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WO2022125673A1 - Peptides de pénétration cellulaire, complexes peptidiques et leurs procédés d'utilisation - Google Patents

Peptides de pénétration cellulaire, complexes peptidiques et leurs procédés d'utilisation Download PDF

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Publication number
WO2022125673A1
WO2022125673A1 PCT/US2021/062422 US2021062422W WO2022125673A1 WO 2022125673 A1 WO2022125673 A1 WO 2022125673A1 US 2021062422 W US2021062422 W US 2021062422W WO 2022125673 A1 WO2022125673 A1 WO 2022125673A1
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WO
WIPO (PCT)
Prior art keywords
seq
peptide
cell
penetrating
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2021/062422
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English (en)
Inventor
Steven Chen
Zachary CROOK
Natalie Winblade Nairn
Scott Presnell
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Blaze Bioscience Inc
Fred Hutchinson Cancer Center
Original Assignee
Blaze Bioscience Inc
Fred Hutchinson Cancer Center
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Filing date
Publication date
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Publication of WO2022125673A1 publication Critical patent/WO2022125673A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43522—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from scorpions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • a major objective of drug development is ensuring that a therapeutic agent can reach its intended target. Frequently, this means designing therapeutics that can reach the cytosol, nucleus, or other subcellular compartments of a cell. Drugs with poor cell permeation may not reach an intended cytosolic, nuclear, or other intracellular drug target, resulting in decreased efficacy. Moreover, drugs with poor cell permeation may also require higher dosages than those with more targeted or more successful cell permeation, resulting in a higher incidence of toxicity or side effects. Biologic drugs, such as those made of peptides, proteins, knotted peptides, or miniproteins, RNA, and DNA frequently are unable to enter the cytosol and therefore these classes of drugs are not able to drug intracellular targets. There is a need for methods to deliver drugs with low permeation or absorption across cellular membranes to reach cytosolic and nuclear drug targets.
  • the present disclosure provides a peptide complex comprising a cellpenetrating peptide comprising a sequence that has: at least 80% sequence identity with any one of SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 20 - SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 44 - SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 75, SEQ ID NO: 77, or SEQ ID NO: 80, or a fragment thereof; at least 85% sequence identity with any one of SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO:
  • the peptide complex comprises a sequence that has at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, or a fragment thereof. In some aspects, the peptide complex comprises a sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84. In some aspects, the peptide complex comprises a sequence of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 8; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 40; SEQ ID NO: 41; or SEQ ID NO: 42. In some aspects, the cell-penetrating peptide is fused or linked to a cargo molecule.
  • the present disclosure provides a peptide complex comprising a cellpenetrating peptide and a cargo molecule fused or linked to the cell-penetrating peptide, wherein the cell-penetrating peptide comprises a sequence that has at least 80%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, or SEQ ID NO: 408 - SEQ ID NO: 457, or a fragment thereof.
  • the present disclosure provides a peptide complex comprising a cellpenetrating peptide and a cargo molecule fused or linked to the cell-penetrating peptide, wherein the cell-penetrating peptide comprises a sequence that has at least 80%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, SEQ ID NO: 195 - SEQ ID NO: 254, or a fragment thereof, and wherein the cargo molecule is a cystine-dense peptide, a DNA- binding protein, an RNA-binding protein, a transcriptional activator, a transcriptional inhibitor, a promotor of protein degradation, a molecular glue, a proteolysis targeting chimera, an oligonucleotide, a protein-protein interaction inhibitor, or combinations thereof.
  • the cargo molecule is
  • the cell-penetrating peptide comprises a sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254.
  • the peptide complex comprises a sequence of: SEQ ID NO: 195; SEQ ID NO: 197; or SEQ ID NO: 198.
  • the cellpenetrating peptide comprises a sequence of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 8; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 40; SEQ ID NO: 41; or SEQ ID NO: 42.
  • the present disclosure provides a peptide complex comprising a cellpenetrating peptide and a cargo molecule fused or linked to the cell-penetrating peptide, wherein the cell-penetrating peptide comprises a sequence of any one of SEQ ID NO: 325 - SEQ ID NO: 342 or SEQ ID NO: 343 - SEQ ID NO: 351, and wherein the cargo molecule wherein the cargo molecule is a cystine-dense peptide, a DNA-binding protein, an RNA-binding protein, a transcriptional activator, a transcriptional inhibitor, a promotor of protein degradation, a molecular glue, a proteolysis targeting chimera, an oligonucleotide, a protein-protein interaction inhibitor, or combinations thereof.
  • the cell-penetrating peptide comprises at least 0.5, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 3.0, at least 3.3, at least 3.5, at least 3.7, or at least 4.0 arginine amino acid residues per ten amino acid residues.
  • the cell-penetrating peptide comprises no more than 0.5, no more than 0.8, no more than 0.9, no more than 1.0, no more than 1.1, no more than 1.2, no more than 1.3, no more than 1.4, no more than 1.5, no more than 1.6, no more than 1.7, no more than 1.8, no more than 1.9, no more than 2.0, no more than 2.1, no more than 2.2, no more than 2.3, no more than 2.4, no more than 2.5, no more than 3.0, no more than 3.3, no more than 3.5, no more than 3.7, or no more than 4.0 arginine amino acid residues per ten amino acid residues.
  • the cell-penetrating peptide comprises no arginine amino acid residues.
  • the cell-penetrating peptide comprises at least 0.5, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 3.0, at least 3.3, at least 3.5, at least 3.7, or at least 4.0 lysine amino acid residues per ten amino acid residues.
  • the cell-penetrating peptide comprises no more than 0.5, no more than 0.8, no more than 0.9, no more than 1.0, no more than 1.1, no more than 1.2, no more than 1.3, no more than 1.4, no more than 1.5, no more than 1.6, no more than 1.7, no more than 1.8, no more than 1.9, no more than 2.0, no more than 2.1, no more than 2.2, no more than 2.3, no more than 2.4, no more than 2.5, no more than 3.0, no more than 3.3, no more than 3.5, no more than 3.7, or no more than 4.0 lysine amino acid residues per ten amino acid residues.
  • the cell-penetrating peptide comprises no lysine amino acid residues. [0012] In some aspects, the cell-penetrating peptide comprises at least 0.5, at least 0.8, at least
  • the cell-penetrating peptide comprises no more than 4.0, no more than 3.9, no more than 3.8, no more than 3.7, no more than 3.6, no more than 3.5, no more than 3.4, no more than 3.3, no more than 3.2, no more than 3.1, no more than 3.0, no more than 2.9, no more than 2.8, no more than 2.7, no more than
  • the cellpenetrating peptide comprises at least 0.03, at least 0.05, at least 0. 1, at least 0.2, at least 0.5, at least 0.8, at least 0.9, at least 1 .0, at least 1 .
  • the cell-penetrating peptide comprises a ratio of positively charged amino acid residues to negatively charged amino acid residues of at least 1.0, at least 1.5, at least 2, at least 2.5, at least 2.75, at least 3, at least 3.5, at least 4, at least 5, at least 6, or at least 7.
  • the cell-penetrating peptide comprises a ratio of negatively charged amino acid residues to positively charged amino acid residues of no more than 1.0, no more than 1.5, no more than 2, no more than 2.5, no more than 2.75, no more than 3, no more than 3.5, or no more than 4. In some aspects, the cell-penetrating peptide comprises at least 1, at least 2, at least 3, or at least 4 negatively charged amino acids.
  • the cell-penetrating comprises at least 1, at least 2, at least 3, or at least 4 histidine amino acid residues. In some aspects, the cell-penetrating comprises at least 1, at least 2, at least 3, or at least 4 proline amino acid residues. In some aspects, the cell-penetrating peptide comprises an amphipathic a-helix. In some aspects, the cell-penetrating peptide comprises at least two, at least three, at least four, at least five, or at least six cysteine amino acid residues. In some aspects, the cell-penetrating peptide comprises no cysteine amino acid residues.
  • the present disclosure provides a peptide complex comprising a cellpenetrating peptide comprising: at least 1.0, at least 1.8, at least 1.9, at least 2.0, at least 2.2, at least 2.5, at least 3.0, at least 3.3, or at least 3.5 arginine amino acid residues per 10 amino acid residues; and at least two, at least three, at least four, at least five, or at least six cysteine amino acid residues; wherein the cell -penetrating peptide is fused or linked to a cargo molecule.
  • the cell-penetrating peptide comprises at least four cysteine amino acid residues. In some aspects, the cell-penetrating peptide comprises no more than 3.3, no more than
  • the present disclosure provides a peptide complex comprising a cellpenetrating peptide comprising: at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, or at least 2.7 arginine amino acid residues per 10 amino acid residues; and no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than
  • the present disclosure provides a peptide complex comprising a cellpenetrating peptide comprising: at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 3.0, at least 3.3, or at least 3.5 arginine amino acid residues per 10 amino acid residues; and no more than 3.3, no more than 3.2, no more than 3.1, no more than 3.0, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2.1, or no more than 2.0 positively charged amino acid residue per 10 amino acid residues at a pH of 7.4; wherein the cell-penetrating peptide is fused or linked to a cargo molecule.
  • the positively charged residues are arginine, lysine, or any combination thereof.
  • the cell-penetrating peptide comprises no cysteine amino acid residues.
  • the cell-penetrating peptide comprises at least two, at least three, at least four, at least five, or at least six cysteine amino acid residues.
  • the cell-penetrating peptide comprises a disulfide through disulfide knot.
  • the cell-penetrating peptide comprises a plurality of disulfide bridges formed between cysteine residues.
  • the cell-penetrating peptide comprises three or more disulfide bridges formed between cysteine residues, wherein one of the disulfide bridges passes through a loop formed by two other disulfide bridges. In some aspects, the cell-penetrating peptide comprises at least 1.1 arginine amino acid residues per 10 amino acid residues. In some aspects, the cell-penetrating peptide comprises no more than 2.7 positively charged amino acid residue per 10 amino acid residues.
  • the cell-penetrating peptide is derived from maurocalcin, imperatoxin, hadrucalcin, hemicalcin, opicalcin-1, opicalcin-2, midkine, MCoTI-II, chlorotoxin, huwentoxin, vejocalcin, intrepicalcin, or urocalcin.
  • the cell-penetrating peptide or the fragment thereof comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least, 8, at least
  • the cell-penetrating peptide or the fragment thereof comprises no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than
  • no more than 11 no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, no more than 30, no more than 31, no more than 32, no more than 33, no more than 34, no more than 35, no more than 40, no more than 50, no more than 60, no more than 70, or no more than 80 residues.
  • the cellpenetrating peptide comprises an isoelectric point within a range from about 6.0 to about 12.0, from about 6.0 to about 10.0, from about 6.5 to about 7.5, from about 7.0 to about 10.0, or from about 8.0 to about 10.0.
  • the cell-penetrating peptide is stable at pH of from 6.5 to 7.5.
  • the cell-penetrating peptide is stable at pH values within a range from pH 5.0 to pH 7.0.
  • the cargo molecule comprises a cargo peptide comprising four or more cysteine amino acid residues and at least two disulfide bonds.
  • the cargo molecule is fused or linked to the cell-penetrating peptide at an N-terminus or a C-terminus of the cell-penetrating peptide.
  • the cargo molecule comprises at least 5, at least 6, at least 7, at least 8, 9, at least 10, at least 15, or at least 20 hydrogen bond donors.
  • the cargo molecule comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20 hydrogen bond acceptors.
  • the cargo molecule comprises a molecular weight of at least 500 Da, at least 600 Da, at least 700 Da, at least 800 Da, at least 900 Da, or at least 1000 Da.
  • the cargo molecule comprises a log of a partition coefficient (logP) of at least 5.0, at least 5.2, at least 5.4, at least 5.5, at least 5.6, at least 5.8, or at least 6.0.
  • logP partition coefficient
  • the cargo molecule comprises an antibody, an antibody fragment, an Fc domain, a single chain Fv, an intrabody, or a nanobody.
  • the cargo molecule comprises a cystine-dense peptide, an affibody, a B-hairpin, an avimer, an adnectin, a stapled peptide, a nanofittin, a kunitz domain, a fynomer, or a bicyclic peptide.
  • the cargo molecule comprises an immunomodulatory imide drug, a Boc3Arg tag, an adamantyl group, or a carborane.
  • the cargo molecule comprises a target-binding molecule.
  • the target-binding molecule comprises a target-binding peptide.
  • the target-binding peptide comprises a cystine-dense peptide.
  • the target-binding peptide is capable of binding a transcription factor or a tyrosine kinase.
  • the target-binding peptide is capable of binding TEAD, cold-inducible RNA-binding protein, androgen receptor, ikaros, aiolos, a nuclear receptor, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-8, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CSNK1A1, GSPT1, ZFP91, ZNF629, ZNF91, ZNF276, ZNF653, ZNF827, WIZ, ZNF692, GZF1, ZNF98, BCL6, cMYC, tau, AKT,
  • the target-binding peptide comprises a sequence with at least 90% or at least 95% sequence identity to any one of SEQ ID NO: 295, SEQ ID NO: 298, or SEQ ID NO: 364 - SEQ ID NO: 407. In some aspects, the targetbinding peptide comprises a sequence of any one of SEQ ID NO: 295, SEQ ID NO: 298, or SEQ ID NO: 364 - SEQ ID NO: 407. In some aspects, the target-binding peptide comprises a sequence of SEQ ID NO: 295.
  • the peptide complex comprises a sequence of SEQ ID NO: 307 or SEQ ID NO: 308.
  • the target-binding molecule binds a ubiquitin ligase.
  • the ubiquitin ligase comprises cereblon, cellular inhibitor of apoptosis protein 1, MDM2, DCAF15, DCAF16, cullin-4A, a Cul2-Rbxl-EloN/C-VHL E3 ubiquitin ligase, APC/C activator protein CDH1, or von Hippel -Lindau protein.
  • the target-binding molecule comprises an immunomodulatory imide drug.
  • the target-binding molecule comprises a thalidomide, a pomalidomide, a lenalidomide, a methyl bestatin, a bestatin, a nutlin-3, or a VHL ligand 1.
  • the cargo molecule comprises an anticancer agent, a transcription factor binding agent, an inhibitor of protein-protein interactions, a promoter of protein-protein interactions, a transcription factor, an RNA, a CRISPR component, a molecular glue, a proteolysis targeting chimera, an oligonucleotide, a protein-protein interaction inhibitor, or an immunomodulating agent.
  • the oligonucleotide comprises a DNA, an RNA, an antisense oligonucleotide, an aptamer, an miRNA, an alternative splicing modulator, an mRNA-binding sequence, an miRNA-binding sequence, an siRNA-binding sequence, an RNaseHl -binding oligonucleotide, a RISC-binding oligonucleotide, a polyadenylation modulator, a gapmer, a RIG-I ligand, an mRNA, an antisense RNA, a small interfering RNA, a guide RNA, a U1 adaptor, a micro RNA, or a combination thereof.
  • the oligonucleotide comprises: a sequence of any one of SEQ ID NO: 488 - SEQ ID NO: 573; a sequence that binds to any one of SEQ ID NO: 574 - SEQ ID NO: 611; a sequence of any one of SEQ ID NO: 574 - SEQ ID NO: 611, or a fragment thereof; or a sequence targeting or encoding a gene target provided in TABLE 12.
  • the CRISPR component is a guide RNA, a tracrRNA, a crRNA, or a Cas nuclease.
  • the cargo molecule comprises a detectable agent or a therapeutic agent.
  • the detectable agent is a fluorophore, a near-infrared dye, a contrast agent, a nanoparticle, a metal-containing nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a radioisotope, or a radionuclide chelator.
  • the peptide complex further comprises one or more chemical modifications.
  • the chemical modification extends the half-life or modifies a pharmacokinetics of the peptide complex.
  • the chemical modification blocks an N-terminus of the peptide complex.
  • the chemical modification comprises methylation, acetylation, or acylation.
  • the chemical modification comprises: methylation of one or more lysine residues or analogue thereof; methylation of the N-terminus; or methylation of one or more lysine residue or analogue thereof and methylation of the N- terminus.
  • the peptide complex further comprises a half-life modifying agent, a nuclear localization signal, or an endosomal escape motif.
  • the half-life modifying agent comprises a polymer, a polyethylene glycol (PEG), a hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer of proline, alanine and serine, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, palmitic acid, an albumin, or a molecule that binds to albumin.
  • PEG polyethylene glycol
  • a hydroxyethyl starch polyvinyl alcohol
  • a water soluble polymer a zwitterionic water soluble polymer
  • a water soluble poly(amino acid) a water soluble poly(amino acid)
  • the peptide complex further comprises an additional active agent.
  • the additional active agent is fused to the cell-penetrating peptide or the cargo molecule at an N-terminus, at the epsilon amine of an internal lysine residue, at the carboxylic acid of an aspartic acid or glutamic acid residue, or a C-terminus of the cell-penetrating peptide or the cargo molecule by a linker.
  • the linker comprises an amide bond, an ester bond, a carbamate bond, a carbonate bond, a hydrazone bond, an oxime bond, a disulfide bond, a thioester bond, a thioether bond, or a carbon-nitrogen bond.
  • the linker comprises a peptide linker.
  • the peptide linker comprises a sequence of any one of SEQ ID NO: 255 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485.
  • the linker is a cleavable linker or a pH sensitive linker.
  • the cleavable linker comprises a cleavage site for matrix metalloproteinases, thrombin, cathepsins, or betaglucuronidase.
  • the linker is a hydrolytically labile linker.
  • the linker is a stable linker.
  • the additional active agent comprises at least 5, at least 6, at least 7, at least 8, 9, at least 10, at least 15, or at least 20 hydrogen bond donors. In some aspects, the additional active agent comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20 hydrogen bond acceptors. In some aspects, the additional active agent comprises a molecular weight of at least 500 Da, at least 600 Da, at least 700 Da, at least 800 Da, at least 900 Da, or at least 1000 Da. In some aspects, the additional active agent comprises a log of a partition coefficient (logP) of at least 5.0, at least 5.2, at least 5.4, at least 5.5, at least 5.6, at least 5.8, or at least 6.0.
  • logP partition coefficient
  • the additional active agent is a detectable agent or a therapeutic agent.
  • the detectable agent is a fluorophore, a near-infrared dye, a contrast agent, a nanoparticle, a metal-containing nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a radioisotope, or a radionuclide chelator.
  • the cell-penetrating peptide is a membrane-penetrating peptide. In some aspects, the cell-penetrating peptide is a nuclear envelope-penetrating peptide. In some aspects, the cell-penetrating peptide is a blood brain barrier-penetrating peptide. In some aspects, the cell-penetrating peptide is arranged in a multimeric structure with at least one other peptide. In some aspects, the cell-penetrating peptide lacks an immunogenic sequence. In some aspects, the cell-penetrating peptide is modified to increase homology to a human protein sequence. In some aspects, the cell-penetrating peptide is modified to increase resistance to degradation.
  • the cell-penetrating peptide is modified to reduce an affinity of the peptide for a human leukocyte antigen complex, a major histocompatibility complex, or both. In some aspects, the cell-penetrating peptide is an active agent.
  • the present disclosure provides a pharmaceutical composition comprising a peptide complex of the present disclosure, or a salt thereof, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is formulated for administration to a subject.
  • the pharmaceutical composition is formulated for oral administration, intravenous administration, subcutaneous administration, intramuscular administration, or a combination thereof.
  • the present disclosure provides a method of delivering a cargo molecule across a cellular layer of a cell, the method comprising: contacting the cell with a peptide complex comprising a cell-penetrating peptide fused or linked to cargo molecule, wherein the cell-penetrating peptide comprises a sequence that has at least 80%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, or SEQ ID NO: 408 - SEQ ID NO: 457, or a fragment thereof; penetrating the cellular layer with the cell -penetrating peptide; and delivering the cargo molecule across the cellular layer.
  • the present disclosure provides a method of delivering a cargo molecule across a cellular layer of a cell, the method comprising: contacting the cell with a peptide complex of the present disclosure comprising a cell-penetrating peptide fused or linked to cargo molecule; penetrating the cellular layer with the cell-penetrating peptide; and delivering the cargo molecule across the cellular layer, thereby treating the condition.
  • the present disclosure provides a method of treating a condition in a subject in need thereof, the method comprising: administering to the subject a composition comprising a peptide complex comprising a cell-penetrating peptide fused or linked to cargo molecule, wherein the cell -penetrating peptide comprises a sequence that has at least 80%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, or SEQ ID NO: 408 - SEQ ID NO: 457, or a fragment thereof; penetrating a cellular layer of a cell of the subject with the cell-penetrating peptide; and delivering the cargo molecule across the cellular layer, thereby treating the condition.
  • the present disclosure provides a method of treating a condition in a subject in need thereof, the method comprising: administering to the subject a composition comprising a peptide complex of the present disclosure comprising a cell-penetrating peptide fused or linked to cargo molecule; penetrating a cellular layer of a cell of the subject with the cell-penetrating peptide; and delivering the cargo molecule across the cellular layer.
  • the disease or condition is cancer, a neurological disorder, an inflammatory disorder, an immune disorder, a neurodegenerative disorder, or a genetic disorder.
  • the cancer is liver cancer, breast cancer, colon cancer, lung cancer, prostate cancer, brain cancer, skin cancer, pancreatic cancer, leukemia, or lymphoma.
  • the composition is administered by inhalation, intranasally, orally, topically, intravenously, subcutaneously, intramuscularly administration, intraperitoneally, intratumorally, intrathecally, intravitreally, or a combination thereof.
  • the composition is administered intravenously as a bolus, injection, infusion, or prolonged infusion.
  • the cellular layer is a cell membrane, a nuclear envelope, an endosomal membrane, a lysosomal membrane, or a blood brain barrier.
  • the method comprises penetrating a cell membrane of the cell. In some aspects, the method comprises penetrating a nuclear envelope of the cell. In some aspects, the method comprises delivering the cargo molecule to a cytosol of the cell. In some aspects, the method comprises producing a cargo molecule concentration of at least 10 nM, at least 50 nM, at least 100 nM, at least 300 nM, at least 500 nM, at least 700 nM, at least 1000 nM, at least 1200 nM, at least 1400 nM, or at least 1600 nM in the cytosol of the cell. In some aspects, the method comprises delivering the cargo molecule to a nucleus of the cell.
  • the method comprises producing a cargo molecule concentration of at least 10 nM, at least 50 nM, at least 100 nM, at least 300 nM, at least 500 nM, at least 700 nM, at least 1000 nM, at least 1200 nM, at least 1400 nM, or at least 1600 nM in the nucleus of the cell.
  • the cargo molecule comprises an oligonucleotide that binds a target sequence
  • the method further comprises modulating alternative splicing of the target sequence, dictating the location of a polyadenylation site of the target sequence, inhibiting translation of the target sequence, inhibiting binding of the target sequence to a secondary target sequence, recruiting RISC to the target sequence, recruiting RNaseHl to the target sequence, inducing cleavage of the target sequence, or regulating the target sequence upon binding of the oligonucleotide to the target sequence.
  • the method comprises delivering the cargo molecule into an intracellular space or a paracellular space.
  • the intracellular space is a nanolumen.
  • the cell has uncontrolled or dysregulated cell growth.
  • the cell is a cancerous cell or a tumor cell.
  • the cell is a pancreatic cell, liver cell, colon cell, smooth muscle cell, ovarian cell, breast cell, lung cell, brain cell, skin cell, ocular cell, blood cell, lymph cell, immune system cell, reproductive cell, reproductive organ cell, prostate cell, fibroblast, kidney cell, adenocarcinoma cell, glioma stem cell, tumor cell, or any combination thereof.
  • the method further comprises binding the cargo molecule to a target molecule.
  • the target molecule comprises a transcription factor or a tyrosine kinase.
  • the target molecule comprises TEAD, cold-inducible RNA-binding
  • -l i protein, androgen receptor ikaros, aiolos, a nuclear receptor, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-5, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CSNK1A1, GSPT1, ZFP91, ZNF629, ZNF91, ZNF276, ZNF653, ZNF827, WIZ, ZNF692, GZF1, ZNF98, BCL6, cMYC, tau, AKT, mTOR, STAT3, RAS, RAF, MEK, ERK, P-caten
  • the method further comprises inhibiting the target molecule.
  • the method further comprises binding the cargo molecule to a ubiquitin ligase.
  • the ubiquitin ligase comprises cereblon, cellular inhibitor of apoptosis protein 1, MDM2, DCAF15, DCAF16, cullin-4A, a Cul2-Rbxl-EloN/C-VHL E3 ubiquitin ligase, APC/C activator protein CDH1, or von Hippel -Lindau protein.
  • the cargo molecule comprises an immunomodulatory imide drug, a thalidomide, a pomalidomide, a lenalidomide, a methyl bestatin, a bestatin, a nutlin-3, or a VHL ligand 1.
  • the method further comprises ubiquitinating the target molecule upon binding of the cargo molecule to the target molecule and the ubiquitin ligase.
  • the cargo molecule comprises a detectable agent.
  • the method further comprises imaging the cell.
  • the method further comprises detecting a presence, absence, location, or a combination thereof of the detectable agent in the cell.
  • the method comprises delivering at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% of the peptide complex across the cellular layer.
  • FIG. 1A schematically illustrates a process of covalently tagging peptides with a benzylguanine SNAP substrate by using BG-GLA-NHS.
  • the NHS-ester of a benzylguanine SNAP substrate (BG-GLA-NHS) reacts with a reactive amine group, such as at the N-terminus or at a lysine residue, of a peptide to generate a SNAP substrate-tagged peptide (referred to as a BG-peptide, a BG-peptide complex, a BG-cystine-dense peptide, or a BG-cell-penetrating peptide).
  • FIG. IB schematically illustrates a SNAP penetration assay (SNAPP A) to quantify cell penetration of a BG-peptide.
  • SNAPP A SNAP penetration assay
  • BG-fluorophore a SNAP substrate- tagged fluorophore that can diffuse across the cell membrane
  • Free SNAP -tag protein in the cell will bind to the BG-fluorophore, while SNAP -tag protein that is bound to BG-peptide cannot bind to the fluorescent SNAP substrate because the BG-peptide already occupies the SNAP -tag.
  • a cell penetrant SNAP substrate-tagged peptide (BG-peptide)
  • BG-peptide cell penetrant SNAP substrate-tagged peptide
  • a cell penetrant peptide blocks the SNAP -tag from binding fluorescent SNAP substrate and therefore reduces the fluorescent signal in the assay.
  • This assay may also be used to assess penetration into other cellular compartments, such as the nucleus, by expressing a SNAP -tag protein fusion that localizes to the cellular compartment of interest.
  • FIG. 2 shows representative images of reversed-phase high performance liquid chromatography (RP-HPLC, top) and size exclusion chromatography (SEC, second, third, and bottom) associated with a typical SNAP substrate conjugation reaction with peptide and subsequent purification process.
  • the bottom two images show RP-HPLC traces of concentrated fractions following SEC, demonstrating separation and purification of BG-peptides and unreacted peptides. Peaks in the top panel correspond to, from left to right, BG-GLA-NHS hydrolyzed to BG-GLA-OH, BG-GLA-NHS ligand, KR CTX (SEQ ID NO: 71), and KR CTX BG-peptide (BG-SEQ ID NO: 71).
  • FIG. 3 shows representative fluorescence images of the SNAPPA assay after contacting various BG-peptides comprising cystine-dense peptides to HeLa cells stably transfected with pSNAPf and expressing SNAP -tag, followed by addition of BG-fluorophore and washing away unbound BG-fluorophore.
  • BG-peptides comprising cystine-dense peptides, KR_KTxl5.8 (BG-SEQ ID NO: 74), KR_KTx2.2 (BG-SEQ ID NO: 73), Tx677 (BG-SEQ ID NO: 93), KR CTI (BG-SEQ ID NO: 72), KR CTX (BG-SEQ ID NO: 71), KR MCa (BG-SEQ ID NO: 67), KR_HwTx-IV, (BG-SEQ ID NO: 66), and KR IpTxa (BG-SEQ ID NO: 59), were assayed for cell penetration.
  • Phosphate buffered saline (PBS) was used as a negative control.
  • FIG. 4A shows the results of a SNAP penetration assay measuring cellular penetration into the cytosol by various BG-peptides comprising cystine-dense peptides exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag protein in the cytosol.
  • the BG-peptides comprising cystine-dense peptides were assayed for cell penetration to reach the cytosol.
  • FIG. 4B shows the results of a SNAP penetration assay measuring cellular penetration into the nucleus by various BG-peptides comprising cystine-dense peptides exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag protein in the nucleus.
  • the BG-peptides comprising cystine-dense peptides were assayed for cell penetration to reach the nucleus.
  • FIG. 5 schematically illustrates BG-peptides complexes comprising cell-penetrating peptides and cargo peptides assayed by SNAPPA in FIG. 6A - FIG. 8D.
  • FIG. 6A shows results of a SNAP penetration assay to measure cellular penetration into the cytosol of the cell-penetrating peptide complexes illustrated in FIG. 5 exposed to NIH3T3, HeLa and HEK293 cells.
  • BG-MCa-KTx3.10 Cellular penetration into the cytosol of penetrating peptide complexes BG-MCa-KTx3.10 (BG-SEQ ID NO: 312), BG-MCa-elafin (BG-SEQ ID NO: 316), BG- KR_IpTxa-KTx3.10 (BG-SEQ ID NO: 313) and BG-KR_IpTxa-elafin (BG-SEQ ID NO: 317) was measured, along with BG-cargo peptides without cell-penetrating peptides BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297).
  • BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297) were used as comparators to assess the basal level of penetration of these cargo peptides with the addition of further cell-penetrating peptides.
  • the cell penetrant moiety BG-GLA-OH formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization.
  • BG-KR_IpTxa (BG-SEQ ID NO: 59) was used as a comparator to assess the level of penetration in the absence of a cargo peptide.
  • FIG. 6B shows results of a SNAP penetration assay to measure cellular penetration into the nucleus of the cell-penetrating peptide complexes illustrated in FIG. 5 exposed to NIH3T3, HeLa and HEK293 cells.
  • BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297) were used as comparators to assess the basal level of penetration.
  • the cell penetrant moiety BG-GLA-OH formed when BG-GLA-NHS (BG- substrate) is hydrolyzed, was used as a positive control and for normalization.
  • BG-KR_IpTxa (BG-SEQ ID NO: 59) was used as a comparator to assess the level of penetration in the absence of a cargo peptide.
  • FIG. 7A shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide MCa-KTx3.10, with increasing concentrations of a BG- MCa-KTx3.10 (BG-SEQ ID NO: 312) cell-penetrating peptide complex exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm. The data was normalized to cells exposed to 10 pM BG-GLA-OH.
  • FIG. 7B shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide KR_IpTxa-KTx3.10, with increasing concentrations of a BG-KR_IpTxa-KTx3.10 (BG-SEQ ID NO: 313) cell-penetrating peptide complex exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm. The data was normalized to cells exposed to 10 pM BG-GLA-OH.
  • FIG. 7C shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide MCa-elafin, with increasing concentrations of a BG- MCa-elafin (BG-SEQ ID NO: 316) cell-penetrating peptide complex exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm.
  • the data was normalized to cells exposed to 10 pM BG-GLA-OH.
  • FIG. 7D shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide KR IpTxa-elafin, with increasing concentrations of a BG-KR_IpTxa-elafin (BG-SEQ ID NO: 317) cell -penetrating peptide complex exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm. The data was normalized to cells exposed to 10 pM BG-GLA-OH.
  • FIG. 8A shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide MCa-KTx3.10, with increasing concentrations of a BG- MCa-KTx3.10 (BG-SEQ ID NO: 312) cell-penetrating peptide complex in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus.
  • the data was normalized to cells exposed to 10 pM BG-GLA-OH.
  • FIG. 8B shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide KR_IpTxa-KTx3.10, with increasing concentrations of a BG-KR_IpTxa-KTx3.10 (BG-SEQ ID NO: 313) cell-penetrating peptide complex in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus. The data was normalized to cells exposed to 10 pM BG-GLA-OH.
  • FIG. 8C shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide MCa-elafin, with increasing concentrations of a BG- MCa-elafin (BG-SEQ ID NO: 316) cell-penetrating peptide complex in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus.
  • the data was normalized to cells exposed to 10 pM BG-GLA-OH.
  • FIG. 8D shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide KR IpTxa-elafin, with increasing concentrations of a BG-KR IpTxa-elafm (BG-SEQ ID NO: 317) cell -penetrating peptide complex in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus.
  • the data was normalized to cells exposed to 10 pM BG-GLA-OH.
  • FIG. 9 schematically illustrates BG-cargo peptides and BG-cell penetrating peptide- cargo peptide complexes assayed by SNAPPA in FIG. 10A and FIG. 10B.
  • FIG. 10A shows results of a SNAP penetration assay to measure cellular penetration to reach the cytosol of the cell-penetrating peptide complexes illustrated in FIG. 9 in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm.
  • BG-peptide comprising CDP peptides complexes BG-MCa(loop)-KTx3.10 (BG-SEQ ID NO: 314), BG-peptide of MCa(loop)-elafin (BG-SEQ ID NO: 318), BG-C3A_MCa(l-9)-KTx3.10 (BG-SEQ ID NO: 315), and BG-C3A_MCa(l-9)-elafin (BG-SEQ ID NO: 319) was measured, along with BG-cargo peptides without cell-penetrating peptides BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297).
  • BG-KTx3 10 (BG-SEQ ID NO: 296) and BG-elafm (BG-SEQ ID NO: 297) were used as comparators to assess the basal level of penetration of these cargo peptides with the addition of further cell-penetrating peptides.
  • the cell penetrant moiety BG-GLA-OH formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization.
  • FIG. 10B shows results of a SNAP penetration assay to measure cellular penetration to reach the nucleus of the cell-penetrating peptide complexes illustrated in FIG. 9 exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus.
  • BG-peptide comprising CDP peptides complexes BG-MCa(loop)-KTx3.10 (BG-SEQ ID NO: 314), BG-MCa(loop)-elafm (BG-SEQ ID NO: 318), BG-C3A_MCa(l-9)-KTx3.10 (BG- SEQ ID NO: 315), and BG-C3A_MCa(l-9)-elafin (BG-SEQ ID NO: 319) was measured, along with BG-cargo peptides without cell-penetrating peptides BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafm (BG-SEQ ID NO: 297).
  • BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafm (BG-SEQ ID NO: 297) were used as comparators to assess the basal level of penetration of these cargo peptides with the addition of further cell-penetrating peptides.
  • the cell penetrant moiety BG-GLA-OH formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization.
  • FIG. 11 schematically illustrates BG-peptides complexes comprising a TEAD-binding peptide (“TEAD-binder”), with or without a cell-penetrating peptide, assayed by SNAPPA in FIG. 12A, FIG. 12B, and FIG. 13
  • FIG. 12A shows results of a SNAP penetration assay to measure cellular penetration to reach the cytosol of the cell-penetrating peptides conjugated to a TEAD-binding peptide illustrated in FIG. 11 contacted to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm.
  • SNAP substrate-tagged peptide comprising CDP TEAD-binding peptides complexes BG-C3A_MCa(l-9)-TEAD-binder (BG-SEQ ID NO: 320) and BG- C5A_Had(l-l l)-TEAD-binder (BG-SEQ ID NO: 321) was measured, along with BG-TEAD- binding peptide without cell-penetrating peptides BG- TEAD-binder (BG-SEQ ID NO: 298).
  • BG- TEAD-binder (BG-SEQ ID NO: 298) was used as a comparator to assess the basal level of penetration of this cargo peptides with the addition of further cell-penetrating peptides.
  • the cell penetrant moiety BG-GLA-OH formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization.
  • the SNAP substrate-tagged peptide (BG- peptide) comprising BG-KR_IpTxa (BG-SEQ ID NO: 59) was used as a comparator to assess the level of penetration in the absence of a cargo peptide.
  • FIG. 12B shows results of a SNAP penetration assay to measure cellular penetration to meet the nucleus of the cell-penetrating peptides conjugated to a TEAD-binding peptide illustrated in FIG. 11 exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus.
  • a SNAP substrate-tagged peptide comprising CDP TEAD-binding peptides complexes BG-C3A_MCa(l-9)-TEAD-binder (BG-SEQ ID NO: 320) and BG- C5A_Had(l-l l)-TEAD-binder (BG-SEQ ID NO: 321) was measured, along with BG-TEAD- binding peptide without cell-penetrating peptides (BG-SEQ ID NO: 298).
  • BG-TEAD-binder (BG-SEQ ID NO: 298) was used as a comparator to assess the basal level of penetration of this cargo peptides with the addition of further cell-penetrating peptides.
  • the cell penetrant moiety BG-GLA-OH formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization.
  • BG-KR_IpTxa (BG-SEQ ID NO: 59) was used as a comparator to assess the level of penetration in the absence of a cargo peptide.
  • FIG. 13 shows results of a SNAP penetration assay to measure cellular penetration into the nucleus of the cell-penetrating peptides conjugated to a TEAD-binding peptide (“TEAD- binder”) illustrated in FIG. 11 exposed to primary GSC cells transiently transfected with pSNAPf-H2B and expressing SNAP -tag in the nucleus.
  • TEAD- binder TEAD-binding peptide
  • SNAP substrate-tagged peptide comprising CDP TEAD-binding peptides complexes BG-C3A_MCa(l-9)-TEAD- binder (BG-SEQ ID NO: 320) and BG-C5A_Had(l-l l)-TEAD-binder (BG-SEQ ID NO: 321) was measured, along with BG-TEAD-binding peptide without cell-penetrating peptides BG- TEAD-binder (BG-SEQ ID NO: 298).
  • BG-TEAD-binder (BG-SEQ ID NO: 298) was used as a comparator to assess the basal level of penetration of this cargo peptides with the addition of further cell-penetrating peptides.
  • FIG. 14A shows the results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising cell-penetrating peptide KR_IpTxa in the presence of a variety of endocytosis inhibitors to assess mechanisms responsible for uptake of a BG-KR_IpTxa cellpenetrating peptide (BG-SEQ ID NO: 59).
  • Cytoplasmic penetration of BG-KR IpTxa was measured when exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm while various inhibitors were added.
  • FIG. 14B shows the results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising cell-penetrating peptide MCa_varl in the presence of a variety of endocytosis inhibitors to assess mechanisms responsible for uptake of a BG-MCa_varl cellpenetrating peptide (BG-SEQ ID NO: 67).
  • BG-MCa_varl Cytoplasmic access of BG-MCa_varl was measured in NIH3T3, HeLa and HEK293 cells expressing SNAP-tag in the cytoplasm.
  • BG-MCa_varl with no inhibitor was used as a comparator to assess the level of penetration in the absence of inhibitor.
  • BG-GLA-OH formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control for normalization.
  • FIG. 15A shows the results of a SNAP penetration assay of various SNAP substrate- tagged peptide (BG-peptide) comprising cell-penetrating peptides to measure cellular penetration into the cytosol of the cell-penetrating peptides fused to additional peptides that may further promote endosomal escape.
  • BG-peptide SNAP substrate- tagged peptide
  • BG-KR_IpTxa-PAS BG-SEQ ID NO: 322
  • BG-KR_IpTxa-S19 BG-SEQ ID NO: 323
  • BG-KR_IpTxa BG-SEQ ID NO: 59
  • BG-GLA-OH formed when BG-GLA-NHS (BG- substrate) is hydrolyzed, was used as a positive control and for normalization.
  • FIG. 15B shows the results of a SNAP penetration assay of various SNAP substrate- tagged peptides (BG-peptides) comprising cell-penetrating peptides to measure cellular penetration to the nucleus of the cell-penetrating peptides fused to additional peptides that may further promote endosomal escape.
  • BG-peptides SNAP substrate- tagged peptides
  • BG-KR_IpTxa-PAS nuclear penetration of BG-KR_IpTxa-PAS (BG-SEQ ID NO: 322) and BG-KR_IpTxa-S19 (BG-SEQ ID NO: 323) was measured in NIH3T3, HeLa and HEK293 cells expressing the SNAP-tag in the nucleus, along with BG-KR_IpTxa (BG-SEQ ID NO: 59).
  • BG-KR_IpTxa without an endosomal escape peptide was used as a comparator to assess the baseline level of penetration in the absence of an endosomal escape peptide.
  • the cell penetrant moiety BG-GLA-OH formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization.
  • FIG. 16A schematically illustrates various types of cargo peptides that may be conjugated to cell -penetrating cystine-dense peptides or peptide fragments.
  • FIG. 16B schematically illustrates various types of antibody fragments that may be conjugated to cell -penetrating cystine-dense peptides or peptide fragments.
  • FIG. 17 illustrates a multiple sequence alignment of calcin variants MCa_varl (SEQ ID NO: 67), KR Urocalcin (SEQ ID NO: 39), KR IpTxa (SEQ ID NO: 59), KR Hemicalcin (SEQ ID NO: 34), KR_Opicalcin-l (SEQ ID NO: 37), KR_Opicalcin-2 (SEQ ID NO: 38), KR Vejocalcin (SEQ ID NO: 35), and KR Intrepicalcin (SEQ ID NO: 36).
  • FIG. 18 shows the results of mass spectrometry analysis identifying tryptic fragments of the SNAP substrate-tagged peptide (BG-peptide) comprising cell-penetrating peptide KR_IpTxa (SEQ ID NO: 59; BG-KR_IpTxa) conjugated to SNAP -tag following a cell penetration assay in HeLa cells.
  • HeLa cells expressing GFP-SNAP-tag protein were incubated with 10 pM BG- KR IpTxa before subsequent trypsinization, lysis in mammalian protein extract reagent, and immunoprecipitation with magnetic beads. The recovered GFP-SNAP-tag conjugates were then submitted for mass spectrometry analysis.
  • FIG. 18 discloses SEQ ID NO: 616, SEQ ID NO: 613, and SEQ ID NO: 613, respectively, in order of appearance.
  • FIG. 19 shows the results of mass spectrometry analysis identifying tryptic fragments of BG-KR_IpTxa conjugated to SNAP -tag following a cell penetration assay in HeLa cells to determine limits of sensitivity.
  • HeLa cells expressing GFP-SNAP-tag were exposed to either BG-KR_IpTxa or BG-GLA-OH.
  • One seventh of the cells were incubated with BG-KR_IpTxa and six sevenths were incubated with BG-GLA-OH.
  • the cells were pooled and prepared as above for mass spectrometry analysis, demonstrating the ability to detect uptake of BG-KR- IpTxa at this ratio.
  • FIG. 19 discloses SEQ ID NO: 617, SEQ ID NO: 613, and SEQ ID NO: 613, respectively, in order of appearance.
  • FIG. 20 shows the results of mass spectrometry analysis identifying tryptic fragments of the SNAP substrate-tagged peptide (BG-peptide) comprising cell-penetrating peptide KR_IpTxa (SEQ ID NO: 59) (BG-KR_IpTxa) conjugated to GFP-SNAP-tag protein.
  • BG-peptide cell-penetrating peptide KR_IpTxa
  • BG-KR_IpTxa cell-penetrating peptide KR_IpTxa conjugated to GFP-SNAP-tag protein.
  • HeLa GFP-SNAP cells were lysed with M-PER supplemented with protease and phosphatase inhibitors before incubating with 10 pM BG-KR_IpTxa for 2 hours at 4C.
  • FIG. 20 discloses SEQ ID NO: 617, SEQ ID NO: 613, and SEQ ID NO: 613, respectively, in order of appearance.
  • FIG. 21 shows the results of mass spectrometry analysis identifying additional tryptic fragments of the SNAP substrate-tagged peptide (BG-peptide) comprising cell-penetrating peptide KR IpTxa (SEQ ID NO: 59; BG-KR_IpTxa) conjugated to GFP-SNAP-tag protein in a cell free environment.
  • BG-peptide SNAP substrate-tagged peptide
  • KR IpTxa SEQ ID NO: 59
  • BG-KR_IpTxa cell-penetrating peptide KR IpTxa conjugated to GFP-SNAP-tag protein in a cell free environment.
  • Prototypic peptides from BG-KR_IpTxa were also detected under the previously described conditions with the tryptic fragment highlighted detected and identified by MS2, indicating the presence of intact BG-KR_IpTxa.
  • FIG. 21 discloses SEQ ID NO: 618 (ADNDCCGR
  • FIG. 22 shows the results of mass spectrometry analysis identifying tryptic fragments of BG-KR_IpTxa (SEQ ID NO: 59) in a cell free environment.
  • HeLa GFP-SNAP cells were lysed with M-PER supplemented with protease and phosphatase inhibitors before incubating with IpM BG-KR_IpTxa for 2 hours at 4 °C.
  • the modified GFP-SNAP-tag was detected and identified by MS2.
  • FIG. 22 discloses SEQ ID NO: 619, SEQ ID NO: 613, and SEQ ID NO: 613, respectively, in order of appearance.
  • FIG. 23 shows representative images of HeLa cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously after incubation with PBS in the SNAPPA. Cells were fluorescently imaged with a Hoescht live-cell stain (left) and with Texas Red (right). Texas Red fluorescence was used to quantify the uptake of BG-fluorophore (SNAP -Cell TMR-Star dye).
  • FIG. 24 shows representative images of HeLa cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously after incubation with 2 pM hydrolyzed BG-GLA-NHS (BG-GLA-OH) in the SNAPPA. Cells were fluorescently imaged with a Hoescht live-cell stain (left) and with Texas Red (right). Texas Red fluorescence was used to quantify the uptake of BG-fluorophore (SNAP-Cell TMR-Star dye).
  • FIG. 25 shows representative images of HeLa cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously after incubation with 10 pM BG-KR_IpTxa in the SNAPPA.
  • Cells were fluorescently imaged with a Hoescht live-cell stain (left) and with Texas Red (right). Texas Red fluorescence was used to quantify the uptake of BG-fluorophore (SNAP- Cell TMR-Star dye).
  • FIG. 26A shows quantification of fluorescence measurements to measure concentrations of GFP-SNAP-tag protein expressed in the cytosol of 3T3 cells.
  • SNAP penetration assays were performed with increasing concentrations of hydrolyzed SNAP -substrate (BG-GLA-OH) to determine the maximum concentration of GFP-SNAP-tag protein expressed in the cytosol, which was quantified as 936 nM based on these results.
  • the maximum concentration calculation assumes that that normalized fluorescence would be zero when GFP-SNAP-tag protein is fully occupied by BG-GLA-OH.
  • FIG. 26B shows quantification of fluorescence measurements to measure concentrations of GFP-SNAP-tag protein expressed in the cytosol of HeLa cells.
  • SNAP penetration assays were performed with increasing concentrations of hydrolyzed SNAP -substrate (BG-GLA-OH) to determine the maximum concentration of GFP-SNAP-tag protein expressed in the cytosol, which was quantified as 700 nM based on these results. The maximum concentration calculation assumes that that normalized fluorescence would be zero when GFP-SNAP-tag protein is fully occupied by BG-GLA-OH.
  • FIG. 26C shows quantification of fluorescence measurements to measure concentrations of GFP-SNAP-tag protein expressed in the cytosol of HEK-293 cells.
  • SNAP penetration assays were performed with increasing concentrations of hydrolyzed SNAP-substrate (BG-GLA-OH) to determine the maximum concentration of GFP-SNAP-tag protein expressed in the cytosol, which was quantified as 1378 nM based on these results.
  • the maximum concentration calculation assumes that that normalized fluorescence would be zero when GFP-SNAP-tag protein is fully occupied by BG-GLA-OH.
  • FIG. 27A shows quantification of fluorescence measurements to measure concentrations of GFP-SNAP-tag protein expressed in the nucleus of 3T3 cells.
  • SNAP penetration assays were performed with increasing concentrations of hydrolyzed SNAP-substrate (BG-GLA-OH) to determine the maximum concentration of GFP-SNAP-tag protein expressed in the nucleus, which was quantified as 1632 nM based on these results.
  • the maximum concentration calculation assumes that that normalized fluorescence would be zero when GFP-SNAP-tag protein is fully occupied by BG-GLA-OH.
  • FIG. 27B shows quantification of fluorescence measurements to measure concentrations of GFP-SNAP-tag protein expressed in the nucleus of HeLa cells.
  • SNAP penetration assays were performed with increasing concentrations of hydrolyzed SNAP-substrate (BG-GLA-OH) to determine the maximum concentration of GFP-SNAP-tag protein expressed in the nucleus, which was quantified as 471 nM based on these results.
  • the maximum concentration calculation assumes that that normalized fluorescence would be zero when GFP-SNAP-tag protein is fully occupied by BG-GLA-OH.
  • FIG. 27C shows quantification of fluorescence measurements to measure concentrations of GFP-SNAP-tag protein expressed in the nucleus of HEK-293 cells.
  • SNAP penetration assays were performed with increasing concentrations of hydrolyzed SNAP-substrate (BG-GLA-OH) to determine the maximum concentration of GFP-SNAP-tag protein expressed in the nucleus, which was quantified as 1523 nM based on these results.
  • the maximum concentration calculation assumes that that normalized fluorescence would be zero when GFP-SNAP-tag protein is fully occupied by BG-GLA-OH.
  • FIG. 28A shows the results of SNAP penetration assays measuring the cytosolic uptake of BG-SEQ ID NO: 320 in the presence of various chemical inhibitors of different mechanisms of endocytosis.
  • Uptake was measured in 3T3 (“3T3 GFP-SNAP”), HeLa (“HeLa GFP-SNAP”), and HEK-293 (“293 GFP-SNAP”) cell lines expressing GFP-SNAP-tag protein in the cytosol. Uptake is shown as the fraction of GFP-SNAP-tag protein that was occupied, where a fraction of 1.0 indicates 100% of the protein was occupied.
  • the cell penetrant moiety BG-GLA-OH was used for normalization to 100% and PBS was used for normalization to 0%.
  • FIG. 28B shows the results of SNAP penetration assays measuring the cytosolic uptake of BG-SEQ ID NO: 321 in the presence of various chemical inhibitors of different mechanisms of endocytosis.
  • Uptake was measured in 3T3 (“3T3 GFP-SNAP”), HeLa (“HeLa GFP-SNAP”), and HEK-293 (“293 GFP-SNAP”) cell lines expressing GFP-SNAP-tag protein in the cytosol. Uptake is shown as the fraction of GFP-SNAP-tag protein that was occupied, where a fraction of 1.0 indicates 100% of the protein was occupied.
  • the cell penetrant moiety BG-GLA-OH was used for normalization to 100% and PBS was used for normalization to 0%.
  • FIG. 29A illustrates examples of components for use in building a targeted degradation complex for targeted degradation of TEAD.
  • Components include a cell-penetrating peptide, a TEAD-binding cystine-dense peptide (CDP), a linker, and an immunomodulatory imide drug (IMiD).
  • the TEAD-binding CDP is capable of binding TEAD and the IMiD is capable of binding cereblon (CRBN), an E3 ubiquitin ligase.
  • FIG. 29B illustrates construction of a targeted degradation complex for targeted degradation of TEAD.
  • the IMiD is linked to the cell-penetrating peptide/TEAD-binding CDP complex via a linker, thereby forming a targeted degradation complex.
  • FIG. 29C illustrates binding of the targeted degradation complex described in FIG. 29B to TEAD.
  • FIG. 29D illustrates ubiquitination and subsequent degradation of TEAD facilitated by the targeted degradation complex described in FIG. 29B.
  • the targeted degradation complex forms a ternary complex with TEAD and CRBN. Recruitment of CRBN to TEAD facilitates ubiquitination of TEAD, which targets TEAD for degradation.
  • FIG. 30 illustrates examples of molecules capable of binding an E3 ubiquitin ligase that may be incorporated into a targeted degradation complex.
  • Molecules capable of binding an E3 ubiquitin ligase include thalidomide or pomalidomide which bind cereblon (CRBN), methyl bestatin or bestatin, which bind cellular inhibitor of apoptosis protein 1 (cIAPl), nutlin-3 which binds MDM2, and VHL ligand 1 which binds von Hippel-Lindau protein (VHL).
  • FIG. 31 shows a western blot of cell lysates generated from HeLa GFP-SNAP cells that were treated with BG-peptides and then washed and lysed, where the blot was stained with an anti-GFP antibody.
  • Intact cells were incubated with 10 pM BG-SEQ ID NO: 320 or 10 pM BG- SEQ ID NO: 321 for 15, 30 or 60 minutes prior to washing, lysis, and sample preparation.
  • the band slightly below the 55 kDa marker may represent GFP-SNAP protein.
  • the band between the 55 kDa and the 72 kDa markers may represent the BG-peptide of SEQ ID NO: 320 or SEQ ID NO: 321 occupying the GFP-SNAP protein, thus causing a shift upward in molecular size versus the GFP-SNAP protein alone.
  • the fainter band between 95 and 130 kDa may represent a dimer of two GFP-SNAP proteins linked and occupied by a BG-peptide that contains at least two BG moieties (Lys residues and the N-terminus may react with BG-GLA-NHS). Occupancy of GFP-SNAP protein indicates the peptides of SEQ ID NO: 320 and SEQ ID NO: 321 penetrated the cells and entered the cytosol.
  • the loss of the shifted GFP-SNAP band suggests some intracellular degradation of exposed BG-peptide (where the cleavage could be on the N-terminal GS, the cell penetrating MCa or Had sequences, the GGS linker, or the TEAD binding peptide) or residues of the SNAP protein proximal to the reactive site may be occurring, resulting in the continued occupation of GFP-SNAP -tag protein and preventing subsequent BG-fluorophore binding.
  • FIG. 32 illustrates examples of structures of various peptide oligonucleotide complexes (e.g., a CPP-oligonucleotide complexes in which the peptide portion comprises a CDP or a fragment thereof) containing alternative and nonconventional bases, as represented in singlestranded, double-stranded, and hairpin structures.
  • oligonucleotides include an aptamer, a gapmer, an anti-miR, an siRNA, a splice blocker ASO, and a U1 adapter.
  • the CPP portion of the CPP-oligonucleotide complex can be used to guide the oligonucleotide sequence to a specific tissue, target, or cell, or to cause endocytosis, cytosolic or nuclear penetration, or endosomal escape of the oligonucleotide sequence by a cell.
  • FIG. 33A - FIG. 33E illustrate incorporation of the shown groups on RNA or DNA.
  • FIG. 33A illustrates structures of oligonucleotides containing a 5 ’-thiol (thiohexyl; C6) modification (left), and a 3 ’-thiol (C3) modification (right).
  • FIG. 33B illustrates an MMT-hexylaminolinker phosphoramidite.
  • FIG. 33C illustrates a TFA-pentylaminolinker phosphoramidite.
  • FIG. 33D illustrates RNA residues incorporating amine or thiol residues.
  • FIG. 33E illustrates oligonucleotides with aminohexyl modifications at the 5’ (left) and 3’ ends (right).
  • FIG. 34 illustrates generation of a cleavable disulfide linkage between a peptide (e.g., a cell-penetrating peptide of SEQ ID NO: 1) and a cyclic dinucleotide.
  • a peptide e.g., a cell-penetrating peptide of SEQ ID NO: 1
  • a cyclic dinucleotide e.g., SEQ ID NO: 1
  • Drug design is often limited by the ability of a drug to access intracellular targets.
  • Small molecule therapeutics can often diffuse through the cell membrane to reach intracellular targets, but small molecules may be unable to specifically and potently inhibit protein-protein interactions, or those interactions lacking well-defined binding pockets.
  • Protein- or peptide- based therapeutics such as antibodies, antibody fragments, and peptides, are more capable of specifically and potently disrupting protein-protein interactions, targeting poorly-defined binding pockets, or drive the formation of protein-protein interactions or otherwise have a therapeutic effect, but protein- or peptide-based drugs may have a difficult time reaching the intracellular milieu. Proteins and peptides are often too large or too hydrophilic to diffuse across the cell membrane.
  • RNA and DNA are large, hydrophilic, and charged and are often unable to diffuse across the cell membrane or unable to exit the endosome in order to access the cytosol, nucleus, or other subcellular compartments at levels sufficient for therapeutic effect
  • cell -penetrating peptides capable of penetrating cellular layers and accessing intracellular spaces, such as the cytoplasm, nucleus, or other subcellular compartments and in some aspects, intercellular compartments, such as nanolumen.
  • These cell-penetrating peptides can carry cargo molecules, such as peptide-based therapeutics (e.g., cystine-dense peptide-based therapeutics), protein-based therapeutics, nucleotide-based therapeutics, or small molecule therapeutics with low permeation or absorption, across cell membranes, thereby delivering the cargo into intracellular or intercellular spaces.
  • cell-penetrating peptides of the present disclosure and complexes or conjugates thereof, penetrate cells and reach an intracellular concentration, or reach an intracellular space, at a sufficient amount to exert a prophylactic or therapeutic effect.
  • peptides e.g., proteins, peptides, or cystine-dense peptides
  • cargos e.g., target-binding molecules, small molecules, RNAs, mRNAs, DNAs, active agents, macromolecular agents, detectable agents, therapeutic agents, or drugs
  • delivery of therapeutic agents across the cell membrane may increase access of the therapeutic agent to intracellular drug targets.
  • Many therapeutically relevant targets have been deemed “undruggable targets” in part due to their location in the nucleus as well as the nature of their functional interactions.
  • transcription factors responsible for driving many types of cancer such as KRAS, MYC, MYB, FOS, JUN, ABL, NF-KB, RAS, RHO, RAN, RAB, and TEAD reside in the nucleus, and their functional protein-protein and protein-nucleic acid interactions may be difficult to target using small molecule therapeutics, which may penetrate cells but may not specifically or potently interact with the transcription factor or block proteinprotein interactions (PPIs).
  • PPIs transcription factor or block proteinprotein interactions
  • Transcription factors residing in the nucleus may be inaccessible to protein and peptide drugs that are capable of specifically and potentially interacting with the transcription factor or blocking protein-protein interactions.
  • Exemplary transcription factors used for targeting the cell -penetrating peptides of the disclosure include transcription factors disclosed in “Targeting Transcription Factors for Cancer Treatment” Molecules. 2018 Jun; 23(6): 1479, which is hereby incorporated by reference. Dysregulation of these transcription factors is responsible for driving many cancers and have been difficult to target with small molecules as they interact and coordinate large protein complexes. Peptide therapeutics that modulate transcription factor interactions may be delivered to the nucleus using the cellpenetrating peptides of disclosed herein, enabling modulation of targets previously considered “undruggable”. Scaffold proteins in the cytoplasm coordinate multiple proteins into complexes through similar mechanisms and are also difficult to target using small molecule therapeutics.
  • the cell-penetrating peptides of the present disclosure can be used to deliver agents that modulate these protein complex interactions to drug targets in the cytoplasm.
  • a cell-penetrating peptide of the present disclosure may deliver a therapeutic agent into the cytoplasm to target the inflammasome, which may be responsible for activating inflammatory responses and coordinated by the Nod-like receptor (NLR) family of proteins.
  • NLR Nod-like receptor
  • Low-grade inflammation driven by NLR family pyrin domain-containing protein 3 (NLRP3) contributes to diabetes and aging.
  • the ability to modulate inflammasome activation is an appealing cytoplasmic target.
  • the cell-penetrating peptides of the present disclosure may facilitate delivery of small peptides or other therapeutic molecules into intracellular cellular compartments (e.g., the cytoplasm, the nucleus, lysosomes, or other subcellular compartments) or intercellular compartments (e.g., nanolumen, intercellular space, or paracellular space).
  • intracellular cellular compartments e.g., the cytoplasm, the nucleus, lysosomes, or other subcellular compartments
  • intercellular compartments e.g., nanolumen, intercellular space, or paracellular space.
  • Drug makers often adhere to the “rule of five” to design small molecule drugs with acceptable pharmacological properties, including permeation or absorption, restricting drug design to molecules with five or fewer hydrogen bond donors, ten or fewer hydrogen bond acceptors, a molecular weight of less than 500 Da (i.e., 500 g/mol), and a log of the partition coefficient of less than 5.
  • the cellpenetrating peptides of the present disclosure may be used to deliver drugs that do not adhere to the “rule of five” and may not otherwise be able to reach intracellular drug targets.
  • the peptides of the present disclosure may be used to deliver heterobifunctional targeted degradation molecules, such as PROTAC molecules, which often do not follow the rule of five.
  • a targeted degradation molecule may be around 1 kDa in size and comprise an E3 ubitiquitin ligase recruiting ligand linked to a target-binding molecule that binds to a protein of interest. These targeted degradation molecules can recognize and promote the ubiquitination and subsequence degradation of specific proteins.
  • cystine-dense peptides including knottins and hitchins and cyclotides
  • adnectins including knottins and hitchins and cyclotides
  • adnectins including knottins and hitchins and cyclotides
  • affibodies including knottins and hitchins and cyclotides
  • affilins includingcalins and atrimers, avimers, fynomers, kunitz domains, humabodies, Obodies, nanofittins, centyrins, DARPins, stapled peptides, cyclic peptides, bicyclic peptides, pronectins, macroclycics, solomers, VNARs, IgNARs, and lasso peptides
  • PPIs protein-protein interactions
  • CRISPR Cas9 proteins ⁇ 160kDa
  • CRISPR components e.g., guide RNAs, tracrRNAs, or crRNAs
  • Delivery of antisense RNA (>30kDa) using the cellpenetrating peptides of the present disclosure could be used to inhibit translation of problematic proteins.
  • the active agent is an anti-cancer agent, a molecule that binds a transcription factor, a molecule that blocks intracellular protein-protein interactions, a molecule that causes the formation or stabilization of intracellular protein-protein interactions, a transcription factor, an RNA, a Cas enzyme or other CRISPR component, an immunomodulating agent, a molecular glue, a targeted degradation molecule, an inhibitor of protein-protein interactions, an inhibitor of enzymatic activity, or a neurotransmitter.
  • a cell-penetrating peptide may be complexed with a RIG-I ligand or a receptor for a RIG-I-like receptor (e.g., MDA5 or TLR3).
  • the ligand may be a double stranded RNA (dsRNA) and may be delivered to a desired cellular compartment (e.g., the cytoplasm or an endosome) to activate the receptor. Delivery of RIG-I ligands across a cellular layer may promote anti -tumor or anti-viral activity in a subject and may be used to treat a cancer or viral infection in a subject.
  • dsRNA double stranded RNA
  • the cell-penetrating peptides of the present disclosure may deliver agents (e.g., cargos, cargo peptides, cargo proteins, or cargo molecules) that drive the formation of protein-protein interactions or that block the formation of protein-protein interactions.
  • the cell-penetrating peptides may deliver agents to the cytoplasm.
  • the cell-penetrating peptides may deliver agents to the nucleus.
  • the cellpenetrating peptides may deliver agents to a nanolumen.
  • a cellpenetrating peptide may deliver an agent to the nucleus that promotes formation of a transcription factor complex or disrupts formation of a transcription factor complex.
  • the agent may activate or inhibit the transcription factor.
  • a cellpenetrating peptide of the present disclosure may deliver an agent to the nucleus that promotes or disrupts protein-protein interactions within transcription factor complexes or protein-nucleic acid interactions AP-2, ARID/BRIGHT, ARID/B RIGHT (RFX), AT hook, BED ZF, bHLH, Brinker, bZIP, C2H2 ZF, C2H2 ZF (KRAB), C2H2 ZF (non-KRAB), C2H2 ZF (AT hook), C2H2 ZF (BED ZF), C2H2 ZF (Homeodomain), C2H2 ZF (Myb/SANT), CBF/NF-Y, CCCH ZF, CENPB, CG-1, CSD, CSL, CUT (Homeodomain), CxxC, CxxC (AT hook), DM, E2F, EBF1, Ets, Ets (AT hook), FLYWCH,
  • a cell-penetrating peptide of the present disclosure may deliver an agent to the nucleus that promotes or disrupts protein-protein interactions with MLL, GABP, RUNX1, KLF8, SIX1, RUNX2, PML, RARu, FOXO, TALI, or Myc [oni]
  • the cell-penetrating peptides of the present disclosure may deliver agents (e g., cargos, cargo peptides, cargo proteins, or cargo molecules) that can cause targeted protein degradation, for example by functioning as molecular glues, proteolysis-targeting chimeras (PROTACs), inhibitors of protein-protein interactions, or targeted degradation complexes.
  • agents e g., cargos, cargo peptides, cargo proteins, or cargo molecules
  • PROTACs proteolysis-targeting chimeras
  • a cell-penetrating peptide may deliver a thalidomide-like molecule to the cytosol.
  • Thalidomidelike molecules that may function as immunomodulatory imide drugs (IMiDs), such as thalidomide, pthalidomide (a-(N-phthalimide)glutarimide), phthalimide derivatives, thalidomide analogues, thalidomide hybrids (e.g., N-phenyl-phthalimide sulfonamides (3a-e), isosters biphenyl -phthalimide amides (4a-e)), pomalidomide, and lenalidomide, can bind to cereblon, cause a conformational change in cereblon, and drive the formation of a complex between cereblon and proteins such as Ikaros and Aiolos, whereby they are ubiquitinated by the CRL4 complex and undergo degradation by the ubiquitin-proteosome system (UPS).
  • IiDs immunomodul
  • IMiDs may be called molecular glues, which cause the formation of PPIs and cause the targeted degradation of proteins.
  • the cell-penetrating peptides of the present disclosure may deliver agents to the cytosol that bind to cereblon, VHL, DCAF15, DCAF16, other molecules in the CRL4 E3 ligase family, other molecules in the E3 ligase family, or other molecules in the UPS system, drive the formation of a complex with another substrate or neosubstrate, and thereby cause ubiquitination and degradation of the substrate or neosubstrate by the UPS, acting as molecular glues.
  • Non-limiting examples of agents that can cause targeted protein degradation and can be delivered by a cell -penetrating peptide of the present disclosure may be found in Collins, et al (Biochem J. 2017 Apr 1; 474(7): 1127-1147), Chopra, et al (Drug Discov Today Technol, 2019 Apr; 31 :5-13), and Chamberlain, et al (Nat Chem Biol, 2019 Oct; 15(10):937- 944) each of which is incorporated by reference in its entirety.
  • Molecular glues can be small molecules, but small molecules may not be able to induce formation of ternary complexes between proteins desired for degradation and the E3 ligase complex.
  • a cell-penetrating peptide can deliver an agent that functions as a molecular glue to the cytosol. In some embodiments, a cell-penetrating peptide can deliver an agent that functions as a targeted degradation complex to the cytosol.
  • Targeted degradation complexes may comprise a molecule that binds to a ubiquitin ligase (e.g., cereblon, VHL, DCAF15, DCAF16, other molecules in the CRL4 E3 ligase family, other molecules of the Cul2-Rbxl-EloN/C-VHL E3 ligase, the MDM2 E3 ligase, cIAPl, cIAP, APCZC(CDHl), or other molecules in the E3 ligase family), a linker, and target-binding molecule that binds to a protein targeted for degradation.
  • a ubiquitin ligase e.g., cereblon, VHL, DCAF15, DCAF16, other molecules in the CRL4 E3 ligase family, other molecules of the Cul2-Rbxl-EloN/C-VHL E3 ligase, the MDM2 E3 ligase, cIAPl, cI
  • a targeted degradation complex may comprise a small molecule that does not fit the “rule of five” often used to constrain molecule size and solubility parameters for successful small molecule therapeutics.
  • Protein degradation can also be caused by molecules that bind the protein targeted for degradation and that also contain an IMiD, a Boc3-Arg tag, an adamantyl group, or a carborane as hydrophobic tag that causes binding to the 20S proteosome or to HSP70.
  • the cell-penetrating peptides of this disclosure may act as molecular glues or as PROTACS or otherwise cause targeted protein degradation.
  • the cell-penetrating peptides of this disclosure may serve to deliver active agents that act as molecular glues or targeted degradation complexes or that otherwise drive targeted degradation to the cytosol, thereby allowing those active agents to reach their targets and cause targeted protein degradation.
  • Proteins that can targeted for degradation by a cell-penetrating peptide or a cell-penetrating peptide complex may include TEAD, cold-inducible RNA-binding protein (also referred to as CIRP or CIRBP), androgen receptor, ikaros, aiolos, nuclear receptors, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-8, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CSNK1A1, GSPT1, ZFP91, ZNF629, ZNF91, ZNF276, ZNF653, ZNF8
  • a cellpenetrating peptide complex of the present disclosure may target AKT, mTOR, STAT3, RAS, RAF, MEK, ERK, P-catenin, IKK, TEAD, NF-KB, PKC, AP-1, Jun, Fos, REL or a transcription factor for degradation.
  • a cell -penetrating peptide or an active agent for targeted protein degradation can have one or more lysine residues removed or replaced with other amino acids to prevent labeling of the lysine residues with ubiquitin. By targeting proteins for degradation, they may be reduced or removed from cells, thereby removing functions that contribute to disease.
  • Proteins that can be degraded using the cell -penetrating peptides or cellpenetrating peptide complexes of the present disclosure may be transcription factors, kinases, zinc finger domain containing, may perform a scaffolding function, or may be adaptor proteins.
  • the cell-penetrating peptides of the presence disclosure may themselves be agents that cause a therapeutic effect by binding to or interacting with various molecules within the cytosol, nucleus, or other subcellular, intracellular, or paracellular compartments.
  • a cell-penetrating peptide may promote or disrupt a protein-protein interaction in the subcellular, intracellular, or paracellular compartment.
  • a cell -penetrating peptide may deliver an agent that cause a therapeutic effect to the cytosol, nucleus, or other subcellular compartment.
  • a cell -penetrating peptide including, but not limited to, designed or engineered peptides, recombinant peptides, synthetic peptides, and small cystine-dense peptides (or disulfide-knotted peptides, knottins, knotted peptides, or hitchins), that are capable of penetrating cellular layers (e.g., cell membranes, nuclear envelopes, endosomes, lysosomes, or other subcellular compartments) to access intracellular or intercellular spaces.
  • cellular layers e.g., cell membranes, nuclear envelopes, endosomes, lysosomes, or other subcellular compartments
  • Cell-penetrating peptides can include calcines (e.g., imperatoxin A (IpTxa), maurocalcin (MCa), hadrucalcin (Had), hemicalcin, vejocalcin, intrepicalcin, opicalcin-1, opicalcin-2, or urocalcin), toxins (e.g., chlorotoxin (CTX), huwentoxin IV (HwTx-IV), potassium channel toxin-like Tx677, potassium channel toxin KTx2.2, potassium channel toxin KTxl5.8), CTI, and variants and fragments thereof.
  • calcines e.g., imperatoxin A (IpTxa), maurocalcin (MCa), hadrucalcin (Had), hemicalcin, vejocalcin, intrepicalcin, opicalcin-1, opicalcin-2, or urocalcin
  • toxins e.g
  • a cell-penetrating peptide of the present disclosure may be conjugated to, linked to, or fused with a cargo molecule (e g., a peptide, a cystine-dense peptide, a small molecule, a protein, an RNA, an mRNA, a DNA, an active agent, a detectable agent, a therapeutic agent, or a drug) to generate a cell-penetrating peptide complex.
  • a cargo molecule e e a peptide, a cystine-dense peptide, a small molecule, a protein, an RNA, an mRNA, a DNA, an active agent, a detectable agent, a therapeutic agent, or a drug
  • the cellpenetrating peptide may itself be an active agent.
  • cell-penetrating peptide complexes may be used to deliver cargo molecules across cellular layers (e.g., cell membranes, nuclear envelopes, intercellular spaces, paracellular spaces, endosomal membranes, lysosomal membranes, blood brain barriers, or nanolumen) and into cellular, intercellular, or paracellular compartments or spaces (e.g., cytosols, nuclei, or nanolumen).
  • cellular layers e.g., cell membranes, nuclear envelopes, intercellular spaces, paracellular spaces, endosomal membranes, lysosomal membranes, blood brain barriers, or nanolumen
  • cytosols e.g., cytosols, nuclei, or nanolumen
  • the cell-penetrating peptides and cell-penetrating peptide complexes of the present disclosure may be used in various methods to deliver cargo molecules to intracellular and intercellular targets (e.g., drug targets).
  • a cell-penetrating peptide of the present disclosure may be conjugated to a therapeutic agent (e.g., a small molecule drug, a peptide drug, a protein drug, a biologic drug, a transcription factor, a gene editing agent, or a disruptor of PPIs or a driver of PPIs) that would otherwise be excluded from intracellular spaces, and the resulting cell-penetrating peptide complex may be administered to a subject (e.g., a human subject) to deliver the therapeutic agent to a target of the therapeutic agent (e g., a protein or a nucleic acid) located in a cellular, intercellular, or paracellular compartment or space (e.g., cytoplasm, nucleus, or nanolumen).
  • a therapeutic agent e.g., a small molecule drug, a peptide drug, a protein drug, a biologic drug, a transcription factor, a gene editing agent, or a disruptor of PPIs or a driver of PPIs
  • alanine A, Ala
  • arginine R, Arg
  • asparagine N, Asn
  • aspartic acid D, Asp
  • cysteine C, Cys
  • glutamic acid E, Glu
  • glutamine Q, Gin
  • glycine G, Gly
  • histidine H, His
  • isoleucine I, He
  • leucine L, Leu
  • lysine K, Lys
  • methionine M, Met
  • phenylalanine F, Phe
  • proline P, Pro
  • serine S, Ser
  • threonine T, Thr
  • tryptophan W, Trp
  • tyrosine Y, Tyr
  • valine V, Vai
  • Xaa can indicate any amino acid.
  • X can be asparagine (N), glutamine (Q), histidine (H), lysine (K), or arginine (R).
  • a peptide of the present disclosure can comprise a nonnatural amino acid, wherein the non-natural amino acid can be an insertion, appendage, or substitution for another amino acid.
  • a non-natural amino acid may comprise a D-isomer, a homo-amino acid, a B-homo-amino acid, an N-methyl amino acid, an a-methyl amino acid.
  • a non-natural amino acid can comprise citrulline (Cit), hydroxyproline (Hyp), norleucine (Nle), 3-nitrotyrosine, nitroarginine, ornithine (Om), naphtylalanine (Nal), aminobutyric acid (a, Abu), di aminobutyric acid (Dab), methionine sulfoxide, or methionine sulfone.
  • a position can be referred to as “position X,” wherein X is the position number within a sequence not counting, if present, the N-terminal GS.
  • position 5 in SEQ ID NO: 59 refers to the “H” residue at position 5 within the sequence, wherein position 1 does not count the N-terminal “GS,” and is, thus, the first D residue.
  • position 9 in SEQ ID NO: 66 refers to the “A” residue within the sequence at position 9, wherein position 1 does not count the N-terminal “GS,” and is, thus, the first E residue.
  • the N-terminal GS is included in the sequence as a byproduct of proteolytic cleavage during recombinant protein production or a spacer.
  • position 11 in SEQ ID NO: 18 refers to the “K” residue at position 11 within the sequence, wherein position 1 is the very first G residue and there is no N-terminal GS.
  • a position can be referred to as “XY,” wherein X is the single-letter abbreviation for an amino acid and wherein Y is the position number within a sequence not counting, if present, the N-terminal GS.
  • G15 refers to the “G” residue at position 15 within a sequence, not counting, if present, an N-terminal GS.
  • Y23 refers to the “Y” residue at position 23 within a sequence, not counting, if present, an N-terminal GS.
  • E25 refers to the “E” residue at position 25 within a sequence, not counting, if present, an N-terminal GS.
  • a position can be referred to as “XYZ,” wherein X and Z are the single-letter abbreviation for an amino acid and wherein Y is the position number within a sequence not counting, if present, the N-terminal GS.
  • L23A refers to a “L” residue at position 23 within a sequence (not counting, if present, an N- terminal GS), which has been substituted with a “A” residue.
  • E25A refers to an “E” residue at position 25 within a sequence (not counting, if present, an N-terminal GS), which has been substituted with an “A” residue.
  • F27A refers to an “F” residue at position 27 within a sequence (not counting, if present, an N-terminal GS), which has been substituted with an “A” residue.
  • Some embodiments of the disclosure contemplate D-amino acid residues of any standard or non-standard amino acid or analogue thereof.
  • an amino acid sequence is represented as a series of three-letter or one-letter amino acid abbreviations, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy terminal direction, in accordance with standard usage and convention.
  • peptide complex may refer to a peptide that is fused to, recombinantly fused to, linked to, conjugated to, chemically conjugated to, recombinantly expressed with, complexed with, or is otherwise connected to an additional agent (e g., a protein, a peptide, a cystine-dense peptide, a small molecule, an RNA, an mRNA, a DNA, an active agent, macromolecular agent, a detectable agent, a therapeutic agent, or a drug).
  • a peptide complex may be a peptide construct.
  • a peptide complex may be a peptide conjugate.
  • a peptide complex of the present disclosure may comprise a cell-penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254, or a variant or fragment thereof) that is fused, linked, conjugated, recombinantly expressed with, or is otherwise connected to a cargo molecule (e.g., a peptide, a peptide, a cystine-dense peptide, a small molecule, an RNA, an mRNA, a DNA, an active agent, macromolecular agent, a detectable agent, a therapeutic agent, or a drug), and may be referred to herein as a cell-penetrating peptide complex.
  • a cargo molecule e.g., a peptide, a peptide
  • a peptide complex may comprise a linker (e.g., a peptide linker of any one SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485, or a small molecule linker as described herein).
  • a linker e.g., a peptide linker of any one SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485, or a small molecule linker as described herein.
  • a “cargo,” “cargo peptide,” “cargo protein,” or “cargo molecule” may be any protein, peptide, or molecule that is fused, linked, conjugated, recombinantly expressed with, or is otherwise connected to a cell-penetrating peptide.
  • a cargo, cargo peptide, cargo protein, or cargo molecule may be carried across a cellular layer (e.g., a plasma membrane, a vesicular membrane, an endosomal membrane, a nuclear envelope, or a blood brain barrier) by a cell-penetrating peptide of the present disclosure.
  • a cargo, cargo peptide, cargo protein, or cargo molecule may be carried within the vesicles of a cell's secretory system (e.g., within the endoplasmic reticulum, Golgi apparatus, lysosomes, or other subcellular compartment, whether to or from the plasma membrane) or within a vesicle as a structure within or outside a cell, comprising liquid or cytoplasm enclosed by a lipid bilayer.
  • a cell's secretory system e.g., within the endoplasmic reticulum, Golgi apparatus, lysosomes, or other subcellular compartment, whether to or from the plasma membrane
  • a vesicle as a structure within or outside a cell, comprising liquid or cytoplasm enclosed by a lipid bilayer.
  • a cargo peptide, cargo protein, or cargo molecule may be a protein, a peptide, a cystine-dense peptide, a small molecule, an RNA, an mRNA, a DNA, an active agent, macromolecular agent, a detectable agent, a therapeutic agent, or a drug.
  • penetration includes movement of a molecule from the extracellular space to the cytosol, the nucleus, or other subcellular compartments by any means, and may also be described as cytosolic access or cytosolic delivery.
  • Cell penetration may also include endosomal uptake, such as by macropinocytosis, clathrin-mediated endocytosis, calveolae-dependent endocytosis, or other endocytosis, followed by endosomal escape such that the molecule leaves the endosome and enters the cytosol. It is understood that cell penetration is not limited to a molecule solely entering endosomes not further accessing or entering the cytosol.
  • a peptide capable of penetrating a cellular layer e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, a cell membrane, an endosomal membrane, a lysosomal membrane, a nuclear envelope, or other subcellular compartment membrane, a blood brain barrier, or a nanolumen
  • a cellpenetrating peptide may also be referred to herein as a cellpenetrating peptide.
  • a cell -penetrating peptide may enter the cytosol. Alternatively or in addition, a cell-penetrating peptide may enter the nucleus.
  • the cellpenetrating peptides disclosed herein can penetrate or enter target cells, such as cancerous or tumor cells, liver cells, pancreas cells, colon cells, ovarian cells, breast cells, and/or lung cells, or any combination thereof.
  • target cells such as cancerous or tumor cells, liver cells, pancreas cells, colon cells, ovarian cells, breast cells, and/or lung cells, or any combination thereof.
  • cell penetration includes movement of a molecule from the extracellular space to the cytosol, the nucleus, or other subcellular compartments by any means, and may also be described as cytosolic access or cytosolic delivery.
  • Cell penetration may also include endosomal uptake, such as by macropinocytosis, clathrin-mediated endocytosis, calveolae-dependent endocytosis, or other endocytosis, followed by endosomal escape such that the molecule leaves the endosome and enters the cytosol. It is understood that cell penetration is not limited to a molecule solely entering endosomes not further accessing or entering the cytosol.
  • a peptide or a library of peptides is designed in silica without derivation from a naturally occurring knottin scaffold.
  • a peptide or a library of peptides is designed in silica by derivation, grafting relevant or important proteinbinding residues, or conserved residues, in the protein-binding interface, or structural modeling based on a naturally occurring peptide or protein known to bind to a protein or receptor of interest.
  • a library of peptides is screened for the ability to access a cellular compartment, such as the cytoplasm or the nucleus, using a fluorescence-based screening method or a mass spectrometry -based screening method, as described herein.
  • a peptide with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 is used as a scaffold or base sequence for further modifications, including addition, deletion, or amino acid substitution.
  • a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 may be modified by performing one or more amino acid substitutions to introduce one or more lysine, arginine, proline, or histidine amino acids.
  • residues GS are added at the N-terminus of a peptide.
  • peptides lack GS at the N-terminus.
  • peptides undergo one or more post-translational modifications.
  • peptides are truncated so that they contain fewer amino acids, or are less than 50, less than 40, less than 30, less than 20, less than 15, less than 12, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, or less than 4 amino acids long.
  • TABLE 1 lists exemplary peptide sequences according to the present disclosure.
  • a cell-penetrating peptide of the present disclosure may comprise one or more positively charged amino acid residues (e.g., lysine or arginine). Positively charged amino acid residues may mediate interactions between the cell-penetrating peptide and negatively charged cellular layers (e.g., cell membranes, nuclear envelopes, intercellular spaces, paracellular spaces, endosomal membranes, lysosomal membranes, or other subcellular compartment membrane, blood brain barriers, or nanolumen).
  • positively charged amino acid residues e.g., lysine or arginine
  • Positively charged amino acid residues may mediate interactions between the cell-penetrating peptide and negatively charged cellular layers (e.g., cell membranes, nuclear envelopes, intercellular spaces, paracellular spaces, endosomal membranes, lysosomal membranes, or other subcellular compartment membrane, blood brain barriers, or nanolumen).
  • a cell-penetrating peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 positively charged amino acid residues.
  • a cell-penetrating peptide may comprise no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 positively charged amino acid residues.
  • a cell-penetrating peptide may comprise from 1 to 30, from 2 to 20, from 3 to 30, from 4 to 30, from 5 to 30, from 1 to 20, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 2 to 15, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, or from 10 to 15 positively charged amino acid residues.
  • Positively charged amino acid residues may include lysine residues and arginine residues.
  • a cell-penetrating peptide may comprise at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1. 1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1 .6, at least 1 .7, at least 1.8, at least 1 .9, at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3, at least 3.1, at least 3.2, at least 3.3, at least 3.4, at least 3.5, at least 3.6, at least 3.7, at least 3.8, at least 3.9, or at least 4 positively charged amino acid residues per 10 amino acid residues.
  • a cell-penetrating peptide may comprise no more than 8, no more than 7.5, no more than 7, no more than 6.5, no more than 6, no more than 5.5, no more than 5, no more than 4.5, no more than 4, no more than 3.8, no more than 3.6, no more than 3.4, no more than 3.2, no more than 3, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2.
  • no more than 2 no more than 1.9, no more than 1.8, no more than 1.7, no more than 1.6, no more than 1.5, no more than 1.4, no more than 1.3, no more than 1.2, no more than 1.1, or no more than 1 positively charged amino acid residues per 10 amino acid residues.
  • a cell-penetrating peptide may comprise no more than 0.5, no more than 0.6, no more than 0.7, no more than 0.8, no more than 0.9, no more than 1, no more than 1.1, no more than 1.2, no more than 1.3, no more than 1.4, no more than 1.5, no more than 1.6, no more than 1.7, no more than 1.8, no more than 1.9, no more than 2, no more than 2.1, no more than 2.2, no more than 2.3, no more than 2.4, no more than 2.5, no more than 2.6, no more than 2.7, no more than 2.8, no more than 2.9, no more than 3, no more than 3.
  • a cellpenetrating peptide may comprise from 0.5 to 8, from 0.5 to 7, from 0.5 to 6, from 0.5 to 5, from 0.5 to 4, from 0.5 to 3, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 1.5 to 5, from 1.5 to 4, from 1.5 to 3, from 1.5 to 2.8, from 1.5 to 2.6, from 1.5 to 2.4, from 1.5 to 2.2, from 1.5 to 2, from 2 to 3, from 2 to 2.8, from 2 to 2.6, from 2 to 2.4, or from 2 to 2.2 positively charged amino acid residues per 10 amino acid residues.
  • Positively charged amino acid residues may include lysine residues and arginine residues.
  • a cell-penetrating peptide of the present disclosure may comprise one or more arginine amino acid residues.
  • a cell-penetrating peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 arginine amino acid residues.
  • a cell-penetrating peptide may comprise no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 arginine amino acid residues.
  • a cell-penetrating peptide may comprise from 1 to 30, from 2 to 20, from 3 to 30, from 4 to 30, from 5 to 30, from 1 to 20, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 2 to 15, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, or from 10 to 15 arginine amino acid residues.
  • a cell-penetrating peptide may comprise at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1. 1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1 .6, at least 1 .7, at least 1.8, at least 1 .9, at least 2, at least 2. 1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3, at least 3.2, at least 3.4, at least 3.6, at least 3.8, or at least 4 arginine amino acid residues per 10 amino acid residues.
  • a cell -penetrating peptide may comprise no more than 8, no more than 7.5, no more than 7, no more than 6.5, no more than 6, no more than 5.5, no more than 5, no more than 4.5, no more than 4, no more than 3.8, no more than 3.6, no more than 3.4, no more than 3.2, no more than 3, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2.
  • a cell-penetrating peptide may comprise from 0.5 to 8, from 0.5 to 7, from 0.5 to 6, from 0.5 to 5, from 0.5 to 4, from 0.5 to 3, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 1.5 to 5, from 1.5 to 4, from 1.5 to 3, from 1.5 to 2.8, from 1.5 to 2.6, from 1.5 to 2.4, from 1.5 to 2.2, from 1.5 to 2, from 2 to 3, from 2 to 2.8, from 2 to 2.6, from 2 to 2.4, or from 2 to 2.2 arginine amino acid residues per 10 amino acid residues.
  • the cell-penetrating peptide comprises no arginine amino acids.
  • a cell-penetrating peptide of the present disclosure may comprise one or more lysine amino acid residues.
  • a cell-penetrating peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 lysine amino acid residues.
  • a cell-penetrating peptide may comprise no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 lysine amino acid residues.
  • a cell-penetrating peptide may comprise from 1 to 30, from 2 to 20, from 3 to 30, from 4 to 30, from 5 to 30, from 1 to 20, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 2 to 15, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, or from 10 to 15 lysine amino acid residues.
  • the cellpenetrating peptide comprises no lysine amino acid residues.
  • a cell-penetrating peptide may comprise at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1. 1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1 .6, at least 1 .7, at least 1.8, at least 1 .9, at least 2, at least 2. 1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3, at least 3.2, at least 3.4, at least 3.6, at least 3.8, or at least 4 lysine amino acid residues per 10 amino acid residues.
  • a cell -penetrating peptide may comprise no more than 8, no more than 7.5, no more than 7, no more than 6.5, no more than 6, no more than 5.5, no more than 5, no more than 4.5, no more than 4, no more than 3.8, no more than 3.6, no more than 3.4, no more than 3.2, no more than 3, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2.
  • a cellpenetrating peptide may comprise from 0.5 to 8, from 0.5 to 7, from 0.5 to 6, from 0.5 to 5, from 0.5 to 4, from 0.5 to 3, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 1.5 to 5, from 1.5 to 4, from 1.5 to 3, from 1.5 to 2.8, from 1.5 to 2.6, from 1.5 to 2.4, from 1.5 to 2.2, from 1.5 to 2, from 2 to 3, from 2 to 2.8, from 2 to 2.6, from 2 to 2.4, or from 2 to 2.2 lysine amino acid residues per 10 amino acid residues.
  • a cell-penetrating peptide of the present disclosure may comprise one or more negatively charged amino acid residues (e g., aspartic acid or glutamic acid).
  • a cellpenetrating peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 negatively charged amino acid residues.
  • a cell-penetrating peptide may comprise no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 negatively charged amino acid residues.
  • a cellpenetrating peptide may comprise from 1 to 30, from 2 to 20, from 3 to 30, from 4 to 30, from 5 to 30, from 1 to 20, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 2 to 15, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, or from 10 to 15 negatively charged amino acid residues.
  • Negatively charged amino acid residues may include aspartic acid residues (or aspartate residues) and glutamic acid residues (or glutamate residues).
  • a cell-penetrating peptide may comprise at least 0.03, at least 0.05, at least 0.1, at least 0.2, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least
  • a cell-penetrating peptide may comprise no more than 8, no more than 7.5, no more than 7, no more than 6.5, no more than 6, no more than 5.5, no more than 5, no more than 4.5, no more than 4, no more than 3.8, no more than 3.6, no more than 3.4, no more than 3.2, no more than 3, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2.1, no more than 2, no more than 1.9, no more than 1.8, no more than 1.7, no more than 1.6, no more than 1.5, no more than 1.4, no more than 1.3, no more than 1.2, no more than 1.1, or no more than 1 negatively charged amino acid residues per 10 amino acid residues.
  • a cell-penetrating peptide may comprise from 0.5 to 8, from 0.5 to 7, from 0.5 to 6, from 0.5 to 5, from 0.5 to 4, from 0.5 to 3, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 1.5 to 5, from 1.5 to 4, from 1.5 to 3, from 1.5 to 2.8, from 1.5 to 2.6, from 1.5 to 2.4, from 1.5 to 2.2, from 1.5 to 2, from 2 to 3, from 2 to 2.8, from 2 to 2.6, from 2 to 2.4, or from 2 to 2.2 negatively charged amino acid residues per 10 amino acid residues.
  • Positively charged amino acid residues may include lysine residues and arginine residues.
  • Negatively charged amino acid residues may include aspartic acid residues (or aspartate residues) and glutamic acid residues (or glutamate residues).
  • a cell-penetrating peptide of the present disclosure may comprise more positively charged amino acid residues than negatively charged amino acid residues.
  • a cell-penetrating peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 more positively charged amino acid residues than negatively charged amino acid residues.
  • a cellpenetrating peptide may comprise no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 more positively charged amino acid residues than negatively charged amino acid residues.
  • a cell-penetrating peptide may comprise from 1 to 30, from 1 to 20, from 1 to 10, from 5 to 30, from 5 to 20, from 5 to 10, from 3 to 30, from 3 to 20, from 3 to 10, from 4 to 30, from 4 to 20, from 4 to 10, from 5 to 30, from 5 to 20, or from 5 to 10 more positively charged amino acid residues than negatively charged amino acid residues.
  • a cell-penetrating peptide of the present disclosure may comprise more negatively charged amino acid residues than positively charged amino acid residues.
  • a cell-penetrating peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 more negatively charged amino acid residues than positively charged amino acid residues.
  • a cellpenetrating peptide may comprise no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 more negatively charged amino acid residues than positively charged amino acid residues.
  • a cell-penetrating peptide may comprise from 1 to 30, from 1 to 20, from 1 to 10, from 5 to 30, from 5 to 20, from 5 to 10, from 3 to 30, from 3 to 20, from 3 to 10, from 4 to 30, from 4 to 20, from 4 to 10, from 5 to 30, from 5 to 20, or from 5 to 10 more negatively charged amino acid residues than positively charged amino acid residues.
  • a cell-penetrating peptide may comprise a ratio of positively charged amino acid residues to negatively charged amino acid residues that is at least 1.0, at least 1.5, at least 2, at least 2.5, at least 2.75, at least 3, at least 3.5, at least 4, at least 5, at least 6, or at least 7.
  • a cell-penetrating peptide may comprise a ratio of negatively charged amino acid residues to positively charged amino acid residues that is no more than 1.0, no more than 1.5, no more than 2, no more than 2.5, no more than 2.75, no more than 3, no more than 3.5, or no more than 4.
  • Proline amino acid residues may provide structural stability or specific conformations for the cell-penetrating peptides of the present disclosure.
  • proline residues may provide a conformation or stability to short cell-penetrating peptide sequences (e g., cellpenetrating peptides of less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, or less than 4 amino acid residues).
  • a cell-penetrating peptide of the present disclosure e.g., a short cell-penetrating peptide
  • a cell-penetrating peptide may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 proline amino acid residues.
  • Histidine amino acid residues may facilitate membrane penetration of the cellpenetrating peptides of the present disclosure.
  • the side chain of a histidine amino acid may be, on average, neutral at an extracellular pH such as pH 7.4, pH 7.3, pH 7.2, pH 7.1, or pH 7.0.
  • the side chain of a histidine amino acid may, on average, become increasingly positively charged as the pH drops in an early endosome, in a late endosome, or in a lysosome. Histidine residue side chains may contribute more positive charge to the net charge of a peptide as pH decreases, for example as the pH of an endosome goes below pH 6.0.
  • a histidine residue side chain may have a pK a of 6.0, such that, as the pH drops below pH 7.4, the amount of positive charge increases.
  • a histidine residue may develop a positive charge in an endosome or a lysosome, thereby causing a change in the conformation of the molecule or a change in interaction with the endosomal membrane such that the cell-penetrating peptide containing one or more histidine residues disrupts the endosome or otherwise escapes the endosome or lysosome as the pH in the endosome or lysosomes drops below about pH 7.4, pH 7.3, pH 7.2, pH 7.1, pH 7.0, pH 6.9, pH 6.8, pH 6.7, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7,
  • a cellpenetrating peptide of the present disclosure may contain a histidine amino acid residue.
  • a cell-penetrating peptide may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 histidine amino acid residues.
  • a short cell-penetrating peptide sequences e.g., any one of SEQ ID NO: 4 - SEQ ID NO: 7, SEQ ID NO: 9 - SEQ ID NO: 17, SEQ ID NO: 21 - SEQ ID NO: 29, SEQ ID NO: 41 - SEQ ID NO: 58, SEQ ID NO: 62 - SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 82 SEQ ID NO: 84, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 214, or
  • the penetration properties of a cell-penetrating peptide increase as the pH decreases.
  • a cell-penetrating peptide of the present disclosure may penetrate membranes, including the endosomal membrane or lysosomal membrane, when the pH is below pH 7.4, pH 7.3, pH 7.2, pH 7.1, pH 7.0, pH 6.9, pH 6.8, pH 6.7, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, or pH 4.5.
  • the pH change or the ionic strength change can cause changes in conformation of the cell-penetrating peptide or interaction of the cell-penetrating peptide with the endosomal or lysosomal membrane.
  • CDPs or knotted peptides including engineered, non-naturally occurring CDPs and those found in nature (e g., a target-binding peptide), can be conjugated to, linked to, or fused to the cell-penetrating peptides of the present disclosure, such as those described in TABLE 1, TABLE 2, or TABLE 3, to deliver a target molecule to cellular, intracellular, or paracellular compartment or space.
  • the cell can be a cancer cell, pancreatic cell, liver cell, colon cell, ovarian cell, breast cell, lung cell, spleen cell, bone marrow cell, or any combination thereof.
  • An engineered peptide can be a peptide that is non-naturally occurring, artificial, synthetic, designed, or recombinantly expressed.
  • a cellpenetrating peptide of the present disclosure, or a peptide complex comprising a cell- penetrating enables cell-penetrating, and an additional CDP or knotted peptide that is conjugated to, linked to, or fused to the cell-penetrating peptide can be delivered to the cellular, intracellular, or paracellular compartment or space in a cell associated with a disease or condition.
  • the cell is a cancer cell.
  • Cancers can include breast cancer, liver cancer, colon cancer, brain cancer, leukemia, acute myeloid leukemia (AML), lymphoma, nonHodgkin lymphoma, myeloma, blood-cell-derived cancer, spleen cancer, cancers of the salivary gland, kidney cancer, muscle cancers, ovarian cancer, prostate cancer, pancreatic cancer, gastric cancer, sarcoma, glioblastoma, astrocytoma, glioma, medulloblastoma, ependymoma, choroid plexus carcinoma, midline glioma, diffuse intrinsic pontine glioma, lung cancer, bone marrow cell cancers, skin cancer, genitourinary cancer, osteosarcoma, muscle-derived sarcoma, melanoma, head and neck cancer, a neuroblastoma, glioblastoma, astrocytoma, glioma, medulloblastom
  • TABLE 2 lists exemplary peptide sequences according to the present disclosure.
  • a peptide can be modified with biotin, for example at a lysine residue (e.g., in SEQ ID NO: 102 - SEQ ID NO: 117).
  • a peptide can be modified with a fluorophore, for example at the N-terminus or the C-terminus of the peptide (e.g., Cy5.5 in SEQ ID NO: 135 - SEQ ID NO: 154).
  • a peptide can contain a non-proteinogenic amino acid, for example an a-aminobutyric acid, abbreviated “a” or “Abu” (e.g., in SEQ ID NO: 118 - SEQ ID NO: 132, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 140 - SEQ ID NO: 154, SEQ ID NO: 163 - SEQ ID NO: 166, or SEQ ID NO: 168).
  • a a-aminobutyric acid
  • peptides of the present disclosure may comprise one or more cellpenetrating peptides.
  • peptides can comprise at least one or multiple peptides having cell penetration properties (e.g., one or more of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254).
  • a cell-penetrating peptide may comprise one or more Arg residues (e g., SEQ ID NO: 1 - SEQ ID NO: 8, SEQ ID NO: 16 - SEQ ID NO: 41, SEQ ID NO: 43 - SEQ ID NO: 82, SEQ ID NO: 84 - SEQ ID NO: 120, SEQ ID NO: 124 - SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 153 - SEQ ID NO: 194, SEQ ID NO: 197 SEQ ID NO: 209, SEQ ID NO: 212 SEQ ID NO: 235, SEQ ID NO: 237 - SEQ ID NO: 243, SEQ ID NO: 250 - SEQ ID NO: 254, SEQ ID NO: 408 - SEQ ID NO: 431, or SEQ ID NO: 433 - SEQ ID NO: 456).
  • a cell -penetrating peptide of the present disclosure may comprise one or more Lys residues. In some embodiments, a cellpenetrating peptide of the present disclosure may comprise one or more Asp residues. In some embodiments, a cell-penetrating peptide of the present disclosure may comprise one or more Glu residues. In some embodiments, a cell-penetrating peptide of the present disclosure may comprise one or more His residues. In some embodiments, a cell-penetrating peptide of the present disclosure may comprise one or more Pro residues.
  • a cellpenetrating peptide of the present disclosure may comprise one or more Cys residues (e g., SEQ ID NO: 1 - SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 18 - SEQ ID NO: 20, SEQ ID NO: 30 - SEQ ID NO: 40, SEQ ID NO: 59 - SEQ ID NO: 61, SEQ ID NO: 64 - SEQ ID NO: 69, SEQ ID NO: 71 - SEQ ID NO: 81, SEQ ID NO: 85 - SEQ ID NO: 117, SEQ ID NO: 119 - SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 169 - SEQ ID NO: 193, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 202 - SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 211
  • one or more Lys residues may be substituted with Arg residues. In some embodiments, one or more Arg residues may be substituted with Lys residues. In some embodiments, one or more Cys residues may be substituted with Ala residues.
  • a cell-penetrating peptide may be a short cell-penetrating peptide (e.g., SEQ ID NO: 4 - SEQ ID NO: 7, SEQ ID NO: 9 - SEQ ID NO: 17, SEQ ID NO: 21 - SEQ ID NO: 29, SEQ ID NO: 41 - SEQ ID NO: 58, SEQ ID NO: 62 - SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 82 - SEQ ID NO: 84, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 214, or SEQ ID NO: 216).
  • a cell -penetrating peptide may be a fragment of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254.
  • a cell-penetrating peptide may be any consecutive fragment of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, or SEQ ID NO: 195 SEQ ID NO: 254 that is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 amino acids long.
  • a cell -penetrating peptide may comprise a functional fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254.
  • the functional fragment may be a consecutive fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254.
  • a functional fragment may be a fragment that has cell -penetrating properties.
  • a functional fragment may be capable of penetrating a cellular layer, cell membrane, a nuclear envelope, an endosomal membrane, a lysosomal membrane, other cellular or subcellular membrane, or a blood brain barrier.
  • a functional fragment may be a fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 18 - SEQ ID NO: 20, SEQ ID NO: 30 - SEQ ID NO: 40, SEQ ID NO: 59 - SEQ ID NO: 61, SEQ ID NO: 64 - SEQ ID NO: 69, SEQ ID NO: 71 - SEQ ID NO: 81, SEQ ID NO: 85 - SEQ ID NO: 117, SEQ ID NO: 119 - SEQ ID NO: 162, SEQ ID NO 166,
  • SEQ ID NO: 224 SEQ ID NO: 226, SEQ ID NO: 244, or SEQ ID NO: 247 comprising at least
  • a functional fragment may be a fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 positively charged amino acid residues.
  • a functional fragment may be a fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 8, SEQ ID NO: 16 - SEQ ID NO: 41, SEQ ID NO: 43 - SEQ ID NO: 82, SEQ ID NO: 84 SEQ ID NO: 120, SEQ ID NO: 124 SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 153 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, SEQ ID NO: 197 - SEQ ID NO: 209, SEQ ID NO: 212 - SEQ ID NO: 235, SEQ ID NO: 237 SEQ ID NO: 243, or SEQ ID NO: 250 SEQ ID NO: 254 comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 arginine amino acid residues.
  • a functional fragment may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 lysine amino acid residues. In some embodiments, a functional fragment may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 histidine amino acid residues. In some embodiments, a functional fragment may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 proline amino acid residues. In some embodiments, a functional fragment may comprise an amphipathic a-helix.
  • a short cell-penetrating peptide may have a sequence of any one of GSXDALPHLKL (SEQ ID NO: 334) or XDALPHLKL (SEQ ID NO: 325), wherein X is any amino acid.
  • a short cell -penetrating peptide may have a sequence of any one of GSGXALPHLKL (SEQ ID NO: 335) or GXALPHLKL (SEQ ID NO: 326), wherein X is any amino acid.
  • a short cell -penetrating peptide may have a sequence of any one of GDXLPHLKL (SEQ ID NO: 327) or GSGDXLPHLKL (SEQ ID NO: 336), wherein X is any amino acid.
  • a short cell -penetrating peptide may have a sequence of any one of GDAXPHLKL (SEQ ID NO: 328) or GSGDAXPHLKL (SEQ ID NO: 337), wherein X is any amino acid.
  • a short cell-penetrating peptide may have a sequence of any one of GDALXHLKL (SEQ ID NO: 329) or GSGDALXHLKL (SEQ ID NO: 338), wherein X is any amino acid
  • a short cell -penetrating peptide may have a sequence of any one of GDALPXLKL (SEQ ID NO: 330) or GSGDALPXLKL (SEQ ID NO: 339), wherein X is any amino acid.
  • a short cell-penetrating peptide may have a sequence of any one of GDALPHXKL (SEQ ID NO: 331) or GSGDALPHXKL (SEQ ID NO: 340), wherein X is any amino acid.
  • a short cell-penetrating peptide may have a sequence of any one of GDALPHLXL (SEQ ID NO: 332) or GSGDALPHLXL (SEQ ID NO: 341), wherein X is any amino acid.
  • a short cell -penetrating peptide may have a sequence of any one of GDALPHLKX (SEQ ID NO: 333) or GSGDALPHLKX (SEQ ID NO: 342), wherein X is any amino acid.
  • An amino acid may be a natural amino acid or a non-natural amino acid.
  • Cell-penetrating peptides may be derived from calcins or calcin variants. For example, the peptides may be derived from the calcin variants shown in FIG. 17.
  • a cell-penetrating peptide derived from a calcin may have a sequence of DCLX 1 X 2 LRX 3 CX 4 X 5 X 6 X 7 DCCX 8 RX 9 CX 10 RR, wherein X'-X 1 ' 1 are each independently any amino acid or no amino acid (SEQ ID NO: 343).
  • X ⁇ X 10 in SEQ ID NO: 343 can be deleted, resulting in no amino acid in the position with reference to SEQ ID NO: 343.
  • a cell-penetrating peptide derived from a calcin may have a sequence of DCLX 1 X 2 LRX 3 CX 4 X 5 X 6 X 7 DCCX 8 RX 9 CX 10 RR, wherein X 1 is selected from P or A, X 2 is selected from H or R, X 3 is selected from L or R, X 4 is selected from R or K, X 5 is selected from E, R, or A, X 6 is selected from N or D, X 7 is selected from N or R, X 8 is selected from S or G, X 9 is selected from R or S, and X 10 is selected from R, S, or K (SEQ ID NO: 344).
  • known calcin sequences may be excluded from the cell-penetrating peptides encompassed by SEQ ID NO: 344.
  • a cell-penetrating peptide derived from a calcin may have a sequence of DCLPHLRLCRX 1 X 2 X 3 DCCSRRCRRRGT X 4 ERRCR, wherein X'-X 4 are each independently any amino acid (SEQ ID NO: 345), or wherein X 1 is E, D, A, R, K, or G, X 2 is N or D or E, X 3 is R, K, N, and X 4 is I, A, P, or G (SEQ ID NO: 346).
  • known calcin sequences may be excluded from the cell-penetrating peptides encompassed by SEQ ID NO: 345 or SEQ ID NO: 346.
  • a cell-penetrating peptide derived from a calcin may have a sequence of X 1 DCLX 2 X 3 LRX 4 CX 5 X 6 X 7 X 8 DCCX 9 RX 10 CX 11 RRX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 , wherein X 4 -X 21 are each independently any amino acid or no amino acid (SEQ ID NO: 347).
  • a cellpenetrating peptide derived from a calcin may have a sequence of X 1 DX 2 LX 3 X 4 LRX 5 , wherein X 1 any amino acid or no amino acid and X 2 -X 5 are each independently any amino acid (SEQ ID NO: 348), or wherein X 1 is G, A, S, R, or K, X 2 is C, A, G, S, X 3 is P, R, K, A, or G, X 4 is H, R, or K, and X 5 is L, R, or K (SEQ ID NO: 349).
  • known calcin sequences may be excluded from the cell-penetrating peptides encompassed by SEQ ID NO: 348 or SEQ ID NO: 349.
  • a cell-penetrating peptide derived from a calcin may have a sequence of LX L X 2 LRX 3 , wherein X 4 -X 3 are each independently any amino acid (SEQ ID NO: 350), or wherein X 1 is P, R, K, A, or G, X 2 is H, R, or K, and X 3 is L, R, or K (SEQ ID NO: 351).
  • known calcin sequences may be excluded from the cell-penetrating peptides encompassed by SEQ ID NO: 350 or SEQ ID NO: 351.
  • a cell-penetrating peptide described herein as a consensus sequence or as variants with respect to percent identity may exclude known cell-penetrating sequences.
  • Additional cell-penetrating tag peptide sequences can include Tat (GRKKRRQRRR; SEQ ID NO: 225), CysTat (CYRKKRRQRRR; SEQ ID NO: 226), S19-TAT (PFVIGAGVLGALGTGIGGIGRKKRRQRRR; SEQ ID NO: 227), R8 (RRRRRRRR; SEQ ID NO: 228), pAntp (RQIKIWFQNRRMKWKK; SEQ ID NO: 229), Pas-TAT (FFLIPKGGRKKRRQRRR; SEQ ID NO: 230), Pas-R8 (FFLIPKGRRRRRRRR; SEQ ID NO: 231), PasFHV (FFLIPKGRRRRNRTRRNRRRVR; SEQ ID NO: 232), Pas-pAntP (FFLIPKGRQIKIWFQNRRMKWKK; SEQ ID NO: 233), F2R4 (FFRRRR; SEQ ID NO: 234), B55 (KAV
  • a cell-penetrating peptide can comprise an Arginine patch (Arg patch), for example, an RRRRRRRR (SEQ ID NO: 228), or a variant or fragment thereof
  • the Arg patch comprises two or more Arg residues, or Argn wherein n is a whole number and can be 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 250).
  • the cell -penetrating peptide can comprise a Tat peptide (Tat proteins are reviewed in Gump et al. TAT transduction: the molecular mechanism and therapeutic prospects. Trends Mol Med. 2007 Oct;13(10):443-8 and Harada et al.
  • the Tat peptide can have a sequence of, for example, YGRKKRRQRRR (SEQ ID NO: 251), GRKKRRQRRR (SEQ ID NO: 225), or any modification, variant, or fragment thereof.
  • the Tat peptide sequence can be GRKKRRQRRRPQ (SEQ ID NO: 252), GRKKRRQRRR (SEQ ID NO: 225), or a fragment or variant thereof.
  • Cell-penetrating peptides may be capable of penetrating and transferring a cargo, either covalently or non-covalently attached to the peptides, into a cell.
  • Such cellpenetrating peptides can be synthesized or derived from known proteins, such as penetratin (RQIKIWFQNRRMKWKKGG; SEQ ID NO: 254), VP22 (MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFV QYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTT APRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKL HFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDL NELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAAS
  • a cell-penetrating peptide can may be derived from maurocaline (GDCLPHLKLCKENKDCCSKKCKRRGTNIEKRCR; SEQ ID NO: 197), imperatoxin (GDCLPHLKRCKADNDCCGKKCKRRGTNAEKRCR; SEQ ID NO: 202), hadrucalcin (SEKDCIKHLQRCRENKDCCSKKCSRRGTNPEKRCR; SEQ ID NO: 200), hemicalcin (GDCLPHLKLCKADKDCCSKKCKRRGTNPEKRCR; SEQ ID NO: 205), opicalcin-1 (GDCLPHLKRCKENNDCCSKKCKRRGTNPEKRCR; SEQ ID NO: 206), opicalcin-2 (GDCLPHLKRCKENNDCCSKKCKRRGANPEKRCR; SEQ ID NO: 207), huwentoxin (ECLEIFKACNPSNDQCCKSSKLVCSRKTRWCKYQIG
  • the cell-penetrating peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any sequence of SEQ ID NO: 1 - SEQ ID NO: 84. In some embodiments, the cell-penetrating peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any sequence of SEQ ID NO: 85 - SEQ ID NO: 194 or SEQ ID NO: 408 - SEQ ID NO: 457.
  • the cell-penetrating peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any sequence of SEQ ID NO: 195 - SEQ ID NO: 254. In some embodiments, the cell-penetrating peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a fragment of SEQ ID NO: 1 - SEQ ID NO: 84.
  • the cell-penetrating peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a fragment of SEQ ID NO: 85 - SEQ ID NO: 194 or SEQ ID NO: 408 - SEQ ID NO: 457. In some embodiments, the cell-penetrating peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a fragment of SEQ ID NO: 195 - SEQ ID NO: 254.
  • a variant may comprise one or more Lys residues replaced with Arg. In some embodiments, a variant may comprise one or more Arg residues replaced with Lys. In some embodiments, a variant may comprise one or more Cys residues replaced with Ala.
  • a fragment may be a contiguous fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 that is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46 at least 47, at least 48, at least 49, at least 50, at least 51
  • a fragment may be a contiguous fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 that is no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, no more than 30, no more than 31, no more than 32, no more than 33, no more than 34, no more than 35, no more than 36, no more than 37, no more than 38, no more than 39, no more than 40, no more than 41, no more than
  • the peptide sequence is flanked by additional amino acids.
  • additional amino acids can, for example, confer a desired in vivo charge, isoelectric point, chemical conjugation site, stability, or physiologic property to a peptide.
  • nuclear localization signals can be coupled to, conjugated to, linked to, or fused to a cell-penetrating peptide or a cell-penetrating peptide complex described herein to promote nuclear localization.
  • cell-penetrating peptides are conjugated to, linked to, or fused to a nuclear localization signal, such as a four-residue sequence of K-K/R-X-K/R (SEQ ID NO: 352), wherein X can be any amino acid, or a variant thereof.
  • cell-penetrating peptides are conjugated to, linked to, or fused to a nuclear localization signal as described in Lange et al, J Biol Chem. 2007 Feb 23 ;282(8): 5101 - 5, such as PKKKRRV (SEQ ID NO: 353) or KRPAATKKAGQAKKKK (SEQ ID NO: 354)
  • a cell-penetrating peptide described herein is conjugated to, linked to, or fused to a nuclear localization signal comprising KxRy (SEQ ID NO: 355), wherein x and y independently can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, such as KKRR (SEQ ID NO: 356), KKKRR (SEQ ID NO: 357), or KKKK (SEQ ID NO: 358).
  • cell-penetrating moieties can also be linked to, conjugated to, linked to, or fused to the peptides described herein, including, but not limited to, polycations, polyorganic acids, endosomal releasing polymers, poly(2- propylacrylic acid), poly(2-ethylacrylic acid), or any combination thereof.
  • cell penetration can be increased by using high dosage of a peptide described here, such as up to 10 pM, or 10 pM or more of the cell-penetrating peptide.
  • a peptide described here such as up to 10 pM, or 10 pM or more of the cell-penetrating peptide.
  • Up 10 pM, or 10 pM or more cell-penetrating peptide e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254
  • Protein transfection agents can also be used to increase cell penetration of a peptide.
  • direct cytosolic expression of a peptide can be used.
  • physical disruption methods such as electroporation can be used to improve delivery of a peptide into a cell.
  • cell penetrance of a peptide described herein can be improved.
  • the binding interface of a peptide described herein can modified from a cellpenetrating peptide, such as a calcine.
  • calcines can be imperatoxin-A, maurocalcine, hemicalcin, opiclacin 1, opicalcin 2, and hadrucalcin.
  • the cellpenetrating peptide can comprise at least 60%, 70%, 80%, 90%, 95%, or 98% with any one of SEQ ID NO: 1 - SEQ ID NO: 100, SEQ ID NO: 102 - SEQ ID NO: 224.
  • the cell penetrance peptide can be calcines, modified calcines, derivatives of calcines, or fragments thereof, or variants thereof, which can be used to increase cell penetration.
  • Modified calcines, derivatives of calcines, or fragments can be screened for cell penetration activity such as activation of sarcoplasmic reticulum ryanodine receptors, activity on ryanodine-sensitive Ca 2+ channels RyRl, Ryr2, or both, or as a selective agonist of the foregoing.
  • modified calcines can include substitution, addition or reduction of Lysine residues, addition or reduction of Arginine residues, or other charged residues, within a native calcine in order to modify activity and optimize such calcine cell -penetration activity or activity on the RyRl or RyR2 receptors.
  • residues of a cell-penetrating peptide of this disclosure are modified so they do not act on or have reduced activity on RyRl, RyR2, or other receptors.
  • cell-penetrating peptides can penetrate into a target cell or into the nucleus of a target cell.
  • target cells include cancerous cells, tumors, neurons, inflammatory cells, cells of metastases, cancerous stem cells, and other cell types.
  • the target cell can be a human cell, a mammalian cell, a human or mammalian cell line, a cancer cell line, a cell extracted from a subject, in vivo, or in vitro.
  • the peptide can contain only one lysine residue, or no lysine residues. In some instances, some or all of the lysine residues in the peptide are replaced with arginine residues. In some instances, some or all of the methionine residues in the peptide are replaced by leucine or isoleucine. Some or all of the tryptophan residues in the peptide can be replaced by phenylalanine or tyrosine. In some instances, some or all of the asparagine residues in the peptide are replaced by glutamine. In some embodiments, some or all of the aspartic acid residues can be replaced by glutamic acid residues.
  • cysteine residues are replaced by alanine residues. In some embodiments, some or all of the cysteine residues are replaced by serine residues. In some embodiments, some or all of the cysteine residues are replaced by glycine residues.
  • the C-terminal Arg residues of a peptide is modified to another residue such as Ala, Asn, Asp, Gin, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai.
  • the C-terminal Arg residue of a peptide can be modified to He.
  • the C-terminal Arg residue of a peptide can be modified to any non-natural amino acid. This modification can prevent clipping of the C-terminal residue during expression, synthesis, processing, storage, in vitro, or in vivo including during treatment, while still allowing maintenance of a key hydrogen bond.
  • a key hydrogen bond can be the hydrogen bond formed during the initial folding nucleation and is critical for forming the initial hairpin.
  • the N-terminus of the peptide is blocked, such as by an acetyl group.
  • the C-terminus of the peptide is blocked, such as by an amide group.
  • the peptide is modified by methylation on free amines. For example, full methylation can be accomplished through the use of reductive methylation with formaldehyde and sodium cyanoborohydride.
  • GS can be added as the first two N-terminal amino acids, as shown in SEQ ID NO: 59 - SEQ ID NO: 84, SEQ ID NO: 93 - SEQ ID NO: 101, or SEQ ID NO: 210 - SEQ ID NO: 224, or such N-terminal amino acids (GS) can be absent as shown in SEQ ID NO: 1 - SEQ ID NO: 58, SEQ ID NO: 85 - SEQ ID NO: 92, SEQ ID NO: 102 - SEQ ID NO: 209, or SEQ ID NO: 225 - SEQ ID NO: 254, or can be substituted by any other one or two amino acids.
  • GS is used as a linker or used to couple to a linker to make a protein conjugate or fusion.
  • the linker comprises a G x S y (SEQ ID NO: 256) peptide, wherein x and y independently can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the peptide linker comprises (GS)x (SEQ ID NO: 258), wherein x can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the peptide linker comprises GGSSG (SEQ ID NO: 259), GGGGG (SEQ ID NO: 260), GSGSGSGS (SEQ ID NO: 261), GGGGS (SEQ ID NO: 263), GGGS (SEQ ID NO: 264), or a variant or fragment thereof or any number of repeats and combinations thereof.
  • KKYKPYVPVTTN (SEQ ID NO: 266) from DkTx
  • EPKSSDKTHT (SEQ ID NO: 267) from human IgG3
  • the peptide linker comprises GGGSGGSGGGS (SEQ ID NO: 292) or a variant or fragment thereof or any number of repeats and combinations thereof. It is understood that any of the foregoing linkers or a variant or fragment thereof can be used with any number of repeats or any combinations thereof. It is also understood that other peptide linkers in the art or a variant or fragment thereof can be used with any number of repeats or any combinations thereof.
  • the linker between the cell -penetrating peptides in the protein conjugate or fusion within the selective depletion complex is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36 at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least
  • the C-terminal Arg residues of a peptide is modified to another residue such as Ala, Asn, Asp, Gin, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai.
  • the C-terminal Arg residue of a peptide can be modified to He.
  • the C-terminal Arg residue of a peptide can be modified to any non-natural amino acid. This modification can prevent clipping of the C-terminal residue during expression, synthesis, processing, storage, in vitro, or in vivo including during treatment, while still allowing maintenance of a key hydrogen bond.
  • a key hydrogen bond can be the hydrogen bond formed during the initial folding nucleation and is critical for forming the initial hairpin.
  • the peptide comprises the sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254.
  • a peptide can be a fragment comprising a contiguous fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 that is at least 3, at least 4, at least 5, at least 6, at least 7, at least, 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46 at least 47, at least 48, at least 49, at least 50, at least 51
  • the peptide sequence is flanked by additional amino acids.
  • additional amino acids can, for example, confer a desired in vivo charge, isoelectric point, chemical conjugation site, stability, or physiologic property to a peptide.
  • a peptide can be a fragment comprising a contiguous fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 that is not greater than 3, not greater than 4, not greater than 5, not greater than 6, not greater than 7, not greater than, 8, not greater than 9, not greater than 10, not greater than 11, not greater than 12, not greater than 13, not greater than 14, not greater than 15, not greater than 16, not greater than 17, not greater than 18, not greater than 19, not greater than 20, not greater than 21, not greater than 22, not greater than 23, not greater than 24, not greater than 25, not greater than 26, not greater than 27, not greater than 28, not greater than 29, not greater than 30, not greater than 31, not greater than 32, not greater than 33, not greater than 34, not greater than 35, not greater than 36, not greater than 37, not greater than 38, not greater than 39,
  • the peptide sequence is flanked by additional amino acids.
  • additional amino acids can, for example, confer a desired in vivo charge, isoelectric point, chemical conjugation site, stability, or physiologic property to a peptide.
  • peptide sequences comprise primarily beta-sheets and/or alphahelix structures.
  • cell-penetrating peptides of the present disclosure are small, compact mini-proteins stabilized by intra-chain disulfide bonds (mediated by cysteines) and a well-packed hydrophobic core.
  • cell-penetrating peptides e.g., SEQ ID NO: 1 SEQ ID NO: 84, SEQ ID NO: 85 SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254
  • structures comprising helical bundles with at least one disulfide bridge between each of the alpha helices, thereby stabilizing the peptides.
  • a cell-penetrating peptide can be amphipathic, comprising a hydrophilic region and a hydrophobic region, which can facilitate interactions with cell membranes and insertion dynamics.
  • a cell-penetrating peptide can comprise an amphipathic a- helix, which can facilitate interactions with cellular membranes.
  • the amphipathicity of a cell-penetrating peptide is pH dependent and is present to a higher degree at endosomal or lysosomal pHs than at extracellular pH.
  • cell-penetrating peptides contain one or more positively charged residues or carry a net positive charge.
  • a cell-penetrating peptide is cyclized peptide or a stapled peptide.
  • peptides can be conjugated to, linked to, or fused to a cargo peptide or cargo molecule to deliver the cargo peptide or cargo molecule to a target of interest in a cell.
  • peptides can be conjugated to, linked to, or fused to a molecule that extends half-life or modifies the pharmacodynamic and/or pharmacokinetic properties of the peptides, or any combination thereof.
  • a peptide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 positively charged residues, such as Arg or Lys, or any combination thereof.
  • one or more lysine residues in the peptide are replaced with arginine residues.
  • one or more arginine residues in the peptide are replaced with lysine residues.
  • 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more Arg or Lys residues are solvent exposed on a peptide.
  • the peptides of the present disclosure can further comprise neutral amino acid residues.
  • the peptide has 35 or fewer neutral amino acid residues.
  • the peptide has 81 or fewer neutral amino acid residues, 70 or fewer neutral amino acid residues, 60 or fewer neutral amino acid residues, 50 or fewer neutral amino acid residues, 40 or fewer neutral amino acid residues, 36 or fewer neutral amino acid residues, 33 or fewer neutral amino acid residues, 30 or fewer neutral amino acid residues, 25 or fewer neutral amino acid residues, or 10 or fewer neutral amino acid residues.
  • the peptides of the present disclosure can further comprise negative amino acid residues.
  • the peptide has 6 or fewer negative amino acid residues, 5 or fewer negative amino acid residues, 4 or fewer negative amino acid residues, 3 or fewer negative amino acid residues, 2 or fewer negative amino acid residues, or 1 or fewer negative amino acid residues.
  • negative amino acid residues can be selected from any negatively charged amino acid residues, in some embodiments, the negative amino acid residues are either E, or D, or a combination of both E and D.
  • a peptide comprises no Cys or disulfides. In some cases, a peptide comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more Cys or disulfides. In other embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues have been replaced with Ser residues.
  • 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues have been replaced with Thr residues. In some embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues have been replaced with Ala, Gly, or Met residues.
  • the NMR solution or x-ray crystallography structures of related structural homologs can be used to inform mutational strategies that may improve the folding, stability, manufacturability, while maintaining a particular biological function. They can be used to predict the 3D pharmacophore of a group of structurally homologous scaffolds, as wells as to predict possible graft regions of related proteins to create chimeras with improved properties. For example, this strategy was used to identify critical amino acid positions and loops that may be used to design peptides with improved cell-penetrating properties, high expression, high stability in vivo, or any combination thereof.
  • a cell-penetrating peptide capable of penetrating a cellular layer comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with with a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, as provided in TABLE 1, or a fragment thereof.
  • a cell-penetrating peptide capable of penetrating a cellular layer comprises a sequence with no more than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a peptide of any one of SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, as provided in TABLE 2, or a fragment thereof.
  • a cell -penetrating peptide capable of penetrating a cellular layer comprises a sequence with no more than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a peptide of any one of SEQ ID NO: 195 — SEQ ID NO: 254, as provided in TABLE 3, or a fragment thereof.
  • Two or more peptides can share a degree of sequence identity or homology and share similar properties in vivo.
  • a peptide can share a degree of sequence identity or homology with any one of the peptides of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254.
  • one or more peptides of the disclosure can have up to about 20% pairwise sequence identity or homology, up to about 25% pairwise sequence identity or homology, up to about 30% pairwise sequence identity or homology, up to about 35% pairwise sequence identity or homology, up to about 40% pairwise sequence identity or homology, up to about 45% pairwise sequence identity or homology, up to about 50% pairwise sequence identity or homology, up to about 55% pairwise sequence identity or homology, up to about 60% pairwise sequence identity or homology, up to about 65% pairwise sequence identity or homology, up to about 70% pairwise sequence identity or homology, up to about 75% pairwise sequence identity or homology, up to about 80% pairwise sequence identity or homology, up to about 85% pairwise sequence identity or homology, up to about 90% pairwise sequence identity or homology, up to about 95% pairwise sequence identity or homology, up to about 96% pairwise sequence identity or homology, up to about 97% pairwise sequence identity or homology, up to about 98% pairwise sequence identity or homology,
  • one or more peptides of the disclosure can have at least about 20% pairwise sequence identity or homology, at least about 25% pairwise sequence identity or homology, at least about 30% pairwise sequence identity or homology, at least about 35% pairwise sequence identity or homology, at least about 40% pairwise sequence identity or homology, at least about 45% pairwise sequence identity or homology, at least about 50% pairwise sequence identity or homology, at least about 55% pairwise sequence identity or homology, at least about 60% pairwise sequence identity or homology, at least about 65% pairwise sequence identity or homology, at least about 70% pairwise sequence identity or homology, at least about 75% pairwise sequence identity or homology, at least about 80% pairwise sequence identity or homology, at least about 85% pairwise sequence identity or homology, at least about 90% pairwise sequence identity or homology, at least about 95% pairwise sequence identity or homology, at least about 96% pairwise sequence identity or homology, at least about 97% pairwise sequence identity or homology, at least about 98% pairwise sequence identity or homology,
  • Various methods and software programs can be used to determine the homology between two or more peptides, such as NCBI BLAST, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, or another suitable method or algorithm. Pairwise sequence alignment is used to identify regions of similarity that may indicate functional, structural and/or evolutionary relationships between two biological sequences (protein or nucleic acid) By contrast, multiple sequence alignment (MSA) is the alignment of three or more biological sequences. From the output of .MSA applications, homology can be inferred and the evolutionary relationship between the sequences assessed.
  • MSA multiple sequence alignment
  • sequence homology and “sequence identity” and “percent (%) sequence identity” and “percent (%) sequence homology” have been used interchangeably to mean the sequence relatedness or variation, as appropriate, to a reference polynucleotide or amino acid sequence.
  • the peptide is any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 or a functional fragment thereof.
  • the peptide of the disclosure further comprises a peptide with 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99%, 97%, 95%, 90%, 85%, or 80% sequence identity or homology to any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 or fragment thereof.
  • the peptide can be a peptide that is homologous to any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 or a functional fragment thereof.
  • homologous is used herein to denote peptides having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity or homology to a sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 or a functional fragment thereof.
  • variant nucleic acid molecules that encode a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408
  • SEQ ID NO: 457 or SEQ ID NO: 195 - SEQ ID NO: 254 can be identified by either a determination of the sequence identity or homology of the encoded peptide amino acid sequence with the amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254, or by a nucleic acid hybridization assay.
  • SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 can be characterized as nucleic acid molecules (1) that remain hybridized with a nucleic acid molecule having the nucleotide sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 (or its complement) under highly stringent washing conditions, in which the wash stringency is equivalent to 0.1 x-0.2xSSC with 0.1% SDS at 50-65° C., and (2) that encode a peptide having at least 70%, at least 80%, at least 90*Eo, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least
  • a peptide is modified to increase homology to a human protein sequence.
  • a peptide is modified to increase resistance to degradation.
  • a peptide is modified to reduce an affinity of the peptide for a human leukocyte antigen complex, a major histocompatibility complex, or both.
  • peptides that have cell-penetrating capabilities can be aligned where amino acids that are the same or similar are aligned.
  • Positional amino acid variants at one position in a given peptide may be placed in the same position in a different aligned peptide to create a new peptide.
  • fragments of one peptide that is penetrant may identify fragments in aligned peptides that may also be cell penetrant.
  • Amino acids of like charge can be varied, such as changing Lys to Arg and vice versa, and changing Asp to Glu and vice versa.
  • Cysteine residues may be replaced in peptide fragments or where unpaired with other Cys residues to improve manufacturability, shelf-life stability, purity, in vivo stability, in vivo safety, and in vivo activity.
  • one or more cysteine residues may be mutated to alanine.
  • one or more cysteine residues may be mutated to any other amino acid, or to glycine or aminobutyric acid.
  • Histidine residues may be added to facilitate endosomal escape.
  • Proline residues may be added to facilitate endosomal escape.
  • An alignment may be generated using R language and an “MSA” software package, which codes for R language specific for multiple alignments (Bodenhofer, U et al.
  • peptides of the family of sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9 - SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO 22 - SEQ ID NO: 29, SEQ ID NO: 44 - SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, or SEQ ID NO: 68 may retain essential properties such as structure, function, peptide folding, biodistribution, or stability.
  • peptides comprising a segment corresponding to SEQ ID NO: 350 or SEQ ID NO: 351 may have structure, function, peptide folding, biodistribution, binding, accumulation, retention, or stability favorable for cell penetration.
  • a positively charged region for example as seen in residues 30-33 of SEQ ID NO: 197 or SEQ ID NO: 202 or residues 32-35 of SEQ ID NO: 200, may improve cell-penetrating properties of a peptide (e.g., a cystine-dense peptide).
  • Other conserved regions within sequences of the present disclosure can be identified.
  • an alignment may be generated using R language and an “msa” software package, which codes for R language specific for multiple alignments (Bodenhofer, U et al.
  • cell-penetrating peptides, cargo peptides, or cell -penetrating peptide complexes of the present disclosure comprise one or more Cys, or one or more disulfide bonds.
  • the cell-penetrating peptides are derived from cystine-dense peptides (CDPs), knotted peptides, or hitchins.
  • the cargo peptides are derived from cystine-dense peptides (CDPs), knotted peptides, or hitchins.
  • peptide is considered to be interchangeable with the terms “knotted peptide”, “cystine- dense peptide”, “CDP”, and “hitchin”. (See e.g., Correnti et al. Screening, large-scale production, and structure-based classification for cystine-dense peptides. Nat Struct Mol Biol. 2018 Mar; 25(3): 270-278).
  • the peptides of the present disclosure are cystine-dense peptides (CDPs), related to knotted peptides or hitchin-derived peptides or knottin-derived peptides.
  • CDPs cystine-dense peptides
  • the cellpenetrating peptides can be cystine-dense peptides (CDPs).
  • Hitchins can be a subclass of CDPs wherein six cysteine residues form disulfide bonds according to the connectivity [1-4], 2-5, 3-6 indicating that the first cysteine residue forms a disulfide bond with the fourth residue, the second with the fifth, and the third cysteine residue with the sixth.
  • the brackets in this nomenclature indicate cysteine residues form the knotting disulfide bond.
  • Knotting can be a subclass of CDPs wherein six cysteine residues form disulfide bonds according to the connectivity 1-4, 2-5, [3-6], Knotting are a class of peptides, usually ranging from about 20 to about 80 amino acids in length that are often folded into a compact structure. Knottins are typically assembled into a complex tertiary structure that is characterized by a number of intramolecular disulfide crosslinks and can contain beta strands and other secondary structures.
  • the peptides described herein can be derived from knotted peptides.
  • the amino acid sequences of peptides as disclosed herein can comprise a plurality of cysteine residues. In some cases, at least six cysteine residues of the plurality of cysteine residues present within the amino acid sequence of a peptide participate in the formation of disulfide bonds. In some cases, all cysteine residues of the plurality of cysteine residues present within the amino acid sequence of a peptide participate in the formation of disulfide bonds.
  • the term “knotted peptide” can be used interchangeably with the terms “cystine-dense peptide”, “CDP”, or “peptide”.
  • CDPs comprise alternatives to existing biologies, primarily antibodies, which can bypass some of the liabilities of the immunoglobulin scaffold, including poor tissue permeability, immunogenicity, and long serum half-life that can become problematic if toxicities arise.
  • Peptides of the present disclosure in the 20-80 amino acid range represent medically relevant therapeutics that are mid-sized, with many of the favorable binding specificity and affinity characteristics of antibodies but with improved cell penetration, stability, reduced immunogenicity, and simpler manufacturing methods.
  • the intramolecular disulfide architecture of CDPs provides particularly high stability metrics, reducing fragmentation and immunogenicity, while their smaller size could improve tissue penetration or cell penetration and facilitate tunable serum half-life.
  • peptides representing candidate peptides that can serve as vehicles for delivering target molecules to cellular, intracellular, and paracellular compartments or spaces.
  • cell-penetrating peptides can be engineered peptides.
  • An engineered peptide can be a peptide that is non-naturally occurring, artificial, isolated, synthetic, designed, or recombinantly expressed.
  • the cell-penetrating peptides of the present disclosure comprise one or more properties of CDPs, knotted peptides, or hitchins, such as stability, resistance to proteolysis, resistance to heat, resistance to denaturation, resistance to reducing conditions, and/or ability to cross the blood brain barrier.
  • cell-penetrating peptides as described herein contain no disulfides or Cys (e g., SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9 - SEQ ID NO: 17, SEQ ID NO: 21 - SEQ ID NO: 29, SEQ ID NO: 41 - SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 70, SEQ ID NO: 82 - SEQ ID NO: 84, SEQ ID NO: 188, SEQ ID NO: 163 - SEQ ID NO: 165, SEQ ID NO: 168, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 198 - SEQ ID NO: 201, SEQ ID NO: 210, SEQ ID NO: 213, SEQ ID NO: 225, SEQ ID NO: 227 - SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 246, or S
  • cell-penetrating peptides may comprise one or more Cys, or one or more disulfide bond (e g., SEQ ID NO: 1 - SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 18 - SEQ ID NO: 20, SEQ ID NO: 30 - SEQ ID NO: 40, SEQ ID NO: 59 - SEQ ID NO: 61, SEQ ID NO: 64 - SEQ ID NO: 69, SEQ ID NO: 71 - SEQ ID NO: 81, SEQ ID NO: 85 - SEQ ID NO: 117, SEQ ID NO: 119 SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 169 - SEQ ID NO: 193, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 202 - SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO:
  • the cell-penetrating peptides are derived from knotted or knottin peptides.
  • such knotted peptides can penetrate a cellular layer (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen).
  • a peptide that penetrates a cellular layer comprises one or more properties of some knottin peptides, such as stability, resistance to heat, resistance to denaturation, resistance to proteolysis, and/or resistance to reducing conditions.
  • knotted peptides can be conjugated to, linked to, or fused to cellpenetrating peptides, such as those described in TABLE 1, TABLE 2, or TABLE 3, to deliver the knotted peptide to a target cell, such as a cancer cell, a pancreatic cell, liver cell, colon cell, a smooth muscle cell, ovarian cell, breast cell, lung cell, brain cell, skin cell, ocular cell, blood cell, lymph cell, immune system cell, reproductive cell, reproductive organ cell, prostate cell, fibroblast, kidney cell, adenocarcinoma cell, glioma stem cell, tumor cell, or any combination thereof.
  • a target cell such as a cancer cell, a pancreatic cell, liver cell, colon cell, a smooth muscle cell, ovarian cell, breast cell, lung cell, brain cell, skin cell, ocular cell, blood cell, lymph cell, immune system cell, reproductive cell, reproductive organ cell, prostate cell, fibroblast, kidney cell, adenocarcinoma cell, gliom
  • knotted peptides may perform a function inside a target cell, such a cancer cell, such as a breast cancer, liver cancer, colon cancer, brain cancer, pancreatic cancer, lung cancer, leukemia, lymphoma, myeloma, skin cancer, fibroblastic cancer, kidney cell cancer, adenocarcinoma cell, glioma stem cell, or tumor cell.
  • a knotted peptide may promote or inhibit a protein-protein interaction within a target cell, or a knotted peptide may promote or inhibit transcription of a target gene.
  • knotted peptides are conjugated to, linked to, or fused to cell-penetrating peptides that are capable of delivering the knotted peptide across a cellular layer (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen) to target cells in the central nervous system.
  • cell-penetrating peptides can be used to deliver a cytotoxic agent to the target cell.
  • a knotted cargo peptide may be conjugated to a knotted cell-penetrating peptide to form a diknottin (e g., SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 316, or SEQ ID NO: 317).
  • a diknottin e g., SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 316, or SEQ ID NO: 317.
  • Knottins are a class of peptides, usually ranging from about 11 to about 81 amino acids in length that are often folded into a compact structure. Knottins are typically assembled into a complex tertiary structure that is characterized by a number of intramolecular disulfide crosslinks and may contain beta strands, alpha helices, and other secondary structures. The presence of the disulfide bonds gives knottins remarkable environmental stability, allowing them to withstand extremes of temperature and pH and to resist the proteolytic enzymes of the blood stream. The presence of a disulfide knot may provide resistance to reduction by reducing agents.
  • knottins also allows them to bind to targets without paying the “entropic penalty” that a floppy peptide accrues upon binding a target.
  • binding is adversely affected by the loss of entropy that occurs when a peptide binds a target to form a complex. Therefore, “entropic penalty” is the adverse effect on binding, and the greater the entropic loss that occurs upon this binding, the greater the “entropic penalty.”
  • unbound molecules that are flexible lose more entropy when forming a complex than molecules that are rigidly structured, because of the loss of flexibility when bound up in a complex.
  • rigidity in the unbound molecule also generally increases specificity by limiting the number of complexes that molecule can form.
  • knotted peptides can bind targets with antibody -like affinity, or with nanomolar or picomolar affinity.
  • a wider examination of the sequence structure and sequence identity or homology of knottins reveals that they have arisen by convergent evolution in all kinds of animals and plants. In animals, they are often found in venoms, for example, the venoms of spiders and scorpions and have been implicated in the modulation of ion channels.
  • the knottin proteins of plants can inhibit the proteolytic enzymes of animals or have antimicrobial activity, suggesting that knottins can function in the native defense of plants.
  • the present disclosure provides peptides that comprise or are derived from these knotted peptides (or knottins).
  • the term “knotted peptide” is considered to be interchangeable with the terms “knottin.”
  • Knotted peptides of the present disclosure comprise cysteine amino acid residues.
  • the peptide has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 cysteine amino acid residues.
  • the peptide has at least 8 cysteine amino acid residues.
  • the peptide has at least 10 cysteine amino acid residues, at least 12 cysteine amino acid residues, at least 14 cysteine amino acid residues or at least 16 cysteine amino acid residues.
  • a knotted peptide can comprise disulfide bridges.
  • a knotted peptide can be a peptide wherein 5% or more of the residues are cysteines forming intramolecular disulfide bonds.
  • a disulfide-linked peptide can be a drug scaffold.
  • the disulfide bridges form a knot.
  • a disulfide bridge can be formed between cysteine residues, for example, between cysteines 1 and 4, 2 and 5, or, 3 and 6. In some cases, one disulfide bridge passes through a loop formed by the other two disulfide bridges, for example, to form the knot. In other cases, the disulfide bridges can be formed between any two cysteine residues.
  • the present disclosure further includes peptide scaffolds that, e g., can be used as a starting point for generating additional peptides.
  • these scaffolds can be derived from a variety of knotted peptides (or knottins).
  • knotted peptides are assembled into a complex tertiary structure that is characterized by a number of intramolecular disulfide crosslinks, and optionally contain beta strands and other secondary structures such as an alpha helix.
  • knotted peptides include, in some embodiments, small disulfide-rich proteins characterized by a disulfide through disulfide knot.
  • This knot can be, for example, obtained when one disulfide bridge crosses the macrocycle formed by two other disulfides and the interconnecting backbone.
  • the knotted peptides can include growth factor cysteine knots or inhibitor cysteine knots.
  • Other possible peptide structures include peptide having two parallel helices linked by two disulfide bridges without p- sheets (e g., hefutoxin).
  • a knotted peptide can comprise at least one amino acid residue in an L configuration.
  • a knotted peptide can comprise at least one amino acid residue in a D configuration.
  • a knotted peptide is 15-40 amino acid residues long.
  • a knotted peptide is 11-57 amino acid residues long.
  • a knotted peptide is 11-81 amino acid residues long.
  • a knotted peptide is at least 20 amino acid residues long.
  • Knotted peptides or peptides can be derived from a class of proteins known to be present or associated with toxins or venoms.
  • the peptide can be derived from toxins or venoms associated with scorpions or spiders.
  • the peptide can be derived from venoms and toxins of spiders and scorpions of various genus and species.
  • the peptide can be derived from a venom or toxin of the Leiurus quinquestriatus hebraeus, Buthus occitanus tunetanus, Hottentotta judaicus, Mesobuthus eupeus, Buthus occitanus Israelis, Hadrurus gertschi, Androctonus australis, Centruroides noxius, Heteroflowers laoticus, Opistophthalmus carinatus, Haplopelma schmidti, Isometrus maculatus, Haplopelma huwenum, Haplopelma hainanum, Haplopelma schmidti, Agelenopsis aperta, Haydronyche
  • a peptide of the present disclosure can comprise a sequence having cysteine residues at one or more of corresponding positions 11, 12, 13, 14, 19, 20, 21, 22, 36, 38, 39, 41, for example with reference to SEQ ID NO: 295.
  • a peptide comprises Cys at corresponding positions 11, 12, 19, 20, 36, 39, or any combination thereof.
  • a peptide can comprise a sequence having a cysteine residue at corresponding position 11.
  • a peptide can comprise a sequence having a cysteine residue at corresponding position 12.
  • a peptide can comprise a sequence having a cysteine residue at corresponding position 13. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 14. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 19. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 20. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 21. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 22.
  • a peptide can comprise a sequence having a cysteine residue at corresponding position 36. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 38. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 39. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 41.
  • the first cysteine residue in the sequence can be disulfide bonded with the 4th cysteine residue in the sequence
  • the 2nd cysteine residue in the sequence can be disulfide bonded to the 5th cysteine residue in the sequence
  • the 3rd cysteine residue in the sequence can be disulfide bonded to the 6th cysteine residue in the sequence.
  • a peptide can comprise one disulfide bridge that passes through a ring formed by two other disulfide bridges, also known as a “two-and-through” structure system.
  • the peptides disclosed herein can have one or more cysteines mutated to serine.
  • peptides of the present disclosure comprise at least one cysteine residue. In some embodiments, peptides of the present disclosure comprise at least two cysteine residues. In some embodiments, peptides of the present disclosure comprise at least three cysteine residues. In some embodiments, peptides of the present disclosure comprise at least four cysteine residues. In some embodiments, peptides of the present disclosure comprise at least five cysteine residues. In some embodiments, peptides of the present disclosure comprise at least six cysteine residues.
  • peptides of the present disclosure comprise at least ten cysteine residues. In some embodiments, a peptide of the present disclosure comprises six cysteine residues. In some embodiments, a peptide of the present disclosure comprises seven cysteine residues. In some embodiments, a peptide of the present disclosure comprises eight cysteine residues. In some embodiments, a peptide of the present disclosure comprises an even number of cysteine residues.
  • a peptide of the present disclosure comprises an amino acid sequence having cysteine residues at one or more positions, for example with reference to SEQ ID NO: 295.
  • the one or more cysteine residues are located at any one of the corresponding amino acid positions 6, 10, 20, 34, 44, 48, or any combination thereof.
  • the one or more cysteine (C) residues participate in disulfide bonds with various pairing patterns (e.g., C10-C20).
  • the corresponding pairing patterns are C6-C48, C10-C44, and C20-C34.
  • the peptides as described herein comprise at least one, at least two, or at least three disulfide bonds. In some embodiments, at least one, at least two, or at least three disulfide bonds are arranged according to the corresponding C6-C48, C10-C44, and C20-C34 pairing patterns, or a combination thereof. In some embodiments, peptides as described herein comprise three disulfide bonds with the corresponding pairing patterns C6-C48, C10-C44, and C20-C34.
  • a peptide (e.g., a cell-penetrating peptide or a cell-penetrating peptide complex) comprises a sequence having a cysteine residue at corresponding position 6. In certain embodiments, a peptide comprises a sequence having a cysteine residue at corresponding position 10. In certain embodiments, a peptide comprises a sequence having a cysteine residue at corresponding position 20. In certain embodiments, a peptide comprises a sequence having a cysteine residue at corresponding position 34. In certain embodiments, a peptide comprises a sequence having a cysteine residue at corresponding position 44.
  • a peptide comprises a sequence having a cysteine residue at corresponding position 50.
  • the first cysteine residue in the sequence is disulfide bonded with the last cysteine residue in the sequence.
  • the second cysteine residue in the sequence is disulfide bonded with the second to the last cysteine residue in the sequence.
  • the third cysteine residue in the sequence is disulfide bonded with the third to the last cysteine residue in the sequence and so forth.
  • the first cysteine residue in the sequence is disulfide bonded with the 6th cysteine residue in the sequence
  • the 2nd cysteine residue in the sequence is disulfide bonded to the 5th cysteine residue in the sequence
  • the 3rd cysteine residue in the sequence is disulfide bonded to the 4th cysteine residue in the sequence.
  • a peptide can comprise one disulfide bridge that passes through a ring formed by two other disulfide bridges, also known as a “two-and-through” structure system.
  • the peptides disclosed herein have one or more cysteines mutated to serine.
  • a peptide of the present disclosure has an even number of cysteine residues.
  • a peptide of the present disclosure comprises no cysteine residues.
  • a peptide (e.g., a cell-penetrating peptide or a cell-penetrating peptide complex) comprises no cysteine or disulfides.
  • a peptide comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more cysteine or disulfides.
  • 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more cysteine residues have been replaced with serine residues. In some embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more cysteine residues have been replaced with threonine residues.
  • a peptide (e.g., a cell-penetrating peptide or a cell-penetrating peptide complex) comprises no Cys or disulfides.
  • a peptide comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more Cys or disulfides.
  • 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues have been replaced with Ser residues.
  • 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues have been replaced with Thr residues.
  • one or more or all of the methionine residues in the peptide are replaced by leucine or isoleucine.
  • one or more or all of the tryptophan residues in the peptide are replaced by phenylalanine or tyrosine.
  • one or more or all of the asparagine residues in the peptide are replaced by glutamine.
  • the N-terminus of the peptide is blocked, such as by an acetyl group.
  • the C-terminus of the peptide is blocked, such as by an amide group.
  • the peptide is modified by methylation on free amines.
  • Percent sequence identity or homology is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (Id.). The sequence identity or homology is then calculated as: ([Total number of identical matches]/[length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences])(100).
  • FASTA similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of sequence identity or homology shared by an amino acid sequence of a peptide disclosed herein and the amino acid sequence of a peptide variant
  • the FASTA algorithm is described by Pearson and Lipman, Proc. Nat'lAcad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63 (1990).
  • the ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are “trimmed” to include only those residues that contribute to the highest score.
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch- Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, Siam J. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions.
  • FASTA can also be used to determine the sequence identity or homology of nucleic acid molecules using a ratio as disclosed above.
  • the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as described above.
  • ⁇ amino acids that are a “conservative amino acid substitution” are illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.
  • the BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat'lAcad. Sci.
  • the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention.
  • conservative amino acid substitution preferably refers to a substitution represented by a BLOSUM62 value of greater than -1.
  • an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
  • preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
  • Determination of amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can determine specific residues that can be more or less tolerant of change and maintain the overall tertiary structure of the molecule.
  • Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity or homology and computer analysis using available software (e.g., the Insight II.RTM. viewer and homology modeling tools; MSI, San Diego, Calif), secondary structure propensities, binary patterns, complementary packing and buried polar interactions (Barton, G.J., Current Opin. Struct. Biol. 5'312-6 (1995) and Cordes, M.H. et al., Current Opin. Struct. Biol. 6:3-10 (1996)).
  • modifications to molecules or identifying specific fragments determination of structure can typically be accompanied by evaluating activity of modified molecules.
  • peptides can have a net charge, for example, of -5, -4, -3, -2, -1, 0, +1, +2, +3, +4, or +5.
  • the net charge is zero, the peptide can be uncharged or zwitterionic.
  • the peptide contains one or more disulfide bonds and has a positive net charge at physiological pH where the net charge can be +0.5 or less than +0.5, +1 or less than +1, +1.5 or less than +1.5, +2 or less than +2, +2.5 or less than +2.5, +3 or less than +3, +3.5 or less than +3.5, +4 or less than +4, +4.5 or less than +4.5, +5 or less than +5, +5.5 or less than +5.5, +6 or less than +6, +6.5 or less than +6.5, +7 or less than +7, +7.5 or less than +7.5, +8 or less than +8, +8.5 or less than +8.5, +9 or less than +9.5, +10 or less than +10.
  • the peptide has a negative net charge at physiological pH where the net charge can be -0.5 or less than -0.5, -1 or less than -1, -1.5 or less than -1.5, -2 or less than -2, -2.5 or less than -2.5, -3 or less than -3, -3.5 or less than -3.5, -4 or less than -4, -4.5 or less than -4.5, -5 or less than -5, -5.5 or less than -5.5, -6 or less than -6, -6.5 or less than -6.5, -7 or less than -7, - 7.5 or less than -7.5, -8 or less than -8, -8.5 or less than -8.5, -9 or less than -9.5, -10 or less than -10.
  • peptides of the present disclosure can have an isoelectric point (pl) value from 3 and 10. In other embodiments, peptides of the present disclosure can have a pl value from 4.3 and 8.9. In some embodiments, peptides of the present disclosure can have a pl value from 3-4. In some embodiments, peptides of the present disclosure can have a pl value from 3-5. In some embodiments, peptides of the present disclosure can have a pl value from 3-6. In some embodiments, peptides of the present disclosure can have a pl value from 3-7. In some embodiments, peptides of the present disclosure can have a pl value from 3-8.
  • pl isoelectric point
  • peptides of the present disclosure can have a pl value from 3-9. In some embodiments, peptides of the present disclosure can have a pl value from 4-5. In some embodiments, peptides of the present disclosure can have a pl value from 4-6. In some embodiments, peptides of the present disclosure can have a pl value from 4-7. In some embodiments, peptides of the present disclosure can have a pl value from 4-8. In some embodiments, peptides of the present disclosure can have a pl value from 4-9. In some embodiments, peptides of the present disclosure can have a pl value from 4-10 In some embodiments, peptides of the present disclosure can have a pl value from 5-6.
  • peptides of the present disclosure can have a pl value from 5-7. In some embodiments, peptides of the present disclosure can have a pl value from 5-8. In some embodiments, peptides of the present disclosure can have a pl value from 5-9. In some embodiments, peptides of the present disclosure can have a pl value from 5-10. In some embodiments, peptides of the present disclosure can have a pl value from 6-7. In some embodiments, peptides of the present disclosure can have a pl value from 6-8. In some embodiments, peptides of the present disclosure can have a pl value from 6-9. In some embodiments, peptides of the present disclosure can have a pl value from 6-10.
  • peptides of the present disclosure can have a pl value from 7-8. In some embodiments, peptides of the present disclosure can have a pl value from 7-9. In some embodiments, peptides of the present disclosure can have a pl value from 7-10. In some embodiments, peptides of the present disclosure can have a pl value from 8-9. In some embodiments, peptides of the present disclosure can have a pl value from 8-10. In some embodiments, peptides of the present disclosure can have a pl value from 9-10.
  • the engineering of one or more mutations within a peptide yields a peptide with an altered isoelectric point, charge, surface charge, or rheology at physiological pH.
  • Such engineering of a mutation to a peptide derived from a scorpion or spider can change the net charge of the peptide, for example, by decreasing the net charge by 1, 2, 3, 4, or 5, or by increasing the net charge by 1, 2, 3, 4, or 5.
  • the engineered mutation can facilitate the ability of the peptide to penetrate a cell, an endosome, or the nucleus.
  • Suitable amino acid modifications for improving the rheology and potency of a peptide can include conservative or non-conservative mutations.
  • a peptide can comprise at most 1 amino acid mutation, at most 2 amino acid mutations, at most 3 amino acid mutations, at most 4 amino acid mutations, at most 5 amino acid mutations, at most 6 amino acid mutations, at most 7 amino acid mutations, at most 8 amino acid mutations, at most 9 amino acid mutations, at most 10 amino acid mutations, or another suitable number as compared to the sequence of the venom or toxin component that the peptide is derived from.
  • a peptide, or a functional fragment thereof comprises at least 1 amino acid mutation, at least 2 amino acid mutations, at least 3 amino acid mutations, at least 4 amino acid mutations, at least 5 amino acid mutations, at least 6 amino acid mutations, at least 7 amino acid mutations, at least 8 amino acid mutations, at least 9 amino acid mutations, at least 10 amino acid mutations, or another suitable number as compared to the sequence of the venom or toxin component that the peptide is derived from.
  • mutations can be engineered within a peptide to provide a peptide that has a desired charge or stability at physiological pH.
  • the NMR solution structures, the x-ray crystal structures, as well as the primary structure sequence alignment of related structural homologs or in silico design can be used to inform mutational strategies that can improve the folding, stability, and/or manufacturability, while maintaining a particular biological function.
  • the general strategy for producing homologs or in silico designed peptides or proteins can include identification of a charged surface patch or conserved residues of a protein, mutation of critical amino acid positions and loops, and testing of sequences. This strategy can be used to design peptides with improved properties or to correct deleterious mutations that complicate folding and manufacturability.
  • the present disclosure also encompasses multimers of the various peptides described herein.
  • multimers include dimers, trimers, tetramers, pentamers, hexamers, heptamers, and so on.
  • a multimer may be a homomer formed from a plurality of identical subunits or a heteromer formed from a plurality of different subunits.
  • a peptide of the present disclosure is arranged in a multimeric structure with at least one other peptide, or two, three, four, five, six, seven, eight, nine, ten, or more other peptides.
  • the peptides of a multimeric structure each have the same sequence.
  • the present disclosure further includes peptide scaffolds that, e.g., can be used as a starting point for generating additional peptides.
  • these scaffolds can be derived from a variety of knotted peptides or knottins.
  • Suitable peptides for scaffolds can include, but are not limited to, CDPS, chlorotoxin, brazzein, circulin, stecrisp, hanatoxin, midkine, hefutoxin, potato carboxypeptidase inhibitor, bubble protein, attractin, a-GI, a-GID, p- PIIIA, co-MVIIA, ®-CVID, %-MrIA, p-TIA, conantokin G, conantokin G, conantokin G, conantakin G, GsMTx4, margatoxin, shK, toxin K, chymotrypsin inhibitor (CTI), and EGF epiregulin core.
  • the peptide sequence is flanked by additional amino acids.
  • One or more additional amino acids can, for example, confer a desired in vivo charge, isoelectric point, chemical conjugation site, stability, or physiologic property to a peptide.
  • a cell-penetrating peptide sequence (e.g., a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) can be fused or linked to a cargo molecule, such a small molecule or peptide.
  • a cell-penetrating peptide may be appended to either the N- terminus or the C-terminus of a peptide (e.g., a cargo peptide, an active agent peptide, a therapeutic peptide, or a detectable peptide).
  • the cell -penetrating peptide can be appended to the N-terminus of a peptide (e.g., a cargo peptide, an active agent peptide, a therapeutic peptide, or a detectable peptide) following an N-terminal GS dipeptide and preceding, for example, a GGGS (SEQ ID NO: 257) linker.
  • a peptide e.g., a cargo peptide, an active agent peptide, a therapeutic peptide, or a detectable peptide
  • cellpenetrating peptide sequence e.g., a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254
  • a peptide e.g., a cargo peptide, an active agent peptide, a therapeutic peptide, or a detectable peptide
  • a peptide linker such as G x S y (SEQ ID NO: 256) peptide linker, wherein x and y can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the peptide linker comprises (GS)x (SEQ ID NO: 258), wherein x can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the peptide linker comprises GGSSG (SEQ ID NO: 259), GGGGG (SEQ ID NO: 260), GSGSGSGS (SEQ ID NO: 261), GSGG (SEQ ID NO: 262), GGGGS (SEQ ID NO: 263), GGGS (SEQ ID NO: 264), GGS (SEQ ID NO: 265), GGGSGGGSGGGS (SEQ ID NO: 255), or a variant or fragment thereof.
  • KKYKPYVPVTTN (SEQ ID NO: 266) from DkTx
  • EPKSSDKTHT (SEQ ID NO: 267) from human IgG3
  • the cell-penetrating peptide can be appended to the peptide at any amino acid residue.
  • the cell-penetrating peptide can be appended to the peptide at any amino acid residue without interfering with an activity of the peptide (e.g., a therapeutic activity, an enzymatic activity, a binding activity, or a transcription factor activity).
  • the tag peptide is appended via conjugation, linking, or fusion techniques. In some embodiments, the tag peptide is placed between two other peptides with active functions. In other embodiments, the cell-penetrating peptide can be appended to the peptide at any amino acid residue. In further embodiments, the cell-penetrating peptide can be appended to the peptide at any amino acid residue without interfering with peptide activity (e.g., therapeutic activity, enzymatic activity, binding activity, or transcription factor activity). In some embodiments, the cell-penetrating peptide is appended via conjugation, linking, or fusion techniques. Examples of cell -penetrating peptide complexes that may improve cell penetration of the cargo peptide are provided in TABLE 4.
  • a cargo molecule is conjugated to, linked to, or fused to one or more cell-penetrating peptides (e.g., a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) for delivery of a cargo molecule across a cellular layer or into the cytoplasm or nucleus of a cell.
  • Conjugation, linking, or fusion can be direct or with a spacer in between (chemical or peptide-based).
  • a spacer can be any peptide linker.
  • a spacer can be GGGSGGGSGGGS (SEQ ID NO: 255), KKYKPYVPVTTN (SEQ ID NO: 266) from DkTx, EPKSSDKTHT (SEQ ID NO: 267) from human IgG3 or any variant or fragment thereof.
  • a cell-penetrating peptide may be linked to a cargo peptide via a peptide linker (e.g., any of SEQ ID NO: 255 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485).
  • a cell-penetrating peptide may be linked to a cargo molecule via a small molecule linker (e.g., a linker provided in TABLE 11).
  • a cargo molecule can be delivered carried across a cellular layer (e g., a plasma membrane, a vesicular membrane, an endosomal membrane, a nuclear envelope, or a blood brain barrier) to a cellular, intracellular, or paracellular space (e.g., a cytosol, a nucleus, or a nanolumen) when linked, fused, conjugated, or otherwise connected to a cell -penetrating peptide of the present disclosure.
  • a cellular layer e.g., a plasma membrane, a vesicular membrane, an endosomal membrane, a nuclear envelope, or a blood brain barrier
  • a cellular, intracellular, or paracellular space e.g., a cytosol, a nucleus, or a nanolumen
  • a cargo molecule can be an active agent (e.g., a therapeutic agent or a detectable agent).
  • a cargo molecule can be a cystine-dense peptide, an anti-cancer agent, a transcription factor binding agent, an inhibitor of protein-protein interactions, a promoter of protein-protein interactions, a transcription factor, an RNA, a Cas enzyme or other CRISPR component, a DNA-binding protein, an RNA-binding protein, a transcriptional activator, a transcriptional inhibitor, a promotor of protein degradation, a molecular glue, a proteolysis targeting chimera, a protein (e.g., a protein that inhibits proteinprotein interactions), an oligonucleotide, or an immunomodulating agent.
  • an active agent e.g., a therapeutic agent or a detectable agent.
  • a cargo molecule can be a cystine-dense peptide, an anti-cancer agent, a transcription factor binding agent, an inhibitor of protein-protein interactions,
  • a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254, or a fragment thereof can be conjugated, fused, linked, or otherwise connected to an active agent comprising a cystine- dense peptide, an anti-cancer agent, a transcription factor binding agent, an inhibitor of proteinprotein interactions, a promoter of protein-protein interactions, a transcription factor, an RNA, a Cas enzyme or other CRISPR component, a DNA-binding protein, an RNA-binding protein, a transcriptional activator, a transcriptional inhibitor, a promotor of protein degradation, a molecular glue, a proteolysis targeting chimera, a protein (e g., a protein that inhibits proteinprotein interactions), an oligonucleotide, or an immunomodulating agent for delivery
  • one or more cell-penetrating peptides can be fused, linked, or conjugated to a cargo peptide or cargo molecule for improved cell penetration.
  • a cargo molecule may be a target-binding molecule that binds a target of interest, such as a target peptide.
  • the cargo molecule inhibits the target upon binding. For example, biding of the cargo molecule may inhibit formation of protein-protein or protein-nucleic acid interactions with the target. In another example, binding of the cargo molecule may inhibit a conformation change or enzymatic activity of the target molecule.
  • target molecules include TEAD, coldinducible RNA-binding protein, androgen receptor, ikaros, aiolos, a nuclear receptor, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-8, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CSNK1A1, GSPT1, ZFP91, ZNF629, ZNF91, ZNF276, ZNF653, ZNF827, WIZ, ZNF692, GZF1, ZNF98, BCL6, cMYC, tau, AKT, mTOR, STAT3, RAS, RAF
  • a cargo molecule that binds a target may comprise a sequence of any one of SEQ ID NO: 293 - SEQ ID NO: 298 or SEQ ID NO: 364 - SEQ ID NO: 407.
  • a cargo molecule that binds a target may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID NO: 293 - SEQ ID NO: 298 or SEQ ID NO: 364 - SEQ ID NO: 407. Examples of cargo-binding molecules are provided in TABLE 5.
  • a cargo molecule may comprise a small molecule (e.g., a small molecule ligand) that binds a target.
  • a cargo molecule may comprise a small molecule that binds a ubiquitin ligase.
  • cargo molecules that binds ubiquitin ligase include thalidomide, pomalidomide, lenalidomide, methyl bestatin, bestatin, nutlin-3, and VHL ligand 1, as well as other immunomodulatory drugs (IMiDs).
  • a cargo molecule such as a cargo molecule of a targeted degradation complex, may comprise a target-binding peptide (e.g., a peptide that binds TEAD, cold-inducible RNA-binding protein, androgen receptor, ikaros, aiolos, nuclear receptors, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-8, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CSNK1A1, GSPT1, ZFP91, ZNF629, ZNF91, ZNF276, ZNF653, ZNF827, WI
  • a peptide complex of the present disclosure may be delivered across a cellular layer of a cell by way of the cell-penetrating peptide.
  • the peptide complex may be delivered to a cellular compartment (e g., the cytosol or nucleus) of the cell.
  • the peptide complex may be delivered to an intercellular compartment (e.g., a nanolumen, intercellular space, or paracellular space) across the cellular layer.
  • At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 22%, at least about 25%, at least about 27%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% of the peptide complex is delivered across the cellular layer.
  • the cell-penetrating peptides described herein are attached to another molecule (e.g., a cargo molecule), such as a cystine-dense peptide (CDP) that provides a functional capability.
  • a cell-penetrating peptide attached to a CDP may have a sequence of any one of SEQ ID NO: 299 - SEQ ID NO: 308 or SEQ ID NO: 312 - SEQ ID NO: 321.
  • cell-penetrating peptides can direct the CDP into the cell.
  • cell-penetrating peptides can direct the CDP into the nucleus.
  • cell-penetrating peptides can direct the CDP cross the blood brain barrier. In further embodiments, cell-penetrating peptides can direct the CDP into a nanolumen between cells (e.g., cancer cells). In some embodiments, the CDP has a therapeutic effect inside the cell. In further embodiments, cell-penetrating peptides can direct the CDP into a nanolumen between cells (e.g., cancer cells).
  • cell-penetrating peptides can direct the CDP into or across a cellular space or compartment (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, or other subcellular compartment membrane, a blood brain barrier, or a nanolumen).
  • a cellular space or compartment e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, or other subcellular compartment membrane, a blood brain barrier, or a nanolumen.
  • the CDP may bind to a cytosolic, nuclear, intracellular, or paracellular protein and modulate an activity or a protein-protein interaction of the protein.
  • the CDP may bind to and modulate a transcription factor, cereblon, VHL, DCAF15, DCAF16, other molecules in the CRL4 E3 ligase family, other molecules in the E3 ligase family, or other molecules in the UPS system.
  • the CDP may bind to target molecule, such as a target peptide.
  • Binding of the CDP to the target molecule may inhibit the target molecule (e g., inhibit a conformational change, inhibit an enzymatic activity, inhibit ligand binding, inhibit a protein-protein interaction, or inhibit a protein-nucleic acid interaction of the target).
  • binding of the CDP may recruit additional agents to the target, such as a ubiquitin ligase.
  • the CDP is a DNA-binding protein, an RNA-binding protein, a transcriptional activator, a transcriptional inhibitor, a promotor of protein degradation, a molecular glue, a protein (e.g., a protein that inhibits protein-protein interactions), or a proteolysis targeting chimera.
  • the cell-penetrating peptides or peptide complexes of the present disclosure can be conjugated to, linked to, or fused with an active agent (e.g., a cargo molecule, a therapeutic agent, a drug, a biologic, or a peptide).
  • an active agent e.g., a cargo molecule, a therapeutic agent, a drug, a biologic, or a peptide.
  • the cell-penetrating peptide may be linked to more than one active agent.
  • the cell-penetrating peptide may be linked to a cargo peptide (e.g., a targetbinding peptide) and an additional active agent (e.g., a target-binding ligand, a therapeutic agent, a drug, a biologic, or a peptide).
  • a cargo peptide e.g., a targetbinding peptide
  • an additional active agent e.g., a target-binding ligand, a therapeutic agent, a drug, a biologic, or a peptide.
  • the cell-penetrating peptide may deliver the active agent into a cellular compartment (e.g., across a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, a blood brain barrier, or a nanolumen).
  • Peptides according to the present disclosure can be conjugated to, linked to, or fused to an agent for use in the treatment of a disease or a condition (e.g., cancer, neurodegeneration, over-expression or under-expression of a gene, inflammation, or protein over-expression or accumulation).
  • a disease or a condition e.g., cancer, neurodegeneration, over-expression or under-expression of a gene, inflammation, or protein over-expression or accumulation.
  • the peptides described herein are fused to another molecule, such as an active agent that provides an additional functional capability.
  • An active agent may be a small molecule active agent, a peptide active agent, or a nucleic acid active agent.
  • a cell-penetrating peptide may be conjugated to a small molecule therapeutic agent.
  • the cell-penetrating peptides of the present disclosure may be fused to an active agent that may otherwise be excluded from a cell or a cellular compartment to facilitate absorption or permeation of the active agent.
  • a cell-penetrating peptide of the present disclosure may facilitate cytosolic delivery of an active agent with multiple hydrogen bond donors or hydrogen bond acceptors, a large molecular weight, or a high partition coefficient, which may lead to poor cytosolic delivery of the active agent in the absence of the cell -penetrating peptide.
  • a cell-penetrating peptide may be conjugated to an active agent comprising at least 5, at least 6, at least 7, at least 8, 9, at least 10, at least 15, or at least 20 hydrogen bond donors. In some embodiments, the cell-penetrating peptide may be conjugated to an active agent comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20 hydrogen bond acceptors.
  • the cell-penetrating peptide may be conjugated to an active agent comprising a molecular weight of at least 200 Da, at least 500 Da, at least 600 Da, at least 700 Da, at least 800 Da, at least 900 Da, at least 1000 Da, at least 1500 Da, at least 2000 Da, at least 3000 Da, at least 4000 Da, at least 5000 Da, at least 6000 Da, at least 7000 Da, at least 8000 Da, at least 9000 Da, or at least 10,000 Da.
  • an active agent comprising a molecular weight of at least 200 Da, at least 500 Da, at least 600 Da, at least 700 Da, at least 800 Da, at least 900 Da, at least 1000 Da, at least 1500 Da, at least 2000 Da, at least 3000 Da, at least 4000 Da, at least 5000 Da, at least 6000 Da, at least 7000 Da, at least 8000 Da, at least 9000 Da, or at least 10,000 Da.
  • the cell-penetrating peptide may be conjugated to an active agent comprising a molecular weight of up to 1000 Da, up to 2000, up to 3000 Da, up to 4000 Da, up to 5000 Da, up to 6000 Da, up to 7000 Da, up to 8000 Da, up to 9000 Da, up to 10,000 Da, up to 12,000 Da, up to 15,000 Da, up to 20,000 Da, or up to 25,000 Da.
  • the cell-penetrating peptide may be conjugated to an active agent comprising a log of a partition coefficient (logP) of at least 5.0, at least 5.2, at least 5.4, at least 5.5, at least 5.6, at least 5.8, or at least 6.0.
  • the partition coefficient may be a measure of a ratio of the solubility of the agent in a hydrophobic solvent (e.g., octanol) relative to water.
  • a peptide can be fused with an active agent through expression of a vector containing the sequence of the peptide with the sequence of the active agent.
  • the sequence of the peptide and the sequence of the active agent can be expressed from the same Open Reading Frame (ORF).
  • ORF Open Reading Frame
  • the sequence of the peptide and the sequence of the active agent can comprise a contiguous sequence.
  • the peptide and the active agent can each retain similar functional capabilities in the fusion peptide compared with their functional capabilities when expressed separately.
  • examples of active agents can include other peptides.
  • the peptides described herein are attached to another molecule, such as an active agent that provides a functional capability.
  • cell-penetrating peptides can direct the active agent into the cell.
  • cell-penetrating peptides can direct the active agent into the nucleus.
  • cell-penetrating peptides can direct the active agent across the blood brain barrier.
  • cell-penetrating peptides can direct the active agent into a nanolumen between cells (e.g., cancer cells).
  • cell-penetrating peptides can direct the active agent into or across a cellular space or compartment (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen).
  • the active agent has a therapeutic effect inside the cell.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 active agents can be linked to a peptide.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell-penetrating peptides of this disclosure can be linked to an active agent.
  • Multiple active agents can be attached by methods such as conjugating to multiple lysine residues and/or the N-terminus, or by linking the multiple active agents to a scaffold, such as a polymer or dendrimer and then attaching that agent-scaffold to the peptide (such as described in Yurkovetskiy, A. V., Cancer Res 75(16): 3365-72 (2015)) or by recombinant fusion.
  • a scaffold such as a polymer or dendrimer
  • active agents include but are not limited to: a peptide, an oligopeptide, a polypeptide, a peptidomimetic, a polynucleotide, a polyribonucleotide, a cysteine-dense peptide, an affibody, an avimer, an adnectin, a B-hairpin, a stapled peptide, a kunitz domain, a nanofttin, a fynomer, a bicycle peptide, a Cas protein, a transcription factor, a DNA, a cDNA, a ssDNA, a RNA, a dsRNA, a micro RNA, a guide RNA, a U1 adaptor, a crRNA, a tracrRNA, an oligonucleotide, an antibody, a single chain variable fragment (scFv), a nanobody, an antibody fragment, an aptamer, a cytokine, an
  • the peptide is covalently or non-covalently linked to an active agent, e.g., directly or via a linker.
  • cytotoxic molecules that can be used include auristatins, MMAE, MMAF, dolostatin, auristatin F, monomethylaurstatin D, DM1, DM4, maytansinoids, maytansine, calicheamicins, N-acetyl-y-calicheamicin, pyrrolobenzodiazepines, PBD dimers, doxorubicin, vinca alkaloids (4-deacetylvinblastine), duocarmycins, cyclic octapeptide analogs of mushroom amatoxins, epothilones, and anthracy lines, CC-1065, taxanes, paclitaxel, cabazitaxel, docetaxel, SN-38, irinotecan, vincristine, vinblastine, platinum compounds,
  • the peptides or fusion peptides of the present disclosure can also be conjugated to, linked to, or fused to other moieties that can serve other roles, such as providing an affinity handle (e.g., biotin) for retrieval of the peptides from tissues or fluids.
  • an affinity handle e.g., biotin
  • peptides or fusion peptides of the present disclosure can also be conjugated to, linked to, or fused to biotin.
  • biotin can also act as an affinity handle for retrieval of peptides or fusion peptides from tissues or other locations.
  • fluorescent biotin conjugates that can act both as a detectable label and an affinity handle can be used.
  • Non limiting examples of commercially available fluorescent biotin conjugates include Atto 425- Biotin, Atto 488-Biotin, Atto 520-Biotin, Atto-550 Biotin, Atto 565-Biotin, Atto 590-Biotin, Atto 610-Biotin, Atto 620-Biotin, Atto 655-Biotin, Atto 680-Biotin, Atto 700-Biotin, Atto 725- Biotin, Atto 740-Biotin, fluorescein biotin, biotin-4-fluorescein, biotin-(5-fluorescein) conjugate, and biotin-B-phycoerythrin, ALEXA FLUOR 488 biocytin, ALEXA FLUOR 546, ALEXA FLUOR 549, lucifer yellow cadaverine biotin-X, Lucifer yellow biocytin, Oregon green 488 biocytin, biotin-rhodamine
  • the conjugates could include chemiluminescent compounds, colloidal metals, luminescent compounds, enzymes, radioisotopes, and paramagnetic labels.
  • the peptide-active agent fusions described herein can be attached to another molecule.
  • the peptide sequence also can be attached to another active agent (e.g., small molecule, peptide, polypeptide, polynucleotide, antibody, aptamer, cytokine, growth factor, neurotransmitter, an active fragment or modification of any of the preceding, fluorophore, radioisotope, radionuclide chelator, acyl adduct, chemical linker, or sugar, etc.).
  • the peptide can be fused with, or covalently or non-covalently linked to an active agent.
  • the conjugates can include chemiluminescent compounds, colloidal metals, luminescent compounds, enzymes, radioisotopes, and paramagnetic labels.
  • the peptide described herein can also be attached to another molecule.
  • the peptide sequence also can be attached to another active agent (e.g., small molecule, peptide, polypeptide, polynucleotide, antibody, aptamer, cytokine, growth factor, neurotransmitter, an active fragment or modification of any of the preceding agents, fluorophore, radioisotope, radionuclide chelator, acyl adduct, chemical linker, or sugar).
  • another active agent e.g., small molecule, peptide, polypeptide, polynucleotide, antibody, aptamer, cytokine, growth factor, neurotransmitter, an active fragment or modification of any of the preceding agents, fluorophore, radioisotope, radionuclide chelator, acyl adduct, chemical linker, or sugar.
  • the peptide can be conjugated to, linked to, or fused with, or covalently or non- covalently linked to an active agent.
  • a peptide sequence derived from a toxin or venom knottin protein can be present on, conjugated to, linked to, or fused with a particular peptide.
  • a peptide can be incorporated into a biomolecule by various techniques.
  • a peptide can be incorporated by a chemical transformation, such as the formation of a covalent bond, such as an amide bond.
  • a peptide can be incorporated, for example, by solid phase or solution phase peptide synthesis.
  • a peptide can be incorporated by preparing a nucleic acid sequence encoding the biomolecule, wherein the nucleic acid sequence includes a subsequence that encodes the peptide.
  • the subsequence can be in addition to the sequence that encodes the biomolecule or can substitute for a subsequence of the sequence that encodes the biomolecule.
  • Active agents that may be delivered using the cell -penetrating peptides of the present disclosure include a RIG-I ligand, or ligands targeting a related receptor, such as melanoma differentiation-associated protein 5 (MDA5) or toll-like receptor 3 (TLR3).
  • Ligands for RIG-I, MDA5, and TLR3 that may be complexed with a cell -penetrating peptide include abnormal double stranded RNA (dsRNA) comprising a 5’ diphosphate or, in the case of RIG-I, a 5’ triphosphate, such as a dsRNA associated with viral infection.
  • dsRNA abnormal double stranded RNA
  • Activation of RIG-I and MDA5 using RIG-I or MDA5 ligand can be effective at promoting antitumor immunity and apoptosis.
  • RIG-I, MDA5, or TLR3 ligands that may be complexed with a cell-penetrating peptide of the present disclosure include dsRNA, 5’ diphosphate dsRNA, 5’ triphosphate dsRNA, double stranded hairpin RNA, a benzobisthiazole compound, and polyinosinic:polycytidylic acid. Delivery of a RIG-I, MDA5, or TLR3 ligand across a cellular layer may promote anti -tumor or anti-viral activity in a subject, thereby treating a cancer or viral infection in a subject.
  • RIG-I is active intracellularly
  • the present disclosure provides a peptide-RIG-I ligand complex in which the peptide can be capable of cell penetration such that the RIG-I ligand can access the cytoplasm of the target cell.
  • RIG-I ligands by themselves may be able to activate the RIG-I helicase when in contact with it, but when applied to cells in vivo or in vitro, may access the cytoplasm at only low levels and thus not be able to access and activate RIG-I.
  • RIG-I ligands may require formulation with transfection reagents or other components to access the cytoplasm, which may not be feasible for human cancer therapy, due to toxicity, stability, safety issues, or inability to apply the formulation systemically (such as by intravenous or subcutaneous administration) and deliver sufficient amounts of active agent to the tumor.
  • RIG-I can be activated in vivo for anti-cancer therapy.
  • the RIG-I ligand in the peptide- RIG-I complex can access the cytoplasm of the target cell via cleavage of the peptide- RIG-I complex in the endosome or after exit from the endosome into the cytosol and dissociated RIG-I ligand therefrom can access the cytoplasm, or via any other mechanism as described herein.
  • the RIG-I ligand can also optionally access the cytoplasm and RIG-I without cleavage. Cleavage can also occur in the cytosol.
  • the peptide- RIG-I ligand complex can be designed such that the peptide is distal from the end of the RIG-I ligand that activates the helicase, and as such the peptide-RIG-I ligand complex may be active without cleavage.
  • the interaction between RIG-I ligands and the RIG-I helicase, such as shown in the crystal structure in Devarker et al., Proc Natl Acad Sci, 113(3): 596-601, 2016, can be analyzed to design peptide-RIG-I ligand complexes.
  • the peptides of this disclosure can be located a number of base pairs away from the 5’ triphosphate end of the RIG-I ligand, such as 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs away from the 5 ’ triphosphate end. In some embodiments, the peptide of this disclosure is conjugated 7-20 base pairs away from the 5’ triphosphate end end. In some embodiments, the peptide of this disclosure is conjugated more than 20 base pairs away from the 5’ triphosphate end. In some embodiments, the RIG-I ligand can be chemically conjugated to the peptide.
  • the peptide and RIG-I ligand can be linked with a cleavable linker, such that the linker can be cleaved selectively once intracellular, such as in the endosome or cytosol, thereby releasing the RIG-I ligand adequately in high concentrations within a cell in order to target intracellular RIG-I.
  • the peptide and RIG-I ligand can be linked such that the RIG-I is inactive or blocked from binding to its receptor by the peptide until the peptide is removed, thereby reducing exposure of noncancerous tissues to the RIG-I ligand.
  • the linker can be a disulfide bond. In other embodiments, the linker can be acid labile.
  • the linker can be enzymatically cleavable, such that it is cleaved by enzymes in the endosomal- lysosomal pathway, or within the cytosol.
  • the peptide-RIG-I ligand complex can be co-formulated.
  • the peptide-RIG-I ligand complex can be formulated in a delivery vehicle, such as a liposome.
  • the peptide-RIG-I ligand complex comprises a RIG-I ligand that can be encapsulated in a liposome, which can be further coated with a peptide of the present disclosure.
  • the peptide-RIG-I ligand complex can be linked by a stable linker and is active as a complex.
  • the linker can comprise additional functions as peptides or chemical structures that enhance endosomal escape, endosomal uptake, tissue biodistribution to the tumor, or cell penetration. Cell penetrating or endosomal escape peptide sequences can be added to the linker or to the other end of the peptide.
  • Receptor-specificity of an RNA ligand-cell-penetrating peptide complex may be achieved by selective delivery of the complex to a compartment where the receptor is found (e.g., the cytoplasm or an endosome), by using a receptor-specific ligand, or a combination thereof.
  • RIG-I and MDA5 are both RNA helicases present in the cytoplasm, while various tolllike receptors (TLRs) may be found in the endosome.
  • ligand recognized by two or more receptors found in different cellular compartments may be complexed with a cell-penetrating peptide of the present disclosure to specifically deliver the ligand to a desired cellular compartment (e.g., the cytosol or an endosome) to specifically activate one of the receptors.
  • a desired cellular compartment e.g., the cytosol or an endosome
  • delivery of polyinosinic: poly cytidylic acid to the cytoplasm may specifically activate MDA5, which is present in the cytoplasm, rather than a toll-like receptor found in endosomes (e g., TLR3, TLR7, TLR8, or TLR10).
  • an RNA ligand can be conjugated to a peptide of this disclosure to form a peptide complex that enters the cytoplasm and activates a cytoplasmic receptor (e g., RIG-I or MDA5).
  • a cytoplasmic receptor e g., RIG-I or MDA5
  • complexing the ligand to a cell-penetrating peptide capable of endosomal delivery may enable activation of an endosomal RNA-sensing receptor (e.g., an RNA-sensing TLR).
  • RIG-I and MDA5 each have a C-terminal domain involved in ligand specificity and two N-terminal CARD domains, which enable mitochondrial antiviral -signaling protein (MAVS)- mediated signal transduction, and both recognize the internal RNA duplex structure.
  • MAVS mitochondrial antiviral -signaling protein
  • RIG-I can also recognize the 5’ end of dsRNA.
  • Ligand binding of RIG-I or MDA5 may lead to activation of the MAVS -dependent signaling pathway, stimulating production of proinflammatory substances, including Type I interferons that contribute to antiviral and antitumor immunity, and are distinct from the gene expression induced by TLR3 activation.
  • Activation of RIG-I or MDA5 can additionally lead to the activation of the inflammasome resulting in changes to the tumor microenvironment that promotes antitumor immunity, such as secretion of IL-1, IL- 18, and damage-associated molecular patterns (DAMPs).
  • DAMPs damage-associated molecular patterns
  • RIG-I and MDA5 both signal through mitochondrial antiviral-signaling (MAVS) proteins, which initiates signaling via IRF3/7 and NFKB factors. MAVS is also important for initiation of tumor cell apoptosis, via RIG-I-like receptor (RLR) activation, which leads to immunogenic cell death (ICD). Additionally, RIG-I can utilize multiple interferon regulatory factors (IRF). Thus, engagement of RIG-I or MDA5 with a RIG-I-specific ligand can activate anti-viral immune mechanisms, which can have therapeutic effects against tumors. In some embodiments, engagement of RIG-I or MDA5 with a RIG-I ligand can induce direct immunogenic cell death (ICD) of tumor cells, but not normal cells.
  • ICD interferon regulatory factors
  • a RIG-I or MDA5 ligand can stimulate DC activation including inflammasome activity.
  • a RIG-I ligand can induce tumor cells to produce IFN and CXCL10 via the IRF3 pathway.
  • a RIG-I or mDA5 ligand can induce tumor regression in a subject, such as a human, non-human primate, or any other animal.
  • a RIG-I ligand can additionally inhibit a Thl7 and Treg responses.
  • cell penetrating peptide complexes of this disclosure can target MDA5, a related cytoplasmic sensor for dsRNA, which shares many of the same functions as RIG-I.
  • a RIG-I ligand may comprise a short dsRNA.
  • the short dsRNA may have a length of from 5 base pairs to 60 base pairs, from 5 base pairs to 10 base pairs, from 7 base pairs to 10 base pairs, from 11 base pairs to 18 base pairs, from 14 base pairs to 120 base pairs, from 5 base pairs to 15 base pairs, from 15 base pairs to 25 base pairs, from 25 base pairs to 40 base pairs, from 40 base pairs to 60 base pairs, from 60 base pairs to 80 base pairs, from 80 base pairs to 100 base pairs, from 100 base pairs to 120 base pairs, from 120 base pairs to 140 base pairs, from 140 base pairs to 160 base pairs, for from 19 base pairs to 60 base pairs.
  • a short dsRNA may comprise a length of from 19 base pairs to 60 base pairs and at least one 5’ triphosphate or 5’ diphosphate.
  • the short dsRNA may further comprise an uncapped 5’ A or G nucleotide, a 5’ triphosphate on a blunt end, a 1 nucleotide 5’ overlap, a 1 nucleotide overlap at the 5 ’ end with the triphosphate, or combinations thereof.
  • at least one 5 ’ triphosphate or 5 ’ diphosphate is located on the 5 ’ end of the sense strand of the dsRNA.
  • the short dsRNA may comprise a mismatch, for example a mismatch that is 8 or more base pairs from the 5’ end of the dsRNA.
  • a dsRNA RIG-I ligand may be complexed with a cellpenetrating peptide of the present disclosure via an RNA modification.
  • the RNA modification may be positioned 8 or more base pairs from the 5 ’ end of the dsRNA.
  • Additional types of dsRNA that may function as RIG-I ligands may include dsRNA hairpin RNA (e.g., in which some ribosides are paired with a partner within the same hairpin), or any other short dsRNA comprising a 5’ triphosphate.
  • a dsRNA described herein may be a based paired region within a longer single RNA sequence or may a base paired region of two separate RNA strands.
  • Double stranded RNA RIG-I ligands can be made by a variety of techniques that are used to combine the sense and antisense strands of the RNAs into a double stranded form.
  • the sense and antisense strands of the dsRNA can be separately transcribed or synthesized and combined into dsRNA structures using a variety of recombinant or synthetic techniques.
  • the sense and antisense strands of the dsRNA can be transcribed or synthesized in a single RNA that contains a loop structure (hairpin) that is optionally later cleaved by an RNAse to obtain the dsRNA.
  • a RIG-I ligand may comprise two RNA strands complexed together as a double strand, comprising a 5’ diphosphate or a 5’ triphosphate group on one or both strands, or a single RNA strand complexed together in a hairpin that is double stranded at one or more locations in the molecule.
  • the double strand may extend throughout the sequence or there may be regions of mismatch, and there may be one or more locations of hairpin self-association within one or both strands.
  • the ends of the double strand may be at the same blunt location, or one or the other end may overhang. Hairpins and other structures within the RNA complex can be more immunogenic and activate the RIG-I pathway at higher levels.
  • MDA5 ligands may also exhibit all the above structural variations but may also contain no or one 5’ phosphate.
  • RNA backbone or bases of a RIG-I ligand can be modified to improve in vitro and in vivo stability (e.g., serum stability, manufacturability, shelf stability) or other properties of the molecule including base pairing affinity and immune system activation.
  • Pyrimidines can be 2'- fluoro-modified, which can increase stability to nucleases as well as increase immune system activation.
  • the RNA backbone can be phosphorothioate-substituted (where the non-bridging oxygen is replaced with sulfur), which can increase resistance to nuclease digestion as well as altering the biodistribution and tissue retention and increasing the pharmacokinetics such as by increasing protein binding, and can also induce more immune stimulation.
  • Methyl phosphonate modification of an RNA can also be used.
  • 2’-Omethyl and 2’-F RNA bases can be used, which can protect against base hydrolysis and nucleases and increase the melting temperature of duplexes.
  • the modification can also comprise a bridged nucleic acid, a morpholino nucleic acid, a PNA, an LNA, an ethyl cEt nucleic acid. Bridged, Locked, and other similar forms of Bridged Nucleic Acids (BNA, LNA, cEt) where any chemical bridge such as an N-0 linkage between the 2' oxygen and 4' carbons in ribose can be incorporated to increase resistance to exo- and endonucleases and enhance biostability.
  • BNA, LNA, cEt Bridged Nucleic Acids
  • RNA backbone or base modifications can be placed anywhere in the RNA sequence, at one, multiple, or all base locations.
  • the modifications may be distal from the end of the dsRNA complex that contains the 5’ triphosphate and interacts with the helicase.
  • the RNA backbone or base modifications may enhance, decrease, or have no effect on the level of RIG-I activation by the peptide-RIG-I ligand complex.
  • phosophorothioate nucleic acids may be used at the 2-3 terminal nucleic acids of one or both sequences.
  • 2’F modified nucleic acids may be used at least at 5%, at least at 10%, at least at 25%, at least at 50%, at least at 75%, or at up to 100% of internal positions.
  • 2’F modified nucleic acids may be used at from 2 to 4 positions, at all internal positions, or at all positions.
  • the RIG-I ligand may have additional modified nucleotides or bases present within the sequence, such as to allow chemical modification such as conjugation to a linker and peptide or conjugation to an additional delivery agent such as a lipid, cholesterol, or hydrocarbon chain.
  • a cell -penetrating peptide of the present disclosure may be complexed with an oligonucleotide.
  • the oligonucleotide may function as an active agent, a therapeutic agent, a detectable agent, or a combination thereof.
  • a cell-penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) may be complexed with an oligonucleotide to form a peptide oligonucleotide complex, also referred to as a peptidenucleotide agent conjugate, a peptide oligonucleotide complex, or a peptide target-binding agent complex, may comprise a peptide complexed with a nucleotide (e g., an oligonucleotide).
  • a nucleotide e g., an oligonucleotide
  • the peptide of the peptide oligonucleotide complex may comprise a cell-penetrating peptide, as described herein (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254).
  • a cellpenetrating peptide of a peptide oligonucleotide complex may facilitate cell penetration of the peptide oligonucleotide complex.
  • a peptide oligonucleotide complex comprising a cell-penetrating peptide may cross a cellular membrane, enabling delivery of the peptide oligonucleotide complex to a cytoplasm or a nucleus of the cell and interaction between the nucleotide of the peptide oligonucleotide complex and various cytosolic or nuclear components (e.g., genomic DNA, an ORF, mRNA, pre-mRNA, or DNA).
  • cytosolic or nuclear components e.g., genomic DNA, an ORF, mRNA, pre-mRNA, or DNA.
  • the nucleotide of the peptide oligonucleotide complex may be a target-binding agent comprising single stranded DNA, single stranded RNA, double stranded DNA, double stranded RNA, or a combination thereof.
  • the term “nucleotide” may refer to an oligonucleotide or polynucleotide molecule or to a single nucleotide base.
  • a nucleotide of a peptide complex may comprise a DNA or RNA oligonucleotide.
  • the nucleotide may be a small interfering RNA (siRNA), a micro RNA (miRNA, or miR), an anti-miR, an antisense RNA, an antisense oligonucleotide (ASO), a complementary RNA, a complementary DNA, an interfering RNA, a small nuclear RNA (snRNA), a spliceosomal RNA, an inhibitory RNA, a nuclear RNA, an oligonucleotide complementary to a natural antisense transcript (NAT), an aptamer, a gapmer, a splice blocker ASO, or a U1 adapter.
  • siRNA small interfering RNA
  • miRNA micro RNA
  • ASO anti-miR
  • ASO antisense oligonucleotide
  • a complementary RNA a complementary DNA
  • an interfering RNA a small nuclear RNA (snRNA), a spliceosomal RNA
  • a nucleotide of the peptide oligonucleotide complex may comprise a sequence of any one of any one of SEQ ID NO: 488 - SEQ ID NO: 573 or a sequence complementary to a portion of any sequence provided in SEQ ID NO: 574 - SEQ ID NO: 611 or an open reading frame listed in TABLE 6.
  • the nucleotide may be an siRNA that inhibits translation of a target mRNA by promoting degradation of the target mRNA.
  • the nucleotide may be an miRNA that inhibits translation of a target mRNA by promoting cleavage or destabilization of the target mRNA.
  • the nucleotide may be an aptamer that binds to a target protein, thereby inhibiting protein-protein interactions with the target protein, inhibiting enzymatic activity of the target protein, or activating the target protein.
  • oligonucleotide complexes examples include an aptamer, a gapmer, an anti-miR, an siRNA, a splice blocker ASO, and a U1 adapter.
  • the peptide portion of the peptide oligonucleotide complex (e.g., a CDP of a CDP- oligonucleotide complex) can be used to guide the nucleotide sequence (e g., an oligonucleotide of a CDP- oligonucleotide complex) to a specific tissue, target, or cell, or to deliver the oligonucleotide to the cytosol or nucleus of the cell, or to cause endosomal escape of the oligonucleotide.
  • a CDP of a CDP- oligonucleotide complex can be used to guide the nucleotide sequence (e g., an oligonucleotide of a CDP- oligonucleotide complex) to a specific tissue, target, or cell, or to deliver the oligonucleotide to the cytosol or nucleus of the cell, or to cause endosomal escape of the
  • the peptide oligonucleotide complexes of the present disclosure may include nucleotide and nucleotide variants within the peptide oligonucleotide complex wherein the nucleotide portion is targeted to specific target molecule for modulation.
  • Modulation of a target molecule may comprise degradation, inhibiting translation, decreasing expression, increasing expression, enhancing a binding interaction (e.g., a protein-protein interaction), or inhibiting a binding interaction (e.g., a protein-protein interaction).
  • nucleotide sequences that may be used in the nucleotide portion of the peptide oligonucleotide complex, such as those targeting or complementary to nucleotides (e.g., DNA or RNA molecules) listed in SEQ ID NO: 574 - SEQ ID NO: 611, TABLE 12, or TABLE 6, or to nucleotides (e.g., DNA or RNA molecules) encoding the proteins listed in SEQ ID NO: 574 - SEQ ID NO: 611, TABLE 12, or TABLE 6, or otherwise described herein.
  • nucleotide sequences that may be used in the nucleotide portion of the peptide oligonucleotide complex include SEQ ID NO: 488 - SEQ ID NO: 573.
  • nucleic acid sequences, variants, and properties of the nucleic acids that are used in the nucleic acid portion of the peptide oligonucleotide complex may be referred to as nucleic acids of the present disclosure, nucleotides of the present disclosure, or like terminology.
  • nucleic acids or nucleotides are described in the context of the peptide oligonucleotide complexes disclosed, such as a nucleotide sequence comprising single stranded (ssDNA, ssRNA), double stranded (dsDNA, dsRNA), or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or U1 Adapter within the peptide oligonucleotide complex, with the accorded alterations, functions and uses described.
  • sDNA single stranded
  • the nucleotide sequence (e.g., a target binding agent capable of binding a target molecule) is single stranded (ssDNA, ssRNA), double stranded (dsDNA, dsRNA), or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or Ul Adapter.
  • ssDNA, ssRNA double stranded
  • dsDNA double stranded
  • dsRNA double stranded
  • a combination of single and double stranded for example with a mis
  • Peptides according to the present disclosure can be conjugated to, linked to, or fused to such nucleotide sequences to make a peptide oligonucleotide complex.
  • other active agents e.g., small molecule, protein, or peptide active agents
  • active agents as described herein can be conjugated to, linked to, complexed with, or fused to such nucleotide sequences, peptides or peptide oligonucleotide complex to form peptide oligonucleotide complex conjugates.
  • a nucleotide (e.g., a nucleotide of a peptide oligonucleotide complex) may be fully or partially reverse complementary to all or a portion of a target molecule (e.g., a target DNA or RNA sequence).
  • a target molecule expresses or encodes a protein (e.g., an mRNA encoding a protein associated with a disease).
  • a nucleotide may be fully or partially reverse complementary to a portion of an open reading frame encoding a gene or protein of interest.
  • a nucleotide may be reverse complementary to any portion of an RNA or open reading frame encoding a transcript or protein of interest.
  • a target molecule may comprise a fragment of any of the sequences provided in TABLE 6 along any portion of its length.
  • a target molecule may comprise a fragment of any of the sequences provided in SEQ ID NO: 574 - SEQ ID NO: 611.
  • a target molecule may comprise a sequence with one or more T residues replaced with U or one or more U residues replaced with T. TABLE 6 - Examples of Open Reading Frame Reference Sequences
  • a number of technologies can be used to generate therapeutically active nucleotide sequences for use in peptide oligonucleotide complexes that include the cell-penetrating peptides (e g., SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) disclosed herein.
  • cell-penetrating peptides e g., SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254
  • a nucleotide of a peptide oligonucleotide complex may bind to a target molecule (e.g., a target DNA, RNA, or protein) and modulate an activity of the target molecule.
  • a target molecule e.g., a target DNA, RNA, or protein
  • the nucleotide may function as a target-binding agent, also referred to as a targeted agent.
  • nucleotides that may function as target-binding agents include nucleotide antisense RNAs, complementary RNAs, inhibitory RNAs, interfering RNAs, nuclear RNAs, antisense oligonucleotides, microRNAs, oligonucleotides complementary to natural antisense transcripts, small interfering RNAs, small nuclear RNAs, aptamers, gapmers, anti-miRs, splice blocker antisense oligonucleotides, and Ul adapters.
  • Nucleotides may enter into cells through complexation with a cell-penetrating peptide to form a cell-penetrating peptide oligonucleotide complex.
  • the cell-penetrating peptide oligonucleotide complex may be delivered into a cytoplasm or nucleus of a cell.
  • the oligonucleotide, the peptide oligonucleotide complex, or any fragment thereof may enter the cytosol and may enter the nucleus.
  • oligonucleotides upon entry into the nucleus, can bind directly to mRNA structures and prevent the maturation (e g., capping or splicing) of the targeted sequence, modulate alternative splicing of a targeted sequence, and recruit RNaseHl to induce cleavage of a targeted sequence.
  • oligonucleotides in the cytoplasm can bind directly to the target mRNA and sterically block the ribosomal subunits from attaching and/or running along the mRNA transcript during translation hence resulting in lack of translation of the target sequence.
  • oligonucleotides can also be designed to directly bind to microRNA (miRNA) sequences or natural antisense transcripts (NATs) sequences, each of the foregoing thereby prohibiting miRNAs and NATs from inhibiting their own specific RNA targets, which ultimately leads to reduced degradation or increased translation of one or more sequences themselves targeted by the miRNA or NAT.
  • miRNA microRNA
  • NATs natural antisense transcripts
  • siRNA (which may be targeted to a specific sequence and regulate expression of the target sequence) may alternatively be used to bind and regulate a targeted sequence in the cytoplasm, engaging an RNA-induced silencing complex (RISC), which is a multiprotein complex that incorporates one strand of a small interfering RNA (siRNA) or micro RNA (miRNA), using the siRNA or miRNA as a template for recognizing complementary mRNA of the targeted sequence. When it finds a complementary strand, its RNase domain cleaves the targeted sequence.
  • RISC RNA-induced silencing complex
  • siRNA small interfering RNA
  • miRNA micro RNA
  • an aptamer e.g., a nucleotide that modulates a specific protein or other target
  • An aptamer (e.g., extracellular or intracellular) may function by directly binding and modulating activity of a protein target, for example by forming aptamer-protein interactions rather than through base pairing or hybridization interactions.
  • ASO antisense oligonucleotides
  • nt nucleotides
  • the first ASOs are sometimes called “Gapmers” because they have a central region with DNA-based-sugar nucleotides that are often (but not always) flanked by non-DNA-sugar nucleotides with greater resistance to nucleases.
  • the DNA region at least 4 nt in length but typically >6, causes a DNA/RNA hybrid that engages RNase H endonuclease to cleave the target RNA.
  • a DNA region of a gapmer may comprise from about 4 to about 30, from about 4 to about 25, from about 4 to about 20, from about 4 to about 15, from about 4 to about 10, from about 6 to about 30, from about 6 to about 25, from about 6 to about 20, from about 6 to about 15, or from about 6 to about 10 nucleotide residues.
  • a non-DNA region of a gapmer may comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide residues 3’ of the DNA region.
  • a non-DNA region of a gapmer may comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide residues 5’ of the DNA region. Examples of gapmers are provided in TABLE 7.
  • the second conventional ASO simply serves to bind to the target transcript, but not induce RNase degradation, so no DNA-based-sugars are used. Instead, binding is designed to disrupt processing into mature mRNA.
  • One such activity relies on binding to the mRNA at or near splice sites to drive particular splice isoforms in the target RNA, resulting in modulating target RNA by disrupting mRNA splicing and resulting in exon skipping. These are commonly called “splice blocking” or “splice blocker” ASOs amongst other known names.
  • DMD distrophin
  • Duchenne Muscular Dystrophy patients One example is eteplirsen, designed to alter splicing of DMD (dystrophin) gene in Duchenne Muscular Dystrophy patients, correcting a mutation that would otherwise create a truncated and nonfunctional dystrophin by splicing out the mutant exons and creating a different truncated (but functional) protein to appear.
  • DMD distrophin
  • siRNA molecules which specifically interact with the canonical RNAi pathway (the RISC complex) to drive cleavage or steric blocking of hybridized transcripts; cleavage-vs-blocking depends on whether the match is perfect (cleavage) or imperfect but still stable (blocking).
  • Length is typically a double-stranded RNA where the overlapping region is 19-22 and each strand has two extra nt at their 3’ ends.
  • Chemistry is largely RNA-based-sugars, with some DNA-based sugars at the 3’ overhangs.
  • Clinical examples include patisiran (targets TTR) and givosiran (targets ALAS ).
  • an overlapping region of a siRNA may comprise from about 10 to about 40, from about 10 to about 35, from about 10 to about 30, from about 10 to about 25, from about 10 to about 22, from about 10 to about 21, from about 10 to about 20, from about 15 to about 40, from about 15 to about 35, from about 15 to about 30, from about 15 to about 25, from about 15 to about 22, from about 15 to about 21, from about 15 to about 20, from about 17 to about 40, from about 17 to about 35, from about 17 to about 30, from about 17 to about 25, from about 17 to about 22, from about 17 to about 21, from about 17 to about 20, from about 18 to about 40, from about 18 to about 35, from about 18 to about 30, from about 18 to about 25, from about 18 to about 22, from about 18 to about 21, from about 18 to about 20, from about 19 to about 40, from about 19 to about 35, from about 19 to about 30, from about 19 to about 25, from about 19 to about 22, from about 19 to about 21, or from about 19 to about 20 nucleotide residues.
  • an overhang region may be selected from about 19 to about
  • Anti-miRs may function as steric blockers designed against miRNAs that would block a RISC complex loaded with a specific disease-associated miRNA without being subject to cleavage by the RISC complex RNase subunit.
  • One clinical example is miravirsen, a 15-base oligo with a mixture of DNA and LNA sugars that targets miR- 122 in hepatitis C patients.
  • An anti-miR nucleotide may be of sufficient length to anneal specifically and stably to the target miR, but the length of the sequence may vary.
  • an anti-miR may have a length of up to about 21 nt, corresponding to the maximum size loaded into RISC.
  • an anti-miR nucleotide may comprise from about 10 to about 25, from about 10 to about 23, from about 10 to about 21, from about 10 to about 20, from about 10 to about 19, from about 10 to about 18, from about 13 to about 25, from about 13 to about 23, from about 13 to about 21, from about 13 to about 20, from about 13 to about 19, from about 13 to about 18, from about 15 to about 25, from about 15 to about 23, from about 15 to about 21, from about 15 to about 20, from about 15 to about 19, from about 15 to about 18, from about 16 to about 25, from about 16 to about 23, from about 16 to about 21, from about 16 to about 20, from about 16 to about 19, or from about 16 to about 18 nucleotide residues.
  • U1 adapters which have two parts. One anneals to the Ul-snRNA of the Ul-snRNP complex, and the other binds to the target RNA, bringing the Ul-snRNP to the polyA site and inhibiting polyadenylation; absence of a polyA tail causes the mRNA to be degraded.
  • the Ul-binding region is at least 10 nt but up to 19 nt.
  • Target binding region can be from about 15 nt to about 25 nt. Chemistry in early studies made heavy use of LNA and 2’-O- Methyl sugars.
  • a U1 binding region may comprise from about 10 to about 25, from about 10 to about 23, from about 10 to about 21, from about 10 to about 20, from about 10 to about 19, from about 10 to about 18, from about 13 to about 25, from about 13 to about 23, from about 13 to about 21, from about 13 to about 20, from about 13 to about 19, from about 13 to about 18, from about 15 to about 25, from about 15 to about 23, from about 15 to about 21, from about 15 to about 20, from about 15 to about 19, from about 15 to about 18, from about 16 to about 25, from about 16 to about 23, from about 16 to about 21, from about 16 to about 20, from about 16 to about 19, or from about 16 to about 18 nucleotide residues.
  • a target binding region may comprise from about 10 to about 40, from about 10 to about 35, from about 10 to about 30, from about 10 to about 25, from about 10 to about 22, from about 10 to about 21, from about 10 to about 20, from about 15 to about 40, from about 15 to about 35, from about 15 to about 30, from about 15 to about 25, from about 15 to about 22, from about 15 to about 21, from about 15 to about 20, from about 17 to about 40, from about 17 to about 35, from about 17 to about 30, from about 17 to about 25, from about 17 to about 22, from about 17 to about 21, from about 17 to about 20, from about 18 to about 40, from about 18 to about 35, from about 18 to about 30, from about 18 to about 25, from about 18 to about 22, from about 18 to about 21, from about 18 to about 20, from about 19 to about 40, from about 19 to about 35, from about 19 to about 30, from about 19 to about 25, from about 19 to about 22, from about 19 to about 21, or from about 19 to about 20 nucleotide residues.
  • An exemplary nucleic acid sequence contains a U1 adapter for modulating BCL2 mRNA that is highly active against BCL2 can include: 5 ’GCCGUACAGUUCCACAAAGGGCCAGGUzL4GG4 U-3 ’ (SEQ ID NO: 504), wherein the underlined portion (GCCGUACAGUUCCACAAAGG (SEQ ID NO: 573)) corresponds to the BCL2 recognition sequence and the italicized portion (GCCAGGUAAGUAU (SEQ ID NO: 492)) corresponds to the U1 recognition sequence.
  • Another example of a nucleotide of the present disclosure is an aptamer.
  • Aptamers disrupt target activity using a mechanism that differs from other nucleotides described herein that form base pairing interactions with a target nucleotide.
  • Aptamers are nucleic acids that form secondary structures (e.g., where a single strand of nucleic acid base-pairs with itself upon folding, creating loops in various locations).
  • Aptamers may be screened for interaction with target proteins.
  • Aptamers may have varied nucleotide chemistry and may include a mixture of conventional RNA and/or DNA sugars and modified sugars (e.g., 2’-O-Methyl (2’-O-Me) RNA or 2’-Fluoro (2’-F) RNA sugars).
  • pegaptanib a VEGF-binding aptamer
  • pegaptanib has a mixture of 2’-O-Methyl (2’-O-Me) RNA and 2’-Fluoro (2’-F) RNA sugars and regular RNA and DNA sugars.
  • An aptamer sequence may be long enough to form a stable secondary structure (e g., through intramolecular base pairing), but the length may vary.
  • an aptamer sequence may comprise from about 20 nt to about 40 nt. For example, experiments that identified pegaptanib used oligos of 20-40 nt in length.
  • an aptamer may comprise from about 15 to about 60, from about 15 to about 50, from about 15 to about 40, from about 15 to about 35, from about 15 to about 30, from about 20 to about 60, from about 20 to about 50, from about 20 to about 40, from about 20 to about 35, from about 20 to about 30, from about 25 to about 60, from about 25 to about 50, from about 25 to about 40, from about 25 to about 35, or from about 25 to about 30 nucleotide residues.
  • Nucleotides may be designed for use in the peptide nucleotide complexes of the present disclosure.
  • nucleotides that modify processing, translation, or other RNA functions e.g., a gapmer, splice blocker, siRNA, anti-miR, or U1 adapter
  • RNA functions e.g., a gapmer, splice blocker, siRNA, anti-miR, or U1 adapter
  • any length of a nucleotide (nt) can be used within the foregoing ranges; (b) cross-species homology (e g., by targeting highly-conserved motifs) is often a desirable feature but is not necessary for activity or clinical development; (c) avoidance of common SNPs in humans unless that SNP is involved in disease pathology (e.g., an allele-specific oligo) is often a desirable feature but is not necessary for activity or clinical development; (d) gene specificity (they have minimal homology to other sequences; for example, a sequence may have 3 or more mismatches to every other sequence), (e) avoid predicted secondary structures in both the oligo and the target region (there are software tools available to screen in silico for such secondary structure formation); (f) higher G/C content may be preferable, as G/C-rich sequences (e.g.
  • CCAC, TCCC, GCCA may be helpful for increasing affinity of the nucleotide to its target, whereas A/T-rich sequences (e.g. TAA) or runs of 4+ G (GGGG) may exhibit low or result in structural (G-quadruplex) formation.
  • An oligonucleotide sequence can be 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity or match to the target sequence. In some situations, an oligonucleotide with 100% complementarity will result in the target RNA being degraded. In some situations, an oligonucleotide that is less than 100% complementarity may not lead to degradation of the target RNA but may prevent translation and production of the encoded protein.
  • gapmers have one or more of the following properties: (a) 12-30 nt in length. It is understood that any length of a nucleotide (nt) can be used within the foregoing range, (b) target sites are anywhere in the pre-mRNA, including UTRs, exons, or introns (c) central DNA region: minimum of 4 contiguous DNA nucleotides, often 10 or more are used. No artificial substitutions at 2’ site (e.g.
  • flanking region can be DNA- or RNA-based-sugars. 2’ substitutions such as 2’-0-ME or 2’-0-M0E are tolerated.
  • LNA Linked nucleic acids
  • morpholino phosphorodiamidate morpholino oligo
  • Backbone can be natural phosphodiester (PO) or nonnatural phosphorothioate (PS) linkages.
  • a clinical example is fomivirsen, a 21 nt gapmer wherein the whole oligo is PS-backbone DNA.
  • Another example is mipomersen, a 20 nt gapmer wherein the entire backbone is PS linkages, and the central region uses DNA sugar flanked by 2’-0-M0E modified RNA.
  • all C bases are 5-methyl-C, though this is not a strict requirement for engagement of RNase Hl.
  • thiophosphorodiamidate chemistries may be used.
  • steric blockers have one or more of the following properties; (a) as the molecule does not need to engage RNase H or any other enzyme, backbone and sugar chemistry can be more varied, (g) target sites for the nucleotide are complementary to one or more splice sites in the target RNA.
  • a clinical example is eteplirsen, a 30 nt splice blocking ASO wherein whole oligonucleotide uses morpholino (Phosphorodiamidate morpholino oligo) chemistry.
  • nusinersen an 18 nt ASO, whose backbone is entirely PS linked and uses 2’-0-M0E RNA chemistry.
  • siRNA have one or more of the following properties: (a) can be between 15 and 25 nt in length (between 13 to 23 nt overlap respectively), or up to 25 nt (23 nt overlap) per strand, but 21 nt (19 nt overlap) is common.
  • any length of a nucleotide (nt) overlap can be used within the foregoing ranges; (b) complements a sequence typically but not exclusively of 21 -nt length in the target mRNA that typically but not exclusively begins with “AA” (c) target sites are ideally found in the mature spliced mRNA as the RISC complex for RNA cleavage is primarily cytosolic; (d) preferably but not exclusively avoids sequences within 100 nt of the mRNA start site, as the transcript at start site is more likely to be occupied by RNA polymerase, (e) successful siRNA constructs typically have more G/C at 5’ end of sense strand, more A/T at 3’ end of sense strand, and are roughly 30-60% in G/C content.
  • anti-miR have one or more of the following properties: (a) a perfect match to target sequence (specifically the 5 ’ end of the guide strand of the miRNA); (b) length can vary and can even be greater than the length of the mature guide strand. Screening for effective anti-miR constructs may begin with the shortest sequence that achieves specificity (no off-target homology) and increase length from there to empirically determine ideal minimal length for strong miRNA inhibition; (c) 2’ sugar modifications (2’-O- Me, 2’-0-M0E, 2’-F) and LNA sugars are commonly used. Sugars can be a mixture.
  • a clinical example of an anti-miR is miravirsen, which uses a mixture of DNA and LNA sugars
  • PS linkages in backbone are common. PS linkages may reduce affinity, but sugar modifications may increase affinity.
  • aptamers have one or more of the following properties: (a) length of aptamers can vary widely, as there is no biological complex (e.g., RISC) they interact with to function. Although composed of nucleic acids, they are more protein-like in function (e.g., bind to a target protein, etc.). The minimum length may be determined empirically to maintain sufficient stability of intra-strand hybridization to fold into a secondary structure, the upper limit on size is limited only by pharmacology, as longer sequences have a higher risk of engaging inflammatory pathways.
  • RISC biological complex
  • the minimum length may be determined empirically to maintain sufficient stability of intra-strand hybridization to fold into a secondary structure, the upper limit on size is limited only by pharmacology, as longer sequences have a higher risk of engaging inflammatory pathways.
  • Aptamer screening typically begins with libraries of 20-40 nt in length (not including flanking regions required for library amplification during screening); (b) as they form interactions via secondary structure rather than base pairing interactions, there are few limitations for their base patterns, since secondary structures are not only desirable but essential to their function. Design may be empirical for each target; (c) selection is typically via Systematic Evolution of Ligands by Exponential Enrichment (SELEX): random or semirandom sequences between primer-binding flanking regions are exposed to a target of interest on a solid substrate. The pooled oligonucleotide mixture is rinsed from the substrate, leaving only sequences that interact with the target remaining, and then binding sequences are eluted and amplified by PCR.
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • sugar modifications commonly used include 2 ’-fluoro (2’-F), 2’-0-M0E, and 2’-O-Me, though other chemistries including (but not limited to) LNA and unlocked- nucleic-acids (UNA) are also possible;
  • backbones are typically PO or PS, but other linkages such as methylphosphonate are possible.
  • a clinical aptamer example, pegaptanib is entirely PO backbone, but others in development use other linkages,
  • aptamer termini are typically capped with unnatural nucleotide chemistries (e.g.
  • aptamers can be much more creative with chemical modifications of the bases themselves; these can include bases designed to induce covalent bonds with target proteins to permanently disable them; (h) such modifications are tested after selection of an active, high affinity aptamer, as unmodified bases are required for nucleic acid amplification during SELEX (i) if the target protein is extracellular, less considerations are necessary than for cell penetration capabilities.
  • PK pharmacokinetic
  • other general design considerations aimed at enhancing pharmacokinetic (PK) properties of the nucleotide, peptide, or peptide oligonucleotide complex include one or more of the following properties: (a) building in conjugation to moieties that reduce clearance or increase cellular uptake including cholesterol or other lipids, diacylglycerol, GalNAc, palmitoyl, PEG, an RGD motif, cell penetrating peptides or moieties (e g., a cellpenetrating peptide or cell penetrating peptide as described herein). Adding cholesterol to the peptide oligonucleotide complex can improve biodistribution to the target tissue, increase cellular uptake by endocytosis, and alter the serum pharmacokinetics.
  • the therapeutic activity and molecular method of the peptide oligonucleotide complex may depend on which target molecule (e.g., a DNA or RNA) that the nucleic acid complements, or in the case of an aptamer, which target molecule (e.g., protein or other macromolecule) it binds.
  • Target choice can fall into one or more non-mutually exclusive categories such as tissuetarget-based or disease-selective.
  • Known targets have known mRNA and genomic sequences that can be used to design a variety of complementary nucleic acids for use in the peptide nucleotide complexes described herein depending on the activity (e.g., gene regulation, protein degradation, reduction of cancer cell activators) desired.
  • tissue-targeting may comprise selecting targets acting in the tissues where a cell-penetrating peptide portion of the peptide oligonucleotide complex would preferentially access.
  • Targets for the peptide oligonucleotide complexes can include oncogenes, for example by designing the nucleic acid portion of the complex to target overexpressed genes or those for which the tumor is lacking a redundant ortholog (i.e., normal cells function by using X or Y, tumors do not express Y, so X is targeted).
  • disease-selective targeting can be used to treat conditions where the transcript is selectively found in the diseased tissue, and preferentially accumulate there, to improve safety and reduce off-target effects.
  • the target-binding agent e.g., a nucleotide of a peptide oligonucleotide complex
  • the target-binding agent may be capable of binding the targets described in SEQ ID NO: 574 - SEQ ID NO: 611, TABLE 12, or TABLE 6, or to nucleotides (e.g., DNA or RNA molecules) encoding the proteins listed in SEQ ID NO: 574 - SEQ ID NO: 611, TABLE 12, or TABLE 6, or otherwise described herein.
  • nucleotide sequences that may be used in the nucleotide portion of the peptide oligonucleotide complex include SEQ ID NO: 488 - SEQ ID NO: 573.
  • any oligonucleotide may be used that is complementary to a portion of the target DNA or RNA molecule.
  • target binding agent may comprise a nucleotide sequence is single stranded (ssDNA, ssRNA) or double stranded (dsDNA, dsRNA) or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or U1 Adapter.
  • ssDNA, ssRNA double stranded
  • dsDNA double stranded
  • Such oligos may be about 5 to 30 nt in length, 10 to 25 nt in length, 15 to 25 nt in length, 19 to 23 nt in length, or at least 10 nt in length, at least 15 nt in length, at least 20 nt in length, at least 25 nt in length, or at least 30 nt in length, at least 50 nt in length, at least 100 nucleotides in length across any portion of the target RNA.
  • sequences to which such oligonucleotides may bind include SEQ ID NO: 574 - SEQ ID NO: 611, or any genomic or ORF sequence referenced in TABLE 6.
  • RNA target described herein including for any of the targets or molecules encoding the targets described in TABLE 12, and SEQ ID NO: 574 - SEQ ID NO: 611, or any genomic or ORF sequence referenced in TABLE 6 such target binding agent of any nt length is described.
  • a nucleotide binds to the target molecule with a melting temperature of not less than 37 °C and not more than 99 °C. In some embodiments, a nucleotide binds to the target molecule with a melting temperature of not less than 40 °C and not more than 85 °C, not less than 40 °C and not more than 65 °C, not less than 40 °C and not more than 55 °C, not less than 50 °C and not more than 85 °C, not less than 60 °C and not more than 85 °C, or not less than 55 °C and not more than 65 °C.
  • a nucleotide binds the target molecule with an affinity of not more than 500 nM, not more than 100 nM, not more than 50 nM, not more than 10 nM, not more than 1 nM, not more than 500 pM, not more than 400 pM, not more than 300 pM, not more than 200 pM, or not more than 100 pM.
  • a nucleotide binds the target molecule with an affinity of not more than 500 nM and not less than 100 pM, not more than 100 nM and not less than 200 pM, not more than 50 nM and not less than 300 pM, not more than 10 nM and not less than 400 pM, or not more than 1 nM and not less than 500 pM.
  • a nucleotide comprises at least 90%, 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% sequence identity to any one of SEQ ID NO: 488 - SEQ ID NO: 573.
  • a nucleotide comprises a sequence of any one of SEQ ID NO: 488 - SEQ ID NO: 573, any one of SEQ ID NO: 488 - SEQ ID NO: 573 wherein U is replaced with T, or any one of SEQ ID NO: 488 - SEQ ID NO: 573 wherein T is replaced with U.
  • a nucleotide comprises no more than 1, 2, 3, 4, or 5 base changes relative to a sequence of any one of SEQ ID NO: 488 - SEQ ID NO: 573.
  • a nucleotide is at least 60%, at least 70%, at least 80%, at least 90%, 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% reverse complementary to the target molecule. In some embodiments, a nucleotide is 100% reverse complementary to the target molecule. In some embodiments, a nucleotide comprises no more than 1, 2, 3, 4, or 5 base pair mismatches upon binding to the target molecule. In some embodiments, a nucleotide comprises at least 1, 2, 3, 4, or 5 base pair mismatches upon binding to the target molecule.
  • a nucleotide may modulate an activity of a target molecule.
  • modulating the activity of the target molecule comprises reducing expression of the target molecule, increasing the expression of the target molecule, reducing translation of the target molecule, degrading the target molecule, reducing a level of the target molecule, modifying the processing of the target molecule, modifying the splicing of the target molecule, inhibiting processing of the target molecule, reducing a level of a protein encoded by the target molecule, or blocking an interaction with the target molecule.
  • the expression of the target molecule is reduced by at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99.9%.
  • the translation of the target molecule is reduced by at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99 9%.
  • the expression of the target molecule is reduced by a factor of at least 2, 4, 8, 10, 15, 16, 20, 32, 50, 64, 100, 128, 200, 256, 500, 512, or 1000.
  • the translation of the target molecule is reduced by a factor of at least 2, 4, 8, 10, 15, 16, 20, 32, 50, 64, 100, 128, 200, 256, 500, 512, or 1000. In some embodiments, at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the target molecule is degraded. In some embodiments, the level of the protein encoded by the target molecule is reduced by at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99.9%.
  • modifying the splicing of the target molecule increases a level of a protein encoded by the target molecule by at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99.9%.
  • a peptide oligonucleotide complex of the present disclosure may comprise a nucleotide complexed with a peptide (e.g., a cell-penetrating peptide).
  • the nucleotide may comprise single stranded DNA, single stranded RNA, double stranded DNA, double stranded RNA, or combinations thereof.
  • a nucleotide of a peptide oligonucleotide complex may be non-naturally occurring, also referred to as an “engineered nucleotide”.
  • a nucleotide may comprise a naturally occurring sequence.
  • a nucleotide may be exogenously expressed, enzymatically synthesized in vitro, or chemically synthesized.
  • a nucleotide may be expressed in a bacterial, yeast, or mammalian cell line and purified for use in a peptide oligonucleotide complex of the present disclosure.
  • a nucleotide may be enzymatically synthesized in vitro using an RNA or DNA polymerase.
  • a nucleotide may be chemically synthesized on a solid support using protected nucleotides.
  • phosphoramidite synthesis One example of a chemical synthesis method that may be used to prepare a nucleotide for use in a peptide oligonucleotide complex of the present disclosure is phosphoramidite synthesis. Briefly, single nucleotide residues may be sequentially added from 3’ to 5’ to the growing nucleotide chain by repeating the steps of de-blocking (detrityl ati on), coupling, capping, and oxidation. Phosphoramidite synthesis may be performed on a solid support such as controlled pore glass (CPG) or macroporous polystyrene (MPPS). Similarly, thiophosphorodiamidate may be used.
  • CPG controlled pore glass
  • MPPS macroporous polystyrene
  • thiophosphorodiamidate may be used.
  • a nucleotide of a peptide oligonucleotide complex may bind to a target molecule (e.g., a target DNA, a target RNA, or a target protein).
  • a target molecule e.g., a target DNA, a target RNA, or a target protein
  • binding of the oligonucleotide to the target molecule may alter an activity of the target molecule.
  • binding of an oligonucleotide e.g., an siRNA, an miRNA, a gapmer, or a U1 adaptor
  • binding of a nucleotide to a target DNA may increase or decrease expression of a gene encoded by the target DNA.
  • binding of a nucleotide to an RNA may increase or decrease expression of a gene encoded by the target DNA.
  • binding of an oligonucleotide (e.g., an aptamer) to a target protein may increase or decrease activity (e.g., an enzymatic activity or a binding activity) of the target protein.
  • the target molecule may be associated with a disease or condition and increasing or decreasing the activity of the target molecule may treat the disease or condition.
  • a sequence of the oligonucleotide of a peptide oligonucleotide complex may be selected for its ability to bind to or modulate the activity of a target molecule.
  • an oligonucleotide may be reverse complementary to a target DNA or RNA molecule.
  • an siRNA oligonucleotide may be reverse complementary to a target RNA molecule.
  • am oligonucleotide may be partially reverse complementary (e.g., comprising one or more mis-matched base pairs) to a target DNA or RNA molecule.
  • an siRNA oligonucleotide may comprise a base mismatch relative to a target RNA molecule.
  • a sequence of the oligonucleotide may be selected for its annealing temperature relative to a target DNA or RNA molecule.
  • a preferred annealing temperature may be achieved by selecting the length of the nucleotide, the degree of complementarity of the nucleotide to the target molecule, the chemistry of the nucleotides, or any combination thereof.
  • Nucleotide sequence parameters e.g., complementarity, annealing temperature, melting temperature, base mismatches, and binding affinity
  • an oligonucleotide may adopt a secondary structure that binds to a target DNA, RNA, or protein molecule.
  • an aptamer may adopt a secondary structure to bind to a target protein.
  • the aptamer sequence may be selected to adopt a secondary structure that binds to a target protein.
  • Nucleotide secondary structure may be predicted using any available software, such as RNAfold and the like.
  • a nucleotide sequence may be determined experimentally by selecting for the ability to bind to a target molecule. For example, a nucleotide library may be contacted to a target molecule, and sequences that bind to the target molecule may be identified.
  • a nucleotide comprises a G/C content of not less than 20% and not more than 80%. In some embodiments, a nucleotide comprises a G/C content of not less than 30% and not more than 65%. In some embodiments, the nucleotide comprises a G/C content of not less than 20%, not less than 25%, not less than 30%, not less than 35%, not less than 40%, not less than 45%, or not less than 50%. In some embodiments, the nucleotide comprises a G/C content of not more than 80%, not more than 75%, not more than 70%, not more than 65%, or not more than 50%.
  • a nucleotide comprises an A/T content or A/U content of not less than 20% and not more than 80%. In some embodiments, a nucleotide comprises an A/T content or A/U content of not less than 30% and not more than 65%. In some embodiments, the nucleotide comprises a A/U (or A/T, or combination of A/U and A/T) content of not less than 20%, not less than 25%, not less than 30%, not less than 35%, not less than 40%, not less than 45%, or not less than 50%.
  • the nucleotide comprises a A/U content (or A/T, or combination of A/U and A/T) of not more than 80%, not more than 75%, not more than 70%, not more than 65%, or not more than 50%.
  • a nucleotide has a length of no more than 1000 nt, 600 nt, 200 nt, 100 nt, 60 nt, 56 nt, 52 nt, 50 nt, 48 nt, 46 nt, 44 nt, 22 nt, 40 nt, 38 nt, 36, nt, 34 nt, 32 nt, 30 nt, or 24 nt.
  • a nucleotide has a length of from 24 to 100 nt, from 24 to 60 nt, from 24 to 50 nt, or from 36 to 50 nt. In some embodiments, a nucleotide has a length of about 42 nt.
  • a nucleotide has a length of no more than 500 nt, 300 nt, 100 nt, 50 nt, 30 nt, 28 nt, 26 nt, 25 nt, 24 nt, 23 nt, 22 nt, 21 nt, 20 nt, 19 nt, 18, nt, 17 nt, 16 nt, 15 nt, or 12 nt.
  • a nucleotide has a length of from 12 to 50 nt, from 12 to 30 nt, from 12 to 25 nt, from 18 to 25 nt, from 18 to 25 nt, from 19 to 23 nt, or from 20 to 22 nt. In some embodiments, a nucleotide has a length of about 21 nt.
  • a peptide oligonucleotide complex of the present disclosure may be further conjugated, linked, or fused to an active agent in addition to the nucleotide active agent (e.g., a target-binding agent capable of binding a target molecule).
  • an active agent e.g., a target-binding agent capable of binding a target molecule.
  • Such additional active agent may be complexed, fused, linked or conjugated to one or more of the peptide, nucleotide, or linker within the peptide oligonucleotide complex.
  • the active agent may be directly or indirectly linked to the peptide of the peptide oligonucleotide complex or the nucleotide of the peptide oligonucleotide complex.
  • a peptide nucleic acid complex further comprising an additional active agent may be referred to as a peptide-active agent conjugate or a peptide construct.
  • the peptide oligonucleotide complexes of the present disclosure can also be used to deliver another active agent.
  • Peptides according to the present disclosure can be conjugated to, linked to, or fused to an agent for use in the treatment of tumors and cancers or other diseases.
  • the peptides described herein are fused or conjugated to another molecule, such as an active agent that provides an additional functional capability.
  • a peptide or nucleotide can be fused with an active agent through expression of a vector containing the sequence of the peptide with the sequence of the active agent.
  • the sequence of the peptide and the sequence of the active agent can be expressed from the same Open Reading Frame (ORF).
  • ORF Open Reading Frame
  • sequence of the peptide and the sequence of the active agent can comprise a contiguous sequence.
  • the peptide and the active agent can each retain similar functional capabilities in the peptide construct compared with their functional capabilities when expressed separately.
  • examples of active agents can include other peptides.
  • the peptides or nucleotides described herein are attached to another molecule, such as an active agent that provides a functional capability.
  • the active agent may be any active agent (e.g., therapeutic agent, detectable agent, or binding moiety) described herein.
  • the peptide or nucleotide is covalently or non-covalently linked to an active agent, e.g., directly or via a linker. Exemplary linkers suitable for use with the embodiments herein are discussed in further detail below.
  • a peptide e.g., a cell-penetrating peptide of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254
  • peptide complex e.g., comprising a cell-penetrating peptide and a cargo molecule
  • the cell-penetrating peptide may deliver the agent into a cellular space or compartment or across a cellular layer (e g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen).
  • a cellular layer e g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen.
  • the cell-penetrating peptides or peptide complexes of the present disclosure may be fused to a detectable agent that may otherwise be excluded from a cell or a cellular compartment to facilitate absorption or permeation of the detectable agent.
  • a cell -penetrating peptide of the present disclosure may facilitate absorption or permeation of a detectable agent with multiple hydrogen bond donors or hydrogen bond acceptors, a large molecular weight, or a high partition coefficient, which may lead to poor absorption or permeation of the detectable agent in the absence of the cell -penetrating peptide.
  • a cell -penetrating peptide may be conjugated to a detectable agent comprising at least 5, at least 6, at least 7, at least 8, 9, at least 10, at least 15, or at least 20 hydrogen bond donors. In some embodiments, the cell-penetrating peptide may be conjugated to a detectable agent comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20 hydrogen bond acceptors. In some embodiments, the cell-penetrating peptide may be conjugated to a detectable agent comprising a molecular weight of at least 500 Da, at least 600 Da, at least 700 Da, at least 800 Da, at least 900 Da, or at least 1000 Da.
  • the cell-penetrating peptide may be conjugated to a detectable agent comprising a log of a partition coefficient (logP) of at least 5.0, at least 5.2, at least 5.4, at least 5.5, at least 5.6, at least 5.8, or at least 6.0.
  • the partition coefficient may be a measure of a ratio of the solubility of the agent in a hydrophobic solvent (e.g., octanol) relative to water.
  • a peptide is conjugated to, linked to, or fused with detectable agents, such as a fluorophore, a near-infrared dye, a contrast agent, a nanoparticle, a metalcontaining nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a metal, a radioisotope, a dye, radionuclide chelator, or another suitable material that can be used in imaging.
  • detectable agents such as a fluorophore, a near-infrared dye, a contrast agent, a nanoparticle, a metalcontaining nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a metal, a radioisotope, a dye, radionuclide chelator, or another suitable material that can be used in imaging.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 detectable agents can be linked to a peptide.
  • the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium.
  • the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium.
  • the radioisotope is actinium-225 or lead-212.
  • the near-infrared dyes are not easily quenched by biological tissues and fluids.
  • the fluorophore is a fluorescent agent emitting electromagnetic radiation at a wavelength between 650 nm and 4000 nm, such emissions being used to detect such agent.
  • fluorescent dyes that could be used as a conjugating molecule in the present disclosure include DyLight-680, DyLight-750, VivoTag-750, DyLight-800, IRDye-800, VivoTag-680, Cy5.5, or indocyanine green (ICG).
  • near infrared dyes often include cyanine dyes (e.g., Cy7, Cy5.5, and Cy5).
  • Suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4', 5'-dichloro-2',7' - dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethyl- rhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine (TMR), etc.),
  • radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters.
  • the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium.
  • the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium.
  • the radioisotope is actinium-225 or lead-212.
  • fluorescent biotin conjugates that can act both as a detectable label and an affinity handle can be used.
  • fluorescent biotin conjugates include Atto 425-Biotin, Atto 488-Biotin, Atto 520-Biotin, Atto- 550 Biotin, Atto 565-Biotin, Atto 590-Biotin, Atto 610-Biotin, Atto 620-Biotin, Atto 655-Biotin, Atto 680-Biotin, Atto 700-Biotin, Atto 725-Biotin, Atto 740-Biotin, fluorescein biotin, biotin-4- fluorescein, biotin-(5-fluorescein) conjugate, and biotin-B-phycoerythrin, ALEXA FLUOR 488 biocytin, ALEXA FLUOR 546, ALEXA FLUOR 549, lucifer yellow cadaverine bio
  • the conjugates could include chemiluminescent compounds, colloidal metals, luminescent compounds, enzymes, radioisotopes, and paramagnetic labels.
  • the peptide-active agent fusions described herein can be attached to another molecule.
  • the peptide sequence also can be attached to another active agent (e.g., small molecule, peptide, polypeptide, polynucleotide, antibody, aptamer, cytokine, growth factor, neurotransmitter, an active fragment or modification of any of the preceding, fluorophore, radioisotope, radionuclide chelator, acyl adduct, chemical linker, or sugar, etc.).
  • the peptide can be fused with, or covalently or non-covalently linked to an active agent.
  • the conjugates can include chemiluminescent compounds, colloidal metals, luminescent compounds, enzymes, radioisotopes, and paramagnetic labels.
  • the peptide described herein can also be attached to another molecule.
  • the peptide sequence also can be attached to another active agent (e g., small molecule, peptide, polypeptide, polynucleotide, antibody, aptamer, cytokine, growth factor, neurotransmitter, an active fragment or modification of any of the preceding agents, fluorophore, radioisotope, radionuclide chelator, acyl adduct, chemical linker, or sugar).
  • another active agent e g., small molecule, peptide, polypeptide, polynucleotide, antibody, aptamer, cytokine, growth factor, neurotransmitter, an active fragment or modification of any of the preceding agents, fluorophore, radioisotope, radionuclide chelator, acyl adduct, chemical linker, or sugar.
  • the peptide can be conjugated to, linked to, or fused with, or covalently or non-covalently linked to an active agent.
  • Peptides can be conjugated to, linked to, or fused to a radiosensitizer or photosensitizer.
  • radiosensitizers include but are not limited to: ABT-263, ABT-199, WEHL539, paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, etanidazole, misonidazole, tirapazamine, and nucleic acid base derivatives (e.g., halogenated purines or pyrimidines, such as 5-fluorodeoxyuridine).
  • photosensitizers can include but are not limited to: fluorescent molecules or beads that generate heat when illuminated, nanoparticles, porphyrins and porphyrin derivatives (e.g., chlorins, bacteriochlorins, isobacteriochlorins, phthalocyanines, and naphthalocyanines), metalloporphyrins, metallophthalocyanines, angelicins, chalcogenapyrrillium dyes, chlorophylls, coumarins, flavins and related compounds such as alloxazine and riboflavin, fullerenes, pheophorbides, pyropheophorbides, cyanines (e.g., merocyanine 540), pheophytins, sapphyrins, texaphyrins, purpurins, porphycenes, phenothiaziniums, methylene blue derivatives, naphthalimides, nile blue derivatives, a
  • this approach can allow for highly specific targeting of diseased cells (e.g., cancer cells) using both a therapeutic agent (e.g., drug) and electromagnetic energy (e.g., radiation or light) concurrently.
  • a therapeutic agent e.g., drug
  • electromagnetic energy e.g., radiation or light
  • the peptide is conjugated to, linked to, fused with, or covalently or non-covalently linked to the agent, e g., directly or via a linker.
  • Exemplary linkers suitable for use with the embodiments herein are discussed in further detail below.
  • a cell-penetrating peptide conjugated to, linked to, or fused with a detectable agent may be used in a method of imaging a cell or tissue.
  • a cell-penetrating peptide complex comprising a cell-penetrating peptide and a detectable agent may be contacted to a cell or tissue, and the cell or tissue may be imaged.
  • the cell or tissue may be imaged to detect the presence, absence, location, intensity, distribution of the detectable agent, or combinations thereof.
  • imaging may comprise diagnostic imaging, for example to detect the presence or absence of a disease state.
  • a peptide e.g., a cell-penetrating peptide, a cargo peptide, or a cell-penetrating peptide complex
  • the peptide can be chemically modified one or more of a variety of ways.
  • the peptide can be mutated to add function, delete function, or modify the in vivo behavior.
  • cell -penetrating peptides of the presenting disclosure may be chemically modified with a molecule that would increase the serum half-life of the peptide when administered to a subject.
  • one or more loops between the disulfide linkages of a knotted cellpenetrating peptide can be modified or replaced to include active elements from other peptides (such as described in Moore and Cochran, Methods in Enzymology, 503, p. 223-251, 2012).
  • loops from cell-penetrating peptides may be grafted into peptide active agents.
  • Amino acids can also be mutated, such as to increase half-life, decrease immunogenicity, modify, add or delete binding behavior in vivo, add new targeting function, modify surface charge and hydrophobicity, or allow conjugation sites.
  • N-methylation is one example of methylation that can occur in a peptide of the disclosure.
  • the peptide is modified by methylation on free amines.
  • full methylation may be accomplished through the use of reductive methylation with formaldehyde and sodium cyanoborohydride.
  • a chemical modification can, for instance, extend the half-life of a peptide or change the biodistribution or pharmacokinetic profile.
  • a chemical modification can comprise a polymer, a poly ether, polyethylene glycol, a biopolymer, a polyamino acid, a fatty acid, a dendrimer, an Fc region, a simple saturated carbon chain such as palmitate or myristolate, an albumin binder, or albumin.
  • a polyamino acid can include, for example, a poly amino acid sequence with repeated single amino acids (e.g., poly glycine), and a poly amino acid sequence with mixed poly amino acid sequences (e.g, gly-ala-gly-ala; SEQ ID NO: 614) that may or may not follow a pattern, or any combination of the foregoing.
  • a poly amino acid sequence with repeated single amino acids e.g., poly glycine
  • a poly amino acid sequence with mixed poly amino acid sequences e.gly-ala-gly-ala; SEQ ID NO: 614
  • the peptides of the present disclosure can be modified such that the modification increases the stability and/or the half-life of the peptides.
  • the attachment of a hydrophobic moiety, such as to the N-terminus, the C-terminus, or an internal amino acid, can be used to extend half-life of a peptide of the present disclosure.
  • the peptides can also be modified to increase or decrease the gut permeability or cellular permeability of the peptide.
  • the peptide of the present disclosure can include post-translational modifications (e g., methylation and/or amidation and/or glycosylation), which can affect, e.g., serum half-life.
  • simple carbon chains can be conjugated to, linked to, the fusion proteins or peptides.
  • the simple carbon chains can render the fusion proteins or peptides easily separable from the unconjugated material.
  • methods that can be used to separate the fusion proteins or peptides from the unconjugated material include, but are not limited to, solvent extraction and reverse phase chromatography.
  • Lipophilic moieties can extend half-life through reversible binding to serum albumin.
  • Conjugated moieties can, e.g., be lipophilic moieties that extend half-life of the peptides through reversible binding to serum albumin.
  • the lipophilic moiety can be cholesterol or a cholesterol derivative including cholestenes, cholestanes, cholestadienes and oxysterols.
  • the peptides can be conjugated to, linked to, myristic acid (tetradecanoic acid) or a derivative thereof.
  • the peptides of the present disclosure can be coupled (e g., conjugated, linked, or fused) to a half-life modifying agent.
  • half-life modifying agents can include, but is not limited to: a polymer, a polyethylene glycol (PEG), a hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer of proline, alanine and serine, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, palmitic acid, an albumin, or a molecule that binds to albumin.
  • PEG polyethylene glycol
  • a hydroxyethyl starch polyvinyl alcohol
  • a water soluble polymer a zwitterionic water soluble polymer
  • a water soluble poly(amino acid) a water soluble poly(amino acid)
  • proline a water soluble polymer of proline
  • alanine and serine a water
  • conjugation of the peptide to a near infrared dye, such as Cy5.5, or to an albumin binder such as Albu-tag can extend serum half-life of any peptide as described herein.
  • immunogenicity is reduced by using minimal non-human protein sequences to extend serum half-life of the peptide.
  • the peptides of the present disclosure can be modified to reduce their immunogenicity.
  • Immunogenicity can limit the utility of a therapeutic peptidic molecule, as described in FDA Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products (2014), which is incorporated by reference in its entirety.
  • a molecule that is immunogenic and cause the formation of, or increased levels of anti-drug antibodies (ADA) can lead to complications such as toxicity, immune incompatibility, or hypersensitivity particularly in the context of human therapeutics. Consequently, it is desirable to limit immunogenicity and ADA in therapeutic applications of peptides.
  • ADA can be neutralizing or binding but not neutralizing.
  • the formation of ADA can reduce the efficacy of a therapeutic biologic molecule, including by causing earlier clearance of or reduced exposure to the therapeutic.
  • ADA can reduce the safety of a therapeutic biologic molecule, such as by causing cytokine release syndrome, infusion reactions, or clearance of endogenous proteins.
  • a therapeutic biologic molecule such as by causing cytokine release syndrome, infusion reactions, or clearance of endogenous proteins.
  • There are approaches to predicting and reducing the immunogenicity of therapeutics as described in Preclinical models immunogenicity prediction of therapeutic proteins (Brinks, 2013), which is incorporated by reference in its entirety. Some peptide sequences are more likely to cause immunogenicity. Sequences may be designed for reduced immunogenicity by screening sequences and variants using experimental and in silico methods and selecting sequences that are less immunogenic. When two or more peptides or proteins are fused together, the point of linkage may create new sequences that could cause immunogenicity.
  • Peptides that are resistant to enzymatic degradation can also have reduced immunogenicity, because in some cases peptides must undergo proteolysis into shorter peptide fragments, such as 9-11 amino acids long, in order to be presented on the MHCII complex to immune cells resulting in immunogenic effects.
  • the peptides of the present disclosure include variants with deduced immunogenicity.
  • the first two N-terminal amino acids (GS) of SEQ ID NO: 59 - SEQ ID NO: 84, SEQ ID NO: 93 - SEQ ID NO: 101, or SEQ ID NO: 210 - SEQ ID NO: 224 serve as a spacer or linker in order to facilitate conjugation or fusion to another molecule, as well as to facilitate cleavage of the peptide from such conjugated to, linked to, or fused molecules.
  • the fusion proteins or peptides of the present disclosure can be conjugated to, linked to, or fused to other moieties that, e.g., can modify or effect changes to the properties of the peptides.
  • a cell -penetrating peptide of the present disclosure may be modified with a tag that further promotes endosomal escape (e.g., an endosomal escape motif).
  • a tag that further promotes endosomal escape e.g., an endosomal escape motif
  • S19 PFVIGAGVLGALGTGIGGI; SEQ ID NO: 359
  • CM18 KWKLFKKIGAVLKVLTTG; SEQ ID NO: 360
  • PAS FTLIPKG; SEQ ID NO: 361), Aureinl .2 (GLFDIIKKIAESF; SEQ ID NO: 362), or B18 (LGLLLRHLRHHSNLLANI; SEQ ID NO: 363)
  • LGLLLRHLRHHSNLLANI LGLLLRHLRHHSNLLANI
  • the cell-penetrating peptides of the present disclosure e g., SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 and complexes thereof penetrate cells with and intracellular concentration to exert a prophylactic or therapeutic effect.
  • 0.01% or more, 0.05% or more, 0.1% or more, 0.2% or more, 0.5% or more, 1% or more, 3% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, up to 100% of a molecule or cellpenetrating peptide can enter the cytosol from the extracellular space or circulation.
  • a cell-penetrating peptide may penetrate a cellular layer such that the cytosolic, nuclear, intracellular, or paracellular concentration of the peptide is at least about 0.1%, at least about 0.2% at least about 0.5%, at least about 1%, at least about 3%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about at least about 100% of the extracellular peptide concentration.
  • the nucleic acid portion of a peptide oligonucleotide complex (e g., an oligonucleotide of a cell-penetrating peptide oligonucleotide complex) contains one or more bases within the nucleic acid molecule that are modified.
  • nucleic acid portion a single stranded (ssDNA, ssRNA) or double stranded (dsDNA, dsRNA) or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or U1 Adapter.
  • ssDNA, ssRNA double stranded
  • dsDNA, dsRNA double stranded
  • a combination of single and double stranded for example with a mismatched sequence, hairpin or other structure
  • an antisense RNA complementary RNA, inhibitory RNA,
  • One or more bases in a given nucleotide sequence may be modified to increase in vivo stability, to increase resistance to enzymes such as nucleases, increase protein binding including to serum proteins, increase in vivo half-life, to modify the tissue biodistribution, or to modify how the immune system responds.
  • the phosphonate, the ribose, or the base may be modified.
  • the modification comprises a phosphorothioate modification, a phosphodiester modification, a thio- phosphoramidate modification, a methyl phosphonate modification, a phosphorodi thioate modification, a methoxypropylphosphonate modification, a 5’-(E)-vinylphosphonate modification, a 5 ’methyl phosphonate modification, an (S)-5’-C-methyl with phosphate modification, a 5 ’-phosphorothioate modification, a peptide nucleic acid (PNA), a 2’-0 methyl modification, a 2’-O-methoxyethyl (2’-O-Me) modification, a 2’-fluoro (2’-F) modification, a 2’-deoxy-2’-fluoro modification, a 2’ arabino-fluor modification, a 2’-O-benyzl modification, a 2’-O-methyl-4-pyridine modification, a locked nucle
  • the oligonucleotide may be comprised entirely of a combination of 2’-O-Me and 2’-F modifications. Diastereomers or one or both stereoisomers may be used. Any of the stabilization chemistries or patterns, including STC, ESC, advanced, ESC, ADI-3, AD5, disclosed in Hu Signal Transduction and Targeted Therapy 2020,5: 101 can be used. Pyrimidines can be 2’-fluoro-modified, which can increase stability to nucleases but can also increase immune system activation.
  • the RNA backbone can be phosphorothioate- substituted (where the non-bridging oxygen is replaced with sulfur), which can increase resistance to nuclease digestion as well as altering the biodistribution and tissue retention and increasing the pharmacokinetics such as by increasing protein binding, but can also induce more immune stimulation.
  • Methyl phosphonate modification of an RNA can also be used.
  • 2’-Omethyl and 2’-F RNA bases can be used, which can protect against base hydrolysis and nucleases and increase the melting temperature of duplexes.
  • Bridged, Locked, and other similar forms of Bridged Nucleic Acids where any chemical bridge such as an N-0 linkage between the 2’ oxygen and 4’ carbons in ribose can be incorporated to increase resistance to exo- and endonucleases and enhance biostability.
  • BNA Bridged Nucleic Acids
  • LNA Low-Nodecyl-N-(2-aminoe)
  • cEt Bridged Nucleic Acids
  • phosophorothioate nucleic acid linkages may be used between the 2-4 terminal nucleic acids of one or both sequences.
  • 2’F modified nucleic acids may be used at least at 2-4 positions, at least 5%, at least 10% at least 25% of internal positions, at least 50%, at least 75%, or up to 100% of internal positions, all internal positions or all positions.
  • one or more of 2’F base, an LNA base, a BNA base, an ENA base, a 2’O-MOE base, a morpholino base, a 2’0Me base, a 5 ’-Me base, a (S)-cEt base or combinations thereof may be used at least at 2-4 positions, at least 5%, at least 10% at least 25% of internal positions, at least 50%, at least 75%, or up to 100% of internal positions, all internal positions or all positions.
  • Modified bases can be used to increase in the in vivo half-life of the oligonucleotide. They can allow the oligonucleotide to remaining intact in the serum, endosome, cytosol, or nucleus, including for days, weeks, or months. This can allow ongoing activity, including if the oligonucleotide is slowly released from the endosome over days, weeks, or months within a given cell (such as described in Brown et al., Nucleic Acids Research, 2020, pl 1827-11844).
  • a nucleotide comprises at least one phosphorothioate linkage.
  • a peptide oligonucleotide complex comprises from 1 to 12 phosphorothioate linkages. In some embodiments, a nucleotide comprises at least one thiophosphoroamidate linkage. In some embodiments, a nucleotide comprises from 1 to 12 thiophosphoroamidate linkages. In some embodiments, a nucleotide comprises at least one modified base. In some embodiments, at least modified base comprises a 2’F base, an LNA base, a BNA base, an ENA base, a 2’O-MOE base, a 5’-Me base, a (S)-cEt base, a 2’OMe base, a morpholino base, or combinations thereof.
  • the cell-penetrating peptides of the presented disclosure can be linked to a cargo peptide (e.g., an active agent peptide, a target-binding peptide, a therapeutic peptide, a detectable peptide, or a cystine-dense peptide) in numerous ways.
  • a cargo peptide e.g., an active agent peptide, a target-binding peptide, a therapeutic peptide, a detectable peptide, or a cystine-dense peptide
  • a cell -penetrating peptide can be conjugated to a cargo peptide via a peptide linker to form a cell-penetrating peptide fusion.
  • a peptide linker does not disturb the independent folding of peptide domains (e.g., a cystine-dense peptide).
  • a peptide linker does not negatively impact manufacturability (synthetic or recombinant) of the peptide complex (e g., the cell -penetrating peptide complex).
  • a peptide linker does not impair post-synthesis chemical alteration (e.g. conjugation of a fluorophore or albumin-binding chemical group) of the peptide fusion.
  • a peptide linker can connect the C-terminus of a first peptide (e.g., a cell-penetrating peptide or a cargo peptide) to the N-terminus of a second peptide (e.g., a cell-penetrating peptide or a cargo peptide).
  • a peptide linker can connect the C-terminus of the second peptide (e.g., a cell-penetrating peptide or a cargo peptide) to the N-terminus of a third peptide (e g., a cell -penetrating peptide or a cargo peptide).
  • a linker e.g., any one of SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485
  • a cell-penetrating peptide e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254
  • a cellpenetrating peptide e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254
  • a cellpenetrating peptide fusion e.g., SEQ ID NO: 299 - SEQ ID NO: 3
  • a linker e g., any one of SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485
  • a linker can connect the C-terminus of a cargo peptide to the N- terminus of a cell-penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) to form a cell-penetrating peptide fusion.
  • a cell-penetrating peptide e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 -
  • a peptide linker can comprise a Tau-theraphotoxin-Hsla, also known as DkTx (double-knot toxin), extracted from a native knottin-knottin dimer from Haplopelma schmidti (e.g., SEQ ID NO: 266).
  • the linker can lack structural features that would interfere with dimerizing independently functional CDPs (e.g., a cell-penetrating CDP and a target-binding CDP).
  • a linker can comprise a glycine-serine (Gly-Ser or GS) linker (e.g., SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485).
  • Gly-Ser linkers can have minimal chemical reactivity and can impart flexibility to the linker.
  • Serines can increase the solubility of the linker or the peptide complex, as the hydroxyl on the side chain is hydrophilic.
  • a linker can be derived from a peptide that separates the Fc from the Fv domains in a heavy chain of human immunoglobulin G (e.g., SEQ ID NO: 267).
  • a linker derived from a peptide from the heavy chain of human IgG can comprise a cysteine to serine mutation relative to the native IgG peptide.
  • peptides of the present disclosure can be dimerized using an immunoglobulin heavy chain Fc domain. These Fc domains can be used to dimerize functional domains (e.g., a cell-penetrating peptide and a cargo peptide), either based on antibodies or other otherwise soluble functional domains. In some embodiments, dimerization can be homodimeric via Fc sequences.
  • dimerization can be heterodimeric by mutating the Fc domain to generate a “knob-in-hole” format where one Fc CH3 domain contains novel residues (knob) designed to fit into a cavity (hole) on the other Fc CH3 domain.
  • a first peptide domain e.g., a cell-penetrating peptide
  • a second peptide domain e.g., a TfR-binding peptide or target-binding peptide
  • Knob+knob dimers can be highly energetically unfavorable.
  • a purification tag can be added to the “knob” side to remove hole+hole dimers and select for knob+hole dimers.
  • the peptides of the present disclosure can be linked to another peptide (e.g., a cargo peptide) at the N-terminus or C-terminus.
  • one or more peptides can be linked or fused via a peptide linker (e.g., a peptide linker comprising a sequence of any one of SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485).
  • a cell -penetrating peptide can be fused to a cargo peptide via a peptide linker of any one of SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485.
  • a peptide linker can have a length of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, or about 50 amino acid residues.
  • a peptide linker can have a length of from about 2 to about 5, from about 2 to about 10, from about 2 to about 20, from about 3 to about 5, from about 3 to about 10, from about 3 to about 15, from about 3 to about 20, from about 3 to about 25, from about 5 to about 10, from about 5 to about 15, from about 5 to about 20, from about 5 to about 25, from about 10 to about 15, from about 10 to about 20, from about 10 to about 25, from about 15 to about 20, from about 15 to about 25, from about 20 to about 25, from about 20 to about 30, from about 20 to about 35, from about 20 to about 40, from about 20 to about 45, from about 20 to about 50, from about 3 to about 50, from about 3 to about 40, from about 3 to about 30, from about 10 to about 40, from about 10 to about 30, from about 50 to about 100, from about 100 to about 200, from about 200 to about 300, from about 300 to about 400, from about 400 to about 500, or from about 500 to about 600 amino acid residues.
  • a first peptide e.g., a cell -penetrating peptide
  • a second peptide e.g., a cargo peptide
  • a flexible linker can provide rotational freedom between the first peptide and the second peptide and can allow the first peptide and the second peptide to perform their respective functions with minimal strain.
  • a peptide linker can have a persistence length of no more than 6 A, no more than 7 A, no more than 8 A, no more than 9 A, no more than 10 A, no more than 12 A, no more than 15 A, no more than 20 A, no more than 25 A, no more than 30 A, no more than 40 A, or no more than 50 A.
  • a peptide linker can have a persistence length of from about 4 A to about 100 A, from about 4 A to about 50 A, from about 4 A to about 20 A, from about 4 A to about 10 A, from about 10 A to about 20 A, from about 20 A to about 30 A, from about 30 A to about 50 A, or from about 50 A to about 100 A.
  • the persistence length of the linker can be a measure of the flexibility of the peptide linker and can be quantified as the peptide length over which correlations in the direction of the tangent are lost.
  • a peptide linker can be selected based on a desired linker length, hydrodynamic radius, chromatographic mobility, posttranslational modification propensity, or combinations thereof.
  • a linker separating two or more functional domains of a peptide complex e.g., separating a cell -penetrating peptide and a cargo peptide
  • a linker separating two or more functional domains of a peptide complex can comprise a small, flexible linker, for example to reduce the hydrodynamic radius of the complex for use in tight spaces like dense-core tumor stroma.
  • a peptide linker can support independent folding of the two or more functional domains and may not inhibit functions of the respective functional domains.
  • a peptide can be appended to the N-terminus of any peptide of the present disclosure following an N-terminal GS dipeptide and preceding, for example, a GGGS (SEQ ID NO: 264) spacer.
  • a peptide e.g., a target-binding peptide
  • the peptide linker comprises (GS)x (SEQ ID NO: 258), wherein x can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the peptide linker comprises GGSSG (SEQ ID NO: 259), GGGGG (SEQ ID NO: 260), GSGSGSGS (SEQ ID NO: 261), GSGG (SEQ ID NO: 262), GGGGS (SEQ ID NO: 263), GGGS (SEQ ID NO: 264), GGS (SEQ ID NO: 265), GGGSGGGSGGGS (SEQ ID NO: 255), or a variant or fragment thereof.
  • KKYKPYVPVTTN (SEQ ID NO: 266) from DkTx
  • EPKSSDKTHT (SEQ ID NO: 267) from human IgG3
  • the peptide linker comprises GGGSGGSGGGS (SEQ ID NO: 292).
  • the peptide linker comprises a linker of any one of SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485. Examples of peptide linkers compatible with the cell-penetrating peptides and cell-penetrating peptide fusions of the present disclosure are provided in TABLE 10.
  • any of the foregoing linkers or a variant or fragment thereof can be used with any number of repeats or any combinations thereof. It is also understood that other peptide linkers in the art or a variant or fragment thereof can be used with any number of repeats or any combinations thereof.
  • Peptides according to the present disclosure can be attached to another moiety (e.g., an active agent or an detectable agent), such as a small molecule, a second peptide, a protein, an antibody, an antibody fragment, an aptamer, polypeptide, polynucleotide, a fluorophore, a radioisotope, a radionuclide chelator, a polymer, a biopolymer, a fatty acid, an acyl adduct, a chemical linker, or sugar or other active agent or detectable agent described herein through a linker, or directly in the absence of a linker.
  • an active agent or an detectable agent such as a small molecule, a second peptide, a protein, an antibody, an antibody fragment, an aptamer, polypeptide, polynucleotide, a fluorophore, a radioisotope, a radionuclide chelator, a polymer, a biopol
  • an active agent or a detectable agent can be conjugated to, linked to, or fused to the N-terminus or the C-terminus of a peptide to create an active agent or detectable agent fusion peptide.
  • the link can be made by a peptide fusion via reductive alkylation.
  • a cleavable linker is used for in vivo delivery of the peptide, such as a linker that can be cleaved or degraded upon entry in a cell, endosome, or a nucleus.
  • in vivo delivery of a peptide requires a small linker that does not interfere with penetration of a cell or localization to a nucleus of a cell.
  • a linker can also be used to covalently attach a peptide as described herein to another moiety or molecule having a separate function, such a targeting, cytotoxic, therapeutic, homing, imaging, or diagnostic functions.
  • a peptide can be directly attached to another molecule by a covalent attachment.
  • the peptide is attached to a terminus of the amino acid sequence of a larger polypeptide or peptide molecule, or is attached to a side chain, such as the side chain of a lysine, serine, threonine, cysteine, tyrosine, aspartic acid, a non-natural amino acid residue, or glutamic acid residue.
  • the attachment can be via an amide bond, an ester bond, an ether bond, a carbamate bond, a carbon-nitrogen bond, a triazole, a macrocycle, an oxime bond, a hydrazone bond, a carbon-carbon single double or triple bond, a disulfide bond, or a thioether bond.
  • similar regions of the disclosed peptide(s) itself can be used to link other molecules.
  • an amino acid side chain such as the side chain of a lysine, serine, threonine, cysteine, tyrosine, aspartic acid, a non-natural amino acid residue, or glutamic acid residue
  • an amide bond, an ester bond, an ether bond, a carbamate bond, a carbon-nitrogen bond, a triazole, a macrocycle, an oxime bond, a hydrazone bond, a carbon-carbon single double or triple bond, a disulfide bond, or a thioether bond, or linker as described herein can be used to link other molecules.
  • Attachment via a linker can involve incorporation of a linker moiety between the other molecule and the peptide.
  • the peptide and the other molecule can both be covalently attached to the linker.
  • the linker can be cleavable, labile, non-cleavable, stable, stable self-immolating, hydrophilic, or hydrophobic.
  • non-cleavable or “stable” (such as used in association with an amide, cyclic, or carbamate linker or as otherwise as described herein) is often used by a skilled artisan to distinguish a relatively stable structure from one that is more labile or “cleavable” (e.g., as used in association with cleavable linkers that may be dissociated or cleaved structurally by enzymes, proteases, self-immolation, pH, reduction, hydrolysis, certain physiologic conditions, or as otherwise described herein).
  • Non- cleavable or “stable” linkers offer stability against cleavage or other dissociation as compared to “cleavable” linkers, and the term is not intended to be considered an absolute non-cleavable or non-dissociative structure under any conditions. Consequently, as used herein, a “non- cleavable” linker is also referred to as a “stable” linker.
  • the linker can have at least two functional groups with one bonded to the peptide, the other bonded to the other molecule, and a linking portion between the two functional groups.
  • Non-limiting examples of the functional groups for attachment can include functional groups capable of forming an amide bond, an ester bond, an ether bond, a carbonate bond, a carbamate bond, or a thioether bond.
  • Non-limiting examples of functional groups capable of forming such bonds can include amino groups; carboxyl groups; hydroxyl groups; aldehyde groups; azide groups; alkyne and alkene groups; ketones; hydrazides; acid halides such as acid fluorides, chlorides, bromides, and iodides; acid anhydrides, including symmetrical, mixed, and cyclic anhydrides; carbonates; carbonyl functionalities bonded to leaving groups such as cyano, succinimidyl, and JV-hydroxysuccinimidyl; hydroxyl groups; sulfhydryl groups; and molecules possessing, for example, alkyl, alkenyl, alkynyl, allylic, or benzylic leaving groups, such as halides, such
  • Non-limiting examples of the linking portion can include alkylene, alkenylene, alkynylene, polyether, such as polyethylene glycol (PEG), hydroxy carboxylic acids, polyester, polyamide, polyamino acids, polypeptides, cleavable peptides, valine-citrulline, aminobenzylcarbamates, D-amino acids, and polyamine, any of which being unsubstituted or substituted with any number of substituents, such as halogens, hydroxyl groups, sulfhydryl groups, amino groups, nitro groups, nitroso groups, cyano groups, azido groups, sulfoxide groups, sulfone groups, sulfonamide groups, carboxyl groups, carboxaldehyde groups, imine groups, alkyl groups, halo-alkyl groups, alkenyl groups, halo-alkenyl groups, alkynyl groups, halo-alkynyl groups,
  • a peptide and drug complexed, conjugated, or fused via a linker is described with the formula Peptide-A-B-C-Drug, wherein the linker is A-B-C.
  • A can be a stable amide link, is an amine on the peptide and the linker and can be achieved via a tetrafluorophenyl (TFP) ester or an NHS ester.
  • B can be (-CH2-) X - or a short PEG (-CEECFEO-jx (x is 1-10), and C can be the ester bond to the hydroxyl or carboxylic acid on the drug.
  • C can refer to the “cleavable” or “stable” part of the linker.
  • A can also be the “cleavable” part.
  • A can be amide, carbamate, thioether via maleimide or bromoacetamide, triazole, oxime, or oxacarboline.
  • the cleaved active agent or drug can retain the chemical structure of the active agent before cleavage or can be modified as a result of cleavage.
  • such active agent can be active while linked to the peptide, remain active after cleavage or become inactivated, be inactive while linked to the peptide, or it can be activated upon cleavage.
  • peptide complexes have stable linkers.
  • a peptide of the disclosure can be expressed recombinantly or chemically synthesized.
  • the peptide can be complexed, conjugated, or fused to a detectable agent or an active agent via a stable linker, such as an amide linkage or a carbamate linkage.
  • the peptide can be complexed, conjugated, or fused to a detectable agent or an active agent via a stable linker, such as an amide bond using standard 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or dicylcohexylcarbodiimide (DCC) based chemistry or thionyl chloride or phosphorous chloride-based bioconjugation chemistries.
  • EDC 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • DCC dicylcohexylcarbodiimide
  • a stable linker may or may not be cleaved in buffer over extended periods of time (e.g., hours, days, or weeks).
  • a stable linker may or may not be cleaved in body fluids such as plasma or synovial fluid over extended periods of time (e.g., hours, days, or weeks).
  • a stable linker may or may not be cleaved after exposure to enzymes, reactive oxygen species, other chemicals or enzymes that can be present in cells (e.g., macrophages), cellular compartments (e.g., endosomes and lysosomes), inflamed areas of the body (e g., inflamed joints), tissues or body compartments.
  • a stable linker may be cleaved by unknown mechanisms.
  • a stable linker may or may not be cleaved in vivo but remains an active agent after peptide conjugation.
  • a peptide and drug complexed, conjugated, or fused via a linker can be described with the formula Peptide-A-B-C-Drug, wherein the linker is A-B-C.
  • A can be a stable amide link such as that formed by reacting an amine on the peptide with a linker containing a tetrafluorophenyl (TFP) ester or an NHS ester.
  • A can also be a stable carbamate linker such as that formed by reacting an amine on the peptide with an imidazole carbamate active intermediate formed by reaction of CDI with a hydroxyl on the linker.
  • A can also be a stable secondary amine linkage such as that formed by reductive alkylation of the amine on the peptide with an aldehyde or ketone group on the linker.
  • A can also be a stable thioether linker formed using a maleimide or bromoacetamide in the linker with a thiol in the peptide, a triazole linker, a stable oxime linker, or a oxacarboline linker.
  • B can be (-CH2-) X - or a short PEG (-CH2CH2O-) X (x is 0-20) or other spacers or no spacer.
  • C can be an amide bond formed with an amine or a carboxylic acid on the drug, a thioether formed between a maleimide on the linker and a sulfhydroyl on the drug, a secondary or tertiary amine, a carbamate, or other stable bonds.
  • Any linker chemistry described in “Current ADC Linker Chemistry,” Jain et al., Pharm Res, 2015 DOI 10. 1007/sl 1095-015-1657-7 can be used.
  • the resulting peptide complexes can be administered to a human or animal subcutaneously, intravenously, orally, or injected directly into a joint to treat disease.
  • the peptide is not specifically cleaved from the detectable agent or active agent via a targeted mechanism.
  • the peptide can be degraded by mechanisms such as catabolism, releasing a drug that is modified or not modified form its native form (Antibody-Drug Conjugates: Design, Formulation, and Physicochemical Stability, Singh, Luisi, and Pak. Pharm Res (2015) 32:3541- 3571).
  • the peptide drug conjugate exerts its pharmacological activity while still intact, or while partially or fully degraded, metabolized, or catabolized.
  • peptide complexes can have cleavable linkers.
  • a peptide and drug can be complexed, conjugated, or fused via a linker and can be described with the formula Peptide-A-B-C-Drug, wherein the linker is A-B-C.
  • A can be a stable amide link such as that formed by reacting an amine on the peptide with a linker containing a tetrafluorophenyl (TFP) ester or an NHS ester.
  • TFP tetrafluorophenyl
  • A can also be a stable carbamate linker that is formed by an amine reaction on the peptide with an imidazole carbamate active intermediate formed by reaction of CDI with a hydroxyl on the linker.
  • A can also be a stable secondary amine linkage such as that formed by reductive alkylation of the amine on the peptide with an aldehyde or ketone group on the linker.
  • A can also be a stable thioether linker formed using a maleimide or bromoacetamide in the linker with a thiol in the peptide, a triazole linker, a stable oxime linker, or an oxacarboline linker.
  • B can be (-CH2-) X - or a short PEG (-CEECFLO-jx (x is 0-20) or other spacers or no spacer.
  • C can be an ester bond to the hydroxyl or carboxylic acid on the drug, or a carbonate, hydrazone, or acylhydrazone, designed for hydrolytic cleavage.
  • the hydrolytic rate of cleavage can be varied by varying the local environment around the bond, including carbon length (-CH2-)x, steric hindrance (including adjacent side groups such as methyl, ethyl, cyclic), hydrophilicity or hydrophobicity.
  • peptide complexes can have a linear or cyclic ester linkage, which can include or do not include side chains such as methyl or ethyl groups.
  • a linear ester linkage can be more susceptible to cleavage (such as by hydrolysis, an enzyme such as esterase, or other chemical reaction) than a cyclic ester due to steric hindrance or hydrophobicity /hydrophilicity effects.
  • side chains such as methyl or ethyl groups on the linear ester linkage can optionally make the linkage less susceptible to cleavage than without the side chains.
  • hydrolysis rate can be affected by local pH, such as lower pH in certain compartments of the body or of the cell such as endosomes and lysosomes or diseased tissues.
  • C can also be a pH sensitive group such as a hydrazone or oxime linkage.
  • C can be a disulfide bond designed to be released by reduction, such as by glutathione.
  • (or A-B-C) can be a peptidic linkage design for cleavabe by enzymes.
  • a self-immolating group such as pABC can be included to cause release of a free unmodified drug upon cleavage
  • the linker can be cleaved by enzymes such as esterases, matrix metalloproteinases, cathepsins such as cathepsin B, glucuronidases, a protease, or thrombin.
  • the bond designed for cleavage can be at A, rather than C, and C can be a stable bond or a cleavable bond.
  • An alternative design can be to have stable linkers (such as amide or carbamate) at A and C and have a cleavable linker in B, such as a disulfide bond.
  • the rate of reduction can be modulated by local effects such as steric hindrance from methyl or ethyl groups or modulating hydrophobicity /hydrophilicity.
  • peptide complexes can have an ester carbonyl linkage, a long hydrocarbon linker, or carbamate linker, each of which can include hydrophilic groups, such as alcohols, acids, or ethers, or include a hydrocarbon side chain or other moiety that tunes the rate of cleavage.
  • the rate of hydrolysis can be faster with hydrophilic groups, such as alcohols, acids, or ethers, near an ester carbonyl.
  • hydrophobic groups present as side chains or as a longer hydrocarbon linker can slow the cleavage rate of the ester.
  • cleavage of a carbamate group can also be tuned by hindrance, hydrophobicity, and the like.
  • using a less labile linking group, such as a carbamate rather than an ester can slow the cleavage rate of the linker.
  • a linker can comprise a triazole group, such as any one of the heterocyclic compounds with molecular formula C2H3N3, having a five-membered ring of two carbon atoms and three nitrogen atoms, optionally with a hydrogen atom bonded to N at any position in the ring, such as: r example, a 1, 2, 3-Triazole
  • linkers include linear or non-cyclic linkers such as: ach n is independently 0 to about 1,000; 1 to about 1,000; 0 to about 500; 1 to about 500; 0 to about 250; 1 to about 250; 0 to about 200; 1 to about 200; 0 to about 150; 1 to about 150; 0 to about 100; 1 to about 100; 0 to about 50; 1 to about 50; 0 to about 40; 1 to about 40; 0 to about 30; 1 to about 30; 0 to about 25; 1 to about 25; 0 to about 20; 1 to about 20; 0 to about 15; 1 to about 15; 0 to about 10; 1 to about 10; 0 to about 5; or 1 to about 5.
  • ach n is independently 0 to about 1,000; 1 to about 1,000; 0 to about 500; 1 to about 500; 0 to about 250; 1 to about 250; 0 to about 200; 1 to about 200; 0 to about 150; 1 to about 150; 0 to about 100; 1 to about 100; 0 to about 50;
  • each n is independently 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50.
  • m is 1 to about 1,000; 1 to about 500; 1 to about 250; 1 to about 200; 1 to about 150; 1 to about 100; 1 to about 50; 1 to about 40; 1 to about 30; 1 to about 25; 1 to about 20; 1 to about 15; 1 to about 10; or 1 to about 5.
  • m is 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50, or any linker as disclosed in Jain, N., Pharm Res. 32(11): 3526-40 (2015) or Ducry, L., Antibody Drug Conjugates (2013).
  • a linker can comprise a cyclic group, such as an organic nonaromatic or aromatic ring, optionally with 3-10 carbons in the ring, optionally built from a carboxylic acid, optionally be used to form a carbamate linkage.
  • a carbamate linkage can be more resistant to cleavage, such as by hydrolysis, enzymes such as esterases, or other chemical reactions, than an ester linkage.
  • a linker can comprise a cyclic carboxylic acid, for example a cyclic di carboxylic acid, for example one of the following groups: 1,4-cyclohexane dicarboxylic acid, 1,2-cyclohexane dicarboxylic acid, or 1,3 -cyclohexane dicarboxylic acid, 1,1- cyclopentanediacetic acid, stereoisomer thereof.
  • the linker can comprise one of the following groups. used to form an ester linkage.
  • a cyclic ester linkage can be more sterically resistant to cleavage, such as by hydrolysis by water, enzymes such as esterases, or other chemical reactions, than a noncyclic or linear ester linkage.
  • a linker can comprise an aromatic dicarboxylic acid, for example terephthalic acid, isophthalic acid, phthalic acid substituted analog thereof.
  • a linker can comprise a natural or non-natural amino acid, for example
  • a linker can comprise alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); glutamic acid (E, Glu); glutamine (Q, Gin); glycine (G, Gly); histidine (H, His); isoleucine (I, He); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Vai); or any plurality or combination thereof.
  • the non-natural amino acid can comprise one or more functional groups, e.g., alkene or alkyne, that can
  • a substituted analog or a stereoisomer is a structural analog of a compound disclosed herein, for which one or more hydrogen atoms of the compound can be substituted by one or more groups of halo (e.g., Cl, F, Br), alkyl (e.g., methyl, ethyl, propyl), alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, heterocycloalkyl, or any combination thereof.
  • a stereoisomer can be an enantiomer, a diastereomer, a cis or trans stereoisomer, a E or Z stereoisomer, or a R or S stereoisomer.
  • linear linkers include;
  • each nl, or n2 or m is independently 0 to about 1,000; 1 to about 1,000; 0 to about 500; 1 to about 500; 0 to about 250; 1 to about 250; 0 to about 200; 1 to about 200; 0 to about 150; 1 to about 150; 0 to about 100; 1 to about 100; 0 to about 50; 1 to about 50; 0 to about 40; 1 to about 40; 0 to about 30; 1 to about 30; 0 to about 25; 1 to about 25; 0 to about 20; 1 to about 20; 0 to about 15; 1 to about 15; 0 to about 10; 1 to about 10; 0 to about 5; or 1 to about 5.
  • each n is independently 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50.
  • m is 1 to about 1,000; 1 to about 500; 1 to about 250; 1 to about 200; 1 to about 150; 1 to about 100; 1 to about 50; 1 to about 40; 1 to about 30; 1 to about 25; 1 to about 20; 1 to about 15; 1 to about 10; or 1 to about 5.
  • m is 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50.
  • the linker can comprise a linear dicarboxylic acid, e.g., one of the following groups: succinic acid, 2,3 -dimethylsuccinic acid, glutaric acid, adipic acid, 2,5 -dimethyladipic acid, or a substituted analog or a stereoisomer thereof.
  • the linker can be used to form a carbamate linkage.
  • the carbamate linkage can be more resistant to cleavage, such as by hydrolysis, enzymes such as esterases, or other chemical reactions, than an ester linkage
  • the linker can be used to form a linear ester linkage.
  • the linear ester linkage can be more susceptible to cleavage, such as by hydrolysis, enzymes such as esterases, or other chemical reactions, than a cyclic ester or carbamate linkage.
  • Side chains such as methyl groups on the linear ester linkage can optionally make the linkage less susceptible to cleavage than without the side chains.
  • a linker can be a succinic linker, and a targeting agent (e.g., a single stranded (ssDNA, ssRNA) or double stranded (dsDNA, dsRNA) or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or U1 Adapter) or other active agent or detectable agent can be attached to a peptide via an ester bond or an amide bond with two methylene carbons in between.
  • a linker can be any linker
  • a nucleotide e.g., a single stranded (ssDNA, ssRNA) or double stranded (dsDNA, dsRNA) or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure
  • an antisense RNA complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or Ul Adapter
  • an active agent, or a detectable agent can be attached to a peptide using any one or more of the linkers shown below in TABLE 11.
  • a peptide, an additional active agent, or a detectable agent can be attached to a
  • an active agent is attached to a linker wherein a nucleophilic functional group (e.g., a hydroxyl group) of the active agent molecule acts as the nucleophile and replaces a leaving group on the linker moiety, thereby attaching it to the linker.
  • a nucleophilic functional group e.g., a hydroxyl group
  • an active agent is attached to a linker wherein a nucleophilic functional group (e.g., thiol group, amine group, etc.) of the linker replaces a leaving group on the active agent, thereby attaching it to the linker.
  • a nucleophilic functional group e.g., thiol group, amine group, etc.
  • Such leaving group may be a primary alcohol to form a thioether bond, thereby attaching it to the linker.
  • a primary alcohol can be converted into a leaving group such as a mesylate, a tosylate, or a nosylate in order to accelerate the nucleophilic substitution reaction.
  • the peptide complexes of the present disclosure can comprise a cargo molecule (e g., a target-binding agent, an active agent, a therapeutic agent, or a detectable agent), a linker, and/or a peptide of the present disclosure.
  • a general connectivity between these three components can be active agent-linker-peptide, such that the linker is attached to both the active agent and the peptide.
  • the peptide is attached to a linker via an amide bond.
  • Amide bonds can be relatively stable (e.g., in vivo) compared to other bonds described herein, such as esters, carbonates, etc.
  • the amide bond between the peptide and the linker may thus provide advantageous properties due to its in vivo stability of the active agent is sought to be cleaved from a peptide-active agent-conjugate without the linker being attached to the active agent after such in vivo cleavage.
  • an active agent is attached to the linker-peptide moiety via linkages such as ester, carbonate, carbamate, etc., wherein the peptide or active agent is attached to the linker via an amide bond. This can allow for selective cleavage of the active agent-linker bond (as opposed to the linker-peptide bond) allowing the active agent to be released without a linker moiety attached to it after cleavage.
  • the linker can be a cleavable or a stable linker.
  • the use of a cleavable linker permits release of the conjugated moiety (e.g., a nucleotide targeting agent, a therapeutic agent, a detectable agent, or a combination thereof) from the peptide, e.g., after targeting to the target tissue or cell or subcellular compartment or after endocytosis.
  • the linker is enzyme cleavable, e.g., a valine-citrulline linker that can be cleavable by cathepsin, or an ester linker that can be cleavable by esterase.
  • the linker contains a selfimmolating portion.
  • the linker includes one or more cleavage sites for a specific protease, such as a cleavage site for matrix metalloproteases (MMPs), thrombin, urokinase-type plasminogen activator, or cathepsin (e.g., cathepsin K).
  • MMPs matrix metalloproteases
  • thrombin thrombin
  • urokinase-type plasminogen activator or cathepsin (e.g., cathepsin K).
  • a peptide-active agent complexes of the present disclosure can comprise one or more, about two or more, about three or more, about five or more, about ten or more, or about 15 or more amino acids that can form an amino acid sequence cleavable by an enzyme.
  • enzymes can include proteinases.
  • a peptide-active agent complex can comprise an amino acid sequence that can be cleaved by a Cathepsin, a Chymotrypsin, an Elastase, a Subtilisin, a Thrombin I, or a Urokinas, or any combination thereof.
  • the cleavable linker can be cleaved, dissociated, or broken by other mechanisms, such as via pH, reduction, or hydrolysis. Hydrolysis can occur directly due to water reaction, or be facilitated by an enzyme, or be facilitated by presence of other chemical species.
  • a hydrolytically labile linker, (amongst other cleavable linkers described herein) can be advantageous in terms of releasing active agents from the peptide.
  • an active agent in a conjugate form with the peptide may not be active, but upon release from the conjugate after targeting to the target tissue or cell or subcellular compartment, the active agent is active.
  • the cleaved active agent may retain the chemical structure of the active agent before cleavage or may be modified.
  • a stable linker may optionally not cleave in buffer over extended periods of time (e.g., hours, days, or weeks).
  • a stable linker may optionally not cleave in body fluids such as plasma or synovial fluid over extended periods of time (e.g., hours, days, or weeks).
  • a stable linker optionally may cleave, such as after exposure to enzymes, reactive oxygen species, other chemicals or enzymes that may be present in cells (such as macrophages), cellular compartments (such as endosomes and lysosomes), inflamed areas of the body (such as inflamed joints), or tissues or body compartments.
  • a stable linker may optionally not cleave in vivo but present an active agent that is still active when conjugated to, linked to, or fused to the peptide.
  • the rate of hydrolysis of the linker can be tuned.
  • the rate of hydrolysis of linkers with unhindered esters may be faster compared to the hydrolysis of linkers with bulky groups next an ester carbonyl.
  • a bulky group can be a methyl group, an ethyl group, a phenyl group, a ring, or an isopropyl group, or any group that provides steric bulk.
  • the rate of hydrolysis can be faster with hydrophilic groups, such as alcohols, acids, or ethers, or near an ester carbonyl.
  • hydrophobic groups present as side chains or by having a longer hydrocarbon linker can slow cleavage of the ester.
  • cleavage of a carbamate group can also be tuned by hindrance, hydrophobicity, and the like.
  • using a less labile linker, such as a carbamate rather than an ester can slow the cleavage rate of the linker.
  • the steric bulk can be provided by the drug itself, such as by ketorolac when conjugated via its carboxylic acid.
  • the rate of hydrolysis of the linker can be tuned according to the residency time of the conjugate in the target tissue or cell or subcellular compartment, according to how quickly the peptide accumulates in the target tissue or cell or subcellular compartment, or according to the desired time frame for exposure to the active agent in the target tissue or cell or subcellular compartment. For example, when a peptide is cleared from the target tissue or cell or subcellular compartment relatively quickly, the linker can be tuned to rapidly hydrolyze. In contrast, for example, when a peptide has a longer residence time in the target tissue or cell or subcellular compartment, a slower hydrolysis rate can allow for extended delivery of an active agent.
  • a linker may be tuned for different cleavage rates for similar cleavage rates in different species.
  • a linker can be a succinic linker, and a drug can be attached to a peptide via an ester bond or an amide bond with two methylene carbons in between.
  • a linker can be any linker with both a hydroxyl group and a carboxylic acid, such as hydroxy hexanoic acid or lactic acid.
  • the linker can release the active agent in an unmodified form.
  • the active agent can be released with chemical modification.
  • catabolism can release the active agent still linked to parts of the linker and/or peptide.
  • peptide complexes have stable linkers.
  • a peptide of the disclosure can be expressed recombinantly or chemically synthesized.
  • the peptide can be complexed, conjugated, or fused to a detectable agent or an active agent via a stable linker, such as an amide linkage or a carbamate linkage.
  • the peptide can be complexed, conjugated, or fused to a detectable agent or an active agent via a stable linker, such as an amide bond using standard l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or dicylcohexylcarbodiimide (DCC) based chemistry or thionyl chloride or phosphorous chloride-based bioconjugation chemistries.
  • EDC l-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • DCC dicylcohexylcarbodiimide
  • a stable linker may or may not be cleaved in buffer over extended periods of time (e.g., hours, days, or weeks).
  • a stable linker may or may not be cleaved in a cellular, intracellular, or paracellular space over extended periods of time (e.g., hours, days, or weeks).
  • a stable linker may or may not be cleaved after exposure to enzymes, reactive oxygen species, other chemicals or enzymes that can be present in cells (e.g., cancer cells, pancreatic cells, liver cells, colon cells, smooth muscle cells, ovarian cells, breast cells, lung cells, brain cells, skin cells, ocular cells, blood cells, lymph cells, immune system cells, reproductive cells, reproductive organ cells, prostate cells, fibroblasts, kidney cells, adenocarcinoma cells, glioma stem cells, or tumor cells,), cellular, paracellular, or intracellular compartments (e.g., cytosols, nuclei, or nanolumen), cells, tissues or body compartments.
  • a stable linker may be cleaved by unknown mechanisms.
  • a stable linker may or may not be cleaved in viv
  • the linker can be a stable linker or a cleavable linker.
  • the stable linker can slowly release the conjugated moiety by an exchange of the conjugated moiety onto the free thiols on serum albumin.
  • the use of a cleavable linker can permit release of the conjugated moiety (e.g., a therapeutic agent) from the peptide, e.g., after administration to a subject in need thereof.
  • the use of a cleavable linker can permit the release of the conjugated therapeutic from the peptide.
  • the linker is enzyme cleavable, e.g., a valine-citrulline linker.
  • the linker contains a self-immolating portion.
  • the linker includes one or more cleavage sites for a specific protease, such as a cleavage site for matrix metalloproteases (MMPs), thrombin, cathepsins, peptidases, or beta-glucuronidase.
  • MMPs matrix metalloproteases
  • thrombin cleavage site for matrix metalloproteases
  • cathepsins peptidases
  • beta-glucuronidase beta-glucuronidase
  • the linker is cleavable by other mechanisms, such as via pH, reduction, or hydrolysis.
  • the rate of hydrolysis or reduction of the linker can be fine-tuned or modified depending on an application.
  • the rate of hydrolysis of linkers with unhindered esters can be faster compared to the hydrolysis of linkers with bulky groups next to an ester carbonyl.
  • a bulky group can be a methyl group, an ethyl group, a phenyl group, a ring, or an isopropyl group, or any group that provides steric bulk.
  • the steric bulk can be provided by the drug itself, such as by ketorolac when conjugated, linked, or fused via its carboxylic acid.
  • the rate of hydrolysis of the linker can be tuned according to the residency time of the conjugate or fusion in the target location. For example, when a peptide is cleared from a tumor, or the brain, relatively quickly, the linker can be tuned to rapidly hydrolyze. When a peptide has a longer residence time in the target location, a slower hydrolysis rate would allow for extended delivery of an active agent. “Programmed hydrolysis in designing paclitaxel prodrug for nanocarrier assembly” Sci Rep 2015, 5, 12023 Fu et al., provides an example of modified hydrolysis rates. [0325] The rate of hydrolysis of the linker (e.g., a linker of a peptide conjugate) can be measured.
  • Such measurements can include determining free active agent in plasma, serum, or synovial fluid, or other fluid or tissue of a subject in vivo and/or by incubating a linker or a peptide conjugate comprising a linker of the present disclosure with a buffer (e.g., PBS) or blood plasma from a subject (e.g., rat plasma, human plasma, etc.) or synovial fluid or other fluids or tissues ex vivo.
  • the methods for measuring hydrolysis rates can include taking samples during incubation or after administration and determine free active agent, free peptide, or any other parameter indicate of hydrolysis, including also measuring total peptide, total active agent, or conjugated active agent-peptide.
  • a hydrolysis half-life of a linker can differ depending on the plasma or fluid or species or other conditions used to determine such half-life. This can be due to certain enzymes or other compounds present in a certain plasma (e.g., rat plasma). For instance, different fluids (such as plasma or synovial fluid) can contain different amounts of enzymes such as esterases, and these levels of these compounds can also vary depending on species (such as rat versus human) as well as disease state (such as normal versus arthritic).
  • the complexes of the present disclosure can be described as having a modular structure comprising various components, wherein each of the components (e.g., peptide, linker, active agent and/or detectable agent) can be selected dependently or independently of any other component.
  • each of the components e.g., peptide, linker, active agent and/or detectable agent
  • a targeted degradation peptide complex for targeted degradation of a target peptide can comprise a cell-penetrating peptide of the present disclosure (e.g., those having the amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 254), a targetbinding peptide (e.g., any one of SEQ ID NO: 293 - SEQ ID NO: 298 or SEQ ID NO: 364 - SEQ ID NO: 407), a linker (e.g., any linker described in TABLE 10 or TABLE 11, SEQ ID NO: 255 - SEQ ID NO: 292, SEQ ID NO: 458 - SEQ ID NO: 485, or otherwise described), and a ubiquitin ligase-binding agent (e.g., thalidomide, pomalidomide, lenalidomide, methyl bestatin, bestatin, nutlin-3, or VHL ligand 1).
  • the linker for example, can be selected and/or modified to achieve a certain active agent release (e.g., a certain release rate) via a certain mechanism (e.g., via hydrolysis, such as enzyme and/or pH-dependent hydrolysis) at the target site (e.g., in the brain) and/or to minimize systemic exposure to the active agent.
  • a certain active agent release e.g., a certain release rate
  • a certain mechanism e.g., via hydrolysis, such as enzyme and/or pH-dependent hydrolysis
  • any one or more of the components of the conjugate can be modified and/or altered to achieve certain in vivo properties of the conjugate, e.g., pharmacokinetic (e.g., clearance time, bioavailability, uptake and retention in various organs) and/or pharmacodynamic (e.g., target engagement) properties.
  • pharmacokinetic e.g., clearance time, bioavailability, uptake and retention in various organs
  • pharmacodynamic e.
  • the conjugates of the present disclosure can be modulated to prevent, treat, and/or diagnose a variety of diseases and conditions, while reducing side effects (e.g., side effects that occur if such active agents are administered alone (i.e., not conjugated to a peptide)).
  • side effects e.g., side effects that occur if such active agents are administered alone (i.e., not conjugated to a peptide)
  • the non-natural amino acid can comprise one or more functional groups, e.g., alkene or alkyne, that can be used as functional handles.
  • a multiple bond of such functional groups can be used to add one or more molecules to the conjugate.
  • the one or more molecules can be added using various synthetic strategies, some of which may include addition and/or substitution chemistries.
  • an addition reaction using a multiple bond can comprise the use of hydrobromic acid, wherein the bromine can act as a leaving group and thus be substituted with various moieties, e.g., active agents, detectable agents, agents that can modify or alter the pharmacokinetic (e.g., plasma half-life, retention and/or uptake in central nervous system (CNS) or elsewhere) and/or pharmacodynamic (e.g., hydrolysis rate such as an enzymatic hydrolysis rate) properties of the conjugate.
  • various moieties e.g., active agents, detectable agents, agents that can modify or alter the pharmacokinetic (e.g., plasma half-life, retention and/or uptake in central nervous system (CNS) or elsewhere) and/or pharmacodynamic (e.g., hydrolysis rate such as an enzymatic hydrolysis rate) properties of the conjugate.
  • a conjugate as described herein comprises one or more nonnatural amino acid and/or one or more linkers.
  • Such one or more non-natural amino acid and/or one or more linkers can comprise one or more functional groups, e g., alkene or alkyne (e.g., non-terminal alkenes and alkynes), which can be used as functional handles.
  • a multiple bond of such functional groups can be used to add one or more molecules to the conjugate.
  • the one or more molecules can be added using various synthetic strategies, some of which may include addition and/or substitution chemistries, cycloadditions, etc.
  • an addition reaction using a multiple bond can comprise the use of hydrogen bromide (e g., via hydrohalogenation reactions), wherein the bromide substituent, once attached, can act as a leaving group and thus be substituted with various moieties comprising a nucleophilic functional groups, e.g., active agents, detectable agents, agents.
  • a multiple bond can be used as a functional handle in a cycloaddition reaction.
  • Cycloaddition reactions can comprise 1,3-dipolar cycloadditions, [2+2] -cycloadditions (e.g., photocatalyzed), Di els- Alder reactions, Huisgen cycloadditions, nitrone -olefin cycloadditions, etc.
  • Such cycloaddition reactions can be used to attached various functional groups, functional moieties, active agents, detectable agents, and so forth to the conjugate.
  • a 1,3-dipolar cycloaddition reaction can be used to attach a molecule to a conjugate, wherein the molecule comprises a 1,3-dipole that can react with, e.g., an alkyne to form a 5 -membered ring, thereby attaching said molecule to the conjugate.
  • attaching such molecule or agent can modify or alter the pharmacokinetic (e g., plasma halflife, retention and/or uptake in CNS or biodistribution) and/or pharmacodynamic (e.g., hydrolysis rate such as an enzymatic hydrolysis rate) properties of the conjugate.
  • Attaching such molecule or agent can also alter (e.g., increase) the depot effect of a conjugate, or provide functionality for in vivo tracking, e.g., using fluorescence or other types of detectable agents.
  • a conjugate of the present disclosure can comprise a linker comprising one or more of the following groups: substituted analog or a stereoisomer thereof, wherein each nl and n2 is independently a value from 1 to 10.
  • a group can be used as a handle to attach one or more molecules to a conjugate, e.g., to alter the pharmacokinetic (e.g., plasma half-life, retention and/or uptake in central nervous system (CNS) or elsewhere) and/or pharmacodyna via nucleophilic or electrophilic addition followed by nucleophilic substitution mic properties of the conjugate.
  • Functionalization of such a group can occur using one or more multiple bonds (e.g., double bonds, triple bonds, etc.) of the groups.
  • Such functionalization can comprise addition and/or substitution chemistries.
  • a functional group of a linker such as a double bond
  • a single bond e.g., via an addition reaction such as a nucleophilic addition reaction
  • one or both of the carbon atoms of the newly formed single bond can have a leaving group (e.g., a bromine) attached to them.
  • a leaving group e.g., via nucleophilic substitution reaction
  • a specific molecule e.g., an active agent, a detectable agent, etc.
  • a multiple bond can be used as a functional handle in a cycloaddition reaction.
  • Cycloaddition reactions can comprise 1,3 -dipolar cycloadditions, [2+2]-cycloadditions (e.g., photocatalyzed), Diels-Alder reactions, Huisgen cycloadditions, nitrone-olefin cycloadditions, etc.
  • Such cycloaddition reactions can be used to attached various functional groups, functional moieties, active agents, detectable agents, and so forth to the conjugate.
  • a 1,3 -dipolar cycloaddition reaction can be used to attach a molecule to a conjugate, wherein the molecule comprises a 1,3-dipole that can react with, e.g., an alkyne to form a 5-membered ring, thereby attaching said molecule (e.g., active agent, detectable agent, etc.) to the conjugate.
  • molecules may be attached to a conjugate to e.g., modulate the half-life, increase the depot effect, or provide new functionality of a conjugate, such as fluorescence for tracking.
  • a peptide of the present disclosure can be stable in various biological or physiological conditions, such as the pH or reducing environments inside a cell, in the cytosol, in a cell nucleus, lysosome, or endosome.
  • a cell-penetrating peptide of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 may be conjugated to a peptide (e.g., a cystine-dense peptide) that can exhibit resistance to reducing agents, heat, denaturation, proteases, oxidative conditions, or acidic conditions, for example a cell -penetrating peptide complex of SEQ ID NO: 299 - SEQ ID NO: 308 or SEQ ID NO: 312 - SEQ ID NO: 321.
  • a peptide e.g., a cystine-dense peptide
  • biologic molecules such as peptides and proteins
  • GI tract can contain a region of low pH (e.g.
  • protease-rich environment that can degrade peptides and proteins.
  • Proteolytic activity in other areas of the body such as the mouth, eye, lung, intranasal cavity, joint, skin, vaginal tract, mucous membranes, and serum, can also be an obstacle to the delivery of functionally active peptides and polypeptides.
  • the half-life of peptides in serum can be very short, in part due to proteases, such that the peptide can be degraded too quickly to have a lasting therapeutic effect when administering reasonable dosing regimens.
  • proteolytic activity in cellular compartments such as lysosomes and reduction activity in lysosomes and the cytosol can degrade peptides and proteins such that they may be unable to provide a therapeutic function on intracellular targets. Therefore, peptides that are resistant to reducing agents, proteases, and low pH may be able to provide enhanced therapeutic effects or enhance the therapeutic efficacy of co-formulated or conjugated, linked, or fused active agents in vivo.
  • oral delivery of drugs can be desirable in order to target certain areas of the body (e.g., disease in the GI tract such as colon cancer, irritable bowel disorder, infections, metabolic disorders, and constipation) despite the obstacles to the delivery of functionally active peptides and polypeptides presented by this method of administration.
  • oral delivery of drugs can increase compliance by providing a dosage form that is more convenient for patients to take as compared to parenteral delivery.
  • Oral delivery can be useful in treatment regimens that have a large therapeutic window. Therefore, peptides that are resistant to reducing agents, proteases, and low pH can allow for oral delivery of peptides without nullifying their therapeutic function.
  • Cell-penetrating peptides or peptide complexes of this disclosure can contain one or more cysteines, which can participate in disulfide bridges that can be integral to preserving the folded state of the peptide. Exposure of peptides to biological environments with reducing agents can result in unfolding of the peptide and loss of functionality and bioactivity.
  • glutathione GSH
  • a peptide can become reduced during trafficking of a peptide across the gastrointestinal epithelium after oral administration. A peptide can become reduced upon exposure to various parts of the GI tract.
  • the GI tract can be a reducing environment, which can inhibit the ability of therapeutic molecules with disulfide bonds to have optimal therapeutic efficacy, due to reduction of the disulfide bonds.
  • a peptide can also be reduced upon entry into a cell, such as after internalization by endosomes or lysosomes or into the cytosol, or other cellular compartments. Reduction of the disulfide bonds and unfolding of the peptide can lead to loss of functionality or affect key pharmacokinetic parameters such as bioavailability, peak plasma concentration, bioactivity, and half-life. Reduction of the disulfide bonds can also lead to loss of functionality due to increased susceptibility of the peptide to subsequent degradation by proteases, resulting in rapid loss of intact peptide after administration.
  • a peptide that is resistant to reduction can remain intact and can impart a functional activity for a longer period of time in various compartments of the body and in cells, as compared to a peptide that is more readily reduced.
  • the peptides of this disclosure can be analyzed for the characteristic of resistance to reducing agents to identify stable peptides.
  • the peptides of this disclosure can remain intact after being exposed to different molarities of reducing agents such as 0.00001 M - 0.0001 M, 0.0001 M - 0.001 M, 0.001 M - 0.01 M, 0.01 M - 0.05 M, 0.05 M - 0.1 M, or 0.1 M to 0.2 M for 15 minutes or more.
  • the reducing agent used to determine peptide stability can be dithiothreitol (DTT), Tris(2- carboxyethyl)phosphine HC1 (TCEP), 2-Mercaptoethanol, (reduced) glutathione (GSH), or any combination thereof.
  • DTT dithiothreitol
  • TCEP Tris(2- carboxyethyl)phosphine HC1
  • GSH 2-Mercaptoethanol
  • GSH 2-Mercaptoethanol
  • GSH reduced glutathione
  • peptides are completely resistant to GSH reducing conditions and are partially resistant to degradation in DTT reducing conditions.
  • peptides described herein can withstand or
  • proteases also referred to as peptidases or proteinases, are enzymes that can degrade peptides and proteins by breaking bonds between adjacent amino acids. Families of proteases with specificity for targeting specific amino acids can include serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, and asparagine proteases. Additionally, metalloproteases, matrix metalloproteases, elastase, carboxypeptidases, Cytochrome P450 enzymes, and cathepsins can also digest peptides and proteins.
  • Proteases can be present at high concentration in blood, in mucous membranes, lungs, skin, the GI tract, the mouth, nose, eye, and in compartments of the cell. Misregulation of proteases can also be present in various diseases such as rheumatoid arthritis and other immune disorders. Degradation by proteases can reduce bioavailability, biodistribution, half-life, and bioactivity of therapeutic molecules such that they are unable to perform their therapeutic function. In some embodiments, peptides that are resistant to proteases can better provide therapeutic activity at reasonably tolerated concentrations in vivo.
  • peptides of this disclosure can resist degradation by any class of protease.
  • peptides of this disclosure resist degradation by pepsin (which can be found in the stomach), trypsin (which can be found in the duodenum), serum proteases, or any combination thereof.
  • the proteases used to determine peptide stability can be pepsin, trypsin, chymotrypsin, or any combination thereof.
  • peptides of this disclosure can resist degradation by lung proteases (e.g., serine, cysteinyl, and aspartyl proteases, metalloproteases, neutrophil elastase, alpha-1 antitrypsin, secretory leucoprotease inhibitor, and elafin), or any combination thereof.
  • lung proteases e.g., serine, cysteinyl, and aspartyl proteases, metalloproteases, neutrophil elastase, alpha-1 antitrypsin, secretory leucoprotease inhibitor, and elafin
  • Peptide Stability in Acidic Conditions Peptides of this disclosure can be administered in biological environments that are acidic. For example, after oral administration, peptides can experience acidic environmental conditions in the gastric fluids of the stomach and gastrointestinal (GI) tract.
  • the pH of the stomach can range from about 1-4 and the pH of the GI tract ranges from acidic to normal physiological pH descending from the upper GI tract to the colon.
  • the vagina, late endosomes, and lysosomes can also have acidic pH values, such as less than pH 7. These acidic conditions can lead to denaturation of peptides and proteins into unfolded states.
  • the peptides of this disclosure can resist denaturation and degradation in acidic conditions and in buffers, which simulate acidic conditions.
  • peptides of this disclosure can resist denaturation or degradation in buffer with a pH less than 1, a pH less than 2, a pH less than 3, a pH less than 4, a pH less than 5, a pH less than 6, a pH less than 7, or a pH less than 8.
  • peptides of this disclosure remain intact at a pH of 1-3.
  • At least 5%- 10%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the peptide remains intact after exposure to a buffer with a pH less than 1, a pH less than 2, a pH less than 3, a pH less than 4, a pH less than 5, a pH less than 6, a pH less than 7, or a pH less than 8.
  • the peptides of this disclosure can be resistant to denaturation or degradation in simulated gastric fluid (pH 1-2).
  • low pH solutions such as simulated gastric fluid can be used to determine peptide stability.
  • the peptides described herein are resistant to degradation in vivo, in the serum of a subject, or inside a cell.
  • the peptides are stable at physiological pH ranges, such as about pH 7, about pH 7.5, between about pH 5 to 7.5, between about 6.5 to 7.5, between about pH 5 to 8, or between about pH 5 to 7.
  • the peptides described herein are stable in acidic conditions, such as less than or equal to about pH 5, less than or equal to about pH 3, or within a range from about 3 to about 5.
  • the peptides are stable in conditions of an endosome or lysosome, or inside a nucleus.
  • Peptide Stability at High Temperatures Peptides of this disclosure can be administered in biological environments with high temperatures. For example, after oral administration, peptides can experience high temperatures in the body. Body temperature can range from 36°C to 40°C. High temperatures can lead to denaturation of peptides and proteins into unfolded states. Unfolding of peptides and proteins can lead to increased susceptibility to subsequent digestion by other enzymes as well as loss of biological activity of the peptide. In some embodiments, a peptide of this disclosure can remain intact at temperatures from 25°C to 100°C. High temperatures can lead to faster degradation of peptides. Stability at a higher temperature can allow for storage of the peptide in tropical environments or areas where access to refrigeration is limited.
  • 5%-100% of the peptide can remain intact after exposure to 25°C for 6 months to 5 years. 5%-100% of a peptide can remain intact after exposure to 70°C for 15 minutes to 1 hour. 5%-100% of a peptide can remain intact after exposure to 100°C for 15 minutes to 1 hour. In other embodiments, at least 5%-10%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the peptide remains intact after exposure to 25°C for at least 6 months to 5 years.
  • At least 5%- 10%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%- 60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the peptide remains intact after exposure to 70°C for 15 minutes to 1 hour.
  • Various expression vector/host systems can be utilized for the recombinant expression of peptides described herein.
  • Non-limiting examples of such systems include microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing a nucleic acid sequence encoding peptides or peptide fusion proteins/ chimeric proteins described herein, yeast transformed with recombinant yeast expression vectors containing the aforementioned nucleic acid sequence, insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the aforementioned nucleic acid sequence, plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV), tobacco mosaic virus (TMV)), or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the aforementioned nucleic acid sequence, or animal cell systems infected with
  • a host cell can be adapted to express one or more peptides described herein.
  • the host cells can be prokaryotic, eukaryotic, or insect cells.
  • host cells are capable of modulating the expression of the inserted sequences or modifying and processing the gene or protein product in the specific fashion desired. For example, expression from certain promoters can be elevated in the presence of certain inducers (e.g., zinc and cadmium ions for metallothionine promoters).
  • inducers e.g., zinc and cadmium ions for metallothionine promoters.
  • modifications e.g., phosphorylation
  • processing e.g., cleavage
  • Host cells can have characteristic and specific mechanisms for the post-translational processing and modification of a peptide.
  • the host cells used to express the peptides secrete minimal amounts of proteolytic enzymes.
  • organisms can be treated prior to purification to preserve and/or release a target polypeptide.
  • the cells are fixed using a fixing agent.
  • the cells are lysed.
  • the cellular material can be treated in a manner that does not disrupt a significant proportion of cells, but which removes proteins from the surface of the cellular material, and/or from the interstices between cells.
  • cellular material can be soaked in a liquid buffer, or, in the case of plant material, can be subjected to a vacuum, in order to remove proteins located in the intercellular spaces and/or in the plant cell wall.
  • proteins can be extracted from the microorganism culture medium.
  • the peptides can be packed in inclusion bodies. The inclusion bodies can further be separated from the cellular components in the medium. In some embodiments, the cells are not disrupted.
  • a cellular or viral peptide that is presented by a cell or virus can be used for the attachment and/or purification of intact cells or viral particles.
  • peptides can also be synthesized in a cell-free system prior to extraction using a variety of known techniques employed in protein and peptide synthesis.
  • a host cell produces a peptide that has an attachment point for a drug.
  • An attachment point could comprise a lysine residue, an N-terminus, a cysteine residue, a cysteine disulfide bond, a glutamic acid or aspartic acid residue, a C-terminus, or a non-natural amino acid.
  • the peptide could also be produced synthetically, such as by solid-phase peptide synthesis, or solution-phase peptide synthesis. Peptide synthesis can be performed by fluorenylmethyloxycarbonyl (Fmoc) chemistry or by butyloxycarbonyl (Boc) chemistry.
  • the peptide could be folded (formation of disulfide bonds) during synthesis or after synthesis or both.
  • Peptide fragments could be produced synthetically or recombinantly. Peptide fragments can be then be joined together enzymatically or synthetically.
  • the peptides of the present disclosure can be prepared by conventional solid phase chemical synthesis techniques, for example according to the Fmoc solid phase peptide synthesis method (“Fmoc solid phase peptide synthesis, a practical approach,” edited by W. C. Chan and P. D. White, Oxford University Press, 2000).
  • the peptides of the present disclosure can be prepared by solution phase peptide synthesis.
  • the peptides can be folded by one cystine at a time, two at a time, three at a time, four at a time, or all at a time, either in solution or on the resin.
  • the peptide made by manufactured as two or more fragments that are subsequently ligated or joined together.
  • sequences from a peptide library generated from computational design can be synthesized using expression vectors or solid phase or solution phase peptide synthesis methods.
  • cell-penetrating peptides or cell-penetrating peptide fusions can be cloned into a secreted, soluble protein production/expression vector and purified, as per Bannesayake et al., 2011.
  • Purification methods include, but are not limited to, affinity purification columns, ion exchange (cation and/or anion columns), reversed-phase, hydrophobic interaction, and size exchange columns.
  • SDS-PAGE followed by Coomassie staining and reverse phase HPLC can be used to analyze a sample of the purified protein. Protein concentrations were determined by UV spectral absorption and/or amino acid analysis.
  • the peptides of this disclosure can be more stable during manufacturing.
  • peptides of this disclosure can be more stable during recombinant expression and purification, resulting in lower rates of degradation by proteases that are present in the manufacturing process, a higher purity of peptide, a higher yield of peptide, or any combination thereof.
  • the peptides can also be more stable to degradation at high temperatures and low temperatures during manufacturing, storage, and distribution.
  • peptides of this disclosure can be stable at 25°C.
  • peptides of this disclosure can be stable at 70°C or higher than 70°C.
  • peptides of this disclosure can be stable at 100°C or higher than 100°C.
  • Improved cell -penetrating peptides may be identified using various screening methods to select for peptides with cell-penetrating properties.
  • a peptide library e g., a library comprising amino acid substitution variants of a parent peptide
  • a single peptide may be screened at a time using cell -based assays to quantify cytosolic or nuclear access.
  • Such cell-based assays can utilize interactions between a tag protein (e.g., a SNAP -tag, a Halo-tag protein, a split GFP, or a biotin binding protein) and a small molecule substrate (e.g., a BG-GLA-NHS SNAP substrate, a chloroalkane substrate, or a biotin molecule) to identify peptides able to penetrate a cellular layer (e.g., a cell membrane or a nuclear membrane).
  • a tag protein e.g., a SNAP -tag, a Halo-tag protein, a split GFP, or a biotin binding protein
  • a small molecule substrate e.g., a BG-GLA-NHS SNAP substrate, a chloroalkane substrate, or a biotin molecule
  • the labeled peptide library can be incubated with cells expressing the tag protein in a cellular location of interest (e.g., in the cytosol or in the nucleus). Labeled peptides that are able to penetrate the cellular layer and reach the location of interest can bind to the expressed tag protein. Peptides that penetrated the cellular layer can be identified in a number of ways. For example, peptides may be identified by adding small molecule substrate labeled with a fluorescent molecule to the cells.
  • Cells with high concentrations of available tag protein, indicative of low peptide pentration, will bind higher levels of fluorescently labeled substrate, and cells with lower concentrations of available tag protein, indicative of higher peptide penetration, will bind lower levels of fluorescently labeled substrate.
  • Cells can be fluorescently imaged to quantify cellpenetration, where cells contacted with high cell -penetrating peptides have lower fluorescence and cells contacted with low cell-penetrating peptides have higher fluorescence.
  • peptides can be identified by purifying the tag protein (e.g., by immunoprecipitation) along with bound peptides, and the peptides can be identified by mass spectrometry. Identified peptides can penetrate cell membranes.
  • SNAP penetration assay uses many reagents that are commonly referred to herein in a shorthand manner to aid in understanding.
  • SNAP-tag protein is also referred to as “SNAP-tag”.
  • the SNAP-tag can be expressed in a variety of eukaryotic cells (e.g., NIH3T3, HEK293, HeLa, CHO, COS cells and the like).
  • the SNAP-tag is introduced into cells via transfection of a plasmid vector such as for SNAP-tag protein (e.g., pSNAPf) or H2B- tagged SNAP-tag protein (e.g., pSNAPf-H2B) resulting in a cell line expressing the SNAP-tag (e.g., commonly referred to as “NI3T3 pSNAPf’, “HEK293 pSNAPf’, “HeLa pSNAPf’, generically as “pSNAPf cells” or “pSNAPf-H2B cells”, and the like).
  • a plasmid vector such as for SNAP-tag protein (e.g., pSNAPf) or H2B- tagged SNAP-tag protein (e.g., pSNAPf-H2B) resulting in a cell line expressing the SNAP-tag (e.g., commonly referred to as “NI3T3 pSNAPf’,
  • the BG-substrate (BG- GLA-NHS) as used herein is a reactive benzylguanine reagent chemical species used in a chemical reaction that reacts with a test reagent such as a peptide to generate a benzylguanine- containing product, for example a BG-peptide as described below.
  • BG-substrates may be used as a reactive moiety, and one or more reactive benzylguanine reagent chemical agents (e.g., such agents with various properties, fluorescence, radiolabel, for imaging, other measurable aspects) may be created and used to “tag” a test peptide, for example, for use in assessing cell penetration.
  • reactive benzylguanine reagent chemical agents e.g., such agents with various properties, fluorescence, radiolabel, for imaging, other measurable aspects
  • SNAP substrate-tagged peptide or peptide that has been reacted with or “tagged” with a reactive benzylguanine reagent (e.g., BG- GLA-NHS)
  • BG-peptide reactive benzylguanine reagent
  • BG-peptide is typically a test peptide used with cells expressing the SNAP-tag to assess e.g., cytosolic and nuclear cell penetration of the BG-peptide in the SNAP assay.
  • BG-GLA- NHS reactive BG-substrate
  • BG-GLA-OH non-reactive form
  • a “SNAP substrate-tagged fluorophore”, or fluorophore that has been reacted with or “tagged” with a benzylguanine reagent are also referred to as a “BG-fluorophore”.
  • An exemplary BG- fluorophore is the “SNAP-Cell TMR-Star” reagent from New England Biolabs. The BG- fluorophore can diffuse across the cell membrane to bind any unoccupied SNAP -tag in the cell, resulting in fluorescence, and the fluorescence of the cells can be visualized after washing away any free BG-fluorophore that has not bound to unoccupied SNAP -tag.
  • a decrease in fluorescence relative to the PBS control indicates that the SNAP -tag is bound to the BG-peptide (rather than the BG-fluorophore), indicating that the BG-peptide has penetrated the cell.
  • the level of fluorescence is in inverse relationship, wherein adding a cell penetrant BG-fluorophore and then washing away unbound BG- fluorophore, and then detecting fluorescence, the absence of which, relative to the negative control (PBS), is indicative of the SNAP -tag covalently bound to the BG-peptide.
  • SNAP substrate is a general term that can describe a variety of substrates that are used in the SNAPPA as an enzymatic substrate for the SNAP -tag.
  • SNAP substrate can be used to describe whether a reactive benzylguanine- containing reagent (such as a BG-peptide or BG-fluorophore) will react to the SNAP -tag protein in the SNAP assay, serving as a substrate to the SNAP -tag itself. Consequently, it is understood that a “SNAP substrate” is non-limiting and can be an enzymatic substrate for the SNAP -tag in the SNAP assay.
  • each of the BG-GLA-NHS (BG- substrate), and its hydrolyzed form BG-GLA-OH, BG-peptide, and BG-fluorophore can serve as a substrate (SNAP substrate) for the SNAP-tag in the SNAP assay.
  • a cell-based assay to identify cell-penetrating peptides may comprise contacting a library of BG-peptide variants to cells expressing a SNAP-tag in either the cytosol or the nucleus.
  • BG-peptides capable of penetrating the cell membrane may covalently bind to SNAP-tag expressed in the cytosol
  • BG-peptides capable of penetrating the cell membrane and the nuclear envelope may covalently bind to SNAP-tag expressed in the nucleus.
  • peptides that enter the cell may be subsequently identified by contacting the cells with a BG-fluorophore that can permeate the cell membrane and nuclear envelope and bind to any remaining SNAP-tag that is not bound to a BG-peptide.
  • peptides that enter the cell may be identified by identifying which cells have a lower level of fluorescence, indicating a higher level of cell penetration.
  • peptides that enter the cell may be identified by immunoprecipitating the SNAP-tag and any bound BG-peptides and performing mass spectrometry to identify the bound peptides. In some embodiments, these screening methods may be performed as high throughput assays, for example, in microarrays.
  • a valuable aspect of a SNAP penetration assay is that can measure delivery of a peptide to the cytosol or nucleus, rather than solely measuring its uptake into the cell.
  • Other assays exist that merely measure general uptake into the cell, such as by labeling a peptide with a fluorophore and measuring total cellular fluorescence.
  • general uptake assays may measure peptide that is only present in the endosome or lysosome, rather than distinguish peptide that has entered the cytosol.
  • Peptides may be taken up by endocytosis but remain trapped and never enter the cytosol which makes general uptake assays not very effective in identifying cytosolic or nuclear penetrating peptides, as described in Depray, et al (Bioconjug Chem, 2019 Apr 17; 30(4): 1006-1027).
  • the SNAP penetration assay can assess delivery to the cytosol or nucleus. Additionally, SNAP penetration assay may avoid possible false positives from surface-bound peptides, which may result from using fluorophore labels combined with alternative methods such as fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • the SNAP penetration assay uses SNAP -tag comprising a 20 kDa mutant of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (SNAP-tag) which reacts specifically and rapidly with benzylguanine (BG) derivatives (BG-substrate) (e.g., such BG-substrate conjugated to a peptide (BG-peptide) as a synthetic probe, test peptide, or substrate for the SNAP-tag in the SNAPPA assay, and similarly such BG-substrate can be attached to a small molecule that is known to enter cells for use as a positive control in SNAPPA), leading to irreversible covalent labeling of the SNAP-tag with the synthetic probe, as described in Keppler, et al (Nat.
  • Unoccupied SNAP-tag binding cites may be detected by subsequently incubating with a BG-fluorophore to covalently bind SNAP -tags not bound by BG-peptides.
  • the labeling reaction is measured by detecting fluorescence, the absence of which, relative to the negative control, is indicative of the SNAP-tag covalently bound to the BG- peptide.
  • the small size of the SNAP-tag and relatively neutral charge may be favorable for the SNAP penetration assay. Formation of a covalent bond during the SNAP reaction may facilitate recovery and identification of bound species, for example using mass spectrometry.
  • Such BG derivatives are also referred to herein as BG-substrate or SNAP substrates.
  • SNAP substrates are chemically inert towards other proteins, avoiding nonspecific labeling in cellular applications.
  • a BG-substrate is conjugated to the peptide being tested for cell penetration, creating a BG-peptide.
  • SNAP-tag is expressed in the cell itself, either without organelle targeting or with nuclear localization by way of histone H2B fusion.
  • BG-GLA resembles the enzyme’s native substrate, an alkylated guanine nucleotide.
  • the enzyme can cause the BG-GLA to be covalently bound to the SNAP protein.
  • species covalently attached to the BG- GLA for example via NHS reaction, are also covalently attached to the SNAP -tag, blocking it from further reactions.
  • the BG-peptide for example generated by mixing BG-GLA-NHS with peptides of interest resulting in the BG-GLA to covalently bond with solvent-accessible primary amines in the peptide, can be added to the cell culture media, and if it is cell penetrant, it can enter the cytosol and become bound to a SNAP protein there.
  • the SNAP -tag in the cells may become saturated, and subsequently-dosed fluorescently-labeled BG-GLA, which can penetrate cells due to its small size, may have no SNAP protein with which to react. If, however, the BG-GLA is attached to a peptide without cell penetration capabilities, then the attached BG-GLA may not come into contact with the SNAP protein upon cell media incubation. This leaves the SNAP protein un-saturated and may react with subsequently-dosed BG- fluorophore, rendering the cells fluorescent.
  • plasmid vector for SNAP -tag pSNAPf
  • H2B-tagged SNAP -tag pSNAPf-H2B
  • Plasmids can be sequence validated and purified by Endofree Maxiprep before transfection into cell lines, such as NIH3T3, HeLa or HEK293 cells using Lipofectamine 2000. Following a period of recovery, selection for positive transfectants can be initiated using geneticin over a period of 2-3 weeks. The pool of stable transfectants can be expanded and banked in liquid nitrogen.
  • SNAP conjugation and purification of SNAP-CDPs can be produced and purified, as described in Banechayake et al., Nucleic Acids Res. 2011 Nov; 39(21): el43.
  • lyophilized peptides can be resuspended in PBS to an approximate 1 mg/mL concentration.
  • SNAP substrate can be attached to reactive amine groups on peptides via NHS-ester reaction.
  • Benzylguanine-NHS (BG-GLA-NHS) can be obtained from a commercial vendor and resuspended at 10 pg/uL in anhydrous DMSO.
  • BG-GLA-NHS One molar equivalent of BG-GLA-NHS can be added to resuspended peptide and reaction can be allowed to proceed with stirring at room temperature for one hour. Samples of starting material and crude reaction mixture can be analyzed by HPLC/MS to assess reaction progress. For peptides with only one reactive amine group, a total of three molar equivalents can be added over the course of three hours. For peptides with multiple reactive amine groups, reactions can be halted when the presence of multi-SNAP substrate-tagged species becomes apparent. A molar reaction or reaction time can be chosen to target a single SNAP substrate to be conjugated per peptide. The reaction can occur with the N-terminus of the peptide. The reaction can occur with one or more lysine residues of a peptide. Additionally, if precipitation is noted, addition of BG- GLA-NHS can be halted.
  • BG-GLA- NHS e.g., BG-GLA-OH
  • SEC size exclusion chromatography
  • BG-peptides can be separated and eluted over 1.5 column volumes. Fractions with notable 280 nm/214 nm/200 nm absorbance can be pooled and concentrated through a 3 kDa molecular weight cut off spin filter. Individual BG-peptides may elute in distinct volumes, often around 1 column volume. BG-peptides can be verified using HPLC/MS BG-peptide concentration can be assessed using the bicinchoninic acid assay.
  • BG-peptides and controls can be diluted in standard cell growth media (DMEM +10% FBS +pen/strep). 2 pM hydrolyzed BG-GLA-NHS (hydrolyzed to BG-GLA-OH) can be used as a positive control for cell penetration, while PBS can serve as a negative control. An additional mock conjugation/purification of BG-GLA-NHS can also serve as a negative control for purification.
  • pSNAPf and pSNAPf-H2B cell lines can be plated on collagen-coated 96-well plates, 20,000 cells/well, and can be allowed to recover in growth media overnight.
  • the plates can be rinsed with PBS and incubated with diluted BG-peptides and controls in growth media for 2 hours at 37° C.
  • the cells can then be incubated with labelling media containing 600 nM SNAP-Cell TMR-Star and 1 pg/mL Hoescht 33342 in growth media for 15 minutes at 37° C.
  • the labelling media may contain a SNAP substrate conjugated to a rhodamine dye, an Alexa fluor, an Atto dye, a cyanine dye, or a fluorescein dye.
  • the labelling media can be removed, and cells can be incubated with fresh growth media for 30 minutes at 37° C as a washout phase for excess SNAP-Cell TMR-Star dye.
  • the media can be replaced with phenol red-free OptiMEM for imaging.
  • Imaging can be performed on a Molecular Devices ImageXpress Nano, utilizing DAPI and Texas Red filters.
  • cells can be segmented using ImageXpress software to generate counts for nuclei and average cytoplasmic or nuclear intensity of SNAP-Cell TMR- Star.
  • the average intensity can then be normalized using the positive control of 2 pM hydrolyzed BG-GLA-NHS (BG-GLA-OH) as 100% cell penetration and PBS as 0% penetration to determine normalized SNAP -tag occupancy.
  • the degree of cell penetration is inversely proportional to the level of fluorescence in the cells.
  • pSNAPf cells can be preincubated with a variety of endocytosis inhibitors to assess mechanisms responsible for uptake of BG-peptides.
  • Macropinocytosis can be inhibited using 50 pM ethylisopropyl amiloride (EIP A), which blocks Na+/H+ exchangers and prevents formation of macropinosomes.
  • EIP A ethylisopropyl amiloride
  • Clathrin-mediated endocytosis can be inhibited using either 20 pM nocodazole, which inhibits microtubule polymerization, 3 pM cytochalasin D, which interferes with actin polymerization, or 80 pM dynasore, an inhibitor of dynamin GTPase.
  • Endosomal acidification or lysosomal maturation can be inhibited using 50 nM bafilomycin A, which inhibits the vacuolar ATPase complex, or 50 pM chloroquine, which diffuses into endosomes as a weak base.
  • pSNAPf cells can be preincubated with the specified concentration of inhibitor in growth media for 1 hour prior to SNAPPA, which can be performed as described herein.
  • cells expressing a labeled SNAP -tag in either the cytoplasm or the nucleus can be cultured in a microplate format.
  • the labeled SNAP -tag is labeled with a green fluorescent protein, a yellow florescent protein, a red fluorescent protein, a blue fluorescent protein, or a cyan fluorescent protein.
  • Candidate cell-penetrating peptides can be tagged with SNAP substrate and purified using the method described herein. Each BG-candidate peptide can be added to the cells expressing labeled SNAP -tag in a well of the microplate.
  • Peptides capable of cell penetration may cross the cell membrane and covalently bind to the labeled SNAP -tag expressed in the cytoplasm or the nucleus. Following incubation, excess BG-candidate peptides that did not enter the cells can be washed off. Cells can be pooled and lysed, and labeled SNAP- tag that reacted with cell penetrant BG-peptides can be purified by immunoprecipitation via the label. Candidate peptides that entered the cell can be immunoprecipitated with the SNAP -tag. Cell -penetrating peptides can be identified by mass spectrometry.
  • SDPR protease resistance testing can be used with sequencing-grade enzymes, including Trypsin and Chymotrypsin. Trypsin and trypsin inhibitor can be used for HPLC analysis.
  • screens for cell penetration can be assessed by Illumina sequencing.
  • Illumina sequencing involves collecting cell pellets (1.5E6, 3 technical replicates) resupended in 50 pL Terra Direct PCRMix (from Clontech) and amplified for 16 cycles using the original cloning primers. Up to four aliquots can be diluted 16- fold into 60 pL Phusion DNA Polymerase reactions and amplified using distinct Illumina primers, containing adaptor sequences for flow cell adherence. Forward primers can include a 6 bp barcode for multiplexing.
  • Illumina HiSeq 2500 in rapid mode can be used to run samples, which Bowtie2 software can be used for mapping, and Excel (Microsoft) and MATLAB (MathWorks) used for data analysis.
  • a pharmaceutical composition of the disclosure can be a combination of any peptide as described herein with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, antioxidants, solubilizers, buffers, osmolytes, salts, surfactants, amino acids, encapsulating agents, bulking agents, cryoprotectants, and/or excipients.
  • the pharmaceutical composition facilitates administration of a peptide described herein to an organism.
  • the pharmaceutical composition comprises factors that extend half-life of the peptide and/or help the peptide to penetrate the target cells.
  • compositions can be administered in therapeutically-effective amounts as pharmaceutical compositions by various forms and routes including, for example, intravenous, subcutaneous, intramuscular, rectal, aerosol, parenteral, ophthalmic, pulmonary, transdermal, vaginal, optic, nasal, oral, sublingual, inhalation, dermal, intrathecal, intravitreal, intratumoral, intranasal, and topical administration.
  • a pharmaceutical composition can be administered in a local or systemic manner, for example, via injection of the peptide described herein directly into an organ, optionally in a depot.
  • Parenteral injections can be formulated for bolus injection or continuous infusion.
  • the pharmaceutical compositions can be in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Pharmaceutical formulations for parenteral administration include aqueous solutions of a peptide described herein in water-soluble form. Suspensions of peptide-antibody complexes described herein can be prepared as oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension can also contain suitable stabilizers or agents which increase the solubility and/or reduce the aggregation of such peptide-antibody complexes described herein to allow for the preparation of highly concentrated solutions.
  • the cell-penetrating peptides described herein can be used to increase cell penetration of a cargo molecule (e g., a therapeutic agent) and deliver the cargo molecule to a cellular compartment.
  • a cargo molecule e g., a therapeutic agent
  • protein transfection agents, direct cytosolic expression of the peptide, or electrophoration of the peptide can be used to increase cell penetration.
  • other excipients can be formulated with a peptide in order to increase the cell penetration of the peptide, such as those approaches described in “Protein and Peptide Drug Delivery: Oral Approaches” Indian J. Pharm. Sci., Shaji and Patole, v70(3) 269-277, 2008. Any combination of these formulations or approaches can be used to increase cell penetration of a peptide as described herein.
  • the peptide described herein can be lyophilized or in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a purified peptide is administered intravenously.
  • a peptide described herein can be administered to a subject, home, target, migrate to, or be directed to cancerous cell, a tumor, or a cell with dysregulated HIPPO pathway.
  • a peptide can be conjugated to, linked to, or fused to another peptide that provides a targeting function to a specific target cell type in the central nervous system or across the blood brain barrier.
  • a peptide of the disclosure can be applied directly to an organ, or an organ tissue or cells, such as brain or brain tissue or cells, during a surgical procedure.
  • the recombinant peptide described herein can be administered topically and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams, and ointments.
  • Such pharmaceutical compositions can contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.
  • therapeutically effective amounts of a peptide described herein can be administered in pharmaceutical compositions to a subject suffering from a condition that affects the immune system.
  • the subject is a mammal such as a human or a primate.
  • a therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors.
  • a peptide is cloned into a viral or non-viral expression vector.
  • Such expression vector can be packaged in a viral particle, a virion, or a non-viral carrier or delivery mechanism, which is administered to patients in the form of gene therapy.
  • patient cells are extracted and modified to express a cell-penetrating peptide ex vivo before the modified cells are returned back to the patient in the form of a cell-based therapy, such that the modified cells will express the peptide once transplanted back in the patient.
  • compositions can be formulated using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations that can be used pharmaceutically. Formulation can be modified depending upon the route of administration chosen.
  • Pharmaceutical compositions comprising a peptide described herein can be manufactured, for example, by expressing the peptide in a recombinant system, purifying the peptide, lyophilizing the peptide, mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or compression processes.
  • the pharmaceutical compositions can include at least one pharmaceutically acceptable carrier, diluent, or excipient and compounds described herein as free-base or pharmaceutically acceptable salt form.
  • Methods for the preparation of peptide described herein comprising the compounds described herein include formulating peptide described herein with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid, or liquid composition.
  • Solid compositions include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories. These compositions can also contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically acceptable additives.
  • Non-limiting examples of pharmaceutically-acceptable excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), each of which is incorporated by reference in its entirety.
  • compositions can also include permeation or absorption enhancers (Aungst et al. AAPS J. 14(l):10-8. (2012) and Moroz et al. Adv Drug Deliv Rev 101: 108-21. (2016)).
  • Permeation enhancers can facilitate uptake of molecules from the GI tract into systemic circulation.
  • Permeation enhancers can include salts of medium chain fatty acids, sodium caprate, sodium caprylate, N-(8-[2-hydroxybenzoyl]amino)caprylic acid (SNAC), N-(5- chlorosalicyloyl)-8-aminocaprylic acid (5-CNAC), hydrophilic aromatic alcohols such as phenoxyethanol, benzyl alcohol, and phenyl alcohol, chitosan, alkyl glycosides, dodecyl-2-N,N- dimethylamino propionate (DDAIPP), chelators of divalent cations including EDTA, EGTA, and citric acid, sodium alkyl sulfate, sodium salicylate, lecithin-based, or bile salt-derived agents such as deoxy cholates,
  • SNAC N-(8-[2-hydroxybenzoyl]amino)caprylic acid
  • 5-CNAC N-(5- chlorosalicyloyl)-8-a
  • compositions can also include protease inhibitors including soybean trypsin inhibitor, aprotinin, sodium glycocholate, camostat mesilate, vacitracin, or cyclopentadecalactone, Use of Peptides in Treatments
  • the cell-penetrating peptides described herein may be conjugated to, linked to, or fused with a therapeutic agent for use in a method of treatment.
  • the cell-penetrating peptides or cell-penetrating peptide complexes may be used to deliver a therapeutic agent that may otherwise be excluded from a cellular compartment across a cellular layer (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen) an into a target cell. Delivery of the therapeutic agent using the cell -penetrating peptides described herein may increase access to a target (e.g., a target protein or nucleotide sequence) by the therapeutic agent.
  • a target e.g., a target protein or nucleotide sequence
  • the method includes administering an effective amount of a cell -penetrating peptide-therapeutic agent conjugate as described herein to a subject in need thereof.
  • a cell-penetrating peptide-therapeutic agent conjugate may be administered to the subject in need thereof to treat a disease or a condition in the subject.
  • a therapeutic agent that may be delivered into a cell by a cellpenetrating peptide of the present disclosure may include an antibody, an antibody fragment, an Fc domain, a single chain Fv, an intrabody, or a nanobody.
  • the therapeutic agent may be a cystine-dense peptide, an affibody, a B-hairpin, an avimer, an adnectin, a stapled peptide, a nannofittin, a kunitz domain, a fynomer, or a bicyclic peptide.
  • the therapeutic agent may be an anti -cancer agent and may be delivered into a cancer cell.
  • the therapeutic agent may be a peptide (e.g., a cystine- dense peptide), a transcription factor, an RNA, a Cas enzyme or other CRISPR component, an immunomodulating agent, or a hormone.
  • an active agent such as a cystine-dense peptide, that binds to a transcription factor (such as a forkhead box class O transcription factor (FOXO), Nuclear factor E2 -related factor 2 (NRF2), runt-related transcription factor 1 (RUNX1), methyl CpG binding protein 2 transcription factor (MECP2), maturity onset diabetes of the young transcription factor (MODY), forkhead box P3 (FOXP3), p53, p65, signal transducer and activator of transcription (STAT), homeodomain transcription factor (HOX), or SOX9 transcription factor) may be conjugated to a cell -penetrating peptide and delivered to a nucleus of a cell to alter the expression level of a target gene.
  • a transcription factor such
  • a cellpenetrating peptide is conjugated to NRF2 to carry NRF2 to the nucleus, facilitate transcription of antioxi dative genes, and mediate recovery from acetaminophen-induced hepatotoxicity.
  • a cell-penetrating peptide is conjugated to RUNX1 to carry RUNX1 to the nucleus, regulate gene expression, and mediate cartilage deposition.
  • a cell-penetrating peptide complex of the present disclosure may target AKT, mTOR, STAT3, RAS, RAF, MEK, ERK, -catenin, IKK, TEAD, NF-KB, PKC, AP-1, Jun, Fos, REL, a transcription factor, Ras, Rho, Ran, Rab, Arf, androgen receptor, ikaros, aiolos, nuclear receptors, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-3, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CS
  • ADP- Ribosylation Factor (Arf) GTPases are part of the Ras superfamily used in regulation of vesicular cellular trafficking, lipid modification, cytokinesis and cell adhesion.
  • Arf GTPases are tightly regulated by specific guanine nucleotide exchange factors (GEFs) with a conserved Sec7 domain, and GTPase-activating proteins (GAPs) with a conserved zinc finger domain. Like all Ras superfamily members, the GEFs switch on/activate Arfs while the GAPS switch off/inactivate them.
  • GEFs guanine nucleotide exchange factors
  • GAPs GTPase-activating proteins
  • ADP-Ribosylation Factor (Arf) GTPases, guanine nucleotide exchange factors (GEFs) with a conserved Sec7 domain, and GTPase-activating proteins (GAPs), using the cellpenetrating peptide of the invention can be used in cancer therapy, including metastatic phases of cancer.
  • one or more CRISPR components e g., guide RNA, a tracrRNA, a crRNA, or a Cas nuclease
  • an immunomodulating agent may be conjugated to a cell-penetrating peptide and delivered into a cell to modulate or inhibit an immune response.
  • a therapeutic agent for treatment of neurodegeneration may be conjugated to a cell-penetrating peptide and delivered across the blood brain barrier to treat a neurodegenerative disorder (e.g., Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, or amyotrophic lateral sclerosis (ALS)).
  • a neurodegenerative disorder e.g., Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, or amyotrophic lateral sclerosis (ALS)
  • the therapeutic agent may facilitate degradation of excess proteins in a target cell upon delivery to the target cell by the cell-penetrating peptide.
  • a method of treating a condition may comprise targeted degradation of a target molecule, such as a target protein.
  • the condition is associated with increased activity, increased expression, or novel activity brought on by gene amplification, missense mutation, nonsense mutation, aberrant splicing, or fusion protein brought on by chromosomal translocation of the target molecule.
  • a method of treatment may comprise administering to a subject a cell-penetrating peptide complex comprising a cellpenetrating peptide linked or fused to a cargo molecule comprising a target-binding peptide (e.g., a target-binding CDP) and a ubiquitin ligase-binding molecule.
  • a target-binding peptide e.g., a target-binding CDP
  • a method of treatment can also comprise administering to a subject a cell-penetrating peptide complex comprising a cellpenetrating peptide linked or fused to a cargo molecule comprising a target-binding peptide (e.g., a target-binding CDP) and a molecule that binds any part of the ubiquitin-proteasome system (UPS) or cytosolic protein degradation machinery, such as the 26S proteosome, or any component thereof such as the 19S ubiquitin recognition proteins Rpn-13 and Rpn-10. Binding to the UPS could be through an inhibitor, such as KDT-11, which inhibits Rpn-13.
  • a target-binding peptide e.g., a target-binding CDP
  • UPS ubiquitin-proteasome system
  • cytosolic protein degradation machinery such as the 26S proteosome, or any component thereof such as the 19S ubiquitin recognition proteins Rpn-13 and Rpn-10. Binding
  • the peptide complex may cross a cellular layer of a cell (e.g., a cancer cell, pancreatic cell, liver cell, colon cell, ovarian cell, breast cell, lung cell, spleen cell, or bone marrow cell) to access the target and the ubiquitin ligase in a cellular compartment (e.g., the cytosol or the nucleus) or intercellular compartment (e.g., a nanolumen, intercellular space, or paracellular space).
  • a cell e.g., a cancer cell, pancreatic cell, liver cell, colon cell, ovarian cell, breast cell, lung cell, spleen cell, or bone marrow cell
  • a cellular compartment e.g., the cytosol or the nucleus
  • intercellular compartment e.g., a nanolumen, intercellular space, or paracellular space.
  • the cargo molecule may bind to the target and the ubiquitin ligase, forming a ternary complex.
  • the ubiquitin ligase may ubiquitinate the target, labeling it for proteasomal degradation, thereby treating the condition.
  • a method of treatment may comprise administering to a subject a cell -penetrating peptide complex comprising a cell-penetrating peptide linked or fused to a cargo molecule comprising a PROTAC, a molecular glue, a targetbinding peptide, or other cargos that cause targeted protein degradation.
  • the cargo molecule may comprise an IMiD, a Boc3Arg, an adamantyl group, or a carborane.
  • the peptide complex may cross a cellular layer of a cell, such as by entering the cytosol, and bind, label, target, traffic, or otherwise direct a protein or other target for degradation, such as by the ubiquitin-proteosome system.
  • a method of treating a condition may comprise inhibition of a target molecule.
  • the condition is associated with increased activity, increased expression, or novel activity brought on by gene amplification, missense mutation, nonsense mutation, aberrant splicing, or fusion protein brought on by chromosomal translocation of the target molecule, such as a target protein.
  • Inhibiting the target molecule may comprise inhibiting a conformational change, inhibiting an enzymatic activity, inhibiting ligand binding, inhibiting a protein-protein interaction, or inhibiting a protein-nucleic acid interaction of the target molecule.
  • a method of treating a condition may comprise administering to a subject a cell-penetrating peptide complex comprising a cell -penetrating peptide linked or fused to a cargo molecule that binds to and inhibits a target.
  • the cargo molecule may comprise a target-binding peptide (e.g., a target-binding CDP).
  • the peptide complex may cross a cellular layer of a cell (e g , a cancer cell, pancreatic cell, liver cell, colon cell, ovarian cell, breast cell, lung cell, spleen cell, or bone marrow cell) to access the target in a cellular compartment (e g., the cytosol or the nucleus) or intercellular compartment (e g., a nanolumen, intercellular space, or paracellular space).
  • a cellular compartment e g., the cytosol or the nucleus
  • intercellular compartment e g., a nanolumen, intercellular space, or paracellular space.
  • the term “effective amount,” as used herein, refers to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. Compositions containing such agents or compounds can be administered for prophylactic, enhancing, and/or therapeutic treatments. An appropriate “effective” amount in any individual case may be determined using techniques, such as a dose escalation study.
  • the methods, compositions, and kits of this disclosure may comprise a method to prevent, treat, arrest, reverse, or ameliorate the symptoms of a condition.
  • the treatment may comprise treating a subject (e.g., an individual, a domestic animal, a wild animal, or a lab animal afflicted with a disease or condition) with a peptide of the disclosure.
  • the disease may be a cancer or tumor.
  • the peptide may contact the tumor or cancerous cells.
  • the subject may be a human.
  • Subjects can be humans; non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
  • a subject can be of any age.
  • Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, infants, and fetuses in utero.
  • Treatment may be provided to the subject before clinical onset of disease. Treatment may be provided to the subject after clinical onset of disease. Treatment may be provided to the subject after 1 day, 1 week, 6 months, 12 months, or 2 years or more after clinical onset of the disease. Treatment may be provided to the subject for more than 1 day, 1 week, 1 month, 6 months, 12 months, 2 years or more after clinical onset of disease. Treatment may be provided to the subject for less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment may also include treating a human in a clinical trial.
  • a treatment can comprise administering to a subject a pharmaceutical composition, such as one or more of the pharmaceutical compositions described throughout the disclosure.
  • a treatment can comprise a once daily dosing.
  • a treatment can comprise delivering a peptide of the disclosure to a subject, either intravenously, subcutaneously, intramuscularly, by inhalation, dermally, topically, by intra-articular injection, orally, sublingually, intrathecally, intravitreally, transdermally, intranasally, via a peritoneal route, directly into a tumor, e.g., injection directly into a tumor, directly into the brain, e.g., via and intracerebral ventricle route, or directly onto a joint, e.g., via topical, intra-articular injection route.
  • a treatment can comprise administering a peptide-active agent conjugate to a subject, either intravenously, subcutaneously, intramuscularly, by inhalation, by intra-articular injection, dermally, topically, orally, intrathecally, intravitreally, transdermally, intransally, parenterally, orally, via a peritoneal route, nasally, sublingually, or directly onto cancerous tissues.
  • compositions comprising any one of the cell-penetrating peptides described herein conjugated to a therapeutic agent (e.g., an anti-cancer agent), or a pharmaceutical composition thereof, can be used in a method of treating a cancer, tumor progression, and/or dysregulated cell growth.
  • a therapeutic agent e.g., an anti-cancer agent
  • Exemplary diseases, disorder, or condition include: multiple myeloma, plastic anemia, myelodysplasia, and related bone marrow failure syndromes, myeloproliferative diseases, acute and chronic myeloid leukemia, malignancies of lymphoid cells, hematologic malignancies, plasma cell disorders, skeletal muscle disorder, myopathy, muscular dystrophy (e.g., Becker muscular dystrophy, Duchenne muscular dystrophy, Emery -Dreifuss muscular dystrophy, Facioscapulohumoeral muscular dystrophy, Myotonia congentia, and myotonic dystrophy), Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, or amyotrophic lateral sclerosis (ALS), and chronic obstructive pulmonary disorder.
  • multiple myeloma plastic anemia, myelodysplasia, and related bone marrow failure syndromes
  • myeloproliferative diseases acute and chronic myeloid leukemia, mal
  • compositions/peptides disclosed herein are used to treat dysregulated cell growth, cancer, tumor, and/or metastasis associated with any of the following cell, tissue, or organ types: brain, lung, liver, spleen, kidney, lymph node, small intestine, blood cell, pancreatic, colon, stomach, cervix, breast, endometrial, prostate, testicle, ovarian, skin, head and neck, esophageal, oral tissue, and bone marrow.
  • compositions/peptides disclosed herein are used to treat any of the following: osteosarcoma, hepatocellular carcinoma, malignant mesothelioma, schwannoma, meningioma, renal carcinoma, cholangiocarcinoma, bile duct hamartoma, soft tissue carcinoma, ovarian carcinoma, colonic adenoma, T cell acute lymphoblastic leukaemia, gastrointestinal hyperplasia, fibrosarcoma, pancreatic ductal metaplasia, squamous cell carcinoma, kaposis sarcoma, and HIV-induced non-Hodgkin’s lymphoma.
  • the peptides of this disclosure (e g., SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) conjugated to a therapeutic agent can be used to access and treat these disorders by delivering the therapeutic agent across a cellular layer (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen) to a cellular compartment.
  • a cellular layer e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen
  • Delivery of a therapeutic agent using a cell -penetrating peptide of the present disclosure may increase the efficacy of the therapeutic agent by increasing access of the therapeutic agent to intracellular targets (e g., cytosolic proteins or target nucleic acid sequences) relative to the therapeutic agent alone.
  • intracellular targets e g., cytosolic proteins or target nucleic acid sequences
  • the cell penetrating peptides of this disclosure e g., SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, and SEQ ID NO: 195 - SEQ ID NO: 254
  • complexes described herein can deliver a cargo molecule to the cytosol or the nucleus of a cell.
  • the cargo molecule is delivered to the cytosol to achieve a cytosolic concentration of at least 1 nM, at least 5 nM, at least 10 nM, at least 25 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, at least 1000 nM, at least 1100 nM, at least 1200 nM, at least 1300 nM, at least 1400 nM, at least 1500 nM, at least 1600 nM, at least 1700 nM, at least 1800 nM, or at least 2000 nM.
  • the cargo molecule is delivered to the cytosol to achieve a cytosolic concentration of from about 1 nM to about 50 nM, from about 10 nM to about 100 nM, from about 50 nm to about 100 nm, from about 50 nm to about 200 nm, from about 50 nm to about 500 nm, from about 50 nm to about 700 nm, from about 50 nm to about 1000 nm, from about 50 nm to about 1200 nm, from about 50 nm to about 1500 nm, from about 50 nm to about 2000 nm, from about 100 nm to about 200 nm, from about 100 nm to about 500 nm, from about 100 nm to about 700 nm, from about 100 nm to about 1000 nm, from about 100 nm to about 1200 nm, from about 100 nm to about 1500 nm, from about 100 nm to about 2000 nm, from about 100 nm to about 200
  • the cargo molecule is delivered to the nucleus to achieve a nuclear concentration of at least 1 nM, at least 5 nM, at least 10 nM, at least 25 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, at least 1000 nM, at least 1100 nM, at least 1200 nM, at least 1300 nM, at least 1400 nM, at least 1500 nM, at least 1600 nM, at least 1700 nM, at least 1800 nM, or at least 2000 nM.
  • the cargo molecule is delivered to the nucleus to achieve a nuclear concentration of from about 1 nM to about 50 nM, from about 10 nM to about 100 nM, from about 50 nm to about 100 nm, from about 50 nm to about 200 nm, from about 50 nm to about 500 nm, from about 50 nm to about 700 nm, from about 50 nm to about 1000 nm, from about 50 nm to about 1200 nm, from about 50 nm to about 1500 nm, from about 50 nm to about 2000 nm, from about 100 nm to about 200 nm, from about 100 nm to about 500 nm, from about 100 nm to about 700 nm, from about 100 nm to about 1000 nm, from about 100 nm to about 1200 nm, from about 100 nm to about 1500 nm, from about 100 nm to about 2000 nm, from about 500 nm to about 1000 nm,
  • a peptide (e.g., a cell-penetrating peptide) may be linked, conjugated, complexed with, or fused to a nucleotide via various chemistries resulting in peptide oligonucleotide complexes that may form either a cleavable or stable linkage to deliver the oligonucleotide to a cell.
  • a cell-penetrating peptide may delivery an oligonucleotide to the cytoplasm or nucleus, or both, of a cell.
  • nucleotides within the peptide oligonucleotide complex can function within the nucleus of a cell, including gapmers, ASO splice blockers, and U1 adapters. Others function within the cytosol, including siRNA and anti- miRs. Aptamers are unique in that they do not function through hybridization or base paring interactions with nucleic acid targets. Instead, aptamers form secondary structures to bind to proteins or other macromolecules. Aptamers may function wherever the target protein or macromolecule is located.
  • the nucleotide portion of the peptide oligonucleotide complexes described herein may target specific RNAs (e.g., mRNAs or pre-mRNAs) from genes expressed in cancer and other diseases.
  • the nucleotide sequence in the complex may be complementary to any target provided in SEQ ID NO: 574 - SEQ ID NO: 611, TABLE 12, or TABLE 6.
  • the nucleotide sequence in the complex may be complementary to the target RNA, or in the case of an aptamer, may bind a target protein or other macromolecule.
  • the a nucleotide sequence may be single stranded (ssDNA, ssRNA) or double stranded (dsDNA, dsRNA) or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or Ul Adapter.
  • ssDNA, ssRNA double stranded
  • dsDNA double stranded
  • dsRNA double stranded
  • a combination of single and double stranded for example with a mismatched sequence, hairpin or other structure
  • an antisense RNA
  • a target of the nucleotide in a peptide oligonucleotide complex may be a gastrointestinal target, such as a gene with pro-inflammatory, extracellular matrixmodifying, or immune cell recruitment functionality.
  • Peptide oligonucleotide complexes described herein e.g., a peptide oligonucleotide complex comprising a cell-penetrating peptide and a nucleotide that binds a gene target mRNA
  • target gastrointestinal gene targets may be used to treat various gastrointestinal disorders, including inflammatory bowel disease (IBD), ulcerative colitis, and Crohn’s disease.
  • a target of the nucleotide in a peptide oligonucleotide complex may be a cancer target, such as a gene involved in oncogenic signaling, anti-apoptotic genes, pro-inflammatory signaling genes, protein homeostasis genes, developmental regulatory genes, or adapter protein genes that initiate downstream cell growth signaling.
  • a cancer target such as a gene involved in oncogenic signaling, anti-apoptotic genes, pro-inflammatory signaling genes, protein homeostasis genes, developmental regulatory genes, or adapter protein genes that initiate downstream cell growth signaling.
  • HER2 and other RTK e.g., EGFR, ERBB3
  • Knockdown of Grb2 can halt signaling in a way that is difficult to mutationally compensate as Grb2 loss is epistatic to HER2 activity.
  • a pro-inflammatory cytokine may be delivered via an mRNA in a peptide oligonucleotide complex, or an antisense construct targeting an anti-inflammatory signal may be delivered.
  • Peptide oligonucleotide complexes described herein e.g., a peptide oligonucleotide complex comprising a cell-penetrating peptide and a nucleotide that binds a gene target mRNA
  • target cancer gene targets may be used to treat various cancers, including solid tumors.
  • Developmental regulators such as transcription factors involved in early cell fate and pluripotency, and chromatin remodeling enzymes, may be targeted to specifically harm dedifferentiated cells which may be present in advanced tumors and associated with a more mobile and/or mitotic cell state.
  • a peptide oligonucleotide construct targeting a cancer target may treat or prevent cancer by reducing oncogenic signaling, reducing target over-expression, reducing oncogenic antisense activity (e g., miRNAs targeting tumor suppressors), and/or eliminating the source of the oncogenic signaling cascade.
  • gene targets e.g., gastrointestinal, or cancer gene targets
  • an oligonucleotide may target a gene for downregulation.
  • An example of an antagomir that may be complexed with a cell-penetrating peptide to target a gene includes cobomarsen.
  • An example of an aptamer that may be complexed with a cell-penetrating peptide to target a gene includes pegaptanib.
  • Examples of gapmers that may be complexed with a cell-penetrating peptide to target a gene include fomivirsen, mipomersen, inotersen, volanesorsen, tofersen, tominersen, pelacarsen, alicaforsen, apatorsen, and trabedersen.
  • splice blockers that may be complexed with a cell-penetrating peptide to target a gene include nusinersen, eteplirsen, golodirsen, viltolarsen, casimersen, and sepofarsen.
  • An example of a translation blocker that may be complexed with a cell-penetrating peptide to target a gene includes prexigebersen.
  • any targets for the nucleic acid portion of the peptide oligonucleotide complex described herein can be used in conjunction with a U1 adapter to degrade targeted mRNAs.
  • the target recognition (or complementary nucleic acid to the target mRNA) portion directs the peptide oligonucleotide complex to the targeted mRNA selected for degradation, while the U1 portion prevents the addition of polyA to the mRNA resulting in degradation of the targeted mRNA.
  • U1 adapters can comprise any nucleotide sequence complementary to the ssRNA component of the U1 small nuclear ribonucleoprotein (U1 snRNP). In some embodiments, the U1 adapter sequences engage the U1 snRNP near its poly A site.
  • the length of the U1 adapter is 15 to 25 nt in length, or about 20 nt in length. In some embodiments, the U1 adapter is above 40% in its G/C content.
  • Exemplary U1 adapters are shown in TABLE 13, in conjunction with a target nucleic acid “target recognition” portion which comprises a nucleotide sequence is single stranded (ssDNA, ssRNA) or double stranded (dsDNA, dsRNA) or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, or splice blocker ASO.
  • Exemplary U1 adapters include: UCCCCUGCCAGGUAAGUAU (SEQ ID NO: 488); CCCUGCCAGGUAAGUAU (SEQ ID NO: 489); CUGCCAGGUAAGUAU (SEQ ID NO: 490); UGCCAGGUAAGUAU (SEQ ID NO: 491); GCCAGGUAAGUAU (SEQ ID NO: 492); CCAGGUAAGUAU (SEQ ID NO: 493); CAGGUAAGUAU (SEQ ID NO: 494); and CAGGUAAGUA (SEQ ID NO: 495).
  • peptides described herein can be provided as a kit.
  • peptide complexes described herein can be provided as a kit.
  • a kit comprises amino acids encoding a peptide described herein, a vector, a host organism, and an instruction manual.
  • a kit includes written instructions on the use or administration of the peptides.
  • the peptide sequence was reverse-translated into DNA, synthesized, and cloned in-frame with siderocalin using standard molecular biology techniques (M R. Green, Joseph Sambrook. Molecular Cloning. 2012 Cold Spring Harbor Press). The resulting construct was packaged into a lentivirus, transduced into HEK-293 cells, expanded, isolated by immobilized metal affinity chromatography (IMAC), cleaved with tobacco etch virus protease, and purified to homogeneity by reverse-phase chromatography. Following purification, each peptide was lyophilized and stored frozen.
  • IMAC immobilized metal affinity chromatography
  • This example describes generation of pSNAPf and pSNAPf-H2B cell lines.
  • a plasmid vector for SNAP -tag (pSNAPf) was obtained from New England Biolabs and histone H2B- tagged SNAP -tag (pSNAPf-H2B) was obtained from Addgene. Plasmids were sequence validated and purified by Endofree Maxiprep before transfection into NIH3T3, HeLa or HEK293 cells using Lipofectamine 2000. Following a period of recovery, selection for positive transfectants was initiated using geneticin over a period of 2-3 weeks. The pool of stable transfectants was expanded and banked in liquid nitrogen.
  • This example describes conjugation and purification of BG-peptides and generation of BG-substrate positive control reagents.
  • Peptides were produced and purified as described in EXAMPLE 1.
  • SNAP substrate conjugation lyophilized peptides were resuspended in PBS to an approximate 1 mg/mL concentration.
  • a benzylguanine SNAP substrate (BG-GLA-NHS, also referred to as BG-substrate) was attached to reactive amine groups on peptides viaNHS- ester reaction. Reactive amine groups in peptides may include the N-terminus and may include one or more lysine residues.
  • FIG. 1A schematically illustrates the SNAP substrate tagging reaction between a benzylguanine NHS-ester and a reactive amine group of a peptide to generate a SNAP substrate-tagged peptide (also referred to as BG-peptide).
  • BG- GLA-NHS Benzylguanine-NHS
  • DMSO dimethyl sulfoxide
  • One molar equivalent of BG-GLA-NHS per mole of peptide was added to the resuspended peptide and the reaction was allowed to proceed while stirring at room temperature for one hour. Samples of starting material and crude reaction mixture were analyzed by HPLC/MS to assess reaction progress.
  • BG-GLA-NHS was added as needed. Generally, for peptides with only one reactive amine group, a total of three molar equivalents of BG-GLA-NHS was added over the course of three hours. For peptides with multiple reactive amine groups, reactions were halted when the presence of multi-BG-species became apparent, with of goal of producing a reaction product that was primarily a single SNAP substrate tag per peptide. Additionally, if precipitation was noted, addition of BG-GLA-NHS was halted.
  • BG-peptides were separated and eluted over 1.5 column volumes. Fractions with notable 280 nm/214 nm/200 nm absorbance were pooled and concentrated through a 3 kDa molecular weight cut off spin filter. Individual BG-peptides eluted in distinct volumes, though they generally occurred around 1 column volume. BG-peptides were thus purified and separated from any residual BG-GLA-OH. BG-peptides were verified using HPLC/MS. BG-peptide concentration was then assessed using the bicinchoninic acid assay. [0403] Representative images of RP-HPLC and SEC associated with a typical SNAP substrate conjugation reaction and purification process are shown in FIG.
  • Fhe first image (top) shows multiple elution peaks from an HPLC run on the crude reaction mixture representing SNAP substrate BG-GLA-NHS, hydrolyzed BG-GLA-NHS (hydrolyzed to BG-GLA-OH), unconjugated KR CTX (SEQ ID NO: 71) and KR CTX conjugated to SNAP substrate (BG- KR CTX, BG-SEQ ID NO: 71), showing absorbance at 214 nm. Peaks were validated by mass calculated by mass spectrometry (MS). The crude reaction mixture was then run through a size exclusion column (SEC). The SEC absorbance trace is shown in the second image. Details of the individual SEC peaks are shown in the bottom images. Fractions were pooled and concentrated before verifying identity using HPLC/MS.
  • MS mass spectrometry
  • BG-GLA-NHS Hydrolyzed BG-GLA-NHS, which is BG-GLA-OH, was created by incubating BG- GLA-NHS in PBS, pH 7.4, at room temperature for at least 10 minutes. BG-GLA-OH was used in assays as a positive control.
  • BG-GLA-NHS contains a reactive NHS ester, which can react with amine-containing molecules and with hydroxyl-containing molecules.
  • BG-GLA-NHS can react with amines present in peptides to form a BG-peptide conjugate.
  • BG-GLA-NHS can also react with water, becoming hydrolyzed, yielding BG-GLA-OH, which no longer reacts with amine or hydroxyl groups. If BG-GLA-NHS is added to an aqueous buffer, it will typically hydrolyze to form BG- GLA-OH. If BG-GLA-NHS is added to an amine-containing peptide within an aqueous buffer, some of it may react to form BG -peptide and some of it may hydrolyze to form BG-GLA-OH.
  • SNAPPA SNAP Penetration Assay
  • This example describes a SNAP penetration assay (SNAPPA) to identify cell-penetrating peptides.
  • the SNAP penetration assay is illustrated schematically in FIG. IB.
  • Cell-penetrating peptide candidates were labeled with a benzylguanine SNAP substrate as described in EXAMPLE 4.
  • the BG-peptides were incubated with cells expressing a SNAP-tag-protein, which will covalently bind to BG-peptides that enter the cell and reach the cytosol or nucleus where the SNAP-tag-protein is expressed.
  • Cells expressing the SNAP-tag-protein were generated as described in EXAMPLE 3.
  • BG-peptides and controls were diluted in standard cell growth media (DMEM +10% FBS +pen/strep).
  • FBS means fetal bovine serum.
  • 2 pM hydrolyzed BG-GLA-NHS (hydrolyzed to BG-GLA-OH) was used as a positive control for cell penetration, while PBS served as a negative control.
  • BG-GLA-OH is able to diffuse into cells and reach the cytosol, thus representing a positive control of the level of SNAPPA signal obtained when a molecule is cell penetrant.
  • an additional mock conjugation/purification of BG-GLA-NHS also served as a quality control for purification.
  • the mock reaction was performed by adding 1 molar equivalent (250 pM BG-GLA-NHS) to PBS and incubating at room temperature for 1 hour, which allows hydrolysis of BG-GLA-NHS. This was repeated until the concentration reached 750 pM.
  • This mixture of PBS and BG-GLA-NHS (which is hydrolyzed to BG-GLA-OH) was then run on SEC and equivalent fractions where BG-peptides have been found were pooled and concentrated as would be for BG-peptide production. This served as a control to demonstrate adequate purification of free BG-GLA-OH.
  • pSNAPf and pSNAPf-H2B cell lines were plated on collagen-coated 96-well plates, 20,000 cells/well, and allowed to recover in standard cell growth media overnight. Plates were rinsed with PBS and incubated with diluted BG-peptides and controls in standard cell growth media for 2 hours at 37° C. Peptides capable of cell penetration entered the cytosol and reached the SNAP- tag and then covalently bound to the SNAP-tag-protein expressed in the cells (FIG. IB, left path), while peptides that did not penetrate the cell did not bind to the SNAP-tag-protein (FIG. IB, right path).
  • BG-fluorophore SNAP-Cell TMR-Star dye; or SNAP-dye
  • BG-fluorophore 600nM SNAP-Cell TMR-Star
  • Hoescht 33342 1 pg/mL Hoescht 33342, which labels nuclei and is excited at 377 nm excitation
  • SNAP-tag-protein that was already bound to benzylguanine-peptides (BG-peptide) did not react with the BG-fluorophore.
  • the labelling media was removed, and cells were incubated with fresh growth media for 30 minutes at 37° C as a washout phase for excess SNAP-Cell TMR-Star dye. Excess BG-fluorophore was washed away, and the remaining fluorescence, detected by exciting at 562 nm, was inversely related to the amount of peptide that penetrated the cell membrane. Molecules in cells with unbound SNAP-tag-protein exhibited fluorescence from the reaction between the SNAP-tag-protein and BG-fluorophore.
  • Molecules in cells with SNAP-tag bound to BG-peptide did not exhibit fluorescence as the BG-fluorophore did not bind SNAP-tag-protein and was washed away. The media was replaced one last time with phenol red-free OptiMEM for imaging. Imaging occurred on the Molecular Devices ImageXpress Nano, utilizing DAPI and Texas Red filters. For scoring, cells were segmented using ImageXpress software to generate counts for nuclei and average cytoplasmic or nuclear intensity of SNAP-Cell TMR-Star dye. The average intensity was then normalized using the positive control of 2 pM hydrolyzed BG-GLA-NHS (BG-GLA-OH) as 100% cell penetration and PBS as 0% penetration to determine normalized SNAP-tag-protein occupancy.
  • BG-GLA-OH 2 pM hydrolyzed BG-GLA-NHS
  • BG-peptides comprising cystine-dense peptides after SNAPPA in HeLa cells stably transfected with pSNAPf captured on ImageXpress Nano are shown in FIG. 3.
  • Eight BG-peptides comprising cystine-dense peptides that were tested include: KR_KTxl5.8 (BG-SEQ ID NO: 74), KR_KTx2.2 (BG-SEQ ID NO: 73), Tx677 (BG-SEQ ID NO: 93), KR CTI (BG-SEQ ID NO: 72), KR CTX (BG-SEQ ID NO: 71), KR MCa (BG-SEQ ID NO: 67), KR_HwTx-IV, (BG-SEQ ID NO: 66), and KR IpTxa (BG-SEQ ID NO: 59).
  • BG-KR_IpTxa (BG- SEQ ID NO: 59) showed a higher level of cell penetration and BG-KR_KTxl5.8 (BG-SEQ ID NO: 74) showed a lower level of penetration, as shown in FIG. 3.
  • SNAPPA SNAP penetration assay
  • SNAP -tag expressing cells expressing either pSNAPf to quantify cytosolic access or pSNAPf-H2B to quantify nuclear access, were generated as described in EXAMPLE 3.
  • Hydrolyzed SNAP reagent BG-GLA-OH was used as a positive control and set to 100% (1.0 fraction) penetration or protein occupancy.
  • the cell penetrance of BG-peptides comprising cystine-dense peptides was normalized to the BG-GLA-OH positive control.
  • the SNAPPA measured the relative percentage penetrance of various BG-peptides comprising cystine-dense peptides tested in the various cell lines
  • BG-peptides comprising cystine-dense peptides were tested using the SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously.
  • BG-peptides comprising cystine-dense peptides were tested using the SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf-H2B, which encodes for nuclear expression of the SNAP -tag.
  • BG-peptides comprising cystine- dense peptides were assayed for nuclear penetration including: KR_KTxl5.8 (BG-SEQ ID NO: 74), KR_KTx2.2 (BG-SEQ ID NO: 73), Tx677 (BG-SEQ ID NO: 93), KR CTI (BG-SEQ ID NO: 72), KR CTX (BG-SEQ ID NO: 71), KR MCa (BG-SEQ ID NO: 67), KR_HwTx-IV, (BG-SEQ ID NO: 66), and KR IpTxa (BG-SEQ ID NO: 59).
  • BG-MCa_varl SEQ ID NO: 67
  • BG-KR_HwTx-IV SEQ ID NO: 66
  • BG-KR_IpTxa SEQ ID NO: 59
  • Uptake of BG-KR_Txl5.8 BG- SEQ ID NO: 74
  • BG-KR_KTx2.2 BG-SEQ ID NO: 73
  • BG-Tx677 BG-SEQ ID NO: 93
  • BG-KR CTI BG-SEQ ID NO: 72
  • BG-KR CTX BG-SEQ ID NO: 71
  • This example describes assaying cytosolic and nuclear access of cell-penetrating peptide complexes using SNAPPA.
  • Peptides identified using SNAPPA as described in EXAMPLE 5, were conjugated by recombinant fusion to an additional cystine-dense peptide with lower cell penetration to determine if the cell-penetrating peptide could carry an additional peptide into the cytosol or nucleus.
  • Cytosolic and nuclear access of the cell-penetrating peptide complexes was assayed by SNAPPA in three different SNAP -tag expressing cell lines.
  • SNAP-tag expressing cells expressing either pSNAPf to quantify cytosolic penetration or pSNAPf-H2B to quantify nuclear penetration, were generated as described in EXAMPLE 3.
  • BG-peptides comprising cystine-dense peptide complexes were tested using the SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf and expressing the SNAP-tag ubiquitously.
  • BG-peptides comprising cystine-dense peptide complexes were tested using the SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf-H2B and expressing the SNAP-tag in the nucleus.
  • Cystine-dense peptide complexes were constructed by linking a cell-penetrating cystine-dense peptide to an additional cystine-dense peptide with lower cell penetration using a (GGGS) 3 linker (GGGSGGGSGGGS, SEQ ID NO: 255).
  • Cell-penetrating peptides MCa (SEQ ID NO: 197) and KR IpTxa (SEQ ID NO: 1, corresponding to SEQ ID NO: 59 without an N- terminal GS) were tested, and low-cell-penetrating cystine-dense peptides KTx3.10 (AQEPVKGPVSTKPGSCPIILIRCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQ; SEQ ID NO: 294, corresponding to SEQ ID NO: 296 without an N-terminal GS) and elafin (GVPINVKCRGSRDCLDPCKKAGMRFGKCINSKCHCTP; SEQ ID NO: 293, corresponding to SEQ ID NO: 297 without an N-terminal GS) were conjugated by recombinant fusion to the cell-penetrating peptides, and tagged with SNAP substrate as described in EXAMPLE 4.
  • the peptide complexes that were tested in this assay are show in FIG. 5.
  • FIG. 6A An assay to measure cytosolic access of cell-penetrating peptide complexes is shown in FIG. 6A.
  • uptake of BG-cargo peptides was measured in the absence of conjugation to a cell-penetrating peptide.
  • BG-KR IpTxa was tested as a comparator to assess the level of penetration of the cell-penetrating peptide in the absence of conjugation to a cargo peptide.
  • BG-KR_IpTxa (BG-SEQ ID NO: 59) showed comparable high cytosolic access as the positive control BG-GLA-OH, to which all data was normalized
  • conjugating MCa or KR_IpTxa to KTx3.10 or to elafin greatly increased the level of KTx3.10 or elafin that reached the cytosol of each of the three different cell lines
  • KR_IpTxa reached the cytosol at levels comparable to the cell penetrant small molecule BG-GLA-OH.
  • BG-peptide complexes comprising cell-penetrating peptide were tested at 10 pM, 3 pM, and 1 pM using SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously.
  • Titration of the BG-peptide complexes cell-penetrating peptide demonstrated that peptide complexes incorporating the IpTxa cellpenetrating peptide (FIG. 7D and FIG. 7B) and the peptide complexes incorporating the MCa cell-penetrating peptide (FIG. 7C and FIG.
  • the peptide complexes incorporating the IpTxa cell-penetrating peptide demonstrated higher cytosolic delivery than the peptide complexes incorporating the MCa cell-penetrating peptide (SEQ ID NO: 197).
  • the data demonstrate a dose-dependent and cell-type dependent effect of the various peptide complexes on SNAP tag occupancy.
  • FIG. 6B An assay to measure nuclear access of cell-penetrating peptide complexes is shown in FIG. 6B.
  • BG-KTx3.10 BG-SEQ ID NO: 296
  • BG-elafin BG-SEQ ID NO: 297
  • Peptide complexes incorporating the KR IpTxa cell-penetrating peptide (SEQ ID NO: 1), BG-KR_IpTxa-KTx3.10 (BG-SEQ ID NO: 313) and BG-KR_IpTxa- elafin (BG-SEQ ID NO: 317) also showed higher levels of nuclear access as compared to the lower penetrating peptides alone.
  • BG-KR IpTxa (BG-SEQ ID NO: 59) was tested as a comparator to assess the level of penetration of the cell-penetrating peptide in the absence of conjugation to a cargo peptide.
  • BG-KR_IpTxa (BG-SEQ ID NO: 59) showed comparable high cytosolic access as the positive control BG-GLA-OH, to which all data was normalized.
  • the data showed that conjugating MCa (SEQ ID NO: 212) or KR IpTxa (SEQ ID NO: 59) to KTx3.10 (SEQ ID NO: 293) or to elafin (SEQ ID NO: 294) greatly increased the level of KTx3.10 or elafin that reached the nucleus of each of the three different cell lines.
  • KR IpTxa (SEQ ID NO: 59) reached the nucleus at levels comparable to the cell penetrant small molecule BG-GLA-OH.
  • the peptide complexes incorporating the IpTxa cell-penetrating peptide (SEQ ID NO: 1) exhibited higher cytosolic delivery than the peptide complexes incorporating the MCa cell-penetrating peptide (SEQ ID NO: 197), but in other cell lines or concentrations, the peptides conjugates incorporating the MCa cell-penetrating peptide (SEQ ID NO: 197) showed comparable or higher levels of deliver than those incorporating the IpTxa cell-penetrating peptide (SEQ ID NO: 1).
  • One alternative strategy included protein engineering an existing loop from a MCa peptide into KTx3.10 (SEQ ID NO: 293) via recombinant fusion to generate cell-penetrating peptide fusions such as the following SNAP substrate-peptides: BG-MCa(loop)-KTx3.10 (BG-SEQ ID NO: 314), and to elafin (SEQ ID NO: 294) to generate: BG-MCa(loop)-elafin (BG-SEQ ID NO: 318).
  • FIG. 10A shows results of a SNAP penetration assay to measure cytosolic access of the cell-penetrating peptide complexes illustrated in FIG.
  • BG-cargo peptides without additional cellpenetrating peptides BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297) were used as comparators to assess the basal level of penetration. Data was normalized to positive control BG-GLA-OH. The level of cargo peptide delivered to the cytosol varied with different cell lines, species of cargo peptide, and species of cell-penetrating peptide.
  • FIG. 10B shows results of a SNAP penetration assay to measure nuclear access of the cell-penetrating peptide complexes illustrated in FIG. 9 in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus.
  • BG-cargo peptides without additional cell-penetrating peptides BG- KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297) were used as comparators to assess the basal level of penetration.
  • the level of cargo peptide delivered to the cytosol varied with different cell lines, species of cargo peptide, and species of cell-penetrating peptide.
  • TEAD-binding cystine-dense peptide complexes This example describes cell penetration of TEAD-binding cystine-dense peptide complexes.
  • a TEAD-binding cystine-dense peptide (PDEYIERAKECCKKQDIQCCLRIFDESKDPNVMLICLFCW; SEQ ID NO: 295, corresponding to SEQ ID NO: 298 without an N-terminal GS) was conjugated to various cellpenetrating peptides to determine whether the cell-penetrating peptides were capable of facilitating cytosolic or nuclear localization of the TEAD-binding peptides.
  • the tested peptide complexes are illustrated in FIG. 11.
  • BG-peptides comprising cell-penetrating cystine-dense peptide fusions containing the TEAD-binding peptide were tested using the SNAPPA in NIH3T3, HeLa and HEK293 cells. Cells were stably transfected with pSNAPf expressing the SNAP-tag ubiquitously to test cytosolic access.
  • Cell-penetrating cystine-dense peptide fusions were constructed by linking a short N-terminal sequence derived from either MCa (C3A_MCa(l-9), SEQ ID NO: 210) or Had (C5A_Had(l-l 1), SEQ ID NO: 82) and fused to the N-terminus of the TEAD-binding peptide (GSPDEYIERAKECCKKQDIQCCLRIFDESKDPNVMLICLFCW; SEQ ID NO: 298) to generate a C3A_MCa(l-9)-TEAD-binder fusion (SEQ ID NO: 320) or a C5A_Had(l-l 1)- TEAD-binder fusion (SEQ ID NO: 321).
  • Peptide fusions were labeled with SNAP substrate as described in EXAMPLE 4.
  • the TEAD-binding cystine-dense peptide without an additional cell-penetrating peptide tag was tested as a comparator to assess the basal level of penetration.
  • BG-KR IPTxa (BG-SEQ ID NO: 59) was also tested and BG-GLA-OH was included as a positive control for normalization.
  • the data showed that conjugating C3A_MCa(l- 9) (SEQ ID NO: 195) or C5A_Had(l-l 1) (SEQ ID NO: 41) to the TEAD-binding peptide greatly increased the level of the TEAD-binding peptide that reaches the cytosol of each of three different cell lines.
  • BG-peptides complexes comprising cell-penetrating cystine-dense peptide fusions containing the TEAD-binding peptide were tested as with cytosolic access, but in cells stably transfected with pSNAPf-H2B expressing the SNAP -tag in the nucleus to test nuclear access.
  • BG-TEAD-binder (BG-SEQ ID NO: 298) was low, but nuclear access of BG-C3A_MCa(l-9)-TEAD-binder (BG-SEQ ID NO: 320) and BG-C5A_Had(l-l 1)-TEAD- binder (BG-SEQ ID NO: 321) was significantly increased relative to the TEAD-binding cystine- dense peptide without a cell-penetrating peptide, as shown in FIG. 12B.
  • Penetration of the BG- peptides comprising BG-KR_IPTxa (BG-SEQ ID NO: 59) was also tested and BG-GLA-OH was included as a positive control for normalization.
  • the data showed that conjugating C3A MCa(l-9) (SEQ ID NO: 195) or C5A Had(l-11) (SEQ ID NO: 41) to the TEAD-binding peptide greatly increased the level of the TEAD-binding peptide that reached the nucleus of each of three different cell lines.
  • the data also showed that KR IpTxa (SEQ ID NO: 59) reached the nucleus at levels comparable to the cell penetrant small molecule BG-GLA-OH.
  • This example describes cell penetration of cystine-dense peptides and peptide fragments.
  • the ability of various cell-penetrating cystine-dense peptides and cell-penetrating peptide fragments to enter cells, alone or with cargo cystine-dense peptides was measured as described in EXAMPLE 5 - EXAMPLE 8.
  • Peptides were qualitatively ranked based on their ability to access a cell cytoplasm, nucleus, or both, as shown in TABLE 14. “+++” indicates strong cell uptake, and denote decreasing cell uptake, as measured by SNAPP A, though results varied with different cell lines, cargo peptides, and other conditions.
  • the cell-penetrating cystine-dense peptides KR_HwTx-IV (SEQ ID NO: 8, corresponding to SEQ ID NO: 66 with no N-terminal GS), MCa_varl (SEQ ID NO: 18, corresponding to SEQ ID NO: 67 with no N-terminal GS), and MCa (SEQ ID NO: 212, corresponding to SEQ ID NO: 197 with no N-terminal GS) were also able to access the cytosolic and nuclear compartment of cells at significant levels. Some cystine-dense peptides exhibit lower levels of cellular uptake from the media, and some appeared have little to no access in certain cell lines.
  • This example describes endocytotic profiling via SNAP penetration assay.
  • pSNAPf cells were preincubated with a variety of endocytosis inhibitors to assess mechanisms responsible for uptake and cytosolic access of various BG-peptides.
  • Macropinocytosis was inhibited using 50 pM ethylisopropyl amiloride (EIP A), which may block Na+/H+ exchangers and prevents formation of macropinosomes.
  • EIP A ethylisopropyl amiloride
  • Clathrin-mediated endocytosis was inhibited using either 20 pM nocodazole, which may inhibit microtubule polymerization, 3 pM cytochalasin D, which can interfere with actin polymerization, or 80 pM dynasore, which can inhibit dynamin GTPase.
  • Endosomal acidification or lysosomal maturation can be inhibited using 50 nM bafilomycin A, which can inhibit the vacuolar ATPase complex, or 50 pM chloroquine, which can diffuse into endosomes as a weak base.
  • pSNAPf cells were preincubated with the specified concentration of inhibitor in growth media for 1 hour prior to SNAPP A, which was conducted as described in EXAMPLE 5
  • BG-peptides comprising KR IpTxa (SEQ ID NO: 59) in measured in pSNAPf cells preincubated with various chemicals inhibiting different mechanisms of endocytosis.
  • BG-peptides comprising MCa_varl SEQ ID NO: 67
  • cellular uptake of the BG-peptide comprising cell-penetrating peptide BG-MCa_varl was significantly decreased upon inhibition of macropinocytosis with 50 pM EIPA in HeLa cells, though only moderately with 3T3 cells or 293 cells.
  • This example describes the potential for using additional endosomal escape peptides to further promote cell-penetrating peptides for promoting endosomal escape of cell-penetrating peptide fusions.
  • Peptides S19 PFVIGAGVLGALGTGIGGI; SEQ ID NO: 359), CM18 (KWKLFKKIGAVLKVLTTG; SEQ ID NO: 360), PAS (FFLIPKG; SEQ ID NO: 361), Aureinl.2 (GLFDIIKKIAESF; SEQ ID NO: 362), and B18 (LGLLLRHLRHHSNLLANI; SEQ ID NO: 363) purported to promote endosomal escape were conjugated by fusion to KR IpTxa (SEQ ID NO: 1, corresponding to SEQ ID NO: 59 without an N-terminal GS) in order to potentially further promote delivery of peptide to the cytosol, and then labeled with SNAP substrate as described in EXAMPLE 4.
  • BG-KR_IpTxa-PAS (BG-SEQ ID NO: 322) and BG- KR_IpTxa-S19 (BG-SEQ ID NO: 323) were tested using SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously.
  • the addition of the PAS and S19 sequences did not negatively affect cellular uptake and delivery to the cytosol, as shown in FIG.
  • BG-peptide constructs showed high delivery including BG-peptides comprising KR IpTxa (SEQ ID NO: 59), KR_IpTxA-PAS (SEQ ID NO: 322), and KR_IpTxa-S19 (SEQ ID NO: 323) and as compared to the normalizing positive control BG- GLA-OH.
  • the data showed that KR IpTxa without or with the addition of PAS or S19 had high levels of cell penetration in all cell lines tested.
  • KR_IpTxa SEQ ID NO: 59
  • KR_IpTxA-PAS SEQ ID NO: 322
  • KR_IpTxa-S19 SEQ ID NO: 323
  • KR IpTxa SEQ ID NO: 1
  • FIG. 16A and FIG. 16B illustrates peptide cargos that can be conjugated, such as by chemical conjugation or recombinant fusion, to a cellpenetrating cystine-dense peptide or peptide fragment of the present disclosure.
  • a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 1 4, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 is conjugated to, linked to, or fused to a peptide cargo and expressed recombinantly or chemically synthesized as a cell-penetrating peptide complex.
  • the peptide cargo is conjugated to, linked to, or fused to the N-terminus or the C-terminus of the cell-penetrating cystine-dense peptide or peptide fragment.
  • the peptide cargo and the cell-penetrating cystine-dense peptide or peptide fragment are connected by a linker of any one of SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485, or any other linker, or no linker.
  • the peptide cargo is a cystine-dense peptide, an affibody, a 13-hairpin, an avimer, an adnectin, a stapled peptide, a nannofittin, a kunitz domain, a fynomer, or a bicyclic peptide, as shown in FIG. 16A.
  • the peptide is a nanobody, an antibody scFc fragment, or an antibody FAb fragment, as shown in FIG. 16B.
  • One or more cell-penetrating peptides of this disclosure may be conjugated to any of these cargos.
  • One or more of these cargos may be conjugates to a cell-penetrating peptide of this disclosure.

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Abstract

L'invention concerne des peptides et des variants de ceux-ci capables de pénétrer dans une couche cellulaire. Ces peptides de pénétration cellulaire peuvent être conjugués, liés, ou fusionnés à des agents actifs ou à des agents détectables pour administrer les agents dans un compartiment cellulaire à partir duquel ils peuvent autrement être exclus. Les peptides de pénétration cellulaire peuvent être utilisés pour faciliter l'administration d'un agent thérapeutique à un patient pour traiter une maladie ou un problème de santé chez le patient.
PCT/US2021/062422 2020-12-09 2021-12-08 Peptides de pénétration cellulaire, complexes peptidiques et leurs procédés d'utilisation Ceased WO2022125673A1 (fr)

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CN117487828A (zh) * 2022-10-24 2024-02-02 成都威斯津生物医药科技有限公司 招募配体增强抗原提呈效果的核酸分子、融合蛋白及mRNA疫苗
CN118662625A (zh) * 2024-07-24 2024-09-20 东北农业大学 一种植物源新冠病毒鼻喷疫苗及其制备方法
WO2024211847A1 (fr) * 2023-04-07 2024-10-10 Vanderbilt University Protéines cas9 recombinantes de liaison à l'albumine et leurs utilisations
WO2025017349A1 (fr) * 2023-07-18 2025-01-23 Hosseinkhani Saman Plateforme de vaccins à adn à base de peptides
JPWO2025023312A1 (fr) * 2023-07-26 2025-01-30

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116444678A (zh) * 2022-10-18 2023-07-18 百葵锐(深圳)生物科技有限公司 一种具有真菌抗菌抑菌性能的新型多肽
CN116444678B (zh) * 2022-10-18 2024-02-02 百葵锐(深圳)生物科技有限公司 一种具有真菌抗菌抑菌性能的新型多肽
CN117487828A (zh) * 2022-10-24 2024-02-02 成都威斯津生物医药科技有限公司 招募配体增强抗原提呈效果的核酸分子、融合蛋白及mRNA疫苗
CN117487828B (zh) * 2022-10-24 2025-03-18 成都威斯津生物医药科技有限公司 招募配体增强抗原提呈效果的核酸分子、融合蛋白及mRNA疫苗
WO2024211847A1 (fr) * 2023-04-07 2024-10-10 Vanderbilt University Protéines cas9 recombinantes de liaison à l'albumine et leurs utilisations
WO2025017349A1 (fr) * 2023-07-18 2025-01-23 Hosseinkhani Saman Plateforme de vaccins à adn à base de peptides
JPWO2025023312A1 (fr) * 2023-07-26 2025-01-30
WO2025023312A1 (fr) * 2023-07-26 2025-01-30 株式会社高研 Peptide perméable à la membrane de liaison au collagène, et support qui contient ledit peptide et le collagène ou un dérivé de collagène
JP7725041B2 (ja) 2023-07-26 2025-08-19 株式会社高研 コラーゲン結合型膜透過性ペプチド、並びに、該ペプチド及びコラーゲン若しくはコラーゲン誘導体を含む運搬体
CN118662625A (zh) * 2024-07-24 2024-09-20 东北农业大学 一种植物源新冠病毒鼻喷疫苗及其制备方法

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