surrogate contains a six-membered tetrahydropyran (THP). Such tetrahydropyrans may be further modified or substituted. Nucleosides containing such modified tetrahydropyrans 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. (2002) 10:841-854), and fluoro-HNA (F-HNA).

[0091] Many other bicyclic and tricyclic sugar surrogate ring systems that can be used to modify nucleosides for incorporation into antisense compounds are also known in the art (see, e.g., review article: Leumann, JC, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

[0092] Combinations of modifications are also provided, including, without limitation, 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO2008 / 101157 for other disclosed 5',2'-disubstituted nucleosides) and substitution of the ribosyl ring oxygen atom with S and further substitution at the 2' position (see U.S. Patent Publication US2005 / 0130923) or alternatively 5' substitution of bicyclic nucleic acids (see PCT International Application WO2007 / 134181, in which 4'-CH2-O-2' bicyclic nucleosides are further substituted at the 5' position with a 5'-methyl or 5'-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides have been described, along with their oligomerization and biochemical studies (see, e.g., Srivastava et al., 2007).

[0093] In some embodiments, the present invention provides polynucleotides containing modified nucleosides. These modified nucleotides may contain modified sugars, modified nucleobases, and / or modified linkages. The specific modifications are selected so that the resulting polynucleotide has desirable properties. In some embodiments, the polynucleotide contains one or more RNA-like nucleosides. In some embodiments, the polynucleotide contains one or more DNA-like nucleotides.

[0094] In some embodiments, nucleosides of the invention comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the invention comprise one or more modified nucleobases.

[0095] In some embodiments, the modified nucleobase is selected from universal bases, hydrophobic bases, promiscuous bases, size-extended bases, and fluorinated bases, as defined herein.N-2, N-6, O-6 substituted purines, including 5-substituted pyrimidines, 6-azapyrimidines, and 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-halouracil and cytosine, 5-propynyl CH3)uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, as defined herein. , cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. 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 cytidines (e.g., 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 can also include those in which purine or pyrimidine base is replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Additional nucleobases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859; those disclosed in Englisch et al., 1991; and those disclosed in Sanghvi, YS, 1993.

[0096] Representative United States patents that teach the preparation of the specific modified nucleobases noted above, as well as other modified nucleobases, include, without limitation, U.S. Patent 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; 5,502,17 Nos. 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.

[0097] In some embodiments, the present invention provides polynucleotides comprising linked nucleosides. In such embodiments, the nucleosides can be linked to each other using any internucleoside linkage. Two major classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside 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, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Compared with natural phosphodiester bonds, modified bonds can be used to change, typically increase, the nuclease resistance of polynucleotides.In some embodiments, internucleoside linkages with chiral atoms can be prepared as racemic mixtures or as separate 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.

[0098] The polynucleotides described herein may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined in terms of absolute stereochemistry as (R) or (S), as a or R in the case of, for example, sugar anomers, or (D) or (L) in the case of, for example, amino acids. The antisense compounds provided herein include all such possible isomers, as well as their racemic and optically pure forms.

[0099] Neutral internucleoside linkages include, without limitation, phosphotriester, methylphosphonate, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), and thioformacetal (3'-5-CH2-O-5'). Additional neutral internucleoside linkages include nonionic linkages, including siloxanes (dialkylsiloxanes), carboxylate esters, carboxamides, sulfides, sulfonates, and amides (see, e.g., "Carbohydrate Modifications in Antisense Research"; Y.S. Sanghvi and P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Additional neutral internucleoside linkages include nonionic linkages containing mixed N, O, S, and CH moieties.

[0100] Additional modifications can also be made at other positions on the polynucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of the 5' terminal nucleotide. For example, one additional modification of the polynucleotide of the invention includes chemically linking to the polynucleotide one or more additional moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the polynucleotide. Such moieties include cholesterol moieties (Letsinger et al., 1989), cholic acid (Manoharan et al., 1994), thioethers such as hexyl-5-tritylthiol (Manoharan et al., 1992; Manoharan et al., 1993), thiocholesterol (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 di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., 1995; Shea et al., 1990), polyamines or polyethylene glycol chains (Manoharan et al., 1995). al., 1995), or adamantane acetic acid (Manoharan et al., 1995), palmityl moieties (Mishra et al., 1995), or lipid moieties such as octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., 1996).

[0101] Representative United States patents that teach the preparation of such polynucleotide conjugates include, without limitation, 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; 5,11 No. 8,802; No. 5,138,045; No. 5,414,077; No. 5,486,603; No. 5,512,439; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605, No. 735; No. 4,667,025; No. 4,762,779; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4,958,01 No. 3; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,245,022; No. 5,254,469 No. 5,258,506; No. 5,262,536; No. 5,272,250; No. 5,292,873; No. 5,317,098; No. 5,371,241, No. 5,391,723; No. 5,416,203, No. Nos. 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.

[0102] In some embodiments, the present disclosure also encompasses host cells comprising the polynucleotides disclosed herein. In some embodiments, the host cell or host cell population comprises a polynucleotide comprising a nucleic acid sequence encoding a signal peptide. In some embodiments, the host cell or host cell population comprises a polynucleotide comprising a nucleic acid sequence encoding a heterologous polypeptide in frame with a signal peptide. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is a mammalian cell. In some exemplary embodiments, the host cell is a human cell. In some embodiments, the host cell is an in vitro cell line or an ex vivo cell. In some embodiments, the host cell is present in vivo. In some embodiments, the host cell is a somatic cell. In some embodiments, the host cell is a differentiated cell. In some embodiments, the host cell is a stem cell. In some embodiments, the host cell is a tumor cell. In some embodiments, the host cell is selected from the group including CHO-K1 cells, HEK293 cells, HeLa cells, Caco2 cells, U2-OS cells, NIH 3T3 cells, NS0 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, and Molt4 cells. In some exemplary embodiments, the host cell is selected from HeLa A549 cells, Huh7 cells, or IGROV1 cells.

[0103] Compositions for cell culture, tissue culture, ex vivo and / or in vivo delivery In some embodiments, the present disclosure encompasses compositions that combine a polypeptide or polynucleotide provided herein with a suitable delivery system for cell culture, tissue culture, ex vivo, and / or in vivo delivery. In some embodiments, the suitable delivery system introduces the polypeptide or polynucleotide disclosed herein into a cell.

[0104] In some embodiments, the present disclosure encompasses compositions that combine a polynucleotide comprising a nucleic acid sequence encoding a signal peptide and a recombinant polypeptide with a suitable delivery system, hi some embodiments, the present disclosure encompasses the use of any suitable delivery system known in the art.

[0105] In some embodiments, suitable delivery system can be a viral vector.In some embodiments, the viral vector is an RNA viral vector.In some embodiments, the viral vector is a DNA viral vector.Non-limiting examples of suitable viral vectors include adenovirus, adeno-associated virus (AAV), retrovirus, herpes virus, lentivirus, poxvirus, or papillomavirus vector.

[0106] In some embodiments, the delivery system is a non-viral delivery system.Non-limiting examples of non-viral delivery systems include polymers, polyplexes, lipids, lipidoids, lipoplexes, liposomes, lipid fusion constructs, polymer nanoparticles, nanoparticles, lipid nanoparticles (LNPs), core-shell nanoparticles, solid lipid nanoparticles, metal nanoparticles, self-assembled nucleic acid nanoparticles, hyaluronidase, nanoparticle mimics, ribonucleoproteins, positively charged peptides, small RNA conjugates, aptamer-RNA chimeras, RNA fusion protein complexes, and any combination thereof.

[0107] In some embodiments, the polynucleotides of the present disclosure can be formulated using natural and / or synthetic polymers. Polymers include polyethylene, polyethylene glycol (PEG), poly(l-lysine) (PLL), PEG grafted to PLL, cationic lipopolymers, biodegradable cationic lipopolymers, polyethyleneimine (PEI), cross-linked 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[α-(4-aminobutyl)-L-glycolic acid] (PAGA), biodegradable cross-linked cationic multiblock copolymers, polycarbonates, polyanhydrides, polyhydroxy acids, polypropyl fumarate, polycaprolactone, and polyamides. , polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethyleneimine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), acrylic polymer, methyl methacrylate copolymer, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylate, amine-containing polymer, or combinations thereof.Non-limiting examples of polymers that can be used for delivery include, but are not limited to, Dynamic POLYCONJUGATE™ formulations from MIRUS® Bio (Madison, WI) and Roche Madison (Madison, WI), PHASERX™ polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGY™ (Seattle, WA), DMRI / DOPE, poloxamers, VAXFECTIN® adjuvants from Vical (San Diego, CA), chitosan, cyclodextrins, dendrimers, and poly(lactic-co-glycolic acid) (PLGA) polymers from Calando Pharmaceuticals (Pasadena, CA), RONDEL™ (RNAi / oligonucleotide nanoparticle delivery) polymers (Arrowhead Research Corporation, Pasadena, CA), and pH-responsive coblock polymers such as, without limitation, PHASERX™ (Seattle, WA).

[0108] In some embodiments, the delivery system comprises a liposome. Non-limiting examples include N-[1-(2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), dioleoyldimethylammonium-propane (DODAP), and dipalmitoylphosphatidylethanolamine (DOPE) or dioleoylphosphatidylethanolamine (DPPE), distearoylphosphatidylcholine (DSPC), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPE (1,2-dimyris ... These include, but are not limited to, 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide; and dimethyldioctadecylammonium bromide (DDAB), and any combination thereof.

[0109] In some embodiments, the delivery system comprises one or more nanoparticles.Nanoparticles can be solid and can comprise materials including polysaccharides, lipids, proteins, polymers, biodegradable polymers, metal oxides, and any combination thereof.Other nanoparticles are in liquid form and are liposomes, micelles, or emulsion systems mainly composed of amphiphilic molecules or polymers.Lipid nanoparticles (LNPs) are one of the most promising types of nanoparticles due to their high nucleic acid encapsulation efficiency, high stability, and compatibility with biological environments.

[0110] In some embodiments, LNPs can be made from cationic, anionic, zwitterionic, or neutral lipids, or any combination thereof. LNPs can also be composed of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Any lipid or lipid combination known in the art can be used to produce LNPs. Non-limiting examples of lipids used to produce LNPs include DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Non-limiting examples of frequently used cationic lipids include polyethyleneimine, polyamidoamine (PAMAM) starburst dendrimer, lipofectin (a combination of DOTMA and DOPE), lipofectase, LIPOFECTAMINE™ (e.g., LIPOFECTAMINE™ 2000), DOPE, cytofectin, eufectin, 98N12-5, C12-200, DDAB, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Non-limiting examples of frequently used neutral lipids include DPSC, DPPC, POPC, DOPE, and SM. Non-limiting examples of frequently 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 LNPs to enhance transfection activity and nanoparticle stability. In some embodiments, lipid nanoparticles contain ionizable amino lipids (e.g., heptatriaconta-6, 9, 28,31-tetraen-19-yl 4-(dimethylamino)butanoate, DLin-MC3-DMA), phospholipids such as phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), egg yolk phosphatidylcholine (EPC), egg yolk phosphatidylglycerol (EPG), egg yolk phosphatidylethanolamine (EPE), egg yolk phosphatidylserine (EPS), egg yolk phosphatidic acid (EP A), egg yolk phosphatidylinositol (EPI), soybean phosphatidylcholine (SPC), soybean phosphatidylglycerol (SPG), soybean phosphatidylethanolamine (SPE), soybean phosphatidylserine (SPS), soybean phosphatidic acid (SPA), soybean phosphatidylinositol (SPI), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylglycerol Dioleoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylglycerol (DOPG), dimyristoyl phosphatidylglycerol (DMPG), hexadecylphosphocholine (HEPC), hydrogenated soy phosphatidylcholine (HSPC), distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), dioleyl phosphatidylethanolamine (DOPE), palmitoyl stearoyl phosphatidylcholine (PSPC), palmitoyl stearoyl phosphatidylglycerol (PSPG), monooleoyl phosphatidylcholine (DPPG), di ...choline (DPPG), dioleoyl phosphatidylcholine (DPPG), dioleoyl phosphatidylcholine (DPPG), dioleoyl phosphat 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), polyethylene glycol distearoylphosphatidylethanolamine (PEG-DSPE), dipalmitoylphosphatidylserine (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidic acid (DPPA), 1,These include 2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dipalmitoylphosphatidylinositol (DPPI), 1,2-dioleoyl-sn-glycero-3-phosphatidylinositol (DOPI), dimyristoylphosphatidylinositol (DMPI), distearoylphosphatidylinositol (DSPI), and mixtures thereof, cholesterol, and coating lipids (polyethylene glycol-dimyristoylglycerol, PEG-DMG), as disclosed, for example, in Tam et al. (2013). Advances in Lipid Nanoparticles for siRNA delivery. Pharmaceuticals 5(3):498-507. Various such LNP systems are known and are disclosed, for example, but not limited to, in Hou, X. et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater 6,1078-1094 (2021), U.S. Patent 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 No. WO2016 / 011203, and U.S. Patent Publication No. 2017 / 0107539, all of which are expressly incorporated by reference in their entireties as if fully set forth herein. In some embodiments, the lipid nanoparticles may comprise a combination of lipids, such as, for example, cationic lipids, phospholipids (e.g., and PEGylated lipids, e.g., iPhos LNP (9A1-P9 / cholesterol / DODAP / DMG-PEG, 25:30:30:1 mol / mol; 18:1 9A1-P9:nucleic acid, wt / wt)). In some embodiments, the lipid nanoparticles may comprise a combination of lipids, such as those described in U.S. Patent No. 11,304, incorporated by reference in its entirety.Organ-targeted (SORT) lipid nanoparticles such as those provided in US Pat. No. 911. In some embodiments, the LNPs can be selected from iPhos LNPs, mDLNPs, liver SORT LNPs, lung SORT LNPs, or spleen SORT LNPs, and any combination thereof.

[0111] In some exemplary embodiments, the delivery system comprises one or more lipid nanoparticles (LNPs). In some embodiments, the lipid nanoparticles have an average diameter of between about 10 and about 1000 nm. In some embodiments, the lipid nanoparticles have a diameter of less than 300 nm. In some embodiments, the lipid nanoparticles have a diameter of between about 10 and about 300 nm. In some embodiments, the lipid nanoparticles have a diameter of less than 200 nm. In some embodiments, the lipid nanoparticles have a diameter of between about 25 and about 200 nm. In some embodiments, the lipid nanoparticle preparation (e.g., a composition comprising a plurality of lipid nanoparticles) has a size distribution with an average size (e.g., diameter) of between about 70 nm and about 200 nm, more typically with an average size of about 100 nm or less.

[0112] In some embodiments, compositions comprising LNPs have a molar ratio of LNPs to nucleic acid of about 5:1 to about 1000:1. In some embodiments, the molar ratio of LNPs to nucleic acid is about 100:1 to about 1000:1. In other embodiments, the molar ratio is about 250:1 to about 750:1. In some embodiments, 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, 100:1, or any intermediate ratio.

[0113] In some embodiments, the compositions disclosed herein may be therapeutic compositions and may further comprise one or more pharmaceutically acceptable excipients.The pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, suspending agents, isotonicity adjusting agents, thickening agents, surfactants and / or emulsifying agents, disintegrating agents, binders, preservatives, buffering agents, lubricants, preservatives, and / or oils.Such excipients can be optionally included in pharmaceutical preparations.Excipients such as cocoa butter and suppository wax, coloring agents, coating agents, sweeteners, flavoring agents, and / or fragrances can be present in the composition according to the discretion of the formulator.Various excipients for formulating pharmaceutical compositions and the techniques for preparing said compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, AR Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006). The use of conventional excipient vehicles is contemplated within the scope of this disclosure.

[0114] In some embodiments, 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 method of administering drugs with reduced administration frequency and simultaneously improving therapeutic efficacy and patient compliance. In some embodiments, these formulations are characterized by slower release of therapeutic agents compared with conventional release dosage forms administered by the same route. In some embodiments, the formulations are adjustable and release at a predetermined rate within a therapeutic range for a specific period of time.

[0115] In some embodiments, the compositions disclosed herein are formulated for administration to a subject in need thereof via one or more routes, such as oral, intraadipose, intra-arterial, intra-articular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrarectal, intrathecal, intratracheal, intratumoral, intraumbilical, intravaginal, intravenous, intravascular, intravitreal, liposomal, topical, mucosal, parenteral, rectal, subconjunctival, subcutaneous, sublingual, topical, buccal, transdermal, vaginal, in a cream, in a lipid composition, via a catheter, via lavage, via continuous infusion, via infusion, via inhalation, via injection, via topical delivery, or via local perfusion. In some embodiments, the pharmaceutical composition is formulated for administration by injection. In some embodiments, the pharmaceutical composition is formulated as a unit dose. In some embodiments, the formulation may further comprise an excipient suitable for administration via the routes provided herein.

[0116] In some embodiments, the compositions disclosed herein may further comprise one or more suitable excipients for administration. In some embodiments, the compositions may be formulated as injections, liquids, emulsions, suspensions, syrups, tablets, caplets, creams, ointments, lotions, patches, solutions, suspensions, suppositories, lyophilisates, gels and capsules. Methods for preparing pharmaceutical compositions are well known in the art (see, for example, Remington, The Science and Practice of Pharmacy, Alfonso R. Gennaro (Ed.) Lippincott, Williams & Wilkins (published)). Pharmaceutical compositions may also be formulated to facilitate timed release, sustained release, pulsed release or continuous release. Pharmaceutical compositions may also be administered in devices such as timed release, sustained release, pulsed release or continuous release devices.

[0117] In some aspects, the disclosure also encompasses compositions comprising the polypeptides disclosed herein and suitable delivery systems. Any suitable delivery system for polypeptides known in the art may be used herein. Examples of suitable delivery systems include, but are not limited to, polymers, polyplexes, microspheres, lipids, lipidoids, lipoplexes, liposomes, microparticles, polymeric nanoparticles, nanoparticles, lipid nanoparticles (LNPs), core-shell nanoparticles, solid lipid nanoparticles, metal nanoparticles, nanoparticle mimics, and any combination thereof. Some details of commonly used delivery systems are provided in the disclosure for the delivery of polynucleotides, but may be suitably adapted for the delivery of polypeptides.

[0118] <III. Methods> In some aspects, the disclosure also encompasses methods and uses of using the compositions disclosed herein. In some aspects, these compositions comprising the signal peptides provided herein, or the polynucleotides encoding them provided herein, can be used for any suitable application. In some aspects, the secretion of a target protein from cells can be beneficial in such applications. In some aspects, the disclosure provides several signal peptide sequences, each of which may be suitable for use in 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.

[0119] In some embodiments, the present disclosure provides various engineered polynucleotides, engineered polypeptides, expression cassettes, viral vectors, expression vectors, host cells, and suitable formulations that enable the secretion of a heterologous protein of interest from cells. Post-translational secretion of a polypeptide of interest from cells may be desirable, for example, for systemic or organ-specific delivery of therapeutic, diagnostic, theragnostic, and / or reporter polypeptides in a subject in need thereof. In some embodiments, post-translational secretion of a polypeptide of interest from cells may be desirable for non-therapeutic applications, such as laboratory experiments. In some embodiments, post-translational secretion of a polypeptide of interest may be desirable for industrial applications for easy production and isolation of protein products. All such applications for the disclosed secretory signal peptides are contemplated in the present disclosure.

[0120] In some exemplary embodiments, the present disclosure encompasses methods of diagnosis, prevention, and / or treatment comprising administering an effective amount of a composition disclosed herein. In some embodiments, the composition comprises a therapeutic polypeptide fused to a signal peptide disclosed herein, or a polynucleotide composition encoding the same. In some embodiments, a composition (polypeptide or polynucleotide) corresponding to any therapeutic polypeptide for which secretion is desired can be used in the disclosed methods. For example, the composition can be used in methods of ameliorating the effects of a disease, preventing a disease, treating a disease, or inhibiting disease progression in a subject in need thereof. Such methods include inhibiting cell rolling, inflammation, autoimmune disease, metastasis, tumor cell or leukemia cell proliferation and / or replication, or an increase in tumor cell numbers in a tumor-bearing subject or leukemia cell numbers in a leukemia-bearing subject. Additionally, such methods include increasing the mortality rate of tumor or leukemia cells, altering the susceptibility of diseased cells to damage by anti-disease agents, tumor cell damage by anti-cancer agents, or leukemia cell damage by anti-cancer agents. Such methods also include inhibiting or reducing viral entry into cells. Such methods further include preventing or inhibiting cardiovascular disease. In some embodiments, the therapeutic polypeptide used in the method may have, for example, anti-cancer, anti-inflammatory, immunomodulatory, antiviral, antibacterial, antifungal, antihelminthic, cholesterol-lowering, antidiabetic, antifibrotic, analgesic, anesthetic, anti-aging, antidepressant, neuromodulatory, antidermatitis, anti-edema, anti-allergic, antikeratolytic, antifungal, antipruritic, cardiovascular, chemotherapeutic, or hormonal activity. In some embodiments, the therapeutic polypeptide is a protein replacement therapy, polypeptide therapy, vaccine, viral vaccine, secretory therapy, anti-cancer, anti-inflammatory, antiviral, antibacterial, cholesterol-lowering, antidiabetic, or antifibrotic polypeptide. In some embodiments, the therapeutic polypeptide is an antibody.In some embodiments, 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 mucin-containing protein 3 (TIM-3), B- and T-lymphocyte attenuator (BTLA), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), V-domain Ig suppressor of T-cell activation (VISTA), adenosine A2a receptor (A2aR), killer cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), CD20, CD39, and CD73. In some embodiments, 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 cell death ligand 1 (PD-L1), programmed cell 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. In some embodiments, the heterologous polypeptide is an antibody. In some embodiments, the polypeptide is a chimeric antigen receptor (CAR). In some embodiments, the polypeptide is active as a vaccine. In some embodiments, the method also encompasses the use of a diagnostic polypeptide. In some embodiments, the polypeptide is theragnostic. In some embodiments, the polypeptide is an antibody-based diagnostic agent. Generally, the polypeptide is labeled with a radionuclide (e.g., In, Tc, C, I, H, P, or S) and specifically binds to tumor antigens, allowing tumor localization using immunoscintigraphy. In one embodiment, the polypeptide or a fragment thereof binds to the extracellular domain of a specific cancer biomarker.Diagnostic polypeptides can be labeled with probes suitable for detection by various imaging methods.Probe detection methods include, but are not limited to, fluorescence, optical, 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 lanthanides, paramagnetic iron, fluorine-18 and other positron-emitting radionuclides.

[0121] The effective dosage / amount and schedule for administering the composition may be determined empirically, and making such a determination is within the skill of a person skilled in the art.Those skilled in the art will understand that the dosage of the composition disclosed herein that must be administered will vary depending on, for example, the subject receiving the composition, the route of administration, the specific type of composition used, and other drugs being administered.For example, in the case of anti-cancer therapeutics, the amount of therapeutically administered composition that stops tumor growth, causes tumor shrinkage, and / or prevents the development of new tumors compared to the disease course that would occur in the absence of administration is an effective dose.The composition can be administered as a single dose or may require repeated administration.In some embodiments, the method of treatment can further include the administration of additional drugs, such as anti-inflammatory drugs, analgesics, antibacterial drugs, or additional treatments, including therapy, such as radiation therapy.

[0122] In some embodiments, the subject in the method of treatment may include animals, humans or non-humans, who are provided with treatment according to the methods of the present disclosure. Human and veterinary applications are contemplated by the present disclosure. 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 some embodiments, the subject is a human. Both pediatric and adult subjects are included.

[0123] In some embodiments, the present disclosure also encompasses methods of using the compositions provided herein for in vivo diagnosis. In some embodiments, the compositions comprise or encode diagnostic antibodies. In some exemplary embodiments, the methods disclosed herein are used for tumor detection.

[0124] In some embodiments, the present disclosure also encompasses methods of using the compositions disclosed herein in cell and tissue culture to enable secretion of heterologous polypeptides. In some embodiments, the methods include contacting cells with the compositions disclosed herein. In exemplary embodiments, cells can be transfected with the polynucleotide compositions provided herein. In some embodiments, the heterologous polypeptide can be a reporter polypeptide, such as a fluorescent polypeptide or antibody, and the secreted polypeptide can be used for visualization using microscopy or other suitable techniques.

[0125] In some embodiments, the present disclosure also encompasses methods of using the compositions disclosed herein for industrial applications. Secreted proteins offer several advantages for industrial-scale production of products. In some exemplary embodiments, purification of secreted proteins may be easier and more desirable than extraction of heterologous proteins from cells or tissues. In some exemplary embodiments, systemic secretion of proteins may be useful in the food industry to impart flavor to meat products. The applications disclosed herein are merely exemplary and should not be considered limiting.

[0126] <IV.キット> In some embodiments, the compositions and methods provided herein may also be provided in the form of a kit with instructions for use. In some embodiments, the kit includes at least a composition comprising a polynucleotide encoding a signal peptide provided herein, and optionally, suitable substrates, reagents, buffers, diluents, cells, standards, containers, and instructions for use. In some embodiments, the kit may include at least cells comprising a polynucleotide or polypeptide disclosed herein, and optionally, suitable substrates, reagents, buffers, diluents, cells, standards, containers, and instructions for use.

[0127] In some embodiments, the article of manufacture or kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, microfuge tubes, bottles, vials, assay plates, strips, matrices, etc. The containers may be formed from a variety of materials, such as glass, plastic, paper, etc. The kit may further include other materials desirable from a commercial and user standpoint, other buffers, diluents, filters, needles, and syringes.

[0128] "Package insert" is used to refer to instructions that are customarily included in the commercial packaging of a product and that contain information on usage, etc.

[0129] The instructions included in the kit may be affixed to the packaging material or included as a package insert. The instructions are typically, but not limited to, written or printed. Any medium capable of storing such instructions and transmitting them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROMs), and the like. As used herein, the term "instructions" may include the address of an internet site that provides the instructions. [Example]

[0130] The following examples are included to illustrate preferred embodiments of the present disclosure. The techniques disclosed in the following examples represent techniques that the inventors have found to function well in the implementation of the present disclosure, and thus, it should be understood by those skilled in the art that they may be considered to constitute preferred modes for their implementation. However, those skilled in the art should understand that, in light of the present disclosure, many changes can be made in the specific embodiments disclosed, and still obtain equivalent or similar results without departing from the spirit and scope of the present disclosure.

[0131] [Method] <Construction of <SP-mCherry plasmid (pDNA)>> 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 appropriately designed primers. Several SPs were selected, including hAlb (human albumin, SEQ ID NO: 1), hApoB (human apolipoprotein, SEQ ID NO: 2), gLuc (Gaussia luciferase, SEQ ID NO: 3), and hFVII (human factor VII: SEQ ID NO: 4). The polypeptide sequences of the signal sequences fused to mCherry are provided as SEQ ID NOs: 62-66 and are listed in Table 1. Based on standard protocols, the enzymatically digested SP-mCherry products were cloned into the pCS2-MT vector. The SP-mCherry plasmid was verified by sequencing and prepared for in vitro screening.

[0132]

Table 1

[0133] <In vitro SP screening by transfection of pDNA> To perform SP screening, pDNA transfection was carried out in cells. Hela and Huh7 cells were seeded in a 96-well plate at 1×10 4 cells per well. After 24 hours, the cells were treated with the Lipo2k-pDNA formulation of 50 ng pDNA per well. On the 1st, 2nd, and (or) 3rd day after treatment, the cells were immediately photographed with a Keyence scope. On the other hand, cell lysates and media were further collected, and then the mCherry signal was quantified with a plate reader. To clearly observe the signal of mCherry, the cell lysates and media were further transferred to EP tubes and photographed with an IVIS Lumina system. To test the intracellular signal distribution, confocal microscopy was used. Huh7 cells were treated as described above, and after 3 days, the cells were washed three times with 1×PBS, stained with Hoechst 33342, and photographed with a confocal microscope.

[0134] <mRNA synthesis> All mRNAs used in this study were produced by in vitro transcription (IVT) as previously described (Cheng et al. 2020). Briefly, linear pDNA with optimized 5’(3’)-untranslated region (UTR) and polyA sequence was first obtained by enzymatic digestion, and then the IVT reaction was prepared with a standard protocol using N1-methylpseudouridine-5’-triphosphate modification. Finally, the mRNA was capped (Cap-1) with Vaccinia capping enzyme and 2’-O-methyltransferase (NEB).

[0135] <mRNA-nanoparticle formation> mRNA-loaded LNP formulations were formed using the previously described ethanol dilution method (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 at a specific molar ratio were dissolved in ethanol, and RNA was first dissolved in 10 mM citrate buffer (pH 4.0). Then, the two solutions were rapidly mixed at an aqueous to ethanol volume ratio of 3:1 (3:1, aqueous:ethanol, volume:volume) to meet a final weight ratio of 40:1 (total lipid:mRNA). After incubation at room temperature for 10 minutes, the mRNA LNP formulation was immediately added to cells or dialyzed against PBS for 2 hours for in vivo experiments.

[0136] <In vitro mCherry secretion driven by the optimal hFVII-SP verified by the mRNA formulation> hFVII-mCherry mRNA was transfected into several cell lines including Huh7, 293T, Hela, A549 and IGROV1. Cells were seeded at 1×10 4 cells per well in 96-well plates. The mRNA mDLNP formulation was prepared as described above and then cells were treated at various mRNA doses (0 - 750 ng per well) and time points (24 hours - 72 hours). At a predetermined time, cells were directly imaged by a Keyence scope and then the mCherry signal in the medium and cell lysates was quantified by a plate reader. The WT-mCherry mRNA formulation was used as a control group.

[0137] <In vivo mCherry secretion driven by the optimal hFVII-SP verified by the mRNA formulation> All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center and were also in compliance with applicable local, state, and federal regulations. C57BL / 6 mice were obtained from the mouse breeding core facility at UTSW. For the luciferase mRNA test, mDLNP and SORT LNP (liver, lung, and spleen) formulations were intravenously injected into mice at a dose of 0.1 mg / kg mRNA. Three hours later, the mice were injected with D-luciferin (150 mg / kg, intraperitoneal (IP)) and imaged by an IVIS Lumina system (Perkin Elmer). To test the secretion of mCherry, hFVII-mCherry mRNA was encapsulated in mDLNP and intravenously injected into mice at a dose of 0.5 mg / kg mRNA. Serum was separated at different time points (2 hours, 6 hours, 24 hours, 30 hours, 48 hours, 55 hours, and 72 hours), and the signal of mCherry was quantified by a plate reader. On the other hand, tissues were imaged by IVIS to confirm the secretion of mCherry in the blood. The PBS and WT-mCherry formulation treatment groups were used as controls. To further test the secretion of mCherry, liver, lung, and spleen-targeted SORT LNP were used to deliver hFVII-mCherry mRNA (0.5 mg / kg) to the liver, lung, and spleen, respectively. Then, the tissues were imaged by IVIS 24 hours later.

[0138] <Cytotoxicity Rescue of hFVII-Embrel mRNA> L929 cells were used to evaluate TNF-α-mediated cytotoxicity. Both 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 for 24 hours. Fresh medium containing actinomycin and TNF-α was exchanged to a final concentration of 1 μg / ml of actinomycin and 0 - 0.1 ng / ml of TNF-α. After further incubation for 24 hours, cell viability was detected by a CellTiter-Glo kit based on a standard protocol.

[0139] To evaluate the cytotoxicity rescue of hFVII-Enbrel mRNA formulations, cells were pretreated with hFVII-Enbrel mRNA mDLNPs for 2 days before being challenged with TNF-α. Dose-dependent rescue was tested for both mRNA and TNF-α. For dose-dependent rescue of mRNA formulations, mRNA doses ranging from 0 ng / ml to 1.25 ng / ml per well were tested, followed by a 24-hour challenge with 0.1 ng / ml TNF-α. For dose-dependent rescue of TNF-α, a fixed transfection mRNA concentration of 0.4 ng / ml was used, followed by a challenge with 0 ng / ml to 5 ng / ml TNF-α.

[0140] To further verify the rescue effect of Enbrel secreted into the culture medium, we measured the rescue of cytotoxicity by pretreatment with functional medium. Cells were seeded and treated as described above (mRNA concentrations: 0 ng / ml to 1.25 ng / ml). The medium was then collected and transferred to a new 96-well plate containing adherent L929 cells. Simultaneously, the new plate was challenged with TNF-α (0.1 ng / ml) and actinomycin (1 μg / ml). After an additional 24 hours, cell viability was assessed.

[0141] <Pharmacokinetic study> Male C57BL / 6 mice weighing 20 g were randomly assigned to each group. Enbrel protein and hFVII-Enbrel mRNA mDLNP formulations were intravenously injected at a dose of 0.5 mg / kg. Serum samples were collected between 2 and 216 hours, and serum Enbrel was quantified using an ELISA kit (MyBioSource).

[0142] <Psoriasis treatment> Regarding the imiquimod-induced psoriasis-like hyperplasia model, 8-week-old female C57BL / 6 mice were shaved and chemically depilated near the nape (designated as day 1). On day 3, the mice were intravenously injected with an hFVII-Enbrel formulation at a dose of 0.5 mg / kg. Subsequently, a total of 60 mg of Aldara cream (5% imiquimod) (Aldara, 3M Pharmaceuticals) was topically applied to the shaved back skin samples daily for a total of 5 days. On day 9, the whole body was photographed with a camera to show differences between groups, where the lanolin + PBS-treated mice and imiquimod + mCherry mDLNP-treated mice were used as control groups. At the final time point, the back skin was collected, the thickness was measured by H&E staining, and cell proliferation was analyzed by immunohistochemistry of Ki-67 and Gr-1.

[0143] <Evaluation of PDL1 expression> In vivo tumor immunotherapy was studied using the MC38 and B16F10 cell lines. Flow cytometry was used to evaluate PDL1 expression in the cell membrane. The cells were seeded at a density of 3×10 5 cells per well in 6-well plates for 24 hours. The cells were further incubated with IFN-γ (100 ng / ml) for another 24 hours, and after staining with a primary anti-PDL1 antibody and an Alexa Fluor 647-labeled secondary antibody, PDL1 expression was analyzed by flow cytometry. Isotype antibody-stained cells were used for gating.

[0144] <Tumor immunotherapy> MC38 or MC38-Luc (stably expressing luciferase) cells were grown in DMEM medium containing 10% FBS. On day 0, a total of 1×10 6Cells were suspended in 100 μl PBS and subcutaneously injected into the right flank of C57BL / j mice. The hFVII-anti-PDL1 mRNA mDLNP formulation was continuously intravenously injected at a dose of 0.5 mg / kg mRNA every 4 days from day 3. For the MC38 model, tumors were measured 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 the luciferase signal was quantified by IVIS software. For the B16F10-Luc model, a total of 4 × 10 cells were injected. 5 Cells were injected subcutaneously, and mRNA formulations were injected intravenously as described above. Luciferase signal, tumor size, and survival were monitored from day 0 to day 32. For both tumor models, mCherry mDLNP formulations were used as controls. Tumors were measured with digital calipers, and tumor size was calculated using the formula: volume = 0.5 × length × width. When tumor volumes were 1500 cm, tumor size was 1.5 × ... 3 Upon reaching this age, the mice were sacrificed and recorded as dead.

[0145] Example 1: Selection of signal peptides for therapeutic applications We first screened multiple naturally occurring signal peptides (SPs) for their ability to drive reporter protein secretion in cell culture. To this end, several different SPs from three known endogenously secreted proteins (albumin, hAlb; apolipoprotein B, hApoB; and factor VII, hFVII) and one known synthetic secreted protein (Gaussia luciferase, gLuc) were cloned into the pCS2-MT plasmid backbone directly upstream of the reporter mCherry mRNA sequence, along with a negative control (NC) SP leader sequence.

[0146] The construct was preceded by an SP6 promoter and an optimized 5' UTR, followed by an optimized 3' UTR and poly(A) tail (Figure 1A). First, HeLa cells were transfected with wild-type (WT) mCherry pDNA and gLuc-mCherry pDNA lacking SP via Lipofectamine 2000. Both intracellular and extracellular fluorescence were quantified by fluorescence microscopy at 24, 48, and 72 hours posttransfection. gLuc SP induced high levels of mCherry secretion into the culture medium (Figure 1B). Furthermore, mCherry protein content in the cell medium and cell lysate was quantified separately at 24, 48, and 72 hours posttransfection using a fluorescent plate reader. The gLuc SP group demonstrated increased mCherry secretion into the culture medium over time, as well as a higher mCherry fluorescence medium-to-cell lysate ratio (Figure 1C). We then expanded the set of SPs to include a negative control (scrambled sequence), hAlb, hApoB, and hFVII in addition to gLuc. HeLa cells were transfected with the pDNA constructs, again using Lipofectamine 2000. Images taken 72 hours after transfection by fluorescence microscopy and IVIS indicated that the SPs hApoB, gLuc, and hFVII all produced high levels of mCherry protein secretion, whereas the NC and hAlb constructs effectively mediated intracellular mCherry expression but did not promote significant extracellular secretion (Figure 1D-F). When the same set of SPs was evaluated in the hepatoma cell line Huh7, the trend toward mCherry secretion observed in HeLa cells persisted (Figure 1I-J); however, analysis of transfected cells using confocal microscopy revealed morphological differences in the mCherry signal between the SPs that drove extracellular secretion of mCherry and those that only promoted intracellular mCherry expression (Figure 1G-H). Overall, it was determined that hFVII SP was able to extend the highest levels of protein secretion in both cell lines.

[0147] Example 2: Organ delivery of mRNA encoding a signal peptide and secretion of the encoded polypeptide. Based on the above observations, it was interesting to determine whether mRNA containing an integrated SP sequence would yield similar results to those observed with pDNA. To pursue this question, hFVII-mCherry mRNA was generated by in vitro transcription (IVT) from the FVII-mCherry-pCS2-MT plasmid (Figure 2A). mDLNP lipid nanoparticles were tested for use as the initial carrier of RNA. Transfection of several different cell lines with mDLNPs containing FVII-mCherry mRNA showed that protein export into the medium was positively correlated with dose and time post-transfection, with greater fluorescent signal intensity observed across cell lines at longer time intervals and higher doses (Figure 2B-D).

[0148] Next, liver-targeting mDLNPs were tested to deliver FVII-mRNA to the liver in mice. The liver-targeting ability of mDLNPs was first verified by intravenous injection of luciferase mRNA-encapsulated mDLNPs, which showed bright luminescence 6 h after injection as analyzed by IVIS (Figure 2E). Then, hFVII-mCherry mRNA and WT-mCherry mRNA were encapsulated in mDLNPs and administered intravenously (IV) to mice. To determine whether mDLNPs containing hFVII-mCherry mRNA could secrete mCherry into the systemic circulation, thereby enabling the liver to function as a protein factory, blood was collected 2, 6, 24, 30, 48, 55, and 72 h after injection.

[0149] Fluorescence analysis of serum from mice revealed that mCherry secretion did not occur in the WT-mCherry group, but mCherry signal was present in the serum of the FVII-mCherry group at all time points, with peak concentrations occurring at 6 h post-injection (Figure 2F). IVIS images were also taken at 55 and 72 h post-injection for WT-mCherry and hFVII-mCherry-injected mice. Interestingly, in the WT group, mCherry fluorescence was visible in the liver at 55 h, but the signal had completely disappeared by 72 h. However, at both time points, bright mCherry fluorescence was observed in the kidneys of mice injected with mDLNPs containing hFVII-mCherry mRNA, indicating that mCherry protein was removed from the systemic circulation via renal filtration (Figure 2G).

[0150] Next, we tested whether the liver and extrahepatic organs, including the lung and spleen, could be targeted and transfected with liver, lung, and spleen SORT LNP formulations containing hFVII mCherry mRNA, respectively, to promote mCherry secretion. To further explore this hypothesis, we first confirmed the targeting capabilities of the SORT technology using liver, lung, and spleen SORT LNPs containing IV-administered luciferase.

[0151] Indeed, bright luminescence was present in each organ after injection of the corresponding SORT LNP. Each SORT LNP formulation was then loaded with hFVII mCherry mRNA, injected IV into mice, and imaged by IVIS 24 hours post-injection. Due to the inclusion of hFVII SP, mCherry protein was secreted from each tissue by all SORT LNPs. In all cases, signal was present in the kidney in all groups, indicating systemic clearance of mCherry protein via the kidney. Signal was significantly higher in the liver SORT group. In summary, after transfection with liver, lung, and spleen SORT LNPs loaded with hFVII mCherry mRNA, it can be concluded that the liver, lung, and spleen are all capable of mediating intracellular mCherry protein production and extracellular protein secretion into the systemic circulation (Figure 2H).

[0152] [Example 3: Treatment of psoriasis with hFVII-Enbrel mRNA formulation] To test the use of signal peptides in a therapeutic context, mRNA encoding hFVII SP followed by the therapeutic synthetic dimeric fusion protein Enbrel (etanercept), encapsulated in mDLNPs, was tested in L929 cells and an imiquimod-induced psoriasis in vivo model (Figure 3A). L929 cells were first treated with mouse and human TNF-α at doses ranging from 0.001 to 0.1 ng / mL. Even at concentrations as low as 0.02 ng / mL, less than 20% of cells survived (Figure 3B). Next, cells were pretreated with 80 ng of hFVII-Enbrel mRNA-encapsulated LNPs and then challenged 48 hours after hFVII-Enbrel mRNA LNP treatment with either mouse 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 mDLNPs resulted in significantly higher cell viability across the TNF-α dose range when compared to PBS and mCherry mRNA controls (Figure 3C). Furthermore, L929 cells were pretreated for 48 hours with hFVII-Enbrel mRNA mDLNPs at doses ranging from 0.05 ng / mL to 1.25 ng / mL and then administered either 0.1 ng / mL of mouse or human TNF-α. As expected, there was a dose-dependent increase in cell viability associated with increasing pretreatment concentrations of hFVII-Enbrel mRNA (Figure 3D). Finally, after treatment with medium from cells pretreated with hFVII Enbrel mRNA mDLNP at doses ranging from 0.05 ng / mL to 1.25 ng / mL, L929 cells administered 0.1 ng / mL of either mouse or human TNF-α were rescueable in a dose-dependent manner (Fig. 3E, upper panel), with viability recovery reaching nearly 100% in the human TNF-α group at an mRNA dose of only 0.4 ng / mL (Fig. 3E, lower panel).

[0153] 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 that received no LNPs but a small amount of lanolin cream on days 4–8; and two experimental groups, in which one group received IV hFVII Enbrel mRNA mDLNPs 3 days after shaving and depilation, and the other group received IV mCherry mDLNPs. In the two experimental groups, mice were administered imiquimod on days 4–8 to induce a psoriasis-like phenotype. The serum pharmacokinetics of Enbrel was first assessed by blood sampling and Enbrel ELISA after IV injection of 0.5 mg / kg hFVII Enbrel mDLNPs or Enbrel protein. In mice injected with Enbrel protein, peak serum concentrations occurred 2 hours after injection, followed by a rapid decline. However, in the group injected with hFVII Enbrel mRNA mDLNP, serum concentrations continued to increase for up to 48 hours post-injection and remained detectable for up to 168 hours post-injection. hFVII Enbrel mRNA mDLNP showed a >10-fold increase in AUC and a >20-fold increase in Tmax (h) compared with Enbrel protein (107548.73, + / - 4321.8 vs. 8919.09 + / - 4325.51; and 2 hours vs. 40 hours, respectively) (Figure 3G). H&E staining and Ki-67 and Gr-1 IHC staining of skin histological sections were performed in all mice. In addition to the obvious inflammation shown in the mouse images, epidermal thickness and Ki-67 positive cells were also objectively elevated in the mCherry imiquimod-treated group when compared with the lanolin control and hFVII Enbrel mRNA imiquimod groups, thereby suggesting that hFVII Enbrel mRNA mDLNPs can provide significant therapeutic benefit in an in vivo mouse psoriasis model ( Figure 3H-I ).

[0154] Example 4: Tumor immunotherapy with hFVII-anti-PDL1 mRNA Next, we tested the use of the disclosed signal peptides in anticancer polypeptides. For this purpose, we subcutaneously injected two xenograft tumor models: MC38-Luc cells, a highly malignant mouse adenocarcinoma cell line containing a luciferase reporter construct, or B16F10-Luc cells, a mouse melanoma cell line carrying a luciferase reporter construct, into the right hind limb of C57BL6 mice, allowing tumors to grow. To identify the anticancer therapeutic potential of the SP-mRNA LNPs, mice were first inoculated with tumor cells and then intravenously injected with either mDLNPs containing mCherry mRNA or mDLNPs encapsulating mRNA encoding hFVII SP upstream of an anti-PDL1 antibody on days 3, 7, and 11 postinoculation (Figure 4A-B).

[0155] The pharmacokinetic profile of serum anti-PDL1 levels was established, with peak serum concentrations occurring 48 hours after IV injection, essentially mirroring the curve previously observed with hFVII Enbrel mRNA mDLNP. Flow cytometry was also used to determine PDL1 expression on the surface of MC38 cells (Figure 4C-D). Tumor growth was assessed by IVIS imaging for luminescence at days 3, 10, 24, and 32. In the group administered mCherry mDLNP, tumor progression was rapid, with corresponding increases in volume and luminescence clearly evident at each time point. Tumor masses occupied the entire hind limb of each mouse by day 32. However, in the group treated with hFVII anti-PDL1 mRNA mDLNP, tumor growth was significantly reduced by day 24, suggesting that treatment with hFVII anti-PDL1 mRNA mDLNP was effective in inhibiting tumor progression. As expected, this decrease corresponded to decreased luminescence, significantly slower tumor growth, and an overall smaller tumor volume after resection on day 32 (Figure 4E-H). Most notable was the extended survival mediated by hFVII anti-PDL1 mRNA mDLNPs. Mice in this treatment group survived nearly twice as long as mice in the control group (Figure 4I). Similarly, mice inoculated with B16F10-Luc tumors were intravenously injected with mDLNPs containing mCherry mRNA or hFVII anti-PDL1 mRNA. IVIS imaging on days 3 and 16 revealed reduced tumor mass and luminescence, a significantly slower growth rate, and extended overall survival in the hFVII anti-PDL1 mRNA group compared to controls (Figure 4J-N).

[0156] [Example 5: Design of a novel signal peptide sequence] To enable the secretion of proteins that would normally be restricted to the intracellular space into the circulation, we attempted to engineer novel signal peptide (SP) sequences that encode secretion into specific mRNA sequences. To further explore this, we conducted a comprehensive search of known SPs across multiple databases, combined this with current knowledge, and compiled them into a single master database. From there, we classified the known SPs and generated a new list containing the amino acid sequences of 643 naturally occurring SPs identified to date that are associated with secreted proteins. Due to heterogeneity in amino acid sequence length, we sorted the list of 643 sequences by length and grouped sequences of the same length accordingly (i.e., all SP sequences containing 18 amino acids were grouped together, SPs of 19 amino acids in length were grouped together, etc.). After stratifying the signal sequences by length, we created a matrix that allowed us to identify the frequency of each amino acid at each position along the peptide for all sequences of a particular length (see Figure 5). We then selected the most frequent amino acids at each position and concatenated them to form novel amino acid sequences of a particular length. Through this process, we generated 21 novel SP sequences (designated SP1–SP21) that are known not to occur in nature and vary in length from 15 to 35 amino acids (see Table 2 ).

[0157] [Table 2] JPEG2025534282000005.jpg103170

[0158] To determine whether the 21 novel SP sequences could induce secretion, the sequences were integrated upstream of the amino acid sequence encoding human erythropoietin (SEQ ID NO: 58: hEPO without its native signal peptide). However, because hEPO is known to contain its own SP sequence, a pDNA backbone containing hEPO lacking its endogenous SP (NF-NSP-hEPO) was created. Using a reverse codon generation tool, nucleic acid sequences for each of the 21 peptides were synthesized, and then the corresponding oligonucleotides were ordered. Through a series of cloning and PCR reactions, pDNA vectors containing each of the 21 SPs directly upstream of NF-NSP-hEPO were constructed (Figure 6). To ensure that the novel SPs were responsible for potential secretion, a pDNA backbone containing functional hEPO without its SP was constructed as a control. However, the Kozak sequence of hEPO is located upstream of the endogenous SP of hEPO, which was previously removed. Therefore, a Kozak sequence (GCCACCATG) was inserted upstream of the SP-truncated hEPO sequence, which eliminates its secretory ability while allowing hEPO mRNA to be translated (NSP-hEPO). Using the pDNA backbone, in vitro transcription (IVT) reactions were performed for each of the new SP-hEPO and NSP-hEPO sequences to generate mRNAs containing an optimized 5' UTR (SEQ ID NO: 59), a Kozak sequence, a new signal peptide sequence, the hEPO protein sequence, a 3' UTR (SEQ ID NO: 60), and finally an optimized poly(A) tail (SEQ ID NO: 61).

[0159] 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, wt / wt) and administered intravenously to mice at a dose of 0.5 mg / kg. Blood was collected from mice 6, 24, 48, and 72 hours after administration for each construct and analyzed by hEPO enzyme-linked immunosorbent assay (ELISA) to determine serum hEPO concentrations in mIU / mL (Figure 7). Compared to the NSP-hEPO control, the improved differential kinetic profile (C max , t 1 / 2The successful secretion of SP-hEPO with a serotonin concentration (STC) and area under the curve (AUC) was observed.

[0160] array:

[0161] [Table 3] JPEG2025534282000007.jpg255164JPEG2025534282000008.jpg255162JPEG20255342820 00009.jpg255163JPEG2025534282000010.jpg255163JPEG2025534282000011.jpg172170

[0162] References: Hou, X., Zaks, T., Langer, R. et al. Lipid nanoparticles for mRNA delivery. Nature Reviews Materials 6, 1078-1094 (2021). Cheng, Q., Wei, T., Farbiak, L., Johnson, LT, Dilliard, SA & Siegwart, DJ Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nature Nanotechnology 15, 313-320 (2020). 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 30, e1805308 (2018). 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. Nature Materials 20, 701-710 (2021).

Claims

1. A genetically engineered signal peptide comprising the amino acid sequence of any of SEQ ID NOs: 9 to 29, and variants or derivatives thereof.

2. 10. The engineered signal peptide of claim 1 fused to a heterologous polypeptide.

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

4. 4. The engineered signal peptide of claim 3, wherein the heterologous polypeptide is any of an anti-cancer, anti-inflammatory, immunomodulatory, antiviral, antibacterial, antifungal, antihelminthic, cholesterol-lowering, antidiabetic, antifibrotic, analgesic, anesthetic, anti-aging, antidepressant, neuromodulatory, antidermatitis, anti-edema, anti-allergic, antikeratolytic, antifungal, antipruritic, cardiovascular therapeutic, chemotherapeutic, hormone, protein replacement therapy, polypeptide therapeutic, vaccine, viral vaccine, theragnostic, diagnostic, or secreted therapeutic polypeptide.

5. 3. The genetically engineered signal peptide of claim 2, wherein the heterologous polypeptide is an enzyme, a nutraceutical, a food additive, a flavor enhancer, and / or a cosmetic.

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

7. 7. The engineered signal peptide of claim 6, wherein the fluorescent protein is any 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 a signal peptide of any of SEQ ID NOs: 9 to 29.

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

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

11. 10. The recombinant polynucleotide sequence of claim 9, comprising the nucleic acid sequence of any of SEQ ID NOs: 30 to 50, or a variant or derivative thereof.

12. 11. The recombinant polynucleotide sequence of claim 10, comprising a ribonucleic acid sequence corresponding to any of SEQ ID NOs: 30 to 50, or a variant or derivative thereof.

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

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

15. 15. The recombinant polynucleotide of claim 14, wherein the heterologous polypeptide is any anti-cancer, anti-inflammatory, immunomodulatory, antiviral, antibacterial, antifungal, antihelminthic, cholesterol-lowering, antidiabetic, antifibrotic, analgesic, anesthetic, anti-aging, antidepressant, neuromodulatory, antidermatitis, anti-edema, anti-allergic, antikeratolytic, antifungal, antipruritic, cardiovascular therapeutic, chemotherapeutic, hormonal, protein replacement therapy, polypeptide therapeutic, vaccine, viral vaccine, theragnostic, diagnostic or secreted therapeutic polypeptide.

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

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

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

19. 14. The recombinant polynucleotide of 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. a delivery system; and b. i. a signal peptide; and ii. Therapeutic Polypeptides a polynucleotide sequence comprising a nucleic acid sequence encoding A therapeutic composition comprising:

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

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

23. 21. The therapeutic composition of claim 20, wherein the signal peptide is 10 to 50 amino acids in length.

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

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

26. 26. The therapeutic composition of claim 25, wherein the lipid nanoparticles further comprise one or more of a phospholipid, cholesterol, or a polymeric lipid.

27. 27. The therapeutic composition of any one of claims 24-26, wherein the delivery system comprises any of iPhos LNPs, mDLNPs, liver SORT LNPs, lung SORT LNPs, or spleen SORT LNPs.

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

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

30. 21. The therapeutic composition of claim 20, wherein the therapeutic polypeptide is any of an anti-cancer, anti-inflammatory, immunomodulatory, antiviral, antibacterial, antifungal, antihelminthic, cholesterol-lowering, antidiabetic, antifibrotic, analgesic, anesthetic, anti-aging, antidepressant, neuromodulatory, antidermatitis, anti-edema, anti-allergic, antikeratolytic, antifungal, antipruritic, cardiovascular therapeutic, chemotherapeutic, hormone, protein replacement therapy, polypeptide therapeutic, vaccine, viral vaccine, theragnostic, diagnostic, or secreted therapeutic polypeptide.

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

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

33. 32. The diagnostic, preventative, or therapeutic method of claim 31, wherein the administration is via a controlled system selected from an implant, a synthetic material depot, a polymer depot, a lipid depot, a slow-release hydrogel depot, or a slow-release polymer depot.

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

35. 32. The diagnostic, prophylactic, or therapeutic method of claim 31, wherein the subject is in need of prevention.

36. 32. The diagnostic, prophylactic, or therapeutic method of claim 31, wherein the therapeutic polypeptide of claim 15 is secreted systemically in the subject in need thereof.

37. 32. The method of diagnosis, prevention, or treatment of claim 31, wherein the therapeutic polypeptide in the therapeutic composition of claim 21 is directed for expression in either the lung, liver, or spleen.

38. 32. The method of claim 31 , wherein the subject is a mammal.

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

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

41. i. a signal peptide corresponding to any of SEQ ID NOs: 1-4 or a variant or derivative thereof; and ii. Heterologous Polypeptides A recombinant polypeptide comprising:

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

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

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

45. 44. The recombinant polynucleotide sequence of claim 43, comprising a nucleic acid sequence corresponding to any of SEQ ID NOs: 5 to 8, or a variant or derivative thereof.

46. 45. The recombinant polynucleotide sequence of claim 44, comprising a ribonucleic acid sequence corresponding to SEQ ID NOs: 5 to 8, or a variant or derivative thereof.

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

48. 48. The recombinant polynucleotide of claim 47, wherein the heterologous polypeptide is any anti-cancer, anti-inflammatory, immunomodulatory, antiviral, antibacterial, antifungal, antihelminthic, cholesterol-lowering, antidiabetic, antifibrotic, analgesic, anesthetic, anti-aging, antidepressant, neuromodulatory, antidermatitis, antiedema, antiallergic, antikeratolytic, antifungal, antipruritic, cardiovascular therapeutic, chemotherapeutic, hormonal, protein replacement therapy, polypeptide therapeutic, vaccine, viral vaccine, theragnostic, diagnostic or secreted therapeutic polypeptide.

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

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

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

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

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

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

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

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

57. 57. The therapeutic composition of claim 56, wherein the lipid nanoparticles further comprise one or more of a phospholipid, cholesterol, or a polymeric lipid.

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

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

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

61. 53. The therapeutic composition of claim 52, wherein the therapeutic polypeptide is an anti-cancer, anti-inflammatory, immunomodulatory, antiviral, antibacterial, antifungal, antihelminthic, cholesterol-lowering, antidiabetic, antifibrotic, analgesic, anesthetic, anti-aging, antidepressant, neuromodulatory, antidermatitis, anti-edema, anti-allergic, antikeratolytic, antifungal, antipruritic, cardiovascular therapeutic, chemotherapeutic, hormone, protein replacement therapy, polypeptide therapeutic, vaccine, viral vaccine, theragnostic, diagnostic, or secreted therapeutic polypeptide.

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

63. 63. The method of diagnosis, prevention, or treatment of claim 62, wherein the administration is via one or more of parenteral, oral, intraadipose, intra-arterial, intra-articular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrarectal, intrathecal, intratracheal, intratumoral, intraumbilical, intravaginal, intravenous, intravascular, intravitreal, liposomal, topical, mucosal, parenteral, rectal, subconjunctival, subcutaneous, sublingual, topical, transbuccal, or transdermal routes.

64. 64. The diagnostic, preventative, or therapeutic method of claim 63, wherein the administration is via a controlled system selected from an implant, a synthetic material depot, a polymer depot, a lipid depot, a slow-release hydrogel depot, or a slow-release polymer depot.

65. 64. The method of diagnosis, prevention, or treatment of claim 63, wherein the subject is suspected of having or has been diagnosed with any of an autoimmune disease, cancer, diabetes, cardiovascular disease, neurological disease, bacterial infection, fungal infection, viral infection, or fibrosis.

66. 64. The diagnostic, prophylactic, or therapeutic method of claim 63, wherein the subject is in need of prevention.

67. 64. The method of treatment of claim 63, wherein the therapeutic polypeptide of claim 52 is secreted systemically in the subject in need thereof.

68. 64. The diagnostic, prophylactic, or therapeutic method of claim 63, wherein the therapeutic polypeptide of claim 52 is directed for expression in either the lung, liver, or spleen.

69. 64. The method of claim 63, wherein the subject is a mammal.

70. 64. The method of claim 63, wherein the subject is a human.

71. 62. Use of a composition according to any one of claims 52 to 61 for the treatment of a subject in need thereof.

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

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

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

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