Endogenous endoplasmic reticulum targeting signal peptide and its use

Abstract

Novel recombinant polypeptides comprising endogenous signal peptides and payload proteins that target the endoplasmic reticulum of cells, nucleic acid molecules encoding them, pharmaceutical compositions containing them, and methods of using them are provided herein. TIFF2026536591000008.tif92159

Description

[Technical Field]

[0001] Related applications This application claims the benefits of U.S. Provisional Application No. 63 / 592,479 filed on 23 October 2023 and U.S. Provisional Application No. 63 / 605,403 filed on 1 December 2023, each of which is incorporated herein by reference as a whole.

[0002] Sequence List This application includes references to amino acid sequences and / or nucleic acid sequences, which are submitted together with this specification as a sequence listing XML file titled "UTSD 4337_Sequence Listing.xml", created on 23 October 2024, with a file size of 110,592 bytes. The aforementioned sequence listing is incorporated herein by reference in its entirety, in accordance with 37 CFR §1.52(e)(5).

[0003] field This disclosure relates, in general, to endogenous signal peptides, and more specifically, to endogenous signal peptides for targeting encoded proteins to the endoplasmic reticulum. [Background technology]

[0004] background Recent scientific discoveries highlight the countless therapeutic applications of mRNA, stemming from its modularity and ability to provide customizable "directives" to create functional proteins. Clinical trials continue to confirm its favorable efficacy, further highlighting its potential as a next-generation genetic medicine, and scientists are only beginning to explore the surface of its potential applications. Most research so far has focused on amplifying the intracellular expression of encoded polypeptides, while less focus has been placed on the post-translational regulation of encoded proteins and their targeted localization and / or secretion within the body.

[0005] Within cells, there are various specific means and processes that enable the shuttle of proteins to different organelles and the transport of proteins to the extracellular space via efflux pathways. However, to utilize these transport systems, the mRNA encoding each protein must also contain signal peptides (SPs), which are essentially shipping labels, upstream of the protein sequence. Incorporation of known endogenous SPs into therapeutic mRNA could greatly benefit the delivery, localization, and clearance of the encoded therapeutic protein. This disclosure addresses these and other needs. [Overview of the project]

[0006] overview In some embodiments, recombinant polypeptides are provided. In some embodiments, the recombinant polypeptide is of formula X1-(Y1) a -Z1 is included in the formula, where X1 is an endogenous signal peptide, Y1 is a peptide linker, Z1 is a payload protein, and a is an integer selected from 0 and 1.

[0007] In some embodiments, X1 is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, It contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from the group consisting of 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119.

[0008] In some embodiments, the payload protein is a therapeutic peptide or protein.

[0009] In some embodiments, nucleic acid molecules are provided. In some embodiments, the nucleic acid molecules encode recombinant polypeptides provided herein.

[0010] In some embodiments, a vector is provided. In some embodiments, the vector comprises a nucleic acid molecule provided herein.

[0011] In some embodiments, cells are provided. In some embodiments, the cells include nucleic acid molecules provided herein. In some embodiments, the cells include vectors provided herein.

[0012] In some embodiments, a composition is provided. In some embodiments, the composition comprises nucleic acid molecules provided herein. In some embodiments, the composition comprises vectors provided herein.

[0013] In some embodiments, methods are provided for treating a disease or disorder in a subject where such treatment is necessary. In some embodiments, the method comprises the step of administering to a subject an effective amount of a nucleic acid molecule provided herein, thereby treating the disease or disorder.

[0014] In some aspects, the disease or disorder is cancer.

[0015] In some embodiments, methods are provided for treating cancer in subjects where such treatment is needed. In some embodiments, the method comprises the step of administering to a subject a vector comprising a nucleic acid molecule encoding a signal peptide fused to or linked to a payload protein, wherein the signal peptide is an endogenous signal peptide, and the payload protein is a therapeutic peptide or protein useful for treating cancer. In some embodiments, the endogenous signal peptide is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, It contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from the group consisting of 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119.

[0016] In some embodiments, the use of an endogenous signal peptide is provided. In some embodiments, the use comprises the step of targeting a payload protein to a desired intracellular location, the payload protein not naturally containing an amino acid sequence that includes the amino acid sequence of the endogenous signal peptide. In some embodiments, the endogenous signal peptide is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, It contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from the group consisting of 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. [Brief explanation of the drawing]

[0017] [Figure 1A] This paper provides a construct containing mCherry with a signal peptide at its N-terminus, and a schematic diagram of its use for signal peptide screening by in vitro pDNA transfection. [Figure 1B] Representative images of signal peptide-tagged mCherry cells co-localized with ER-Tracker Blue-White DPX are shown. Top: SP is Nat-EMO-18-1 (SEQ ID NO: 10). Bottom: SP is Nat-EMO-21-1 (SEQ ID NO: 40). [Figure 1C]Representative images of signal peptide-tagged mCherry colocalized with ER-Tracker Blue-White DPX are illustrated. Upper: SP is Nat-EMO-19-1 (SEQ ID NO: 21). Lower: SP is Nat-EMO-24-1 (SEQ ID NO: 72). [Figure 1D] Representative images of signal peptide-tagged mCherry colocalized with ER-Tracker Blue-White DPX are illustrated. Upper: SP is Nat-EMO-20-1 (SEQ ID NO: 32). Lower: SP-free control. [Figure 1E] Quantification of experiments corresponding to the representative images of Figures 1B, 1C, and 1D is illustrated. [Figure 1F] Quantification of experiments corresponding to the representative images of Figures 1B, 1C, and 1D is illustrated. [Figure 2A] Quantification of mCherry fluorescence in HeLa cell lysates and culture medium at different time points is provided. mCherry without a signal peptide was used as a control ("WT-mCherry"). gLuc, Gaussia luciferase. [Figure 2B] Quantification of mCherry fluorescence in HeLa cell lysates and culture medium after 72 hours is provided. [Figure 2C] Quantification of mCherry fluorescence in Huh7 cell lysates and culture medium at 72 hours post-transfection is provided. [Figure 3A] Quantification of time-dependent (100 ng mRNA / well) and dose-dependent (at 72 hours) mCherry secretion in Huh7 cells, culture medium, and cell lysates is provided. [Figure 3B] Quantification of mCherry signal in different cell lines is shown. Cells in 96-well plates were treated with mDLNP-mRNA at the indicated time points. MODES FOR CARRYING OUT THE INVENTION

[0018] Detailed Description Prior to describing the compositions and methods herein, it will be understood that the scope of the invention is not limited to the specific processes, compositions, or methodologies described herein, as these may vary. It will also be understood that the terminology used herein is for the purpose of describing only specific versions or embodiments and is not intended to limit the scope of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing embodiments of the methods and systems disclosed herein, and such equivalents will be understood to be within the scope of this disclosure.

[0019] definition The following explanations of terms and methods are provided to accurately describe this disclosure and to guide those skilled in the art in implementing this disclosure.

[0020] Where used herein, “comprising” means “including,” and the singular forms “a,” “an,” or “the” include multiple references unless otherwise explicitly stated in the context. For example, a reference to “including therapeutic agents” includes one or more such therapeutic agents. The term “or” refers to a single element of the alternative elements described unless otherwise explicitly stated in the context. For example, the phrase “A or B” refers to A alone or B alone. The phrase “A, B, or a combination thereof” refers to A alone, B alone, or a combination of A and B. Similarly, “one or more of A and B” refers to a combination of A, B, or both A and B. The phrase “A and B” refers to a combination of A and B. Furthermore, the various elements, features, and processes discussed herein, as well as other known equivalents of each such element, feature, and process, can be combined and adapted by those skilled in the art to carry out the methods according to the principles described herein. Some of the various elements, characteristics, and processes may be specifically included in certain examples, while others may be specifically excluded.

[0021] In some examples, it will be understood that numbers expressing quantities such as components, properties like molecular weight, or reaction conditions, used to describe and claim a particular embodiment, may, in some cases, be modified by the terms “about” or “approximately.” For example, “about” or “approximately” may indicate a variation of + / -10%, + / -5%, or + / -1% of the value it describes. Thus, in some embodiments, the numerical parameters presented herein are approximations that may vary depending on the properties desired for a particular embodiment. In addition, where the phrase “about x ~ y” is used, the term “about” modifies both x and y and can be used interchangeably with the phrase “about x ~ about y” unless otherwise stated in context.

[0022] As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of techniques recognized in the art for introducing foreign nucleic acids into host cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection (e.g., using commercially available reagents such as LIPOFECTIN® (Invitrogen Corp., San Diego, CA), LIPOFECTAMINE® (Invitrogen), FUGENE® (Roche Applied Science, Basel, Switzerland), JETPEI® (Polyplus-transfection Inc., New York, NY), EFFECTENE® (Qiagen, Valencia, CA), DREAMFECT® (OZ Biosciences, France), and similar), or electroporation (e.g., in vivo electroporation). Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals.

[0023] Methods and materials for nonviral delivery of nucleic acids to cells include bioristic methods, virosomes, liposomes, lipid nanoparticles, immunoliposomes, polycationic or lipid-nucleic acid complexes, naked DNA, artificial virions, and drug-enhanced uptake of DNA. Lipofection is described in U.S. Patents 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., TRANSFECTAM® and LIPOFECTIN®). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those disclosed in WO91 / 17424 and WO91 / 16024.

[0024] The chemical composition of peptides is described herein by a series of single-letter amino acid abbreviations or by "amino acid sequence" or "sequence," which are conventional and known to those skilled in the art. Reference sequences are expressly disclosed, but in any aspect and embodiment, reference sequences may be modified to include conservative amino acid substitutions, variants, and fragments while maintaining the characteristics and functionality of the reference sequence.

[0025] As used herein, “payload protein” or “protein of interest” refers to a protein produced by a host cell and chaperoned to a desired intracellular location, for example, to the endoplasmic reticulum. Upon transposition to the appropriate intracellular space, all of the endogenous signal peptides may be fused to the payload protein, some of the endogenous signal peptides may be fused to the payload protein, or none of the endogenous signal peptides may be fused to the payload protein. Optionally, a payload protein that remains partially or completely attached to the endogenous signal peptides may be further processed, for example, to remove any remaining endogenous signal peptides. The payload protein can be any known or unknown protein, such as an enzyme, enzyme inhibitor, growth factor, hormone, antibody, antigen, vaccine, therapeutic agent, or any combination thereof. More specific examples are described below herein.

[0026] As used herein, the terms “fused” or “linked” mean that protein domains connected to each other by either peptide bonds or other covalent bonds are part of the same peptide chain, when used in reference to proteins having different domains or heterologous sequences. Domains or sections may be directly linked or fused to each other, or there may be another domain or peptide sequence between the two domains or sequences, and such sequences will still be considered fused or linked to each other. In some embodiments, the various domains or proteins provided herein may be directly linked or fused to each other, or a linker sequence, such as a glycine / serine sequence described herein, may link the two domains together.

[0027] When used herein, "identity" refers to the subunit sequence identity between two nucleic acid molecules or amino acid molecules, or between two polymer molecules, such as between two polynucleotide molecules or polypeptide molecules. Two amino acid sequences are identical at the same position; for example, if a position in each of two polypeptide molecules is occupied by arginine, then they are identical at that position. The identity or degree to which two amino acid sequences or two nucleic acid sequences have the same residue at the same position in alignment is often expressed as a percentage. The identity between two amino acid sequences or two nucleic acid sequences is a linear function of the number of matching positions or the number of identical positions; for example, if half of the positions in the two sequences are identical, then the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) match or are identical, then the two amino acid sequences are 90% identical.

[0028] "Substantially identical" means that a polypeptide or nucleic acid molecule exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or a reference nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). In some embodiments, such sequences are at least 60%, 80%, or 85%, or 90%, 95%, or even 99%, identical to the sequence used for comparison at the amino acid level or nucleic acid level. Other identity percentages for specific sequences are also described herein.

[0029] Sequence identity can be measured / determined using sequence analysis software (Sequence Analysis Software Package from Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, such as BLAST, BESTFIT, GAP, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTALOMEGA, MUSCLE, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. An exemplary approach to determining the degree of identity may involve using the BLAST program, where probability scores between e3 and e100 indicate closely related sequences. In some embodiments, sequence identity is determined by using BLAST with default settings. In some embodiments, sequence identity is determined using Clustal Omega.

[0030] To the extent that the embodiments provided herein include compositions comprising various proteins, such proteins may, in some cases, include amino acid sequences having sequence identity with the amino acid sequences disclosed herein. Therefore, in certain embodiments, depending on the specific sequence, the degree of sequence identity is preferably greater than 50% (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) with respect to the SEQ ID NO disclosed herein. In addition to these percentages, other identity percentages are also provided herein. Identity between polypeptides can be determined by the Smith-Waterman homology search algorithm performed in the MPSRCH program (Oxford Molecular) using affine gap search with gap-open penalty = 12 and gap-extension penalty = 1 parameters.

[0031] These proteins may contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) conserved amino acid substitutions, i.e., substitutions of certain amino acids with another having the relevant side chain, compared to the proteins of this disclosure. Genetically encoded amino acids can generally be divided into four families: (1) acidic, i.e., aspartic acid, glutamic acid; (2) basic, i.e., lysine, arginine, histidine; (3) nonpolar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) non-charged, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. Generally, single amino acid substitutions within these families do not have a significant impact on biological activity. The protein may have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single amino acid deletions compared to the protein sequence of the disclosure. The protein may also have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) insertions (e.g., each of 1, 2, 3, 4, or 5 amino acids) compared to the protein sequence of the disclosure.

[0032] "Code" refers to the inherent properties of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, and the biological properties that arise therefrom, that enable it to function in biological processes as a template for the synthesis of other polymers and macromolecules having either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence. Therefore, a gene codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, which is identical to the mRNA sequence and is typically provided in a sequence listing, and the non-coding strand, which is used as a template for the transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0033] In this specification, the following abbreviations are used for commonly occurring nucleic acid bases: "A" for adenosine, "C" for cytosine, "G" for guanosine, "T" for thymidine, and "U" for uridine.

[0034] Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" include all nucleotide sequences that encode the same amino acid sequence, including degenerate versions of each other. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns, insofar as the nucleotide sequence encoding a protein may contain introns in some versions.

[0035] The term "oligonucleotide" typically refers to a short polynucleotide. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), it will be understood that this also provides a corresponding RNA sequence (i.e., A, U, C, G) where "U" replaces "T".

[0036] The term "polynucleotide," as used herein, is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, the terms "nucleic acid" and "polynucleotide" are interchangeable as used herein. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any method available in the art, including, but not limited to, recombinant methods, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using cloning techniques and PCR and similar methods, and by synthetic means.

[0037] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to compounds composed of multiple amino acid residues covalently linked by peptide bonds. As used herein, these terms refer to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, generally referred to in the art as proteins, of which there are many types. A “polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0038] As used herein, unless otherwise specified, "antibody fragment" or "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, that is, an antibody fragment that retains the ability to specifically bind to an antigen bound by a full-length antibody, for example, a fragment that retains one or more CDR regions. Examples of antibody-binding fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')₂ fragments, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies (single domain antibodies), as well as multispecific antibodies composed of antibody fragments.

[0039] As used herein, the term "endogenous", when used in reference to a nucleic acid molecule or polypeptide, refers to a nucleic acid molecule or protein that is naturally found in a host organism or cell, or naturally found in an organism or cell of the same species as the host organism.

[0040] A "Fab fragment" is composed of one light chain, as well as the C H 1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0041] An "Fc" region contains two heavy chain fragments comprising the C H 2 domain and C H 3 domain of an antibody. The two heavy chain fragments are held together by more than one disulfide bond and the hydrophobic interactions of the C H 3 domain.

[0042] A "Fab' fragment" contains one light chain, as well as a portion or fragment of one heavy chain that contains the V H domain and the C H 1 domain, and also contains the region between the C H 1 domain and the C H 2 domain, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')₂ molecule.

[0043] The "F(ab')2 fragment" consists of two light chains, as well as C H 1 domain and C H It contains two heavy chains that include a portion of the constant region between the two domains, resulting in the formation of an interchain disulfide bond between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0044] The "Fv region" includes variable regions derived from both heavy and light chains, but lacks a steady region.

[0045] The terms "single-stranded Fv" antibody or "scFv" antibody refer to the V of the antibody. H Domain and V L This refers to antibody fragments containing domains, which are present within a single polypeptide chain. Generally, Fv polypeptides have a polypeptide linker that allows scFv to form a structure desirable for antigen binding. H Domain and V L Further inclusions are made between the domains. For a review of scFv, see Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269–315. See also International Publication No. WO 88 / 01649 and U.S. Patents Nos. 4,946,778 and 5,260,203.

[0046] Antibody molecules may have single specificity (e.g., monovalent or bivalent), bivalent specificity (e.g., bivalent, trivalent, tetravalent, pentavalent, or hexavalent), triple specificity (e.g., trivalent, tetravalent, pentavalent, or hexavalent), or higher specificity (e.g., quadruple specificity) and / or higher titer than hexavalent. Antibody molecules may contain functional fragments of the light chain variable region and functional fragments of the heavy chain variable region, or the heavy and light chains may be fused together to form a single polypeptide.

[0047] As used herein, “comprising” (and any form of “comprising,” such as “comprise,” “comprises,” and “comprised”), “having” (and any form of “having,” such as “have,” and “has”), “including” (and any form of “including,” such as “includes,” and “include”), or “containing” (and any form of “containing,” such as “contains,” and “contain”) are inclusive or non-exclusive and do not exclude any additional unlisted elements or process steps. Any process or composition using the transitional phrase “comprise” or “comprising” can also be said to be described using the transitional phrase “consisting of” or “consists.”

[0048] As used herein, the term “contact” means bringing two elements together in an in vitro or in vivo system. For example, “contact” of a virus or vector described herein with an individual, patient, or cell includes administering the virus to an individual or patient, such as a human, and introducing a compound into a sample containing, for example, a cell preparation or purified preparation containing cells.

[0049] Where used herein, the terms “individual,” “subject,” or “patient” are interchangeable and mean any animal, including mammals such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, or primates such as humans. In some embodiments, the subject is a human. The “subject” that “needs” refers to a condition to be treated, and the subject is identified as one for whom treatment is required, with a particular intention to treat such a condition. The condition may be, for example, any of the conditions described herein.

[0050] The compositions disclosed herein may be administered to a subject in various ways through the administration of the composition to the subject. As used herein, administering or giving means giving or giving the composition to a subject. Oral administration, as used herein, refers to the delivery of the activator by mouth. Topical administration, as used herein, refers to the delivery of the activator to a body surface such as the skin or mucous membrane (e.g., nasal mucosa, vaginal mucosa, oral mucosa, or similar).

[0051] A "disease" is a state of animal health in which the animal is unable to maintain homeostasis and, if the disease does not improve, the animal's health will continuously deteriorate. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but the animal's health is less favorable than what would be expected in the absence of the disorder. Leaving a disorder untreated does not necessarily lead to a further deterioration of the animal's health.

[0052] "Effective dose" or "therapeutic effective dose" is used interchangeably herein and refers to the amount of a compound, formulation, material, or composition described herein that is effective in achieving a particular biological outcome or providing a therapeutic or prophylactic benefit. Such outcomes may include, but are not limited to, an amount that, when administered to a mammal, elicits a detectable level of immune cell activation compared to the immune cell activation detected in the absence of the composition. The immune response can be readily assessed by many methods known in the art. The amount of composition administered herein will vary and can be readily determined based on several factors, including the disease or condition being treated, the age and health and physical condition of the treated mammal, the severity of the disease, the specific compound being administered, and similar factors.

[0053] Scope: Throughout this disclosure, various aspects of the embodiments may be presented in range form. It should be understood that range form descriptions are merely for convenience and conciseness and should not be interpreted as immutable limitations. Therefore, range descriptions should be interpreted as specifically disclosing all possible subranges and individual numbers within that range. For example, a range description such as 1-6 should be considered to specifically disclose subranges within that range, such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and individual numbers such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range. Unless otherwise explicitly stated to be inconsistent, the disclosed range includes the endpoints of the range.

[0054] Endogenous signal peptides While not bound by any particular theory, the embodiments provided herein have been found to demonstrate that by placing an endogenous signal peptide before a desired payload protein, the payload protein can be appropriately guided and expressed in the endoplasmic reticulum. The endogenous signal peptides provided herein can guide the payload protein to any suitable location in or within the rough endoplasmic reticulum, the smooth endoplasmic reticulum, or both. The payload protein may or may not naturally contain a signal peptide that guides the localization of the protein to a specific cellular compartment, such as the endoplasmic reticulum. Thus, the provided endogenous signal peptides can lead to enhanced expression of proteins that are normally delivered to or from the endoplasmic reticulum, or can guide the expression of a payload protein, such as a therapeutic protein, to the endoplasmic reticulum for expression. By using the endogenous signal peptides provided herein, the payload protein can be substantially expressed in the endoplasmic reticulum or further processed and delivered to another cellular compartment.

[0055] In some embodiments, a signal peptide is provided. In some embodiments, the signal peptide comprises the amino acid sequence listed in Table 1 below.

[0056] (Table 1) Exemplary endogenous signal peptides TIFF2026536591000002.tif144128TIFF2026536591000003.tif252127TIFF2026536591000004.tif254127TIFF2026536591000005.tif158128

[0057] In some embodiments, the signal peptide contains an amino acid sequence substantially similar to the amino acid sequences listed in Table 1. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% (e.g., 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%) identical to the amino acid sequences listed in Table 1. In some embodiments, the signal peptide has SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 ,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87 , 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119 contains an amino acid sequence that is at least 90% (e.g., 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%) identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4.In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20.In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 36.In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 39. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 42. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 45. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 49. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 52.In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 54. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 55. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 56. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 59. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 61. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 64. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 65. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 66. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 67. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 68.In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 69. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 70. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 71. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 76. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 79. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 81. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 82. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 83. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 84. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 85. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 86. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 87. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 88. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 90. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 91. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 92. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 94. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 95. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 96.In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 100. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 101. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 102. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 104. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 105. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 106. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 107. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 109. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 110. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 111.In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 114. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 115. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 116. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 117. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 118. In some embodiments, the signal peptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 119.

[0058] Recombinant polypeptides In some embodiments, recombinant polypeptides are provided, comprising formulas X1-Z1, where X1 is a signal peptide provided herein and Z1 is a payload protein.

[0059] In some embodiments, X1 is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, The amino acid sequence includes an amino acid sequence having at least 90% identity (e.g., 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%, or at least 99%) to an amino acid sequence selected from the group consisting of 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 7.In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 13. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 16. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 18. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 19. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 20. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 21. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 22.In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 23. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 24. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 27. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 28. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 29. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 30. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 31. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 32. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 33. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 34. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 36. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 37.In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 38. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 39. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 40. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 41. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 42. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 43. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 44. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 45. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 46. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 47. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 48. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 49. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 50. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 51. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 52.In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 54. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 55. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 56. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 57. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 58. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 59. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 60. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 61. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 62. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 63. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 64. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 65. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 66. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 67.In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 68. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 69. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 70. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 71. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 74. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 75. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 76. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 77. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 78. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 81. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 82. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 83. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 84. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 85. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 86. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 87. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 88. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 89. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 90. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 91. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 92. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 93.In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 94. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 95. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 96. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 97. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 98. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 99. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 100. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 101. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 102. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 103. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 104. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 105. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 106. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 107. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 108.In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 109. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 110. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 111. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 112. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 113. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 114. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 115. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 116. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 117. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 118. In some embodiments, X1 includes an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 119.

[0060] In some embodiments, payload protein Z1 can be any peptide or protein. In some embodiments, payload protein Z1 can be any peptide or protein useful in treating a disease or disorder. In some embodiments, payload protein Z1 can be any peptide or protein useful in treating a disease or disorder associated with the endoplasmic reticulum of a cell. In some embodiments, payload protein Z1 can be any peptide or protein whose expression in the endoplasmic reticulum would be advantageous for the proper folding or production of the protein. For example, a payload protein that may be expressed at low levels due to misfolding may show increased expression if delivered to the endoplasmic reticulum for proper folding and / or production. Therefore, in some embodiments, payload protein Z1 can be any peptide or protein whose increased expression is desirable.

[0061] In some embodiments, X1 is directly linked to Z1. In some embodiments, X1 is indirectly linked to Z1, for example, via a polypeptide linker. Polypeptide linkers are known in the art, and any such linker may be incorporated into the recombinant polypeptide of this disclosure. Thus, in some embodiments, the recombinant polypeptide is of the formula X1-(Y1) a - may be represented by Z1, where X1 is a signal peptide provided herein, Y1 is a linker, such as but not limited to a polypeptide linker, Z1 is a payload protein provided herein, and a is an integer selected from 0 or 1. In some embodiments, X1 is an endogenous signal peptide provided herein.

[0062] nucleic acid molecule In some embodiments, nucleic acid molecules are provided. In some embodiments, the nucleic acid molecule encodes a signal peptide provided herein. In some embodiments, the nucleic acid molecule encodes an endogenous signal peptide provided herein. In some embodiments, the nucleic acid molecule is a deoxyribonucleotide sequence (DNA). In some embodiments, the nucleic acid molecule is a ribonucleic acid sequence (RNA).

[0063] In some embodiments, nucleic acid molecules are SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 9 The signal peptide comprises an amino acid sequence having at least 90% identity (e.g., 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%, or at least 99%) to an amino acid sequence selected from the group consisting of 2, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 5.In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 17.In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 29.In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 39. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 41.In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 42. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 45. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 49. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 52. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 53.In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 54. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 55. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 56. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 57. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 59. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 61. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 64. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 65. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 66.In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 67. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 68. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 69. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 70. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 71. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 76. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 78.In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 81. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 82. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 83. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 84. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 85. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 86. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 87. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 88. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 90.In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 91. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 92. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 94. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 95. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 96. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 100. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 101. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 102.In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 104. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 105. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 106. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 107. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 109. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 110. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 111. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the nucleic acid molecule encodes a signal peptide containing an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 114.In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 115. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 116. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 117. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 118. In some embodiments, the nucleic acid molecule encodes a signal peptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 119.

[0064] In some embodiments, the nucleic acid molecule encodes a recombinant polypeptide provided herein. In some embodiments, the nucleic acid molecule encodes a recombinant polypeptide comprising formula X1-Z1, where X1 is a signal peptide provided herein and Z1 is a payload protein provided herein. In some embodiments, the nucleic acid molecule encodes a recombinant polypeptide comprising formula X1-(Y1) a - Encodes a recombinant polypeptide comprising Z1, where X1 is a signal peptide provided herein, Y1 is a linker, such as but not limited to a polypeptide linker, Z1 is a payload protein provided herein, and a is an integer selected from 0 or 1.

[0065] Those skilled in the art will be able to readily deduce a suitable DNA or RNA sequence based on the amino acid sequences and variant forms provided herein. Furthermore, due to the degenerate nature of codons, those skilled in the art will readily recognize that multiple nucleic acid molecules can be used to encode the same amino acid sequence. Therefore, any nucleic acid sequence capable of encoding the amino acid sequences provided herein is within the scope of this disclosure.

[0066] In some embodiments, the nucleic acid molecules provided herein are One or more elements, selected from, but not limited to, promoters, enhancers, leaders, transcription start sites (TSS), linkers, 5' and 3' untranslated regions (UTR), Kozak sequences, introns, polyadenylation signals, cap sequences, enhancers, viral sequences, IRES sequences, or termination regions, which are suitable or necessary for regulating or enabling the expression of recombinant polypeptides provided herein in cells. This may further include the following. 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 to the start codon, but does not include the start codon; on the other hand, the 3'UTR begins immediately after the stop codon and continues to the transcription termination signal. By incorporating regulatory features of UTRs into the polynucleotides of this disclosure, for example, molecular stability can be enhanced. By incorporating specific features, it may also be possible to ensure control of downregulation when transcripts are mistakenly directed to undesirable organ sites. In some embodiments, any suitable naturally occurring or synthetic UTR sequence can be incorporated into the nucleic acid molecules disclosed herein. Other non-UTR sequences may also be incorporated into the nucleic acid molecules. For example, an intron or a portion of an intron sequence may be incorporated into a region of the nucleic acid molecule of this disclosure. Incorporation of an intron sequence may increase protein production and polynucleotide levels. Combinations of features may be included in adjacent regions or contained within other features. For example, the ORF may be adjacent to a 5'UTR that may contain a potent Kozak translation initiation signal and / or a 3'UTR that may contain an oligo(dT) sequence for template-supporting polyA tail addition. The 5'UTR may contain a first polynucleotide fragment and a second polynucleotide fragment derived from the same and / or different genes.

[0067] In some embodiments, the nucleic acid molecules disclosed herein may be constructed inside a cell. In some embodiments, the nucleic acid molecules may be synthesized in vivo. In some embodiments, the nucleic acid molecules may be synthesized in vitro using methods known in the art, such as in vitro transcription, DNA, RNA, and cDNA synthesis methods. In some embodiments, the nucleic acid molecules disclosed herein may be incorporated into suitable viral vectors, expression cassettes, expression vectors, transposons, extrachromosomal elements, and incorporated into chromosomes, host cells, and delivery systems.

[0068] In some embodiments, the nucleic acid molecule is a chemically modified nucleic acid molecule. In some embodiments, the nucleic acid molecule may contain one or more modified nucleosides containing a modified sugar moiety. Such compounds containing one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability, compared to oligonucleotides containing only 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 substitute. Such a sugar substitute may contain one or more substitutions corresponding to those of the substituted sugar moiety. In some embodiments, the modified nucleic acid molecule may contain a modified skeleton such as, for example, a phosphorothioate, phosphotriester, morpholino, methylphosphonate, short-chain alkyl or cycloalkyl sugar junction, or short-chain heteroatom or heterocyclic sugar junction.

[0069] In some embodiments, the modified sugar moiety is a substituted sugar moiety comprising one or more uncrosslinked sugar substituents, including but not limited to substituents at the 2' and / or 5' positions. Examples of preferred sugar substituents at the 2' position include, but are not limited to, 2'-F, 2-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE"). In certain aspects, the sugar substituent at the 2' position is selected from allyl, amino, azide, thio, O-allyl, O-C1~C10 alkyl, O-C1~C10 substituted alkyl; OCF3, O(CH2)2SCH3, O(CH2)2-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 includes one or more non-crosslinked sugar substituents, such as the TF-5'-methyl sugar moiety.

[0070] Nucleosides containing a 2'-substituted sugar moiety are referred to as 2'-substituted nucleosides. In some embodiments, 2'-substituted nucleosides include 2'-substituted substituents selected from halo, allyl, amino, azide, SH, CN, OCN, CF3, OCF3, 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 CI-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.

[0071] In some embodiments, the 2'-substituted nucleoside comprises a 2'-substituent selected from F, NH2, N3OCF3, O-CH3, O(CH2)3NH2, CH2-CH=CH2, OCH2-CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O-(CH2)2-ON(Rm)(Rn), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (O-CH2-C(=O)-N(Rm)(Rn), where each Rm and R independently comprises H, an amino protecting group, or a substituted or non-substituted group. The substituted C1-C10 alkyl groups are present. In some embodiments, the 2'-substituted nucleoside comprises a sugar moiety containing a 2'-substituent selected from F, OCF3, O-CH3, O2CH2OCH3, 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, the 2'-substituted nucleoside comprises a sugar moiety containing a 2'-substituent selected from F, O-CH3, and OCH2CH2OCH3.

[0072] Certain modified sugar moieties contain bridging sugar substituents that form a second ring resulting in a bicyclic sugar moiety. In some such cases, the bicyclic sugar moiety includes a bridge between the 4'-furanose ring atom and the 2'-furanose ring atom. Examples of such 4'-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)-0-2'(LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-0-2'(ENA); 4'-CH(CH3)-0-2'(cEt) and 4'-CH(CH2OCH3)-0-2', and their analogues (see, for example, U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)-0-2' and its analogues (see, for example, WO2009 / 006478). See also); 4'-CH2-N(OCH3)-2' and its analogues (see, for example, WO2008 / 150729); 4'-CH2-ON(CH3)-2' (see, for example, US2004 / 0171570 published September 2, 2004); 4'-CH2-ON(R)-2' and 4-CH2-N(R)-0-2'-, where each R is independently H, a protecting group, or a C1-C12 alkyl group; 4-CH2-N(R)-0-2', where R is H, a C1-C12 alkyl group, or a protecting group (see, for example, US Patent No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, for example, Chattopadhyaya See et al., J. Org. Chern., 2009, 74, 118-134); as well as 4-CH2-C(=CH2)-2' and its analogues (see PCT international application WO2008 / 154401), but not limited thereto.

[0073] In some embodiments, such 4'-2' crosslinks independently comprise 1-4 linking groups independently selected from -[C(Ra)(Rb)]n-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=NRa)-, -C(=O)-, -C(=S)-, -O-, -Si(Ra)2-S(=O)x-, and -N(Ra)-, where x is 0, 1, or 2; n is 1, 2, 3, or 4; each Ra and Rb independently comprises H, 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, heterocyclic radical J1 and J2 are 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 C5-C20 aryl, heterocyclic radical, substituted heterocyclic radical, O1-O12 aminoalkyl, substituted C1-C12 aminoalkyl, or protecting group.

[0074] Nucleosides containing a bicyclic sugar moiety are called bicyclic nucleosides or BNAs. Bicyclic nucleosides include (A) α-L-methyleneoxy(4-CH2-O-2')BNA, (B) β-D-methyleneoxy(4-CH2-O-2')BNA (also called cross-linked nucleic acid or LNA), (C) ethyleneoxy(4'-(CH2)2-O-2')BNA, (D) aminooxy(4'-CH2-ON(R)-2')BNA, (E) oxyamino(4'-CH2-N(R)-O-2')BNA, and (F) methyl(methyleneoxy)(4'-CH(CH3)-O-2')BNA (bound ethyleneoxy) This includes, but is not limited to, (G) methylene-thio(4-CH2-S-2')BNA, (H) methylene-amino(4'-CH2-N(R)-2')BNA, (I) methyl carboncyclic(4'-CH2-CH(CH3)-2')BNA, (J) propylene carboncyclic(4'-(CH2)3-2')BNA, and (K) methoxy(ethyleneoxy)(4'-CH(CH2OMe)-O-2')BNA (also known as restricted MOE or cMOE). Additional bicyclic sugar moieties are known in the art, and any such bicyclic sugar moiety is within the scope of this disclosure.

[0075] In some embodiments, the bicyclic sugar moiety, and the nucleoside into which such a bicyclic sugar moiety is incorporated, are further defined by their isomer configuration. For example, a nucleoside containing a 4'-2' methylene-oxy bridge may adopt an alpha-L configuration or a beta-D configuration. α-L-methyleneoxy(4-CH2-O-2') bicyclic nucleosides have so far been incorporated into antisense polynucleotides exhibiting antisense activity.

[0076] In some embodiments, the substituted sugar moiety comprises one or more non-crosslinked sugar substituents and one or more crosslinked sugar substituents.

[0077] In some embodiments, the modified sugar moiety is a sugar substitute. In some embodiments, the oxygen atom of a naturally occurring sugar is substituted with, for example, a sulfur, carbon, or nitrogen atom. In some such embodiments, such modified sugar moieties also include the aforementioned bridging and / or non-bridging substituents. For example, certain sugar substitutes include a 4'-sulfur atom, as well as substitutions at the 2' and / or 5' positions. As an additional example, carbocyclic bicyclic nucleosides with 4-2' bridging are described.

[0078] In some embodiments, the sugar substitute includes a ring having more than five atoms. For example, in some embodiments, the sugar substitute includes 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), and fluoroHNA (F-HNA).

[0079] Many other bicyclosaccharide substitute cyclic and tricyclosaccharide substitute cyclic systems that can be used to modify nucleosides for incorporation into antisense compounds are also known in the art, and any such sugar substitute cyclic system is within the scope of this disclosure.

[0080] Combinations of modifications, not limited to those described above, such as 2-F-5'-methyl-substituted nucleosides and substitution of the oxygen atom of the ribosyl ring at S and further substitution at the 2' position, or alternatively, 5'-substitution of bicyclic nucleic acids, are also provided without limitation. The synthesis and preparation of carbocyclic and bicyclic nucleosides are also described in conjunction with their oligomerization and biochemical studies.

[0081] In some embodiments, the disclosure provides nucleic acid molecules comprising modified nucleosides. Such modified nucleotides may include modified sugars, modified nucleic acid bases, and / or modified linkages. Specific modifications are selected, and the resulting polynucleotides possess desirable properties. In some embodiments, the nucleic acid molecule comprises one or more RNA-like nucleosides. In some embodiments, the nucleic acid molecule comprises one or more DNA-like nucleotides.

[0082] In some embodiments, the nucleoside of the Disclosure comprises one or more unmodified nucleic acid bases. In some embodiments, the nucleoside of the Disclosure comprises one or more modified nucleic acid bases.

[0083] In some embodiments, the modified nucleic acid base is selected from universal bases, hydrophobic bases, promiscuous bases, size-extended bases, and fluorinated bases as defined herein. This includes N-2, N-6, and 0-6 substituted purines containing 5-substituted pyrimidines, 6-azapyrimidines, and 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 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, cytosine and thymine, 5-uracil (pseudracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, specifically 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, indiscriminate bases, size-extended bases, and fluorinated bases as defined herein. Further modified nucleic acid bases include tricyclic pyrimidines such as phenoxazinecytidine ([5,4-b][1,4]benzoxazine-2(3H)-one) and phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiadin-2(3H)-one), substituted phenoxazinecytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazine-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indole-2-one), and pyridoindolecytidine (H-pyrimido[3',2':4,5]pyrrolo[2,3-d]pyrimidine-2-one).Modified nucleic acid bases may also include those in which a purine or pyrimidine base is replaced by another heterocycle, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.

[0084] In some embodiments, the disclosure provides nucleic acid molecules comprising linked nucleosides. In some embodiments, the nucleosides may be linked together using any internucleoside linkage groups. Two main classes of internucleoside linkage groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkage groups include, but are not limited to, phosphodiesters (P=O), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Typical non-phosphorus-containing internucleoside linkers include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thionocarbamates (-OC(O)(NH)-S-); siloxanes (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified linkers can be used to alter, typically increase, the nuclease resistance of polynucleotides compared to natural phosphodiester linkers. In some embodiments, internucleoside linkers having chiral atoms can be prepared as racemic mixtures or as separate enantiomers. Typical chiral linkers include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing nucleoside junctions and non-phosphorus-containing nucleoside junctions are well known to those skilled in the art.

[0085] Since the nucleic acid molecules described herein may contain one or more chiral centers, they may result in other stereoisomer configurations that can be defined with respect to absolute stereochemistry, such as (R) or (S) for enantiomers, diastereomers, and sugar anomers, or (D) or (L) for amino acids, and similarly for others. The antisense compounds provided herein include all such possible isomers, as well as their racemic and optically pure forms.

[0086] Neutral nucleoside junctions include, but are not limited to, phosphotriesters, methylphosphonates, 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'). Further neutral nucleoside junctions include nonionic junctions containing siloxanes (dialkylsiloxanes), carboxylate esters, carboxamides, sulfides, sulfonic acid esters, and amides. Further neutral nucleoside junctions include nonionic junctions containing mixed N, O, S, and CH2 constituent parts.

[0087] Additional modifications may be made at other positions on the nucleic acid molecule, specifically at the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of the 5' terminal nucleotide. For example, one of the additional modifications of the nucleic acid molecule of this disclosure involves chemically linking one or more additional moieties or complexes with the polynucleotide to enhance the activity, cell distribution, or cell uptake of the polynucleotide. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties, cholic acid, thioethers, e.g., hexyl-5-tritylthiol, thiocholesterol, aliphatic chains, e.g., dodecanediol or undecyl residues, phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamine or polyethylene glycol chains, or adamantane acetate, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0088] In some embodiments, a vector is provided. In some embodiments, the vector comprises a nucleic acid molecule provided herein. In some embodiments, the nucleic acid molecule encodes a signal peptide provided herein. In some embodiments, the nucleic acid molecule encodes a recombinant polypeptide provided herein.

[0089] cell In some embodiments, cells are provided. In some embodiments, the cells include nucleic acid molecules encoding signal peptides provided herein. In some embodiments, the cells include nucleic acid molecules encoding recombinant polypeptides provided herein. In some embodiments, the cells include vectors provided herein. In some embodiments, the cells are any suitable cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are mammalian cells used in a method for producing a protein product. In some embodiments, the cells are any in vitro cell line. In some embodiments, the cells are any ex vivo cells. In some embodiments, the cells are present in vivo.

[0090] composition In some embodiments, a composition is provided. In some embodiments, the composition comprises a vector encoding a signal peptide provided herein and a delivery system. In some embodiments, the composition comprises a vector encoding a recombinant polypeptide provided herein and a delivery system.

[0091] In some embodiments, the delivery system may 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 adenoviruses, adeno-associated viruses (AAVs), retroviruses, herpesviruses, lentiviruses, poxviruses, or papillomavirus vectors.

[0092] In some embodiments, the delivery system is a nonviral delivery system. Non-limiting examples of nonviral 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, hyaluronidases, nanoparticle mimics, ribonucleoproteins, positively charged peptides, small RNA complexes, aptamer-RNA chimeras, RNA-fusion protein complexes, and any combination thereof.

[0093] The exemplary delivery systems described above should not be construed as limiting in any way. Suitable delivery systems are known in the art, and any such delivery system is within the scope of this disclosure.

[0094] method Methods using endogenous signal peptides and recombinant polypeptides provided herein are also intended herein.

[0095] In some embodiments, methods are provided for treating a disease or disorder. In some embodiments, the method comprises the step of administering a vector containing a nucleic acid molecule encoding a recombinant polypeptide provided herein to a subject in need, thereby treating the disease or disorder. In some embodiments, the payload protein of the recombinant polypeptide is a peptide or protein useful for treating the disease or disorder. In some embodiments, a signal peptide guides the payload protein to be expressed at a specific cellular localization useful for treating the disease or disorder. In some embodiments, the subject in need is administered an effective amount of a vector containing a nucleic acid molecule encoding a recombinant polypeptide provided herein. In some embodiments, the subject in need is administered a therapeutically effective amount of a vector containing a nucleic acid molecule encoding a recombinant polypeptide provided herein. In some embodiments, the disease or disorder is any disease or disorder in which the adequacy or increase of expression of the payload protein is advantageous for treating the disease. In some embodiments, the production of a payload protein useful for treating the disease or disorder is brought about by delivering the payload protein to the endoplasmic reticulum.Non-limiting examples of diseases, disorders, or therapeutic fields in which recombinant polypeptides disclosed herein may be useful include oncology, immunology, cell and immunocytoengineering / reprogramming (ex vivo-engineered macrophage therapy, in situ-engineered macrophage therapy, ex vivo-engineered NK cell therapy, in situ-engineered NK cell therapy, ex vivo-engineered NKT cell therapy, in situ-engineered NKT cell therapy, ex vivo-engineered T cell therapy, in situ-engineered T cell therapy, ex vivo-engineered islet cell therapy, in situ-engineered islet cell therapy, ex vivo-engineered B cell therapy, in situ-engineered B cell therapy, ex vivo-engineered HSPC cell therapy, in situ-engineered HSPC cell therapy, ex vivo-engineered stem cell and progenitor cell therapy, Examples of applications include, but are not limited to, in-situ manipulated stem cell and progenitor cell therapies, cell surface functionalization with receptor-targeting ligands, and similar applications, CAR-T cell engineering, cell targeting, cell signaling, cell surface antigen presentation, cell surface receptor presentation, cell membrane antigen presentation, cell membrane antigen integration, cell membrane receptor presentation, cell membrane receptor integration, inflammatory diseases or disorders, autoimmune diseases or disorders, mitochondrial-related diseases or disorders, genetic diseases or disorders, metabolic disorders, cell storage disorders, protein transport disorders, protein recycling disorders, immunotherapy, enzyme replacement therapy, protein replacement therapy, biologic manufacturing, diabetes, hematological disorders (e.g., hemophilia), hormone replacement therapy, peptide-based drugs (e.g., GLP-1 for obesity), gene therapy, and similar applications. These examples are illustrative and are not intended to limit the application in any way. Those skilled in the art will recognize the applicability of the embodiments provided herein to other diseases and disorders, or other uses not described herein. Such additional diseases or disorders or additional uses are within the scope of this disclosure. In some aspects, the disease or disorder is cancer.

[0096] In some embodiments, a method is provided for treating cancer, the method comprising the step of administering a vector containing a nucleic acid molecule encoding a recombinant polypeptide provided herein to a subject in need, thereby treating the cancer. In some embodiments, the payload protein of the recombinant polypeptide is a peptide or protein useful for treating cancer. In some embodiments, a signal peptide guides the payload protein to be expressed in the endoplasmic reticulum, thereby treating the cancer. In some embodiments, the subject in need is administered an effective amount of a vector containing a nucleic acid molecule encoding a recombinant polypeptide provided herein. In some embodiments, the subject in need is administered a therapeutically effective amount of a vector containing a nucleic acid molecule encoding a recombinant polypeptide provided herein.

[0097] In some embodiments, methods are provided for treating a disease or disorder. In some embodiments, the method comprises administering a composition comprising a vector containing a nucleic acid molecule encoding a recombinant polypeptide provided herein to a subject in need, thereby treating the disease or disorder. In some embodiments, the payload protein of the recombinant polypeptide is a peptide or protein useful for treating the disease or disorder. In some embodiments, a signal peptide directs the payload protein to be expressed at a specific cellular localization useful for treating the disease or disorder. In some embodiments, the subject in need is administered an effective amount of the composition. In some embodiments, the subject in need is administered a therapeutically effective amount of the composition. In some embodiments, the disease or disorder is any disease or disorder in which the adequacy or increase of expression of the payload protein is advantageous for treating the disease. In some embodiments, the production of a payload protein useful for treating the disease or disorder is brought about by delivering the payload protein to the endoplasmic reticulum.Non-limiting examples of diseases, disorders, or therapeutic fields in which the recombinant polypeptides disclosed herein may be useful include oncology, immunology, cell and immunocytoengineering / reprogramming (ex vivo-engineered macrophage therapy, in situ-engineered macrophage therapy, ex vivo-engineered NK cell therapy, in situ-engineered NK cell therapy, ex vivo-engineered NKT cell therapy, in situ-engineered NKT cell therapy, ex vivo-engineered T cell therapy, in situ-engineered T cell therapy, ex vivo-engineered islet cell therapy, in situ-engineered islet cell therapy, ex vivo-engineered B cell therapy, in situ-engineered B cell therapy, ex vivo-engineered HSPC cell therapy, in situ-engineered HSPC cell therapy, ex vivo-engineered stem cell and progenitor cell therapy). Examples include, but are not limited to, in situ-manipulated stem cell and progenitor cell therapies, cell surface functionalization with receptor-targeting ligands, and similar technologies; CAR-T cell engineering; cell targeting; cell signaling; cell surface antigen presentation; cell surface receptor presentation; cell membrane antigen presentation; cell membrane antigen integration; cell membrane receptor presentation; cell membrane receptor integration; inflammatory diseases or disorders; autoimmune diseases or disorders; mitochondrial-related diseases or disorders; genetic diseases or disorders; metabolic disorders; cell storage disorders; protein transport disorders; protein recycling disorders; immunotherapy; enzyme replacement therapy; protein replacement therapy; biologic manufacturing; diabetes; hematological disorders (e.g., hemophilia); hormone replacement therapy; peptide-based drugs (e.g., GLP-1 for obesity); gene therapy; and similar technologies. These examples are illustrative and are not intended to limit the scope in any way. Those skilled in the art will recognize the applicability of the embodiments provided herein to other diseases and disorders, or other uses not described herein. Such additional diseases or disorders or additional uses are within the scope of this disclosure. In some aspects, the disease or disorder is cancer.

[0098] In some embodiments, a method is provided for treating cancer, the method comprising administering a composition comprising a vector containing a nucleic acid molecule encoding a recombinant polypeptide provided herein to a subject in need, thereby treating the cancer. In some embodiments, the payload protein of the recombinant polypeptide is a peptide or protein useful for treating cancer. In some embodiments, a signal peptide guides the payload protein to be expressed in the endoplasmic reticulum, thereby treating the cancer. In some embodiments, the subject in need is administered an effective amount of the composition. In some embodiments, the subject in need is administered a therapeutically effective amount of the composition.

[0099] In some embodiments, a method is provided for producing a protein in a cell, comprising the step of administering a vector containing a nucleic acid molecule encoding a recombinant polypeptide provided herein to a cell, thereby resulting in the production of a protein in the cell. In some embodiments, the protein is a protein that is difficult to express in a cell. Non-limiting examples of proteins that are difficult to express include, but are not limited to, antibodies, as well as proteins and antibodies that require disulfide bond formation. Thus, in some embodiments, a method is provided for producing an antibody in a cell, comprising the step of administering a vector containing a nucleic acid molecule encoding an antibody fused to or linked to an endogenous signal peptide provided herein, thereby resulting in the production of an antibody in the cell. In some embodiments, the antibody is retained in the cell. In some embodiments, the antibody is further processed and secreted from the cell.

[0100] In some embodiments, a method is provided for producing a protein in a cell, comprising the step of administering to a cell a composition comprising a vector containing a nucleic acid molecule encoding a recombinant polypeptide provided herein, thereby resulting in the production of a protein in the cell. In some embodiments, the protein is a protein that is difficult to express in a cell. Non-limiting examples of proteins that are difficult to express include, but are not limited to, antibodies, as well as proteins and antibodies that require disulfide bond formation. Thus, in some embodiments, a method is provided for producing an antibody in a cell, comprising the step of administering to a cell a composition comprising a vector containing a nucleic acid molecule encoding an antibody fused to or linked to an endogenous signal peptide provided herein, thereby resulting in the production of an antibody in the cell. In some embodiments, the antibody is retained in the cell. In some embodiments, the antibody is further processed and secreted from the cell.

[0101] In some embodiments, a method is provided for producing a payload protein, the method comprising administering a vector containing a nucleic acid molecule encoding a recombinant polypeptide provided herein to cells, and culturing the cells under conditions sufficient for producing the payload protein. In some embodiments, the payload protein is secreted from the cells, and the method further comprises collecting the cell supernatant containing the payload protein, and purifying the payload protein from the cell supernatant. In some embodiments, the payload protein is not secreted, and the method further comprises collecting cells containing the payload protein, lysing the cells, and purifying the payload protein from the cell lysates.

[0102] In some embodiments, a method is provided for producing a payload protein, the method comprising administering a vector containing a nucleic acid molecule encoding a recombinant polypeptide provided herein to cells, and culturing the cells under conditions sufficient to produce a payload protein. In some embodiments, the payload protein is secreted from the cells, and the method further comprises collecting a cell supernatant containing the payload protein, and purifying the payload protein from the cell supernatant. In some embodiments, the payload protein is not secreted, and the method further comprises collecting cells containing the payload protein, lysing the cells, and purifying the payload protein from the cell lysates.

[0103] This disclosure, in part, relates to novel recombinant polypeptides comprising endogenous signal peptides and payload proteins. While not bound by any particular theory, the novel recombinant polypeptides of this disclosure are useful in the treatment of disease or disorder through the incorporation of endogenous signal peptides into recombinant polypeptides containing payload proteins useful in the treatment of disease or disorder. Guiding the payload protein into the endoplasmic reticulum is advantageous in the treatment of disease or disorder through the proper production or folding of the payload protein. Furthermore, by utilizing disease- or disordered cells to produce the payload protein, this method avoids the need for timely and inefficient recombinant protein injection, instead allowing the patient's own cells to produce the therapeutic molecule. Similarly, the endogenous signal peptides and novel recombinant polypeptides of this disclosure are useful in the production of payload proteins (e.g., antibodies) in cells through the incorporation of signal peptides into recombinant polypeptides containing the payload protein. Guiding the payload protein into the endoplasmic reticulum facilitates the proper production or folding of the payload protein. The payload protein can then be further processed by the cell so that it is delivered to a suitable location based on the desired utilization of the payload protein. For example, the payload protein may be further processed to be delivered to the cell nucleus, mitochondria, cell membrane, or cytoplasm, or any other suitable organelle, or the payload protein may be further processed to be expelled from the cell. Furthermore, by utilizing the patient's own cells to produce the payload protein, this method avoids the need for timely and inefficient recombinant protein injection, instead allowing the patient's own cells to produce the therapeutic molecule.

[0104] This disclosure also covers, in part, methods for producing or manufacturing payload proteins using endogenous signal peptides provided herein. Without being bound by any particular theory, the endogenous signal peptides provided herein guide payload proteins to specific cellular locations, including, but not limited to, the nucleus, endoplasmic reticulum, mitochondria, cytoplasm, cell membrane, intercellular junctions, cellular processes, synapses, or lysosomes, or to be evicted into the extracellular space. The use of endogenous signal peptides to guide payload proteins to specific cellular locations can enhance the expression and / or production of payload proteins. [Examples]

[0105] The following examples are illustrative of the compounds, compositions, particles, polypeptides, and methods described herein and should not be construed as limiting.

[0106] method Construction of SP-mCherry plasmid (pDNA) Plasmids were constructed in a manner similar to that described in Cheng, Qiang et al. Proceedings of the National Academy of Sciences of the United States of America vol. 120,52 (2023) and International Publication No. WO2024064874A2, which are incorporated herein by reference as a whole. Briefly, the SP-mCherry coding region was directly obtained by PCR using appropriately designed primers. The SPs for the following examples are provided in Table 2 below.

[0107] (Table 2) TIFF2026536591000006.tif38138

[0108] The enzymatically digested SP-mCherry product was cloned into a pCS2-MT vector according to a standard protocol. After sequencing verification, the SP-mCherry plasmid was ready for in vitro screening.

[0109] In vitro SP screening by pDNA transfection To perform SP screening, pDNA transfection was carried out in cells. The pDNA was encapsulated in Lipfectamine 2000 and administered to HeLa cells at 100 ng / well in a 6-well plate, followed by 24-hour incubation. After incubation, the endoplasmic reticulum of living cells was stained using ER-Tracker Blue-White DPX according to the manufacturer's protocol. Subsequently, cells were imaged using confocal microscopy for ER-Tracker Blue-White DPX (endoplasmic reticulum) signaling (DAPI) along with the mCherry signaling. Co-localization of endoplasmic reticulum signaling and mCherry signaling was evaluated and quantified using Fiji software.

[0110] Example 1: Use of endogenous signal peptides to deliver mCherry to the endoplasmic reticulum The ability of the endogenous signal peptide provided herein to deliver payload proteins to the endoplasmic reticulum was tested by constructing a signal peptide-modified mCherry pDNA plasmid containing an endoplasmic reticulum-targeting signal peptide encoded immediately upstream of the mCherry sequence. An unmodified mCherry plasmid without the signal peptide was also used as a control. The overall plasmid construction and schematic method are provided above and in Figure 1A.

[0111] As shown in Figures 1B-1F, two of the five endogenous signal peptides tested were able to significantly drive the mCherry protein to the endoplasmic reticulum (ER), while the mCherry protein without the signal peptide did not localize to the ER. The results for Nat-EMO 19-1 (SEQ ID NO: 21) and Nat-EMO 24-1 (SEQ ID NO: 72) showed a statistically significant increase in ER-localized mCherry compared to the control without the signal peptide, while Nat-EMO 18-1 (SEQ ID NO: 10), Nat-EMO 20-1 (SEQ ID NO: 32), and Nat-EMO 21-1 (SEQ ID NO: 40) each showed a tendency to induce ER-localized mCherry, although not to a statistically significant degree.

[0112] The data from this example demonstrate that an endoplasmic reticulum targeting signal peptide sequence encoded immediately upstream of the mCherry sequence drives the localization of the mCherry protein to the endoplasmic reticulum.

[0113] Example 2: Use of endogenous signal peptides to deliver antibodies to the endoplasmic reticulum Example 2 is carried out using the same method as Example 1, except that the payload protein is an antibody. A plasmid encoding an antibody without an endoplasmic reticulum targeting signal peptide is also used as a control. Instead of mCherry evaluation, appropriate antibody targeting is determined by staining the payload antibody with a suitable secondary antibody and by evaluation via confocal microscopy.

[0114] Example 3: mRNA synthesis and mRNA-nanoparticle formulation mRNA is produced by in vitro transcription as described in Cheng, Qiang et al. Proceedings of the National Academy of Sciences of the United States of America vol. 120,52 (2023); Cheng, Qiang et al. Nature Nanotechnology 15, 313-320 (2020); and International Publication No. WO2024064874A2, each of which is incorporated herein by reference as a whole. Briefly, linear pDNA with an optimized 5'(3')-untranslated region (UTR) and poly(A) sequence was first obtained by enzymatic digestion, followed by preparation of the IVT reaction mixture using a standard protocol with N1-methylpseudridine-5'-triphosphate modification. Finally, the mRNA was capped with vaccinia capping enzyme and 2'-O-methyltransferase (NEB) (Cap-1).

[0115] LNP formulations loaded with mRNA were formed using the ethanol dilution method described in Cheng, Qiang et al. Proceedings of the National Academy of Sciences of the United States of America vol. 120,52 (2023); Cheng, Qiang et al. Nature Nanotechnology 15, 313-320 (2020); and International Publication No. WO2024064874A2, each of which is incorporated herein by reference as a whole. Briefly, all lipids in a specific molar ratio were dissolved in ethanol, and the RNA was first dissolved in 10 mM citrate buffer (pH 4.0). These two solutions were then rapidly mixed in a volume ratio of aqueous solution to ethanol of 3:1 (3:1, aqueous solution:ethanol, vol:vol) to obtain a final weight ratio (total lipids:mRNA) of 40:1. After a 10-minute incubation at room temperature, the mRNA LNP preparation was immediately added to the cells, or the cells were dialyzed in PBS for 2 hours for in vivo experiments.

[0116] Example 4: mCherry targeted by the signal peptide is appropriately secreted from cells transfected with pDNA or cells treated with mRNA-loaded LNPs. The SP of this embodiment is provided in Table 3 below.

[0117] (Table 3) TIFF2026536591000007.tif32138

[0118] We generated pDNA constructs encoding mCherry immediately downstream of the secretory signal peptide and tested them as detailed in Cheng, Qiang et al. Proceedings of the National Academy of Sciences of the United States of America vol. 120,52 (2023); and International Publication No. WO2024064874A2, each of which is incorporated herein by reference as a whole. Briefly, wild-type (WT) mCherry pDNA without SP and gLuc-mCherry pDNA were transfected into HeLa cells via Lipofectamine 2000, and both intracellular and extracellular fluorescence were quantified by fluorescence microscopy at 24, 48, and 72 hours after transfection, indicating that gLuc SP induced high levels of mCherry expression in the culture medium. In addition, at 24, 48, and 72 hours, the content of mCherry protein in the cell culture and cell lysates was individually quantified via a fluorescence plate reader, revealing an increase in mCherry fluorescence in the gLuc SP group (Figure 2A). Subsequently, the SP set was expanded to include a negative control (scrambled sequence), hAlb, hApoB, and hFVII in addition to gLuc. The pDNA constructs were transfected into HeLa cells again using Lipofectamine 2000. Images acquired via fluorescence microscopy and IVIS 72 hours after transfection demonstrated that all SPs, hApoB, gLuc, and hFVII, generated high levels of mCherry protein secretion, while the NC and hAlb constructs efficiently mediated intracellular mCherry expression but did not promote significant extracellular secretion (Figure 2B). The same SP set was examined in the liver cancer cell line Huh7, and the trend of mCherry secretion observed in HeLa cells persisted (Figure 2C).

[0119] Based on the above, we investigated whether mRNA containing the incorporated SP sequence would provide similar insights to those presented with pDNA. hFVII-mCherry mRNA was generated from the FVII-mCherry-pCS2-MT plasmid via in vitro transcription (IVT). mDLNP lipid nanoparticles were tested for use as the primary carrier for RNA. Transfection of multiple different cell lines with mDLNP containing FVII-mCherry mRNA demonstrated that protein transport into the culture medium was positively correlated with the time elapsed since transfection and the dose, with greater fluorescence signal intensity observed across cell lines with longer time intervals and higher doses (Figures 3A and 3B).

[0120] The data from this example demonstrate that, in addition to the ability to transcribe pDNA to mRNA via IVT, equivalent experimental results can be obtained with cell transfection using lipofectamine-mediated pDNA and with LNP-mediated cell transfection using mRNA produced from the same pDNA. Therefore, the data from this example supports the conclusion that the construct tested in Example 1 in pDNA form can be expected to yield similar results to that obtained in the mode in which pDNA is initially transcribed to mRNA via IVT.

Claims

1. formula 1 -(Y 1 ) a -Z 1 A recombinant polypeptide comprising: X 1 However, it is an endogenous signal peptide, Y 1 However, it is a peptide linker, and Z 1 However, it is a payload protein, a is an integer selected from 0 and 1. The recombinant polypeptide.

2. X 1 However, SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 A recombinant polypeptide according to claim 1, comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from the group consisting of 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94%, 95%, 96%, 97%, 98%, 99%, or 119.

3. The recombinant polypeptide according to claim 1 or claim 2, wherein the payload protein is a therapeutic peptide or protein.

4. A nucleic acid molecule encoding a recombinant polypeptide according to any one of claims 1 to 3.

5. A vector comprising the nucleic acid molecule described in claim 4.

6. A cell comprising the nucleic acid molecule according to claim 4 or the vector according to claim 5.

7. A composition comprising the nucleic acid molecule described in claim 4 or the vector described in claim 5.

8. A method for treating a disease or disorder in a subject in need, comprising the step of administering to the subject an effective amount of the nucleic acid molecule described in claim 4, thereby treating the disease or disorder.

9. The method according to claim 8, wherein the disease or disorder is cancer.

10. A method for treating cancer in a subject in need, comprising the step of administering to the subject a vector comprising a nucleic acid molecule encoding a signal peptide fused to or linked to a payload protein, wherein the signal peptide is an endogenous signal peptide and the payload protein is a therapeutic peptide or protein useful for treating the cancer.

11. The aforementioned endogenous signal peptide is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 The method according to claim 10, comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from the group consisting of 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 88, 89, 90, 91, 92, 93, 94, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from the group consisting of 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 88, 89, 90, 91, 92, 93, 94, 95%, 96%, 97%, 98%, 99%, or 119.

12. The use of an endogenous signal peptide for targeting a payload protein to a desired intracellular location, wherein the payload protein does not naturally contain an amino acid sequence comprising the amino acid sequence of the endogenous signal peptide.

13. The aforementioned endogenous signal peptide is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 The use according to claim 12, comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from the group consisting of 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 88, 89, 90, 91, 92, 93, 94, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from the group consisting of 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95%, 96%, 97%, 98%, 99%, or 119.

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