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WO2025096678A1 - Nouveaux peptides de signal ciblant des parties saillantes de cellule et leurs utilisations - Google Patents

Nouveaux peptides de signal ciblant des parties saillantes de cellule et leurs utilisations Download PDF

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Publication number
WO2025096678A1
WO2025096678A1 PCT/US2024/053747 US2024053747W WO2025096678A1 WO 2025096678 A1 WO2025096678 A1 WO 2025096678A1 US 2024053747 W US2024053747 W US 2024053747W WO 2025096678 A1 WO2025096678 A1 WO 2025096678A1
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Prior art keywords
amino acid
signal peptide
seq
acid sequence
cell
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Inventor
Daniel J. Siegwart
Lukas FARBIAK
William Miller
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins

Definitions

  • the present disclosure relates generally to novel signal peptides and more particularly to novel signal peptides for targeting an encoded protein to cell projections.
  • the signal peptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 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 SEQ ID NO: 1, 2, 3, 4, or 5.
  • a signal peptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 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 SEQ ID NO: 1, 2, 3, 4, or 5.
  • a signal peptide as provided for herein is a novel signal peptide.
  • a recombinant polypeptide is provided.
  • the recombinant polypeptide comprises a formula of Xi-(Yi) a -Zi, wherein Xi is a signal peptide, Yi is a peptide linker, and Zi is a payload protein, wherein a is an integer selected from 0 and 1.
  • the signal peptide Xi comprises an amino acid sequence selected from the group consisting of Formula I, II, or III; wherein Formula I is represented as:
  • the signal peptide Xi comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 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 SEQ ID NO: 1, 2, 3, 4, or 5.
  • the payload protein is a therapeutic peptide or protein.
  • nucleic acid molecule encodes for a signal peptide as provided for herein.
  • nucleic acid molecule encodes for a recombinant polypeptide as provided for herein.
  • a vector is provided.
  • the vector comprises a nucleic acid molecule as provided for herein.
  • the nucleic acid molecule encodes for a signal peptide as provided for herein.
  • the nucleic acid molecule encodes for a recombinant polypeptide as provided for herein.
  • a cell comprises a nucleic acid molecule as provided for herein. In some embodiments, the cell comprises a vector as provided for herein. In some embodiments, the nucleic acid molecule encodes for a signal peptide as provided for herein. In some embodiments, the nucleic acid molecule encodes for a recombinant polypeptide as provided for herein.
  • a composition comprising a nucleic acid molecule as provided for herein.
  • the composition comprises a vector as provided for herein.
  • the nucleic acid molecule encodes for a signal peptide as provided for herein.
  • the nucleic acid molecule encodes for a recombinant polypeptide as provided for herein.
  • a method for treating a disease or disorder in a subject in need thereof comprises administering to the subject an effective among of a nucleic acid molecule as provided for herein to the subject, thereby treating the disease or disorder.
  • the nucleic acid molecule encodes for a recombinant polypeptide as provided for herein.
  • the disease or disorder is cancer or a proteinopathy.
  • a method for treating cancer in a subject in need thereof comprises administering to the subject a vector comprising a nucleic acid molecule encoding for a signal peptide fused or linked to a payload protein, wherein the signal peptide is a signal peptide as provided for herein and the payload protein is a therapeutic peptide or protein useful for the treatment of the cancer.
  • a method for treating proteinopathy in a subject in need thereof comprises administering to the subject a vector comprising a nucleic acid molecule encoding for a signal peptide fused or linked to a payload protein, wherein the signal peptide is a signal peptide as provided for herein and the payload protein is a therapeutic peptide or protein useful for the treatment of the proteinopathy.
  • FIG. 1A provides a schematic of the construct comprising mCherry with the signal peptide at the N-terminus and its use for signal peptide screening by pDNA transfection in vitro.
  • FIG. IB illustrates representative images of signal peptide tagged mCherry colocalized with CellMask Deep Red Actin Tracking Stain. Top: SP is Nov-CLP-19-1 (SEQ ID NO: 1). Bottom: SP is Nov-CLP-20-1 (SEQ ID NO: 4).
  • FIG. 1C illustrates representative images of signal peptide tagged mCherry colocalized with CellMask Deep Red Actin Tracking Stain. Top: SP is Nov-CLP-23-1 (SEQ ID NO: 5). Bottom: No SP control.
  • FIG. ID illustrates the quantification of the experiments corresponding to the representative images of FIGs. IB and 1C.
  • FIG. IE illustrates the quantification of the experiments corresponding to the representative images of FIGs. IB and 1C.
  • FIG. 2A provides quantification of mCherry fluorescence in Hela cell lysates and medium at different time points.
  • mCherry with no signal peptide was used as a control (“WT- mCherry”).
  • WT- mCherry mCherry with no signal peptide was used as a control (“WT- mCherry”).
  • gLuc Gaussia luciferase.
  • FIG. 2B provides quantification of mCherry fluorescence in Hela cell lysate and medium after 72 hours.
  • FIG. 2C provides quantification of mCherry fluorescence in Huh7 cell lysates and medium at 72h after transfection.
  • FIG. 3A provides quantification of time-dependent (100 ng mRNA per well) and dose dependent (at 72h) mCherry secretion in Huh7 cells, medium, and cell lysate.
  • FIG. 3B shows quantification of mCherry signal in different cell lines.
  • Cells in 96- well plate were treated with mDLNPs-mRNA, at given time-points.
  • “comprising” means “including” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise.
  • reference to “comprising a therapeutic agent” includes one or a plurality of such therapeutic agents.
  • the term “or” refers to a single element of stated alternative elements, unless the context clearly indicates otherwise.
  • 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.
  • “one or more of A and B” refers to A, B, or a combination of both A and B.
  • a and B refers to a combination of A and B.
  • the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments are to be understood as being modified in some instances by the term "about” or “approximately.”
  • “about” or “approximately” can indicate +/- 10%, +/- 5%, or +/- 1% variation of the value it describes.
  • the numerical parameters set forth herein are approximations that can vary depending upon the desired properties for a particular embodiment. Additionally, where a phrase recites “about x to y,” the term “about” modifies both x and y and can be used interchangeably with the phrase “about x to about y” unless context dictates differently.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection (e.g., using commercially available reagents such as, for example, LIPOFECTIN® (Invitrogen Corp., San Diego, CA), LIPOFECTAMINE® (Invitrogen), FUGENE® (Roche Applied Science, Basel, Switzerland), JETPEITM (Polyplus-transfection Inc., New York, NY), EFFECTENE® (Qiagen, Valencia, CA), DREAMFECTTM (OZ Biosciences, France) and the like), or electroporation (e.g., in vivo electroporation).
  • LIPOFECTIN® Invitrogen Corp., San Diego, CA
  • LIPOFECTAMINE® Invitrogen
  • FUGENE® Roche Applied Science, Basel
  • 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, N.Y., 1989), and other laboratory manuals.
  • Methods and materials of non-viral delivery of nucleic acids to cells further include biolistics, virosomes, liposomes, lipid nanoparticles, immunoliposomes, polycation or lipidnucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
  • Lipofection is described in U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355 and lipofection reagents are sold commercially (e.g., TRANSFECTAMTM and LIPOFECTINTM).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those disclosed in WO91/17424 and WO 91/16024.
  • amino acid sequence/s amino acid sequence/s
  • sequence/s amino acid sequence/s
  • sequence/s amino acid sequence/s
  • reference sequences will be explicitly disclosed, in any aspect and embodiment, a reference sequence may be modified to include conservative amino acid substitutions, as well as variants and fragments, while maintaining the characteristics and functionality of the reference sequence.
  • the term “fused” or “linked” when used in reference to a protein having different domains or heterologous sequences means that the protein domains are part of the same peptide chain that are connected to one another with either peptide bonds or other covalent bonding.
  • the domains or section can be linked or fused directly to one another or another domain or peptide sequence can be between the two domains or sequences and such sequences would still be considered to be fused or linked to one another.
  • the various domains or proteins provided for herein are linked or fused directly to one another or a linker sequences, such as the glycine/serine sequences described herein link the two domains together.
  • Identity refers to the subunit sequence identity between two polymeric molecules such as between two nucleic acid or amino acid molecules, such as, between two polynucleotide or polypeptide molecules.
  • two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an Arginine, then they are identical at that position.
  • the identity or extent to which two amino acid or two nucleic acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage.
  • the identity between two amino acid or two nucleic acid sequences is a direct function of the number of matching or identical positions; e.g., if half of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). In some embodiments, such a sequence is at least 60%, 80% or 85%, or 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison. Other percentages of identity in reference to specific sequences are described herein.
  • Sequence identity can be measured/determined using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, MUSCLE or PILEUP/PRETTYBOX programs).
  • sequence analysis software for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, MUSCLE or PILEUP/PRETTYBOX programs.
  • 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.
  • a BLAST program may be used, with a probability score between e3 and el 00 indicating a closely related sequence.
  • sequence identity is determined by using BLAST with the default settings.
  • sequence identity is determined using Clustal Omega.
  • composition comprising various proteins
  • proteins may, compared to the disclosed proteins, include one or more (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, etc.) conservative amino acid replacements i.e., replacements of one amino acid with another which has a related side chain.
  • conservative amino acid replacements i.e., replacements of one amino acid with another which has a related side chain.
  • amino acids are generally divided into four families: (1) acidic i.e., aspartate, glutamate; (2) basic i.e., lysine, arginine, histidine; (3) non polar i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
  • the proteins may have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single amino acid deletions relative to the disclosed protein sequences.
  • the proteins may also include 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) relative to the disclosed protein sequences.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • A refers to adenosine
  • C refers to cytosine
  • G refers to guanosine
  • T refers to thymidine
  • U refers to uridine
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).
  • oligonucleotide typically refers to short polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also provides the corresponding RNA sequence (i.e., A, U, C, G) in which “U” replaces
  • polynucleotide as used herein is defined as a chain of nucleotides.
  • nucleic acids are polymers of nucleotides.
  • nucleic acids or “nucleic acid molecules” and “polynucleotides” as used herein are interchangeable.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any methods available in the art, including, without limitation, recombinant methods, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using cloning technology and PCR, and the like, and by synthetic means.
  • peptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of a plurality of amino acid residues covalently linked by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • antibody fragment or “antigen binding fragment” refers to antigen binding fragments of antibodies, i.e., antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g., fragments that retain one or more CDR regions.
  • antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; singlechain antibody molecules, e.g., sc-Fv; nanobodies (single domain antibody) and multispecific antibodies formed from antibody fragments.
  • a “Fab fragment” is comprised of one light chain and the CHI and variable regions of one heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • An “Fc” region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
  • a “Fab 1 fragment” contains one light chain and a portion or fragment of one heavy chain that contains the VH domain and the CHI domain and also the region between the CHI and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab') 2 molecule.
  • a “F(ab')2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains.
  • a F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.
  • the “Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions.
  • the term “single-chain Fv” or “scFv” antibody refers to antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • Antibody molecules can be monospecific (e.g., monovalent or bivalent), bispecific (e.g., bivalent, trivalent, tetravalent, pentavalent, or hexavalent), trispecific (e.g., trivalent, tetravalent, pentavalent, or hexavalent), or with higher orders of specificity (e.g, tetraspecific) and/or higher orders of valency beyond hexavalency.
  • An antibody molecule can comprise a functional fragment of a light chain variable region and a functional fragment of a heavy chain variable region, or heavy and light chains may be fused together into a single polypeptide.
  • the terms “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 open-ended and do not exclude additional, unrecited elements or method steps. Any step or composition that uses the transitional phrase of “comprise” or “comprising” can also be said to describe the same with the transitional phase of “consisting of’ or “consists.”
  • contacting means bringing together of two elements in an in vitro system or an in vivo system.
  • “contacting” virus or vector described herein with an individual or patient or cell includes the administration of the virus to an individual or patient, such as a human, as well as, for example, introducing a compound into a sample containing a cellular or purified preparation containing the cell.
  • the term “individual” or “subject,” or “patient” used interchangeably, means any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, such as humans.
  • the subject is a human.
  • a subject that is “in need thereof’ refers to a subject that has been identified as requiring treatment for the condition that is to be treated and is treated with the specific intent of treating such condition.
  • the conditions can be, for example, any of the conditions described herein.
  • the compositions disclosed herein may be provided to a subject in a variety of ways through administration of the composition to the subject.
  • administer or administration means to provide or the providing of a composition to a subject.
  • Oral administration refers to delivery of an active agent through the mouth.
  • Topical administration refers to the delivery of an active agent to a body surface, such as the skin, a mucosal membrane (e.g. , nasal membrane, vaginal membrane, buccal membrane, or the like).
  • Effective amount or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to an amount that when administered to a mammal, causes 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 a plethora of art-recognized methods.
  • the amount of the composition administered herein varies and can be readily determined based on a number of factors such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like.
  • Ranges throughout this disclosure, various aspects of the embodiments can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Unless otherwise explicitly stated to the contrary, a range that is disclosed also includes the endpoints of the range. Novel Signal Peptides
  • novel signal peptides may direct payload proteins as provided for herein to be expressed in cell projections.
  • the payload proteins may or may not natively contain a signal peptide directing localization of said protein to the specific cellular compartment, e.g., the cell projections.
  • the provided novel signal peptides can direct enhanced expression of a proteins natively found in the cell projections to the cell projections, or can direct expression of a payload protein, e.g., a therapeutic protein to the cell projections for expression.
  • a signal peptide is provided.
  • the signal peptide comprises an amino acid sequence as recited in Table 1 below:
  • the signal peptide comprises an amino acid sequence that is substantially similar to an amino acid sequence as recited in Table 1. In some embodiments, the signal peptide comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to an amino acid sequence as recited in Table 1.
  • the signal peptide comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5.
  • the signal peptide comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 1.
  • the signal peptide comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 2.
  • the signal peptide comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 3.
  • the signal peptide comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 4.
  • the signal peptide comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to an amino acid sequence of SEQ ID NO: 5.
  • the signal peptide is a variant of an amino acid sequence as provided in Table 1.
  • the signal peptide comprises an amino acid sequence as defined by Formula I-III below.
  • the signal peptide comprises an amino acid sequence represented by:
  • Ai is methionine (M).
  • A2 is an amino acid selected from the group consisting of H, K, and S.
  • A3 is an amino acid selected from the group consisting of L, H, and A.
  • A4 is an amino acid selected from the group consisting of F, T, and R.
  • As is an amino acid selected from the group consisting of A, L, and E.
  • Ae is an amino acid selected from the group consisting of C, A, and V.
  • A7 is an amino acid selected from the group consisting of L and A.
  • A9 is an amino acid selected from the group consisting of I, A, and L.
  • Alo is an amino acid selected from the group consisting of V, P, and L.
  • An is leucine (L).
  • A12 is an amino acid selected from the group consisting of S, L, and W.
  • A13 an amino acid selected from the group consisting of F, G, and L.
  • A14 is an amino acid selected from the group consisting of L and S.
  • A15 is an amino acid selected from the group consisting of E, G, and C.
  • Ai6 is an amino acid selected from the group consisting of G, L, and Y.
  • A17 is an amino acid selected from the group consisting of G and V. In some embodiments, Ais is an amino acid selected from the group consisting of G, L, and S. In some embodiments, A19 is an amino acid selected from the group consisting of A and C.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at position 2 of SEQ ID NO: 1. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at position 3 of SEQ ID NO: 1. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at position 4 of SEQ ID NO: 1.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at position 5 of SEQ ID NO: 1. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at position 6 of SEQ ID NO: 1. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at position 7 of SEQ ID NO: 1. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at position 8 of SEQ ID NO: 1. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at position 9 of SEQ ID NO: 1.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at position 10 of SEQ ID NO: 1. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at position 12 of SEQ ID NO: 1. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at position 13 of SEQ ID NO: 1. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at position 14 of SEQ ID NO: 1. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at position 15 of SEQ ID NO: 1.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at position 16 of SEQ ID NO: 1. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at position 17 of SEQ ID NO: 1. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at position 18 of SEQ ID NO: 1. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at position 19 of SEQ ID NO: 1. The variant may also comprise any number of substitutions as provided by Formula I.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at at least one position corresponding to position 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, or 19 of SEQ ID NO: 1.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and a substitution at more than one position corresponding to position 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, or 19 of SEQ ID NO: 1.
  • the signal peptide comprises an amino acid sequence represented by: B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20 (Formula II); wherein the identity of each of Bi - B20 are as provided for in Table 3 below.
  • Bi is methionine (M).
  • B2 is an amino acid selected from the group consisting of L and Y.
  • B3 is an amino acid selected from the group consisting of M and L.
  • B4 is an amino acid selected from the group consisting of L and E.
  • B5 is an amino acid selected from the group consisting of M and T.
  • Be is an amino acid selected from the group consisting of L and R.
  • B7 is an amino acid selected from the group consisting of V and R.
  • Bs is alanine (A).
  • B9 is an amino acid selected from the group consisting of A and I.
  • Bio is an amino acid selected from the group consisting of V and F.
  • Bn is an amino acid selected from the group consisting of T and V.
  • B12 is an amino acid selected from the group consisting of M and F.
  • B13 is tryptophan (W).
  • B14 is an amino acid selected from the group consisting of L and I.
  • B15 is an amino acid selected from the group consisting of R and F.
  • Bi6 is an amino acid selected from the group consisting of P and L.
  • B17 is an amino acid selected from the group consisting of L and Q.
  • Bis valine (V).
  • B19 is an amino acid selected from the group consisting of T and Q.
  • B20 is an amino acid selected from the group consisting of A and G.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 2 and a substitution at position 2 of SEQ ID NO: 2.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 2 and a substitution at position 3 of SEQ ID NO: 2.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 2 and a substitution at position 4 of SEQ ID NO: 2.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 2 and a substitution at position 5 of SEQ ID NO: 2. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 2 and a substitution at position 6 of SEQ ID NO: 2. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 2 and a substitution at position 7 of SEQ ID NO: 2. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 2 and a substitution at position 9 of SEQ ID NO: 2. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 2 and a substitution at position 10 of SEQ ID NO: 2.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 2 and a substitution at position 11 of SEQ ID NO: 2. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 2 and a substitution at position 12 of SEQ ID NO: 2. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 2 and a substitution at position 14 of SEQ ID NO: 2. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 2 and a substitution at position 15 of SEQ ID NO: 2. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 2 and a substitution at position 16 of SEQ ID NO: 2.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 2 and a substitution at position 17 of SEQ ID NO: 2. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 2 and a substitution at position 19 of SEQ ID NO: 2. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 2 and a substitution at position 20 of SEQ ID NO: 2. The variant may also comprise any number of substitutions as provided by Formula II. Accordingly, in some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 2 and a substitution at at least one position corresponding to position 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 14, 15, 16, 17, 19, or 20 of SEQ ID NO: 2. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 2 and a substitution at more than one position corresponding to position 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 14, 15, 16, 17, 19, or 20 of SEQ ID NO: 2.
  • the variant of SEQ ID NO: 2 comprises the amino acid sequence of SEQ ID NO: 4.
  • the sequence of SEQ ID NO: 4 is derived from Formula II as follows: Bi is methionine (M), B2 is tyrosine (Y), B3 is methionine (M), B4 is leucine (L), B5 is methionine (M), Be is leucine (L), B7 is valine (V), Bs is alanine (A), B9 is alanine (A), Bio is valine (V), Bn is threonine (T), B12 is methionine (M), B13 is tryptophan (W), B14 is leucine (L), B15 is arginine (R), Bie is proline (P), B17 is leucine (L), Bis is valine (V), B19 is threonine (T), and B20 is alanine (A).
  • the signal peptide comprises an amino acid sequence represented by:
  • Ci is methionine (M).
  • C2 is arginine (R).
  • C3 is an amino acid selected from the group consisting of G and R.
  • C4 is an amino acid selected from the group consisting of S and V.
  • C5 is an amino acid selected from the group consisting of E and L.
  • Ce is an amino acid selected from the group consisting of L and R.
  • C7 is an amino acid selected from the group consisting of P and L.
  • C 8 is leucine (L).
  • C9 is an amino acid selected from the group consisting of V and L.
  • C10 is an amino acid selected from the group consisting of L and G.
  • Cn is an amino acid selected from the group consisting of L and C.
  • C12 is an amino acid selected from the group consisting of A and F.
  • C13 is leucine (L).
  • C14 is an amino acid selected from the group consisting of V and T.
  • C15 is an amino acid selected from the group consisting of L and E.
  • Ci6 is an amino acid selected from the group consisting of C and L.
  • C17 is an amino acid selected from the group consisting of L and C.
  • Cis is alanine (A).
  • C19 is an amino acid selected from the group consisting of P and R.
  • C20 is an amino acid selected from the group consisting of R and V.
  • C21 is an amino acid selected from the group consisting of G and C.
  • C22 is arginine (R).
  • C23 is alanine (A).
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 3 and a substitution at position 4 of SEQ ID NO: 3. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 3 and a substitution at position 5 of SEQ ID NO: 3. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 3 and a substitution at position 6 of SEQ ID NO: 3. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 3 and a substitution at position 7 of SEQ ID NO: 3. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 3 and a substitution at position 9 of SEQ ID NO: 3.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 3 and a substitution at position 10 of SEQ ID NO: 3. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 3 and a substitution at position 11 of SEQ ID NO: 3. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 3 and a substitution at position 12 of SEQ ID NO: 3. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 3 and a substitution at position 14 of SEQ ID NO: 3. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 3 and a substitution at position 15 of SEQ ID NO: 3.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 3 and a substitution at position 16 of SEQ ID NO: 3. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 3 and a substitution at position 17 of SEQ ID NO: 3. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 3 and a substitution at position 19 of SEQ ID NO: 3. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 3 and a substitution at position 20 of SEQ ID NO: 3. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 3 and a substitution at position 21 of SEQ ID NO: 3.
  • the variant may also comprise any number of substitutions as provided by Formula III.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 3 and a substitution at at least one position corresponding to position 3, 4, 5, 6, 7, 9, 10, 11, 12, 14, 15, 16, 17, 19, 20, or 21 of SEQ ID NO: 3.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 3 and a substitution at more than one position corresponding to position 3, 4, 5, 6, 7, 9, 10, 11, 12, 14, 15, 16, 17, 19, 20, or 21 of SEQ ID NO: 3.
  • the variant of SEQ ID NO: 3 comprises the amino acid sequence of SEQ ID NO: 5.
  • the sequence of SEQ ID NO: 5 is derived from Formula II as follows: Ci is methionine (M), C2 is arginine (R), C3 is glycine (G), C4 is serine (S), C5 is glutamate (E), Ce is leucine (L), C7 is proline (P), Cs is leucine (L), C9 is valine (V), C10 is leucine (L), Cn is leucine (L), C12 is alanine (A), C13 is leucine (L), C14 is valine (V), C15 is leucine (L), Ci6 is cysteine (C), C17 is leucine (L), Cis is alanine (A), C19 is proline (P), C20 is arginine (R), C21 is glycine (G), C22 is arginine (R),
  • a “novel” signal peptide is understood to comprise an amino acid sequence not found in nature or not yet known to exist in nature. Accordingly, if an embodiment references a novel signal peptide, it is understood to exclude any corresponding native sequence. For example, in some embodiments, a novel signal peptide is provided, wherein the novel signal peptide comprises an amino acid sequence corresponding to Formula X. In such an embodiment, if a native signal peptide could be construed from the formula of Formula X, the embodiment is understood to exclude said native signal peptide.
  • the novel signal peptide comprises an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, or 5.
  • the novel signal peptide comprises an amino acid sequence having at least 70% identity to an amino acid sequence of SEQ ID NO: 1.
  • the novel signal peptide comprises an amino acid sequence having at least 70% identity to an amino acid sequence of SEQ ID NO: 2. In some embodiments, the novel signal peptide comprises an amino acid sequence having at least 70% identity to an amino acid of SEQ ID NO: 3. In some embodiments, the novel signal peptide comprises an amino acid sequence having at least 70% identity to an amino acid of SEQ ID NO: 4. In some embodiments, the novel signal peptide comprises an amino acid sequence having at least 70% identity to an amino acid of SEQ ID NO: 5.
  • a recombinant polypeptide comprising a formula of Xi-Zi, wherein Xi is a signal peptide as provided for herein and Zi is a payload protein.
  • the recombinant polypeptide comprises a formula of Xi-Zi, wherein Xi is a novel signal peptide as provided for herein and Zi is a payload protein.
  • Xi comprises an amino acid sequence selected from the group consisting of Formula I, Formula II, or Formula III. In some embodiments, Xi comprises an amino acid sequence of Formula I. In some embodiments, Xi comprises an amino acid sequence of Formula II. In some embodiments, Xi comprises an amino acid sequence of Formula III.
  • Xi comprises an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, or 5.
  • Xi comprises an amino acid sequence having at least 70% identity to an amino acid sequence of SEQ ID NO: 1.
  • Xi comprises an amino acid sequence having at least 70% identity to an amino acid sequence of SEQ ID NO: 2. In some embodiments, Xi comprises an amino acid sequence having at least 70% identity to an amino acid sequence of SEQ ID NO: 3. In some embodiments, Xi comprises an amino acid sequence having at least 70% identity to an amino acid sequence of SEQ ID NO: 4. In some embodiments, Xi comprises an amino acid sequence having at least 70% identity to an amino acid sequence of SEQ ID NO: 5.
  • the payload protein, Zi may be any peptide or protein. In some embodiments, the payload protein, Zi, is any peptide or protein that is useful in the treatment of a disease or disorder. In some embodiments, the payload protein, Zi, is any peptide or protein that is useful in the treatment of a disease or disorder associated with cell projections. As a non-limiting example, the disease or disorder may be associated with cytoplasmic vesicles or granules.
  • Xi is linked directly to Zi.
  • Xi is linked indirectly to Zi through, for example, a polypeptide linker.
  • Polypeptide linkers are known in the art, and any such linker may be incorporated into the recombinant polypeptide of the present disclosure.
  • the recombinant polypeptide may be represented by the formula Xi-(Yi) a -Zi, wherein Xi is a signal peptide as provided for herein, Yi is a linker, such as but not limited to a polypeptide linker, Zi is a payload protein as provided for herein, and a is an integer selected from 0 or 1.
  • Xi is a novel signal peptide as provided for herein.
  • a nucleic acid molecule is provided.
  • the nucleic acid molecule encodes for a signal peptide as provided for herein.
  • the nucleic acid molecule encodes for a novel signal peptide as provided for herein.
  • the nucleic acid molecule is a deoxyribonucleotide sequence (DNA).
  • the nucleic acid molecule is a ribonucleic acid sequence (RNA).
  • the nucleic acid molecule encodes for a signal peptide (e.g., a novel signal peptide) comprising an amino acid sequence selected from the group consisting of Formula I, Formula II, or Formula III. In some embodiments, the nucleic acid molecule encodes for a signal peptide (e.g., a novel signal peptide) comprising an amino acid sequence of Formula I. In some embodiments, the nucleic acid molecule encodes for a signal peptide (e.g., a novel signal peptide) comprising an amino acid sequence of Formula II. In some embodiments, the nucleic acid molecule encodes for a signal peptide (e.g., a novel signal peptide) comprising an amino acid sequence of Formula III.
  • a signal peptide e.g., a novel signal peptide
  • the nucleic acid molecule encodes for a signal peptide (e.g., a novel signal peptide) comprising an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, or 5.
  • the nucleic acid molecule encodes for a signal peptide (e.g., a novel signal peptide) comprising an amino acid sequence having at least 70% identity to an amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes for a signal peptide (e.g., a novel signal peptide) comprising an amino acid sequence having at least 70% identity to an amino acid sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid molecule encodes for a signal peptide (e.g., a novel signal peptide) comprising an amino acid sequence having at least 70% identity to an amino acid sequence of SEQ ID NO: 3.
  • a signal peptide e.g., a novel signal peptide
  • the nucleic acid molecule encodes for a signal peptide (e.g., a novel signal peptide) comprising an amino acid sequence having at least 70% identity to an amino acid sequence of SEQ ID NO: 4. In some embodiments, the nucleic acid molecule encodes for a signal peptide (e.g., a novel signal peptide) comprising an amino acid sequence having at least 70% identity to an amino acid sequence of SEQ ID NO: 5.
  • the nucleic acid molecule encodes for a recombinant polypeptide as provided for herein. In some embodiments, the nucleic acid molecule encodes for a recombinant polypeptide comprising a formula of Xi-Zi, wherein Xi is a signal peptide as provided for herein and Zi is a payload protein as provided for herein. In some embodiments, the recombinant polypeptide comprises a formula of Xi-Zi, wherein Xi is a novel signal peptide as provided for herein and Zi is a payload protein as provided for herein.
  • the nucleic acid molecule encodes for a recombinant polypeptide comprising a formula of Xi-(Yi) a -Zi, wherein Xi is a signal peptide (e.g., a novel signal peptide) as provided for herein, Yi is a linker, such as but not limited to a polypeptide linker, Zi is a payload protein as provided for herein, and a is an integer selected from 0 or 1.
  • a nucleic acid molecule as provided for here disclosed may further comprise one or more elements selected from, but not limited to, a promoter, an enhancer, a leader, a transcription start site (TSS), a linker, 5’ and 3’ untranslated regions (UTRs), Kozak sequence, an intron, a polyadenylation signal, cap sequences, enhancers, viral sequences, IRES sequences or a termination region or any element, that is suitable or necessary for regulating or allowing expression of the recombinant polypeptide as provided for herein in a cell.
  • TSS transcription start site
  • UTRs untranslated regions
  • the 5’ UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3’ UTR starts immediately following the stop codon and continues until the transcriptional termination signal.
  • the regulatory features of a UTR can be incorporated into the polynucleotides of the present disclosure to, for example, enhance the stability of the molecule. The specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites.
  • 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 within the nucleic acid molecules.
  • introns or portions of intron sequences may be incorporated into regions of the nucleic acid molecules of the disclosure. Incorporation of intronic sequences may increase protein production as well as polynucleotide levels. Combinations of features may be included in flanking regions and may be contained within other features.
  • the ORF may be flanked by a 5’ UTR which may contain a strong Kozak translational initiation signal and/or a 3’ UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail.
  • 5’ UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes.
  • the nucleic acid molecules disclosed herein may be assembled inside a cell.
  • the nucleic acid molecules may be synthesized in vivo.
  • the nucleic acid molecules may be synthesized in vitro using methods known in the art for example in vitro transcription, DNA, RNA and cDNA synthesis methods.
  • the nucleic acid molecules disclosed herein may be incorporated into a suitable viral vector, expression cassette, expression vector, transposon, extrachromosomal element, integrated into the chromosome, host cell, delivery systems.
  • the nucleic acid molecule is a chemically modified nucleic acid molecule.
  • the nucleic acid molecule e may comprise one or more modified nucleosides comprising a modified sugar moiety.
  • modified sugar moieties are substituted sugar moieties.
  • modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.
  • the modified nucleic acid molecule may comprise a modified backbone, for example, phosphorothioates, phosphotriesters, morpholinos, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic inter sugar linkages.
  • a modified backbone for example, phosphorothioates, phosphotriesters, morpholinos, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic inter sugar linkages.
  • modified sugar moieties are substituted sugar moieties comprising one or more non-bridging sugar substituent, including but not limited to substituents at the 2’ and/or 5’ positions.
  • sugar substituents suitable for the 2’- position include, but are not limited to: 2’-F, 2-OCH3 (“OMe” or “O-methyl”), and 2’- O(CH 2 ) 2 OCH 3 (“MOE”).
  • sugar substituents at the 5 ’-position include, but are not limited to: 5’- methyl (R or S); 5’- vinyl, and 5 ’-methoxy.
  • substituted sugars comprise more than one nonbridging sugar substituent, for example, T-F-5’-methyl sugar moieties.
  • Nucleosides comprising 2 ’-substituted sugar moieties are referred to as 2’ -substituted nucleosides.
  • These 2 ’-substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO 2 ), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
  • a 2 ’-substituted nucleoside comprises a sugar moiety comprising a 2’- substituent group selected from F, O — CH , and OCH 2 CH 2 OCH3.
  • Certain modified sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety.
  • the bicyclic sugar moiety comprises a bridge between the 4’ and the 2’ furanose ring atoms.
  • Examples of such 4’ to 2’ sugar substituents include, but are not limited to: — [C(Ra)(Rb)] — , — [C(Ra)(Rb)]n — O — , — C(RaRb)— N(R)— O— or, — C(RaRb)— O— N(R)— ; 4’-CH 2 -2’, 4’-(CH 2 ) 2 -2’, 4’-(CH 2 )— 0-2’ (LNA); 4’-(CH 2 )— S-2’; 4’-(CH 2 ) 2 — 0-2’ (ENA); 4’-CH(CH 3 )— 0-2’ (cEt) and 4’- CH(CH 2 OCH 3 )— 0-2’, and analogs thereof (see, e.g., U.S.
  • Bicyclic nucleosides comprising bicyclic sugar moieties are referred to as bicyclic nucleosides or BNAs.
  • Bicyclic nucleosides include, but are not limited to, (A) a-L-Methyleneoxy (4- CH2 — O- 2’) BNA, (B) P-D-Methyleneoxy (4-CH2 — 0-2’) BNA (also referred to as locked nucleic acid or LNA), (C) Ethyleneoxy (4’-(CH2)2 — 0-2’) BNA, (D) Aminooxy (4’-CH2 — O — N(R)-2’) BNA, (E) Oxyamino (4’-CH2 — N(R) — 0-2’) BNA, (F) Methyl (methyleneoxy) (4’-CH(CH3) — 0-2’) BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4
  • bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration.
  • a nucleoside comprising a 4’-2’ methylene-oxy bridge may be in the .alpha.-L configuration or in the .beta.-D configuration.
  • a-L-methyleneoxy (4-CH2 — 0-2’) bicyclic nucleosides have previously been incorporated into antisense polynucleotides that showed antisense activity.
  • substituted sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent.
  • modified sugar moieties are sugar surrogates.
  • the oxygen atom of the naturally occurring sugar is substituted, e.g., with a sulfur, carbon or nitrogen atom.
  • such modified sugar moiety also comprises bridging and/or non-bridging substituents as described above.
  • certain sugar surrogates comprise a 4’ -sulfur atom and a substitution at the 2’ -position and/or the 5’ position.
  • carbocyclic bicyclic nucleosides having a 4-2’ bridge have been described.
  • sugar surrogates comprise rings having other than 5-atoms.
  • a sugar surrogate comprises a six-membered tetrahydropyran (THP).
  • THP tetrahydropyran
  • Such tetrahydropyrans may be further modified or substituted.
  • Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), mannitol nucleic acid (MNA), and fluoro HNA (F-HNA).
  • the present disclosure provides nucleic acid molecules comprising modified nucleosides.
  • modified nucleotides may include modified sugars, modified nucleobases, and/or modified linkages. The specific modifications are selected such that the resulting polynucleotides possess desirable characteristics.
  • the nucleic acid molecules comprise one or more RNA-like nucleosides. In some embodiments, the nucleic acid molecules comprise one or more DNA- like nucleotides.
  • nucleosides of the present disclosure comprise one or more unmodified nucleobases. In some embodiments, nucleosides of the present disclosure comprise one or more modified nucleobases.
  • modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein.
  • the present disclosure provides nucleic acid molecules comprising linked nucleosides.
  • the nucleosides may be linked together using any internucleoside linkage.
  • the two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom.
  • Non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino ( — CH2 — N(CH3) — O — CH2 — ), thiodiester ( — O — C(O) — S — ). thionocarbamate ( — O — C(O)(NH) — S — ); siloxane ( — O — Si(H)2 — O — ); and N,N’ -dimethylhydrazine ( — CH2 — N(CH3) — N(CH3) — ).
  • Modified linkages compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the polynucleotide.
  • internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers.
  • Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing intemucleoside linkages are well known to those skilled in the art.
  • nucleic acid molecules described herein may comprise one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), such as for sugar anomers, or as (D) or (L) such as for amino acids etc. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
  • Further neutral intemucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides. Further neutral intemucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2 component parts.
  • nucleic acid molecules of the present disclosure involves chemically linking to the polynucleotide one or more additional moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the polynucleotide.
  • Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl- rac-glycerol or tri ethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.
  • lipid moieties such as a cholesterol moiety, cholic acid, a thioether
  • a vector is provided.
  • the vector comprises a nucleic acid molecule as provided for herein.
  • the nucleic acid molecule encodes for a signal peptide as provided for herein.
  • the nucleic acid molecule encodes for a novel signal peptide as provided for herein.
  • the nucleic acid molecule encodes for a recombinant polypeptide as provided for herein.
  • a cell comprises a nucleic acid molecule encoding for a signal peptide as provided for herein. In some embodiments, the cell comprises a nucleic acid molecule encoding for a recombinant polypeptide as provided for herein. In some embodiments, the cell comprises a vector as provided for herein. In some embodiments, the cell is any appropriate cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a mammalian cell used in a method of manufacturing a protein product. In some embodiments, the cell is any in vitro cell line. In some embodiments, the cell is any ex vivo cell. In some embodiments, the cell is present in vivo.
  • a composition comprising a vector encoding for a signal peptide as provided for herein and a delivery system. In some embodiments, the composition comprises a vector encoding for a recombinant polypeptide as provided for herein and a delivery system.
  • the delivery system may be a viral vector.
  • the viral vector is an RNA viral vector.
  • the viral vector is a DNA viral vector.
  • suitable viral vectors include adenovirus, adeno associated virus (AAV), retrovirus, herpesvirus, lentivirus, poxvirus, or papilloma virus vector.
  • the delivery system is a non-viral delivery system.
  • non-viral delivery systems include polymers, polyplexes, lipids, lipidoids, lipoplexes, liposomes, lipid fusion constructs, polymer nanoparticles, nanoparticles, lipid nanoparticles (LNPs), core-shell nanoparticles, solid lipid nanoparticles, metal nanoparticles, self-assembled nucleic acid nanoparticles, hyaluronidase, nanoparticle mimics, ribonucleoproteins, positively charged peptides, small molecule RNA-conjugates, aptamer- RNA chimeras, RNA-fusion protein complexes and any combination thereof.
  • a method of treating a disease or disorder comprises administering to a subject in need thereof a vector comprising a nucleic acid molecule encoding for a recombinant polypeptide as provided for herein, thereby treating the disease or disorder.
  • the payload protein of the recombinant polypeptide is a peptide or protein useful for the treatment of the disease or disorder.
  • the signal peptide directs the payload protein to be expressed at a particular cellular localization useful for the treatment of the disease or disorder.
  • the subject in need thereof is administered an effective amount of the vector comprising a nucleic acid molecule encoding for a recombinant polypeptide as provided for herein. In some embodiments, the subject in need thereof is administered a therapeutically effective amount of the vector comprising a nucleic acid molecule encoding for a recombinant polypeptide as provided for herein. In some embodiments, the disease or disorder is any disease or disorder that would benefit from the targeting of a payload protein to a desired location via a novel signal peptide as provided for herein.
  • Non-limiting examples of diseases or disorders, or therapeutic fields for which the recombinant polypeptides disclosed herein may be useful include, but are not limited to, oncology, immunology, cell and immune cell engineering/reprogramming (including, but not limited to, 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 and progenitor cell therapy, in situ engineered stem and progenitor cell therapy, cell surface functionalization with receptor targeting ligands, and the like), lysosomal diseases or disorders (
  • the disease or disorder is a disease or disorder associated with cytoplasmic vesicles or cytoplasmic granules, such as, but not limited to, proteinopathies, viral infections, genetic diseases, and tumors.
  • the disease or disorder is cancer.
  • a method of treating a cancer comprising administering to a subject in need thereof a vector comprising a nucleic acid molecule encoding for a recombinant polypeptide as provided for herein, thereby treating the cancer.
  • the payload protein of the recombinant polypeptide is a peptide or protein useful for the treatment of the cancer.
  • the signal peptide directs the payload protein to be expressed at the cell projections, thereby treating the cancer.
  • the subject in need thereof is administered an effective amount of the vector comprising a nucleic acid molecule encoding for a recombinant polypeptide as provided for herein.
  • the subject in need thereof is administered a therapeutically effective amount of the vector comprising a nucleic acid molecule encoding for a recombinant polypeptide as provided for herein.
  • a method of treating a proteinopathy comprising administering to a subject in need thereof a vector comprising a nucleic acid molecule encoding for a recombinant polypeptide as provided for herein, thereby treating the proteinopathy.
  • the payload protein of the recombinant polypeptide is a peptide or protein useful for the treatment of the proteinopathy.
  • the signal peptide directs the payload protein to be expressed at the cell projections, thereby treating the proteinopathy.
  • the signal peptide directs the payload protein to be expressed at a cytoplasmic vesicle or granule which promotes clearance or degradation of the vesicle or granule, thereby treating the proteinopathy.
  • the subject in need thereof is administered an effective amount of the vector comprising a nucleic acid molecule encoding for a recombinant polypeptide as provided for herein.
  • the subject in need thereof is administered a therapeutically effective amount of the vector comprising a nucleic acid molecule encoding for a recombinant polypeptide as provided for herein.
  • a method of treating a disease or disorder comprises administering to a subject in need thereof a composition comprising a vector comprising a nucleic acid molecule encoding for a recombinant polypeptide as provided for herein, thereby treating the disease or disorder.
  • the payload protein of the recombinant polypeptide is a peptide or protein useful for the treatment of the disease or disorder.
  • the signal peptide directs the payload protein to be expressed at a particular cellular localization useful for the treatment of the disease or disorder.
  • the subject in need thereof is administered an effective amount of the composition.
  • the subject in need thereof is administered a therapeutically effective amount of the composition.
  • the disease or disorder is any disease or disorder that would benefit from the targeting of a payload protein to a desired location via an novel signal peptide as provided for herein.
  • Non-limiting examples of diseases or disorders, or therapeutic fields for which the recombinant polypeptides disclosed herein may be useful include, but are not limited to, oncology, immunology, cell and immune cell engineering/reprogramming (including, but not limited to, 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 and progenitor cell therapy, in situ engineered stem and progenitor cell therapy, cell surface functionalization with receptor targeting ligands, and the like), lysosomal diseases or disorders (
  • the disease or disorder is a disease or disorder associated with cytoplasmic vesicles or cytoplasmic granules, such as, but not limited to, proteinopathies, viral infections, genetic diseases, and tumors.
  • the disease or disorder is cancer.
  • a method of treating a cancer comprising administering to a subject in need thereof a composition comprising a vector comprising a nucleic acid molecule encoding for a recombinant polypeptide as provided for herein, thereby treating the cancer.
  • the payload protein of the recombinant polypeptide is a peptide or protein useful for the treatment of the cancer.
  • the signal peptide directs the payload protein to be expressed at the cell projections, thereby treating the cancer.
  • the subject in need thereof is administered an effective amount of the composition.
  • the subject in need thereof is administered a therapeutically effective amount of the composition.
  • a method of treating a proteinopathy comprising administering to a subject in need thereof a composition comprising a vector comprising a nucleic acid molecule encoding for a recombinant polypeptide as provided for herein, thereby treating the proteinopathy.
  • the payload protein of the recombinant polypeptide is a peptide or protein useful for the treatment of the proteinopathy.
  • the signal peptide directs the payload protein to be expressed at the cell projections, thereby treating the proteinopathy.
  • the signal peptide directs the payload protein to be expressed at a cytoplasmic vesicle or granule which promotes clearance or degradation of the vesicle or granule, thereby treating the proteinopathy.
  • the subject in need thereof is administered an effective amount of the composition. In some embodiments, the subject in need thereof is administered a therapeutically effective amount of the composition.
  • a method of producing a payload protein comprising administering to a cell a vector comprising a nucleic acid molecule encoding for a recombinant polypeptide as provided for herein and culturing the cell under conditions sufficient to produce the payload protein.
  • the payload protein is secreted from the cell, and the method further comprises collecting a cell supernatant containing the payload protein and purifying the payload protein from the cell supernatant.
  • the payload protein is not secreted, and the method further comprises collecting the cells comprising the payload protein, lysing the cells, and purifying the payload protein from the cell lysate.
  • a method of manufacturing a payload protein comprising administering to a cell a vector comprising a nucleic acid molecule encoding for a recombinant polypeptide as provided for herein and culturing the cell under conditions sufficient to produce the payload protein.
  • the payload protein is secreted from the cell, and the method further comprises collecting a cell supernatant containing the payload protein and purifying the payload protein from the cell supernatant.
  • the payload protein is not secreted, and the method further comprises collecting the cells comprising the payload protein, lysing the cells, and purifying the payload protein from the cell lysate.
  • novel signal peptides of the present disclosure are useful in the treatment of a disease or disorder, such as those provided for herein, through inclusion of the signal peptide in a recombinant polypeptide comprising a payload protein used in the treatment of the disease or disorder. Correct or, in some instances, enhanced targeting of the payload protein to the cell projections is beneficial in the treatment of the disease or disorder. Furthermore, by utilizing the cells that are afflicted with the disease or disorder to produce the payload protein, the present methods avoid the need of timely and inefficient recombinant protein infusions and instead allow the patient’s own cells to produce the therapeutic molecule.
  • the present disclosure is also directed, in part, toward methods of producing or manufacturing a payload protein using the novel signal peptides as provided for herein.
  • the novel signal peptides as provided for herein direct the payload protein to a particular cellular location, such as, but not limited to, the nucleus, the endoplasmic reticulum, the mitochondria, the cytoplasm, the cell membrane, cell junctions, cell projections, synapses, or lysosomes, or excreted into the extracellular space.
  • a particular cellular location such as, but not limited to, the nucleus, the endoplasmic reticulum, the mitochondria, the cytoplasm, the cell membrane, cell junctions, cell projections, synapses, or lysosomes, or excreted into the extracellular space.
  • Use of the novel signal peptides to direct a payload protein to a particular cellular location can enhance the expression and or production of the payload protein.
  • pDNA transfection was executed in cells. pDNAs were encapsulated in Lipfectamine2000 and administered to HeLa cells at 100 ng/well in 6 well plates and allowed to incubate for 24 hours. Following incubation, CellMask Deep Red Actin Tracking Stain was used to stain cell projections of live cells via manufacturer protocols. The cells were then imaged using confocal microscopy for CellMask Deep Red Actin Tracking Stain (cell projection) signal (Cy-5) with mCherry signal. Fiji software was utilized to assess co-localization of cell projection signal with mCherry signal and quantified.
  • Example 1 Use of novel signal peptides to deliver GFP to cell projections.
  • Example 2 Use of novel signal peptides to deliver an antibody to cell projections.
  • Example 2 is carried out using similar methods to Example 1, with the exception that the payload protein is an antibody.
  • the payload protein is an antibody.
  • plasmids encoding antibodies without the cell projection targeting signal peptides are also used.
  • proper antibody targeting is determined via staining for the payload antibody with an appropriate secondary antibody and assessment via confocal microscopy.
  • Example 3 mRNA synthesis and mRNA-Nanoparticle formulation
  • mRNA are 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 Patent Publication Serial No. WO2024064874A2, each of which is hereby incorporated by reference in their entirety. Briefly, linear pDNA with optimized 5’(3’)-untranslated regions (UTR) and poly A sequences were obtained first by enzyme digestion, then IVT reactions were prepared with standard protocols with Nl-methylpseudouridine-5’ -triphosphate modification. Finally, mRNA was capped (Cap-1) by Vaccinia Capping Enzyme and 2’-O-methyltransferase (NEB).
  • mRNA-loaded LNP formulations are formed using the ethanol dilution method 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 Patent Publication Serial No. WO2024064874A2, each of which is hereby incorporated by reference in their entirety. Briefly, all lipids with specified molar ratios were dissolved in ethanol and RNA was dissolved in 10 mM citrate buffer (pH 4.0) first.
  • Example 4 Signal peptide targeted mCherry is properly secreted from cells transfected with pDNA or treated with mRNA loaded LNPs.
  • SPs of the present example are as provided in Table 3 below:
  • HeLa cells were transfected with wild type (WT) mCherry pDNA containing no SP and gLuc-mCherry pDNA via Lipofectamine2000, and both intracellular and extracellular fluorescence were quantified via fluorescent microscopy at 24-, 48-, and 72-hours post transfection, wherein the gLuc SP induced high levels of mCherry expression into media.
  • WT wild type
  • mCherry protein content present in cell medium and cell lysates were quantified via a fluorescent plate reader individually at 24-, 48-, and 72-hours revealing an increase in mCherry fluorescence in the gLuc SP group (FIG. 2A).
  • the set of SPs was then expanded to include a negative control (scramble sequence), hAlb, hApoB, and hFVII, in addition to gLuc.
  • HeLa cells were again transfected with the pDNA constructs using Lipofectamine2000. Images taken 72h post transfection via fluorescence microscopy and IVIS demonstrated that the SPs hApoB, gLuc, and hFVII all generated high levels of mCherry protein secretion, while the NC and hAlb constructs effectively mediated intracellular mCherry expression but did not promote significant extracellular secretion (FIG. 2B).
  • the data for this example demonstrates that pDNA can be transcribed into mRNA via IVT, and that transfection of cells with pDNA via lipofectamine or transfection of cells with mRNA generated from the same pDNA via LNPs results in comparable experimental outcomes.
  • the data in this example supports the conclusion that the constructs tested in Example 1 in pDNA format would be expected to result in similar outcomes in embodiments where the pDNA is first transcribed into mRNA via IVT.

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Abstract

L'invention concerne de nouveaux peptides de signal qui dirigent une protéine codée vers les parties saillantes d'une cellule, des polypeptides recombinants les comprenant, des molécules d'acide nucléique codant pour ceux-ci, des compositions pharmaceutiques les contenant, et leurs méthodes d'utilisation.
PCT/US2024/053747 2023-10-30 2024-10-30 Nouveaux peptides de signal ciblant des parties saillantes de cellule et leurs utilisations Pending WO2025096678A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006051333A2 (fr) * 2004-11-15 2006-05-18 Ares Trading S.A. Proteines contenant des motifs repetes riches en leucine (lrr)

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006051333A2 (fr) * 2004-11-15 2006-05-18 Ares Trading S.A. Proteines contenant des motifs repetes riches en leucine (lrr)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE UniProt 13 September 2023 (2023-09-13), ANONYMOUS: "Full=Leucine rich repeat, Ig-like and transmembrane domains 3 {ECO:0000313|Ensembl:ENSGGOP00000001364.3}", XP093321938, Database accession no. LRIT3_HUMAN *

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