CN116615205A - Peptide-based transduction of non-anionic polynucleotide analogs for modulation of gene expression - Google Patents
Peptide-based transduction of non-anionic polynucleotide analogs for modulation of gene expression Download PDFInfo
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- CN116615205A CN116615205A CN202180078804.1A CN202180078804A CN116615205A CN 116615205 A CN116615205 A CN 116615205A CN 202180078804 A CN202180078804 A CN 202180078804A CN 116615205 A CN116615205 A CN 116615205A
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Abstract
Described herein are compositions and methods for cargo delivery of non-anionic polynucleotide analogs to the cytosol/nuclear compartment of eukaryotic cells via synthetic peptide shuttles. The non-anionic polynucleotide analogue cargo can be charge neutral or cationic, and the synthetic peptide shuttling agent is a peptide comprising an amphiphilic α -helical motif having both a positively charged hydrophilic outer face and a hydrophobic outer face, wherein the synthetic peptide shuttling agent is not covalently linked or cleavable under physiological conditions to the non-anionic polynucleotide analogue cargo. The non-anionic polynucleotide analog cargo can be a charge neutral or cationic Antisense Synthetic Oligonucleotide (ASO) that hybridizes to an intracellular target RNA for modification of gene expression.
Description
The present description relates to intracellular delivery of non-anionic polynucleotide analog cargo. More specifically, the present description relates to the use of synthetic peptide shuttles for intracellular delivery of non-anionic polynucleotide analog cargo.
The present specification cites a number of documents, the contents of which are incorporated herein by reference in their entirety.
Background
Most non-anionic polynucleotide analog cargo suffer from problems associated with their intracellular delivery, often requiring their covalent conjugation to the delivery moiety, thereby complicating their synthesis and commercialization. Improved methods of increasing cytosolic/nuclear delivery of non-anionic polynucleotide analog cargo are highly desirable.
Disclosure of Invention
Synthetic peptide shuttles represent a recently defined family of peptides that have previously been reported to rapidly and efficiently transduce protein cargo into the cytosol and/or nucleus of a wide variety of target eukaryotic cells. In contrast to traditional cell penetrating peptide-based intracellular delivery strategies, synthetic peptide shuttles are not covalently linked to their polypeptide cargo. Indeed, covalent attachment of shuttle agents to their cargo often has a negative impact on their transduction activity. A first generation of such peptide shuttles is described in WO/2016/161516, wherein the peptide shuttles comprise an Endosomal Leakage Domain (ELD) operably linked to a Cell Penetrating Domain (CPD). WO/2018/068135 subsequently describes other synthetic peptide shuttles rationally designed based on a set of fifteen design parameters, the sole purpose of which is to improve transduction of protein cargo while reducing toxicity of the first generation peptide shuttles.
Although Cell Penetrating Peptides (CPPs) have been used for decades in transfection strategies for intracellular DNA/RNA delivery, first generation synthetic peptide shuttles containing CPD derived from CPPs are not capable of efficiently transducing plasmid DNA cargo into the nucleus for gene expression, where the DNA cargo is largely captured in the endosome. Subsequent experiments revealed that the second generation synthetic peptide shuttle agents are also unsuitable for efficient transduction of plasmid DNA into the nucleus for gene expression. Furthermore, strategies involving negatively charged phosphate backbones that neutralize DNA/RNA by coating with positively charged small molecules have not significantly improved shuttle-mediated cargo transduction, endosomal capture remains a problem. This suggests that shuttle-mediated polynucleotide transduction requires more than just charge neutralization.
The present disclosure relates to the surprising discovery that synthetic peptide shuttles have the ability to rapidly and efficiently transduce a sufficient amount of non-anionic polynucleotide analog cargo into the cytosol/nucleus compartment for modification of gene expression in eukaryotic cells.
In one aspect, described herein is a composition comprising a non-anionic polynucleotide analogue cargo for intracellular delivery and a synthetic peptide shuttling agent independent of or not covalently linked to the non-anionic polynucleotide analogue cargo, the synthetic peptide shuttling agent being a peptide comprising an amphipathic α -helical motif having both a positively charged hydrophilic outer face and a hydrophobic outer face, wherein the synthetic peptide shuttling agent increases cytosolic/nuclear delivery of the non-anionic polynucleotide analogue cargo in eukaryotic cells compared to the absence of the synthetic peptide shuttling agent.
In yet another aspect, described herein is a method for modifying gene expression in a eukaryotic cell, the method comprising: (a) Providing a non-anionic polynucleotide analogue cargo for intracellular delivery, said non-anionic polynucleotide analogue cargo being designed to hybridise to a RNA of interest in said eukaryotic cell; (b) Providing a synthetic peptide shuttle agent that is independent of or not covalently linked to the non-anionic polynucleotide analogue cargo; (c) Contacting the eukaryotic cell with the non-anionic polynucleotide analogue cargo in the presence of the synthetic peptide shuttle agent at a concentration sufficient to increase transduction efficiency and/or cytosol/nucleus delivery of the charge neutral polynucleotide analogue cargo as compared to the absence of the synthetic peptide shuttle agent, wherein the non-anionic polynucleotide analogue cargo hybridizes to the RNA of interest following cytosol/nucleus delivery, thereby effecting modification of gene expression.
General definition
Headings and other identifiers (e.g., (a), (b), (i), (ii), etc.) are provided only for ease of reading the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require that the steps or elements be performed in alphabetical or numerical order or the order in which they are provided.
In the claims and/or the specification, the use of the terms "a" or "an" when used in conjunction with the term "comprising" may mean "one" or "one", but it is also consistent with the meaning of "one/more (or more)", "at least one (at least one)", and "one/one or more than one (one or more than one)".
As used herein, the term "about" indicates that a certain value includes the standard deviation of the error of the device or method employed for determining the value. Generally, the term "about" is intended to designate possible variations of up to 10%. Thus, the term "about" includes variations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10% of a certain value. Unless otherwise indicated, the term "about" when used before a range applies to both ends of the range.
As used in this specification and the claims, the words "comprise" (and any form of comprising, such as "comprises") and "comprising"), "having" (and any form of having, such as "having" and "having"), include "(and any form of comprising, such as" including "and" including ") or" contain "(and any form of containing, such as" contain "and" contain ") are inclusive or open-ended, and do not exclude
Additional, non-recited elements or method steps.
As used herein, "protein" or "polypeptide" or "peptide" means any peptide-linked amino acid chain that may or may not comprise any type of modification (e.g., chemical or post-translational modification such as acetylation, phosphorylation, glycosylation, sulfation, threylation, prenylation, ubiquitination, etc.). For further clarity, protein/polypeptide/peptide modifications are contemplated, provided that the modifications do not disrupt the cargo transduction activity of the shuttle agents described herein. For example, the shuttle agents described herein may be linear or cyclic, may be synthesized with one or more D-or L-amino acids, and/or may be conjugated to a fatty acid (e.g., at the N-terminus thereof). At least one amino acid of the shuttle agents described herein may also be replaced by a corresponding synthetic amino acid having a side chain with physiochemical properties (e.g., structure, hydrophobicity, or charge) similar to the replaced amino acid.
As used herein, "domain" or "protein domain" generally refers to a portion of a protein having a particular functionality or function. Some domains retain their function when separated from the rest of the protein and can therefore be used in a modular fashion. The modular nature of many protein domains may provide flexibility in their location within the shuttle agent of the present description. However, some domains may perform better when engineered at certain locations of the shuttle agent (e.g., at the N-terminal or C-terminal regions, or in between them). The location of a domain in its endogenous protein sometimes indicates where within the shuttle agent the domain should be engineered and what type/length of linker should be used. In view of the present disclosure, the skilled artisan can use standard recombinant DNA techniques to manipulate the position and/or number of domains within the shuttle agents of the present disclosure. In addition, the assays disclosed herein, as well as other assays known in the art, can be used to assess the functionality of each domain in the context of a shuttle agent (e.g., its ability to promote cell penetration across the plasma membrane, endosomal escape, and/or entry into the cytosol). Standard methods can also be used to assess whether the domain of the shuttle agent affects the activity of the cargo to be delivered intracellularly. In this regard, the expression "operably linked" as used herein refers to the ability of the domain to perform one or more of its intended functions (e.g., cell penetration, endosomal escape, and/or subcellular targeting) in the context of the shuttle agent of the present specification. For the sake of clarity, the expression "operably linked" is intended to define a functional linkage between two or more domains, and is not limited to a particular order or distance between them.
As used herein, the term "synthetic" as used in expressions such as "synthetic peptide," "synthetic peptide shuttling agent," or "synthetic polypeptide" is intended to refer to non-naturally occurring molecules that can be produced in vitro (e.g., chemically synthesized and/or produced using recombinant DNA techniques). The purity of the various synthetic formulations can be assessed by, for example, high performance liquid chromatography and mass spectrometry. Chemical synthesis methods may be preferred over cellular expression systems (e.g., yeast or bacterial protein expression systems) because they may eliminate the need for extensive recombinant protein purification steps (e.g., as required for clinical use). In contrast, the production of longer synthetic polypeptides via chemical synthesis methods may be more complex and/or costly, and such polypeptides may be more advantageously produced using cellular expression systems. In some embodiments, the peptides or shuttles of the present disclosure can be synthesized by chemical methods (e.g., solid phase or liquid phase peptide synthesis), as opposed to expression by recombinant host cells. In some embodiments, the peptides or shuttle agents of the present disclosure may lack an N-terminal methionine residue. One skilled in the art can alter the synthetic peptides or shuttling agents of the present description to suit particular needs of stability or other needs by using one or more modified amino acids (e.g., non-naturally occurring amino acids) or by chemically modifying the synthetic peptides or shuttling agents of the present description.
As used herein, the term "independent" is generally intended to mean molecules or agents that are not covalently bound to each other, or that may be transiently covalently linked via cleavable bonds, such that the molecules or agents (e.g., shuttle and cargo) are separated from each other by cleavage of the bonds after administration (e.g., prior to, simultaneously with, or shortly after exposure to a reducing cellular environment and/or intracellular delivery). For example, the expression "self-contained cargo" is intended to refer to cargo to be delivered (transduced) intracellularly that is not covalently bound (e.g., not fused) to the shuttle agent of the present specification upon transduction across the plasma membrane. In some aspects, a shuttle agent that is independent of (not fused to) cargo may be advantageous because it provides increased shuttle agent versatility-e.g., the ratio of shuttle agent to cargo can be easily changed (as opposed to a fixed ratio limited to the case of covalent bonds between shuttle agent and cargo). In some aspects, it may be advantageous from a manufacturing and/or management perspective to covalently attach the shuttle agent to its cargo via a cleavable bond such that they are separated from each other upon contact with the target cell.
As used herein, the expression "is or from" or "from" includes functional variants of a given protein or peptide (e.g., shuttle agents described herein) or domains thereof (e.g., CPD or ELD), such as conservative amino acid substitutions, deletions, modifications, and variants or functional derivatives that do not eliminate the activity of the protein domain.
Other objects, advantages and features of the present description will become more apparent upon reading the following non-limiting description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
Drawings
In the drawings:
FIG. 1 shows intracellular delivery of PMO-FITC cargo in HeLa cells via a first generation "domain-based" and rationally designed synthetic peptide shuttle agent, as assessed by flow cytometry. The results are the average calculated from experiments performed in at least duplicate.
Figure 2 shows the ability of synthetic peptide shuttle FSD10 to transduce antisense PMO into the cytosol of HeLa cells to enable knockdown of GFP gene expression, as assessed by flow cytometry. The results are the average calculated from experiments performed in at least duplicate.
Figure 3 shows the ability of synthetic peptide shuttle FSD250 to transduce antisense PMOs targeting Wnt1 and Gli1 to knockdown Gli1 protein expression in DU145 cells, as assessed by western blotting. The results are the average calculated from experiments performed in at least duplicate.
Figure 4 shows the ability of synthetic peptide shuttle FSD250 to transduce Gli 1-targeted antisense PMOs to knockdown Gli1 protein expression in DU145 cells, as assessed by western blotting. The results are the average calculated from experiments performed in at least duplicate.
Figure 5 shows the results of large-scale screening of candidate peptide shuttles for Propidium Iodide (PI) and GFP-NLS transduction activity. The results are the average calculated from experiments performed in at least duplicate.
Figure 6 shows the results of further screening of candidate peptide shuttles for Propidium Iodide (PI) and/or GFP-NLS transduction activity. The results are the average calculated from experiments performed in at least duplicate.
Figure 7 shows the ability of synthetic peptide shuttle FSD250 to transduce antisense PMO and PNA in HeLa cells. Fig. 7A shows the results of intracellular delivery by flow cytometry. Fig. 7B shows the results of cell viability obtained by flow cytometry. The results are the average calculated from experiments performed in at least duplicate.
FIG. 8 shows the results of inhibition of intracellular delivery of PMO by synthetic peptide shuttle FSD250 by naked DNA (FIG. 8A) and RNA (sgRNA; FIG. 8B) obtained by flow cytometry.
FIG. 9 shows the ability of the second generation synthetic peptide shuttles FSD10 and FSD250 to transduce antisense PMO in RH-30 cells compared to His-CM18-PTD4 (first generation shuttle agent). Fig. 9A shows the results of intracellular delivery by flow cytometry. Fig. 9B shows the results of cell viability by flow cytometry. The results are the average calculated from experiments performed in at least duplicate.
FIG. 10 shows the results of Gli1 knockdown in RH-30 cells after transduction of PMO-Gli1 with synthetic peptide shuttle FSD250, compared to VivoPMO-Gli 1. Fig. 10A shows the results of western blotting for Gli1 and GAPDH (control). Fig. 10B shows the results of densitometry scan analysis of the corresponding western blot of fig. 10A.
FIG. 11 shows a graph of an Endoporter TM In contrast, the synthetic peptide shuttle FSD396 was able to transduce antisense PMO in HeLa cells. Representative immunofluorescence microscopy images of untreated (FIG. 11A), PMO-FITC-treated (FIG. 11B), PMO-FITC+FSD396 (FIG. 11C) and PMO-FITC+endocort (FIG. 11D) are shown.
Figure 12 shows the ability of four different Gli 1-targeting PMOs to knock down Gli1 in human DU145 cells only after transduction with synthetic peptide shuttle FSD 250. Representative western blots and corresponding densitometry scan analyses for Gli1 and actin (controls) are shown.
FIG. 13 shows the results of transduction of PMO-Gli1 into basal cell carcinoma-type tumor explants with synthetic peptide shuttle FSD 250. Representative fluorescence microscopy images of Cy5 (left), cy5+DAPI (nuclear staining; medium), and Cy5+DAPI+Nomarski (i.e., differential interference microscopy [ DIC ]) (right) after treatment with PMO-Gli1-Cy5 and FSD250 or PMO-Gli1-Cy5 alone are shown.
Sequence listing
The application comprises a sequence listing in computer readable form created at 10, 15 of 2021. The computer readable form is incorporated herein by reference.
Detailed Description
In some aspects, described herein are compositions and methods for non-anionic polynucleotide analog cargo transduction. The methods generally comprise contacting a target eukaryotic cell with a composition comprising a non-anionic polynucleotide analog cargo and a synthetic peptide shuttling agent that is independent of or not covalently linked to the non-anionic polynucleotide analog cargo, wherein the synthetic peptide shuttling agent increases cytosolic/nuclear delivery of the non-anionic polynucleotide analog cargo in the eukaryotic cell.
Non-anionic polynucleotide analog cargo
In some embodiments, the non-anionic polynucleotide analog cargo may be a charge neutral or cationic Antisense Synthetic Oligonucleotide (ASO). In some embodiments, the ASO may be a charge neutral or cationic Splice Switching Oligonucleotide (SSO). In some embodiments, the non-anionic polynucleotide analog cargo may be a charge-neutral polynucleotide analog cargo having a phosphodiamide backbone, an amide (e.g., peptide) backbone, a methylphosphonate backbone, a neutral phosphotriester backbone, a sulfone backbone, or a triazole backbone. In some embodiments, the non-anionic polynucleotide analog cargo may be a cationic polynucleotide analog cargo having an aminoalkylated phosphoramidate backbone, a guanidinium backbone, an S-methyl thiourea backbone, or a Nucleoside Amino Acid (NAA) backbone. In some embodiments, the non-anionic polynucleotide analog cargo may be Phosphodiamide Morpholino Oligomer (PMO), peptide Nucleic Acid (PNA), methylphosphonate oligomer, or short interfering ribonucleic acid neutral oligonucleotide (siRNN). In some embodiments, the non-anionic polynucleotide analog cargo may be 5 to 50 mers, 5 to 75 mers, or 5 to 100 mers. In some embodiments, the non-anionic polynucleotide analog cargo is not covalently linked to a cell penetrating peptide, octaguanidine dendrimer, or other intracellular delivery moiety. In some embodiments, the non-anionic polynucleotide analog cargo is cell membrane impermeable or has low membrane permeability (e.g., due to the physicochemical properties of the cargo, preventing free diffusion across the cell membrane), wherein the peptide shuttling agent described herein facilitates or increases intracellular delivery thereof and/or entry into the cytosol/nucleus. In some embodiments, the non-anionic polynucleotide analog cargo may be a cell membrane permeable cargo, wherein the peptide shuttling agent described herein still increases its intracellular delivery and/or entry into the cytosol. In some embodiments, the peptide shuttling agents described herein may reduce the amount or concentration of cargo required to be administered to achieve its intended biological effect compared to administration of the cargo alone.
In some embodiments, the non-anionic polynucleotide analog cargo may be a drug for treating any disease or disorder that modifies gene expression of a therapeutically relevant target RNA. In some embodiments, the non-anionic polynucleotide analog cargo may be a medicament for treating cancer (e.g., skin cancer, basal cell carcinoma, nevus basal cell carcinoma syndrome), inflammation or inflammation-related disorder (e.g., psoriasis, atopic dermatitis, ulcerative colitis, urticaria, dry eye, dry or wet age-related macular degeneration, finger ulcers, light keratosis, idiopathic pulmonary fibrosis), pain (e.g., chronic or acute), or a disorder affecting the lung (e.g., cystic fibrosis, asthma, chronic Obstructive Pulmonary Disease (COPD), or idiopathic pulmonary fibrosis).
In some embodiments, the non-anionic polynucleotide analog cargo described herein may be a Splice Switching Oligonucleotide (SSO), for example, for correcting or modifying splicing of a therapeutically relevant target mRNA. In some embodiments, the target mRNA can be cystic fibrosis transmembrane conductance regulator (CFTR), and the compositions or methods described herein can be used to treat cystic fibrosis (e.g., by administering to the lungs of a cystic fibrosis subject). In this regard, synthetic peptide shuttles have been shown to be capable of delivering recombinant protein cargo to refractory airway epithelial cells with high efficiency (Krishnamurthy et al, 2018).
In some embodiments, the non-anionic polynucleotide analog cargo described herein is not covalently linked to a cell penetrating or cationic peptide, octaguanidine dendrimer, or other intracellular delivery moiety. Such conventional delivery strategies, which have been used for example for peptide conjugation, for phosphodiamide Morpholino oligomers (PPMO) and Vivo-Morpholino add another layer of complexity to the synthetic process of PMO. In contrast, the synthetic peptide shuttles described herein can advantageously transduce unmodified or "naked" non-anionic polynucleotide analog cargo, greatly facilitating manufacture and formulation.
Rational design parameters and peptide shuttles
In some aspects, the shuttle agents described herein may be peptides having transduction activity of non-anionic polynucleotide analog cargo, protein cargo, or both in a target eukaryotic cell. In some embodiments, the shuttle agents described herein preferably meet one or more or any combination of the fifteen rational design parameters below.
(1) In some embodiments, the shuttle agent is a peptide of at least 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. For example, the peptide may comprise a minimum length of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues, and a maximum length of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acid residues. In some embodiments, shorter peptides (e.g., in the range of 17-50 or 20-50 amino acids) may be particularly advantageous because they may be more readily synthesized and purified by chemical synthesis methods, which may be more suitable for clinical use (as opposed to recombinant proteins, which must be purified from cellular expression systems). Although the numbers and ranges in this specification are generally listed as multiples of 5, this specification should not be so limited. For example, the maximum lengths described in this specification should be understood to also include lengths 56, 57, 58 … … 61, 62, etc., and the non-enumeration herein is for brevity only. The same reasoning applies to the% identity listed herein.
(2) In some embodiments, the peptide shuttling agent comprises an amphipathic α -helical motif at neutral pH. As used herein, unless otherwise indicated, the expression "alpha-helical motif" or "alpha-helix" refers to a right-hand coiled or helical conformation (helix) with a rotation angle of 100 degrees between consecutive amino acids and/or an alpha-helix with 3.6 residues per turn. As used herein, the expression "comprising an α -helical motif" or "amphiphilic α -helical motif" or the like refers to a three-dimensional conformation that is predicted to be adopted by the peptides (segments of peptides) of the present specification based on the primary amino acid sequence of the peptides when in a biological environment, whether or not the peptides actually adopt the conformation when used as a shuttle agent in a cell. Furthermore, the peptides of the present description may comprise one or more alpha-helical motifs at different positions of the peptide. For example, the shuttle FSD5 in WO/2018/068135 is predicted to employ one α -helix over its entire length (see fig. 49C of WO/2018/068135), whereas the shuttle FSD18 of WO/2018/068135 is predicted to comprise two separate α -helices towards the N and C terminal regions of the peptide (see fig. 49D of WO/2018/068135). In some embodiments, the shuttle agents of the present disclosure are predicted to not comprise a β -sheet motif, for example as shown in fig. 49E and 49F of WO/2018/068135. Methods for predicting the presence of alpha-helices and beta-sheets in proteins and peptides are well known in the art. For example, one such method is based on 3D modeling using PEP-FOLDTM, which is an online resource for de novo peptide structure prediction (http:// bioserv. Rpbs. Univ-parameters-dide. Fr/services/PEP-FOLD /) (Lamiable et al, 2016; screen et al, 2014; th e venet et al, 2012). Other methods of predicting the presence of alpha-helices in peptides and proteins are known and readily available to the skilled artisan.
As used herein, the expression "amphiphilic" refers to a peptide (e.g., based on a side chain comprising an amino acid of the peptide) having both hydrophobic and hydrophilic elements. For example, the expression "amphiphilic α -helix" or "amphiphilic α -helical motif" refers to a peptide predicted to employ an α -helical motif having a non-polar hydrophobic face and a polar hydrophilic face based on the characteristics of the amino acid side chains forming the helix.
(3) In some embodiments, peptide shuttles of the present description comprise amphiphilic α -helical motifs with positively charged hydrophilic outer faces, such as outer faces enriched in R and/or K residues. As used herein, the expression "positively charged hydrophilic outside" means that based on the alpha-helical projection, there are at least three lysine (K) and/or arginine (R) residues clustered on one side of the amphiphilic alpha-helical motif (see, for example, figure 49A, left of WO/2018/068135). Various procedures, such as the online helical wheel projection tools created by Don Armstrong and Raphael Zidovetzki, may be used to prepare such helical wheel projections. (e.g., accessible: https:// www.donarmstrong.com/cgi-bin/wheel. Pl) or online tools developed by Mdi et al, 2018 (e.g., accessible http:// lbqp. Ub. Br/NetWheels /). In some embodiments, the amphiphilic α -helical motif may comprise a positively charged hydrophilic outer face comprising, based on an α -helix with a rotation angle of 100 degrees between consecutive amino acids and/or an α -helix with 3.6 residues per turn: (a) At least two, three or four adjacent positively charged K and/or R residues upon helical projection; and/or (b) a segment comprising six adjacent residues of three to five K and/or R residues upon helical projection.
In some embodiments, the peptide shuttles of the present specification comprise an amphiphilic α -helical motif comprising a hydrophobic outer face comprising, based on an α -helix having a rotation angle of 100 degrees between consecutive amino acids and/or an α -helix having 3.6 residues per turn: (a) at least two adjacent L residues upon helical projection; and/or (b) a segment comprising ten adjacent residues of at least five hydrophobic residues selected from L, I, F, V, W and M when projected by a helical wheel.
(4) In some embodiments, peptide shuttles of the present description comprise amphiphilic α -helical motifs having a highly hydrophobic core composed of spatially adjacent highly hydrophobic residues (e.g., L, I, F, V, W and/or M). In some embodiments, the highly hydrophobic core may consist of spatially adjacent L, I, F, V, W and/or M amino acids based on an open cylindrical representation of an a-helix of 3.6 residues per turn, which account for 12% to 50% of the amino acids of the peptide excluding any histidine-rich domain (see below), as shown in the right panel of fig. 49A of WO/2018/068135. In some embodiments, the highly hydrophobic core may consist of spatially adjacent L, I, F, V, W and/or M amino acids that account for from 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5% or 20% to 25%, 30%, 35%, 40% or 45% of the amino acids of the peptide. More specifically, the highly hydrophobic core parameters may be calculated by first arranging the amino acids of the peptide in an open cylindrical representation and then delineating regions of consecutive highly hydrophobic residues (L, I, F, V, W, M), as shown in the right-hand graph of fig. 49A of WO/2018/068135. The number of highly hydrophobic residues contained in the depicted highly hydrophobic core is then divided by the total amino acid length of the peptide, excluding any histidine-rich domains (e.g., N-terminal and/or C-terminal histidine-rich domains). For example, for the peptide shown in fig. 49A of WO/2018/068135, there are 8 residues in the highly hydrophobic core depicted, and a total of 25 residues in the peptide (excluding the terminal 12 histidines). Thus, the highly hydrophobic core is 32% (8/25).
(5) The hydrophobic moment relates to a measure of the amphiphilicity of a helix, peptide or part thereof, calculated from the vector sum of the hydrophobicity of the amino acid side chains (Eisenberg et al, 1982). The online tool for calculating the hydrophobic moment of a polypeptide can be obtained from: http:// rzlab. Ucr. Edu/scripts/w heel. Cgi. A high hydrophobic moment indicates a strong amphiphilicity, while a low hydrophobic moment indicates a weak amphiphilicity. In some embodiments, peptide shuttles of the present description may comprise or consist of a peptide or an alpha-helical domain having a hydrophobic moment (μ) of 3.5 to 11. In some embodiments, the shuttle agent may be a peptide comprising an amphiphilic α -helical motif having a hydrophobic moment between a lower limit of 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and an upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.10.0 or 11.0. In some embodiments, the shuttle agent may be a peptide having a hydrophobic moment between a lower limit of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and an upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, or 10.5. In some embodiments, the hydrophobic moment is calculated excluding any histidine-rich domain that may be present in the peptide.
(6) In some embodiments, the peptide shuttles of the present specification may have a predicted net charge of at least +3 or +4 at physiological pH, calculated from K, R, D and the side chain of the E residue. For example, the net charge of the peptide may be at least +5, +6, +7, at least +8, at least +9, at least +10, at least +11, at least +12, at least +13, at least +14, or at least +15 at physiological pH. These positive charges are typically conferred by the increased presence of positively charged lysine and/or arginine residues, in contrast to negatively charged aspartic acid and/or glutamic acid residues.
(7) In some embodiments, the peptide shuttles of the present specification may have a predicted isoelectric point (pI) of from 8 to 13, preferably from 10 to 13. Procedures and methods or proteins for calculating and/or measuring the isoelectric point of a peptide are known in the art. For example, pI may be calculated using the Prot Param software with access to http:// web. Expasy. Org/protParam
(8) In some embodiments, the peptide shuttles of the present specification may be comprised of 35% to 65% hydrophobic residues (A, C, G, I, L, M, F, P, W, Y, V). In particular embodiments, the peptide shuttle agent may consist of any combination of 36% to 64%, 37% to 63%, 38% to 62%, 39% to 61%, or 40% to 60% of the following amino acids: A. c, G, I, L, M, F, P, W, Y and V.
(9) In some embodiments, the peptide shuttles of the present specification may be comprised of 0 to 30% neutral hydrophilic residues (N, Q, S, T). In particular embodiments, the peptide shuttling agent may be comprised of any combination of 1% to 29%, 2% to 28%, 3% to 27%, 4% to 26%, 5% to 25%, 6% to 24%, 7% to 23%, 8% to 22%, 9% to 21%, or 10% to 20% of the following amino acids: n, Q, S and T.
(10) In some embodiments, the peptide shuttles of the present specification may be comprised of 35% to 85% of the following amino acids: A. l, K and/or R. In particular embodiments, the peptide shuttle agent may consist of any combination of 36% to 80%, 37% to 75%, 38% to 70%, 39% to 65%, or 40% to 60% of the following amino acids: A. l, K or R.
(11) In some embodiments, peptide shuttles of the present specification may be comprised of 15% to 45% amino acids a and/or L, provided that at least 5% L is present in the peptide. In particular embodiments, the peptide shuttle agent may consist of 15% to 40%, 20% to 35%, or 20% to 30% of any combination of the following amino acids: a and L, provided that at least 5% L is present in the peptide.
(12) In some embodiments, peptide shuttles of the present description may consist of 20% to 45% of amino acids K and/or R. In particular embodiments, the peptide shuttle agent may consist of 20% to 40%, 20% to 35%, or 20% to 30% of any combination of the following amino acids: k and R.
(13) In some embodiments, peptide shuttles of the present description may be comprised of 0 to 10% amino acids D and/or E. In particular embodiments, the peptide shuttle agent may consist of any combination of 5% to 10% of the following amino acids: d and E.
(14) In some embodiments, the absolute difference between the percentage of a and/or L and the percentage of K and/or R in the peptide shuttling agent may be less than or equal to 10%. In particular embodiments, the absolute difference between the percentage of a and/or L and the percentage of K and/or R in the peptide shuttling agent may be less than or equal to 9%, 8%, 7%, 6% or 5%.
(15) In some embodiments, the peptide shuttles of the present specification may be comprised of 10% to 45% of the following amino acids: q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T or H (i.e., not A, L, K or R). In particular embodiments, the peptide shuttle agent may consist of 15% to 40%, 20% to 35%, or 20% to 30% of any combination of the following amino acids: q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T and H.
In some embodiments, the peptide shuttles of the present specification obey at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, thirteen, at least fourteen, or all of the parameters (1) to (15) described herein. In certain embodiments, the peptide shuttles of the present specification obey all of parameters (1) to (3) and at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or all of parameters (4) to (15) described herein.
In some embodiments, when the peptide shuttles of the present specification comprise only one histidine-rich domain, the residues of said one histidine-rich domain may be included in the calculation/evaluation of parameters (1) to (15) described herein. In some embodiments, when the peptide shuttles of the present specification comprise more than one histidine-rich domain, the residues of only one histidine-rich domain may be included in the calculation/evaluation of parameters (1) to (15) described herein. For example, when the peptide shuttles of the present specification comprise two histidine-rich domains: when a first histidine-rich domain towards the N-terminus and a second histidine-rich domain towards the C-terminus, only the first histidine-rich domain may be included in the calculation/evaluation of parameters (1) to (15) described herein.
In some embodiments, machine learning or computer aided design methods may be implemented to produce peptides that adhere to one or more of parameters (1) through (15) described herein. Some parameters such as parameters (1) and (5) - (15) may be more suitable for implementation in a computer-aided design method, while structural parameters such as parameters (2), (3) and (4) may be more suitable for a manual design method. Thus, in some embodiments, peptides that adhere to one or more of parameters (1) to (15) may be produced by combining computer-aided and manual design methods. For example, multiple sequence alignment analysis of the various peptides (and other peptides) shown herein as effective shuttles revealed the presence of some consensus sequences-i.e., the alternating pattern of hydrophobic, cationic, hydrophilic, alanine and glycine amino acids that is commonly found. The presence of these consensus sequences may lead to adherence to structural parameters (2), (3) and (4) (i.e. amphiphilic α -helix formation, positively charged face and 12% -50% highly hydrophobic core). Thus, these and other consensus sequences can be used in machine learning and/or computer aided design methods to produce peptides that adhere to one or more of parameters (1) - (15).
Thus, in some embodiments, the peptide shuttles described herein may comprise or consist of the following amino acid sequences:
(a) [ X1] - [ X2] - [ linker ] - [ X3] - [ X4] (formula 1);
(b) [ X1] - [ X2] - [ linker ] - [ X4] - [ X3] (formula 2);
(c) [ X2] - [ X1] - [ linker ] - [ X3] - [ X4] (formula 3);
(d) [ X2] - [ X1] - [ linker ] - [ X4] - [ X3] (formula 4);
(e) [ X3] - [ X4] - [ linker ] - [ X1] - [ X2] (formula 5);
(f) [ X3] - [ X4] - [ linker ] - [ X2] - [ X1] (formula 6);
(g) [ X4] - [ X3] - [ linker ] - [ X1] - [ X2] (formula 7);
(h) [ X4] - [ X3] - [ linker ] - [ X2] - [ X1] (formula 8);
(i) [ linker ] - [ X1] - [ X2] - [ linker ] (formula 9);
(j) [ linker ] - [ X2] - [ X1] - [ linker ] (formula 10);
(k) [ X1] - [ X2] - [ linker ] (formula 11);
(l) [ X2] - [ X1] - [ linker ] (formula 12);
(m) [ linker ] - [ X1] - [ X2] (formula 13);
(n) [ linker ] - [ X2] - [ X1] (formula 14);
(o) [ X1] - [ X2] (formula 15); or (b)
(p) [ X2] - [ X1] (formula 16),
wherein:
[ X1] is selected from: 2[ phi ] -1 < + > -2[ phi ] -1[ zeta ] -1 < + > -;2[ phi ] -1 < + > -2[ phi ] -2 < + > -;1 < + > -1[ phi ] -1 < + > -2[ phi ] -1[ zeta ] -1 < + > -; and 1 < + > -2 < + >;
[ X2] is selected from: -2[ phi ] -1+ ] -2[ phi ] -2[ zeta ] -; -2[ phi ] -1 < + > -2[ phi ] -2 < + > -; -2[ phi ] -1 < + > -1[ ζ ] -; -2[ phi ] -1 + ] -2[ phi ] -1[ zeta ] -1 + ]; -2[ phi ] -2 < + > -1[ phi ] -2 < + > -; -2[ phi ] -2 < + > -1[ phi ] -2[ zeta ] -; -2[ phi ] -2 < + > -1[ phi ] -1 < + > -1[ ζ ] -; and-2 [ phi ] -2 < + > -1[ phi ] -1[ zeta ] -1 < + > -;
[ X3] is selected from: -4 < + > -A-; ext> -ext> 3ext> <ext> +ext> >ext> -ext> Gext> -ext> Aext> -ext>;ext> -3 < + > -A-A-; -2 < + > -1 < + > -A-; ext> -ext> 2ext> <ext> +ext> >ext> -ext> 1ext> [ext> phiext> ]ext> -ext> Gext> -ext> Aext> -ext>;ext> -2 < + > -1[ phi ] -A-A-; or-2 < + > -A-1 < + > -A; ext> -ext> 2ext> <ext> +ext> >ext> -ext> Aext> -ext> Gext> -ext> Aext>;ext> -2 < + > -A-A-A-; -1[ phi ] -3 < + > -A-; ext> -ext> 1ext> [ext> phiext> ]ext> -ext> 2+ext> ]ext> -ext> Gext> -ext> Aext> -ext>;ext> -1[ phi ] -2 < + > -A-A-; -1[ phi ] -1[ phi+ ] -A; ext> -ext> 1ext> [ext> phiext> ]ext> -ext> 1ext> +ext> ]ext> -ext> 1ext> [ext> phiext> ]ext> -ext> Gext> -ext> Aext>;ext> -1[ phi ] -1 + ] -1[ phi ] -A-A; -1[ phi ] -1 < + > -A; ext> -ext> 1ext> [ext> phiext> ]ext> -ext> 1+ext> ]ext> -ext> Aext> -ext> Gext> -ext> Aext>;ext> -1[ phi ] -1 < + > -A-A-A; -A-1 < + > -A; ext> -ext> Aext> -ext> 1ext> <ext> +ext> >ext> -ext> Aext> -ext> Gext> -ext> Aext>;ext> and-A-1 < + > -A-A-A;
[ X4] is selected from: -1[ ζ ] -2A-1+ ] -A; -1[ zeta ] -2A-2+ ]; -1 < + > -2A-1 < + > -A; -1[ zeta ] -2A-1 < + > -1[ zeta ] -A-1 < + >; -1[ zeta ] -A-1+ ]; -2 < + > -A-2 < + >; -2 < + > -A-1 < + > -A; -2 < + > -A-1 < + > -1[ ζ ] -A-1 < + >; -2 < + > -1[ ζ ] -A-1 < + >; -1 < + > -1[ ζ ] -A-1 < + > -A; -1 < + > -1[ ζ ] -A-2 < + >; -1 < + > -1[ zeta ] -A-1 < + >; -1 < + > -2[ ζ ] -A-1 < + >; -1 < + > -2 < + >; -1 < + > -2[ ζ ] -1 < + > -A; -1 < + > -2[ zeta ] -1 < + > -1[ zeta ] -A-1 < + >; -1 < + > -2[ zeta ] -1[ zeta ] -A-1 < + >; -3[ zeta ] -2+ ]; -3[ ζ ] -1+ ] -A; -3[ zeta ] -1 < + > -1[ zeta ] -A-1 < + >; -1[ ζ ] -2A-1+ ] -A; -1[ zeta ] -2A-2+ ]; -1[ zeta ] -2A-1 < + > -1[ zeta ] -A-1 < + >; -2 < + > -A-1 < + > -A; -2 < + > -1[ ζ ] -1 < + > -A; -1 < + > -1[ ζ ] -A-1 < + > -A; -1 < + > -2A-1 < + > -1[ zeta ] -A-1 < + >; and-1 [ zeta ] -A-1+ ]; and is also provided with
[ linker ] is selected from: -Gn-; -Sn-; - (GnSn) n-; - (GnSn) nGn-; - (GnSn) nSn-; - (GnSn) nGn- (GnSn) n-; and- (GnSn) nSn (GnSn) n-;
wherein: [ phi ] is an amino acid which is: leu, phe, trp, ile, met, tyr or Val, preferably Leu, phe, trp or Ile; the [ + ] is an amino acid which is: lys or Arg; [ ζ ] is an amino acid, which is: gln, asn, thr or Ser; a is amino acid Ala; g is amino acid Gly; s is the amino acid Ser; and n is an integer of 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, or 1 to 3.
In some embodiments, peptide shuttles of the present description may comprise or consist of a peptide or functional variant thereof that is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 74%, 75%, 82%, 98%, 91%, 81%, or 93%, or an amino acid sequence of any of SEQ ID NOs 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311, 313 to 324, 326 to 332, 338 to 342, 344, or 353 to 364, or an amino acid sequence of any of SEQ ID NOs 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145, 148, 151, 152, 169 to 242, and 243-10, 242 as disclosed in WO/2018/068135. In some embodiments, peptide shuttles of the present description may comprise the amino acid sequence motifs of SEQ ID NOs 158 and/or 159 of WO/2018/068135 found in each of peptides FSD5, FSD16, FSD18, FSD19, FSD20, FSD22 and FSD 23. In some embodiments, peptide shuttles of the present specification may comprise the amino acid sequence motif of SEQ ID NO:159 of WO/2018/068135 operably linked to the amino acid sequence motif of SEQ ID NO:158 of WO/2018/068135. As used herein, "functional variant" refers to a peptide having cargo transduction activity that differs from a reference peptide by one or more conservative amino acid substitutions. As used herein in the context of functional variants, a "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been well defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and optionally proline), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
In some embodiments, the peptide shuttles of the present specification do not comprise one or more of the amino acid sequences of any of SEQ ID NOs 57-59, 66-72 or 82-102 of WO/2018/068135. In some embodiments, the peptide shuttles of the present specification do not comprise one or more of the amino acid sequences of any of SEQ ID NOs 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145, 148, 151, 152, 169-242 and 243-10 242 of WO/2018/068135. More specifically, in some embodiments, the peptide shuttles of the present specification may involve variants of such previously described shuttles peptides, wherein the variants are further engineered for improved transduction activity (i.e., are capable of more robustly transducing non-anionic polynucleotide analog cargo).
In some embodiments, peptide shuttles of the present disclosure may have a minimum threshold value for transduction efficiency and/or cargo delivery score of "surrogate" cargo, as measured in a eukaryotic cell model system (e.g., an immortalized eukaryotic cell line) or in a model organism. The expression "transduction efficiency" refers to the percentage or proportion of the target cell population into which the cargo of interest is delivered intracellularly, which can be determined by, for example, flow cytometry, immunofluorescence microscopy, and other suitable methods that can be used to assess the transduction efficiency of the cargo (e.g., as described in WO/2018/068135). In some embodiments, transduction efficiency may be expressed as a percentage of cargo positive cells. In some embodiments, transduction efficiency may be expressed as fold increase (or fold decrease) compared to a suitable negative control assessed under the same conditions except in the absence of cargo and shuttle agent ("no treatment"; NT) or in the absence of shuttle agent ("cargo only").
In some embodiments, the shuttle agents described herein comprise or consist of:
(i) The amino acid sequence of any of SEQ ID NOs 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311, 313 to 324, 326 to 332, 338 to 342, 344, or 353 to 364;
(ii) Amino acid sequences that differ from any of SEQ ID NOs 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311, 313 to 324, 326 to 332, 338 to 342, 344, or 353 to 364 by NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., do not include any linker domains);
(iii) An amino acid sequence that is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (e.g., calculated without any linker domain) to any of SEQ ID NOs 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311 to 324, 326 to 332, 338 to 342, 344, or 353 to 364;
(iv) Amino acid sequence differing from any of SEQ ID NOs 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311, 313 to 324, 326 to 332, 338 to 342, 344, or 353 to 364 by only conservative amino acid substitutions (e.g., by NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, preferably not including any linker domain), wherein each conservative amino acid substitution is selected from amino acids within the same amino acid class, the amino acids being: aliphatic: G. a, V, L and I; hydroxyl-containing or sulfur/selenium: s, C, U, T and M; aromatic: F. y and W; alkaline: H. k and R; acidic and its amides: D. e, N and Q; or (b)
(v) Any combination of (i) to (iv).
In some embodiments, the shuttle agent described herein for delivering a non-anionic polynucleotide analog cargo is preferably a second generation shuttle agent lacking a cell penetrating domain or lacking a cell penetrating domain fused to an endosomal leakage domain. In some embodiments, the shuttle agents described herein that are particularly useful for delivering non-anionic polynucleotide analog cargo are preferably those having a relatively high delivery score, meaning that the shuttle agent delivers a greater total number of cargo molecules per cell. Because the synthetic polynucleotide analogs described herein function by steric hindrance when hybridized to their target intracellular RNA molecules (i.e., one cargo molecule binds to one intracellular RNA molecule), shuttles with higher delivery scores are expected to be particularly advantageous for such applications. In some embodiments, the shuttle agents described herein (and/or the SEQ ID NOs listed above in the preceding paragraph) are those listed in fig. 5 that deliver PI and/or GFP cargo with a normalized average delivery score of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200.
In some embodiments, the shuttle agents described herein comprise or consist of a variant of the synthetic peptide shuttle agent that is identical to the synthetic peptide shuttle agent as defined herein except that at least one amino acid is replaced with a corresponding synthetic amino acid having a side chain with physiochemical properties (e.g., structure, hydrophobicity, or charge) similar to that of the replaced amino acid, wherein the variant increases cytosolic/nuclear delivery of the non-anionic polynucleotide analog cargo in eukaryotic cells as compared to the absence of the synthetic peptide shuttle agent.
Chemically modified and synthesized amino acids
In some embodiments, the shuttle agents of the present disclosure may comprise oligomers (e.g., dimers, trimers, etc.) of the peptides described herein. Such oligomers may be constructed by covalently binding the same or different types of shuttle monomers (e.g., using disulfide bridge linkages to introduce cysteine residues in the monomer sequence). In some embodiments, the shuttle agents of the present description may comprise N-terminal and/or C-terminal cysteine residues.
In some embodiments, the shuttle agents of the present description may comprise or consist of a cyclic peptide. In some embodiments, the cyclic peptide may be formed via a covalent linkage between a first residue positioned toward the N-terminus of the shuttle agent and a second residue positioned toward the C-terminus of the shuttle agent. In some embodiments, the first and second residues are flanking residues located at the N and C termini of the shuttle agent. In some embodiments, the first and second residues may be linked via an amide bond to form a cyclic peptide. In some embodiments, the cyclic peptide may be formed from a disulfide bond between two cysteine residues within the shuttle agent, wherein the two cysteine residues are located towards the N and C termini of the shuttle agent. In some embodiments, the shuttle agent may comprise or be designed to comprise flanking cysteine residues at the N and C termini that are linked via disulfide bonds to form a cyclic peptide. In some embodiments, the cyclic shuttling agents described herein may be more resistant to degradation (e.g., by proteases) and/or may have a longer half-life than the corresponding linear peptides.
In some embodiments, the shuttle agent of the present description may comprise one or more D-amino acids. In some embodiments, the shuttle agent of the present disclosure may comprise D-amino acids at the N and/or C terminus of the shuttle agent. In some embodiments, the shuttle agent may be comprised entirely of D-amino acids. In some embodiments, the shuttle agents described herein having one or more D-amino acids may be more resistant to degradation (e.g., by proteases) and/or may have a longer half-life than the corresponding peptide consisting of only L-amino acids.
In some embodiments, the shuttle agents of the present description may comprise chemical modifications to one or more amino acids, wherein the chemical modifications do not disrupt the transduction activity of the synthetic peptide shuttle agent. As used herein in this context, the term "disruption" refers to the irreversible elimination of the cargo transduction activity of the peptide shuttling agents described herein by chemical modification. Chemical modifications that temporarily inhibit, attenuate, or delay the cargo transduction activity of the peptide shuttling agents described herein may be included in the chemical modifications to the shuttling agents of the present description. In some embodiments, the chemical modification to any of the shuttle agents described herein may be at the N-and/or C-terminus of the shuttle agent. Examples of chemical modifications include the addition of acetyl groups (e.g., N-terminal acetyl groups), cysteamine groups (e.g., C-terminal cysteamine groups), or fatty acids (e.g., C4-C16, C6-C14, C6-C12, C6-C8, or C8 fatty acids, preferably N-terminal).
In some embodiments, the shuttle agent of the present disclosure comprises a shuttle agent variant having transduction activity for non-anionic polynucleotide analog cargo in a target eukaryotic cell, the variant being identical to any shuttle agent of the present disclosure except that at least one amino acid is replaced with a corresponding synthetic amino acid or amino acid analog having a side chain with a physiochemical property (e.g., structure, hydrophobicity, or charge) similar to that of the replaced amino acid. In some embodiments, the synthetic amino acid substitutions:
(a) Substituting a basic amino acid with any one of: α -aminoglycine, α, γ -diaminobutyric acid, ornithine, α, β -diaminopropionic acid, 2, 6-diamino-4-hexynoic acid, β - (1-piperazinyl) -alanine, 4, 5-dehydro-lysine, δ -hydroxylysine, ω -dimethylarginine, homoarginine, ω' -dimethylarginine, ω -methylarginine, β - (2-quinolinyl) -alanine, 4-aminopiperidine-4-carboxylic acid, α -methylhistidine, 2, 5-diiodohistidine, 1-methylhistidine, 3-methylhistidine, spinacin, 4-aminophenylalanine, 3-aminotyrosine, β - (2-pyridinyl) -alanine or β - (3-pyridinyl) -alanine;
(b) Substitution of a nonpolar (hydrophobic) amino acid with any of the following: dehydro-alanine, beta-fluoroalanine, beta-chloroalanine, beta-iodoalanine, alpha-aminobutyric acid, alpha-aminoisobutyric acid, beta-cyclopropylalanine, azetidine-2-carboxylic acid, alpha-allylglycine, propargylglycine, t-butylalanine, beta- (2-thiazolyl) -alanine, thioproline, 3, 4-dehydroproline, t-butylglycine, beta-cyclopentylalanine, beta-cyclohexylalanine, alpha-methylproline, norvaline, alpha-methylvaline, penicillamine, beta, beta-dicyclohexylalanine, 4-fluoroproline, 1-aminocyclopentanecarboxylic acid, piperidinecarboxylic acid, 4, 5-dehydroleucine, allo-isoleucine, norleucine, alpha-methylleucine, cyclohexylglycine, cis-octahydroindole-2-carboxylic acid, beta- (2-thienyl) -alanine, phenylglycine, alpha-methylphenylalanine, homophenylalanine, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, beta- (3-benzothienyl) -alanine, 4-nitrophenylalanine, 4-bromophenylalanine, 4-tert-butylphenylalanine, alpha-methyltryptophan, beta- (2-naphthyl) -alanine, beta- (1-naphthyl) -alanine, 4-iodophenylalanine, 3-fluorophenylalanine, 4-methyltryptophan, 4-chlorophenylalanine, 3, 4-dichloro-phenylalanine, 2, 6-difluoro-phenylalanine, n-in-methyltryptophan, 1,2,3, 4-tetrahydronor Ha Erman-3-carboxylic acid, β -diphenylalanine, 4-methylphenylalanine, 4-phenylphenylalanine, 2,3,4,5, 6-pentafluoro-phenylalanine or 4-benzoylphenylalanine;
(c) Substituting a polar uncharged amino acid with any of the following: beta-cyanoalanine, beta-ureidoalanine, homocysteine, allothreonine, pyroglutamic acid, 2-oxothiazolidine-4-carboxylic acid, citrulline, thiocitrulline, homoccitrulline, hydroxyproline, 3, 4-dihydroxyphenylalanine, beta- (1, 2, 4-triazol-1-yl) -alanine, 2-mercaptohistidine, beta- (3, 4-dihydroxyphenyl) -serine, beta- (2-thienyl) -serine, 4-azidophenalanine, 4-cyanophenylalanine, 3-hydroxymethyltyrosine, 3-iodotyrosine, 3-nitrotyrosine, 3, 5-dinitrotyrosine, 3, 5-dibromotyrosine, 3, 5-diiodotyrosine, 7-hydroxy-1, 2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, 5-hydroxytryptophan, thyronine, beta- (7-methoxycoumarin-4-yl) -alanine or 4- (7-hydroxy-4-coumarin) -aminobutyric acid; and/or
(d) Replacing an acidic amino acid with any one of: gamma-hydroxy glutamic acid, gamma-methylene glutamic acid, gamma-carboxy glutamic acid, alpha-amino adipic acid, 2-amino pimelic acid, alpha-amino suberic acid, 4-carboxy phenylalanine, sulfoalanine, 4-phosphonophenylalanine or 4-sulfomethyl phenylalanine.
Histidine-rich domains
In some embodiments, the peptide shuttles of the present specification may further comprise one or more histidine-rich domains. In some embodiments, the histidine-rich domain may be an extension of at least 2, at least 3, at least 4, at least 5 or at least 6 amino acids comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% histidine residues. In some embodiments, the histidine-rich domain may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 consecutive histidine residues. Without being bound by theory, the histidine-rich domain in the shuttle agent may act as a proton sponge in the endosome by protonating its imidazole groups under the acidic conditions of the endosome, providing another mechanism for endosomal membrane instability, further facilitating the ability of endosomal captured cargo to enter the cytosol. In some embodiments, the histidine-rich domain may be located at or towards the N-terminus and/or C-terminus of the peptide shuttling agent.
Joint
In some embodiments, peptide shuttles of the present description may comprise one or more suitable linkers (e.g., flexible polypeptide linkers). In some embodiments, such linkers may separate two or more amphiphilic α -helical motifs (see, e.g., shuttle FSD18 in fig. 49D of WO/2018/068135). In some embodiments, a linker may be used to separate two or more domains (CPD, ELD, or histidine-rich domains) from each other. In some embodiments, the linker can be formed by adding a small hydrophobic amino acid sequence (e.g., glycine) that has no rotational potential and a polar serine residue that imparts stability and flexibility. The linker may be flexible and allow for movement of the shuttle region. In some embodiments, prolines may be avoided because they may increase significant conformational rigidity. In some embodiments, the linker can be a serine/glycine rich linker (e.g., GS, GGS, GGSGGGS, GGSGGGSGGGS, etc.). In some embodiments, the use of a shuttle agent comprising a suitable linker may facilitate delivery of cargo to the suspension cells rather than to the adherent cells. In some embodiments, the linker may comprise or consist of: -Gn-; -Sn-; - (GnSn) n-; - (GnSn) nGn-; - (GnSn) nSn-; - (GnSn) nGn- (GnSn) n-; or- (GnSn) nSn (GnSn) n-, wherein G is the amino acid Gly; s is the amino acid Ser; and n is an integer from 1 to 5. In some embodiments, short stretches or "linkers" of flexible and/or hydrophilic amino acids (e.g., glycine/serine rich stretches) can be added to the N-terminus, C-terminus, or both the N-terminus and C-terminus of the shuttle agents described herein or C-terminal truncated shuttle agents described herein. In some embodiments, such stretches may facilitate dissolution of shuttle agents, particularly shorter shuttle agents (e.g., having amphiphilic alpha helices with strongly hydrophobic moieties), that would otherwise be insoluble or only partially soluble in aqueous solutions. In some embodiments, increasing the solubility of the shuttle peptide may avoid the use of organic solvents (e.g., DMSO) that may obscure the cargo transduction results and/or render the shuttle incompatible with the therapeutic application.
Domain-based peptide shuttling agents
In some aspects, the shuttle agent described herein may be a shuttle agent as described in WO/2016/161516 comprising an Endosomal Leakage Domain (ELD) operably linked to a Cell Penetrating Domain (CPD).
Endosomal Leakage Domain (ELD)
In some aspects, peptide shuttles of the present description may comprise Endosomal Leakage Domains (ELDs) for facilitating endosome escape and entry into the cytoplasmic compartment. As used herein, the expression "endosomal leakage domain" refers to an amino acid sequence that confers the ability of endosomally captured cargo to enter the cytoplasmic compartment. Without being bound by theory, the endosomal leakage domain is a short sequence (typically derived from a viral or bacterial peptide) that is thought to induce destabilization of the endosomal membrane and release of endosomal content into the cytoplasm. As used herein, the expression "endosomolytic peptide" is intended to refer to this general class of peptides having endosomolytic properties. Thus, in some embodiments, a synthetic peptide or polypeptide-based shuttle agent of the present description may comprise ELD as an endosomolytic peptide. The activity of such peptides can be assessed, for example, using the calcein endosome escape assay described in example 2 of WO/2016/161516.
In some embodiments, the ELD may be a peptide that breaks the membrane at an acidic pH, such as a pH-dependent membrane active peptide (PMAP) or a pH-dependent cleavage peptide. For example, peptides GALA and INF-7 are amphiphilic peptides that form an alpha helix when the pH decreases to alter the charge of the amino acids contained therein. More particularly, without being bound by theory, it is shown that ELDs such as GALA induce endosomal leakage through pore formation and turnover of membrane lipids after conformational changes due to pH reduction (Kakudo, chaki et al, 2004, li, nicol et al, 2004). In contrast, ELDs such as INF-7 were shown to induce endosomal leakage by accumulating and destabilizing endosomal membranes (El-safe, futaki et al 2009). Thus, during endosomal maturation, the concomitant decrease in pH causes a change in peptide conformation, and this destabilizes the endosomal membrane, resulting in release of endosomal content. The same principle is considered to apply to toxin A of Pseudomonas (Varkouhi, scholte et al 2011). Upon a decrease in pH, the conformation of the toxin translocation domain changes, allowing it to insert into the endosomal membrane where the pore was formed (London 1992,O'Keefe 1992). This ultimately facilitates destabilization of the endosome and translocation of the complex to the exterior of the endosome. The ELDs described above are encompassed by the ELDs of the present specification and other endosomal leakage mechanisms whose mechanism of action is not well defined.
In some embodiments, the ELD may be an antimicrobial peptide (AMP) such as a linear cationic alpha helical antimicrobial peptide (AMP). Due to their ability to interact strongly with bacterial membranes, these peptides play a key role in the innate immune response. Without being bound by theory, it is believed that these peptides assume a disordered state in aqueous solution, but adopt an α -helical secondary structure in a hydrophobic environment. The latter conformation is believed to contribute to its typical concentration-dependent membrane disruption properties. Some antimicrobial peptides may induce endosomal leakage when accumulated in endosomes at specific concentrations.
In some embodiments, the ELD may be an antimicrobial peptide (AMP) such as cecropin-a/melittin hybrid (CM) peptide. Such peptides are believed to be among the smallest and most effective AMP-derived peptides with membrane disruption capability. Cecropins are a family of antimicrobial peptides that have membrane perturbation ability against both gram-positive and gram-negative bacteria. Cecropin A (CA), the first identified antibacterial peptide, is composed of 37 amino acids with a linear structure. Melittin (M), a 26 amino acid peptide, is a cell membrane cleavage factor found in bee venom. Cecropin-melittin hybrid peptides have been shown to produce short, highly potent antibiotic peptides without cytotoxicity (i.e., non-hemolytic) to eukaryotic cells, a property desirable in any antibacterial agent. These chimeric peptides were constructed from various combinations of the hydrophilic N-terminal domain of cecropin a with the hydrophobic N-terminal domain of melittin, and bacterial model systems have been tested. The two 26-mers, CA (1-13) M (1-13) and CA (1-8) M (1-18) (Boman et al, 1989), have been shown to demonstrate a broad spectrum and improved natural cecropin A efficacy without the cytotoxic effects of melittin.
In the work of producing shorter CM series peptides, andreu et al, 1992 authors constructed hybrid peptides such as 26 mer (CA (1-8) M (1-18)), and compared them to 20 mer (CA (1-8) M (1-12)), 18 mer (CA (1-8) M (1-10)) and six 15 mers ((CA (1-7) M (1-8), CA (1-7) M (2-9), CA (1-7) M (3-10), CA (1-7) M (4-11), CA (1-7) M (5-12) and CA (1-7) M (6-13)), and among the six 15 mers, CA (1-7) M (1-8) showed low antibacterial activity, while the other five showed similar antibiotics compared to 26 mer and no hemolysin and thus peptides such as those derived from the ELD series of peptides may be synthesized as described herein.
In some embodiments, the ELD may be the CM series peptide CM18, [ C (1-7) M (2-12) ], consisting of residues 1-7 of cecropin-A (KWKLFKKIGAVLKVLTTG) fused to residues 2-12 (YGRKRRQRRR) of melittin. CM18, when fused with the cell penetrating peptide TAT, appears to independently cross the plasma membrane and destabilize endosomal membranes, allowing some endosomal captured cargo to be released into the cytosol (Salomone et al 2012). However, in some experiments by the authors, the use of CM18-TAT11 peptide fused to a fluorophore (atto-633) caused uncertainty about the contribution of the peptide compared to the fluorophore, since the fluorophore itself had shown a contribution to endosomolytic-for example, photochemical destruction via endosomal membranes (Erazo-Oliveras et al, 2014).
In some embodiments, the ELD may be CM18 having the amino acid sequence of SEQ ID NO:1 of WO/2016/161516, or a variant thereof having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 90%, 91%, 92%, 93%, 94% or 95% identity with SEQ ID NO:1 of WO/2016/161516 and having endosomolytic activity.
In some embodiments, ELD may be a peptide derived from the N-terminus of the HA2 subunit of influenza Hemagglutinin (HA) that may also cause destabilization of endosomal membranes when accumulated in the endosome.
In some embodiments, the synthetic peptide or polypeptide-based shuttle agent of the present description may comprise ELD as or from ELD set forth in table I, or a variant thereof having endosomal escape activity and/or pH-dependent membrane disruption activity.
Table I: examples of endosomal leakage domains
In some embodiments, the shuttle agent of the present disclosure may comprise one or more ELDs or one or more types of ELDs. More particularly, they may comprise at least 2, at least 3, at least 4, at least 5 or more ELDs. In some embodiments, the shuttle agent may comprise between 1 and 10 ELDs, between 1 and 9 ELDs, between 1 and 8 ELDs, between 1 and 7 ELDs, between 1 and 6 ELDs, between 1 and 5 ELDs, between 1 and 4 ELDs, between 1 and 3 ELDs, and the like.
In some embodiments, the order or position of ELDs relative to other domains (CPD, histidine-rich domains) within the shuttle agents of the present description may vary, so long as the shuttle ability of the shuttle agent is maintained.
In some embodiments, the ELD may be a variant or fragment of any of those listed in table I and having endosomolytic activity. In some embodiments, an ELD may comprise or consist of an amino acid sequence of any of SEQ ID NOs 1-15, 63, or 64 of WO/2016/161516, or a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% identical to any of SEQ ID NOs 1-15, 63, or 64 of WO/2016/161516.
In some embodiments, the shuttle agent of the present description does not comprise one or more of the amino acid sequences of any of SEQ ID NOs 1-15, 63 or 64 of WO/2016/161516.
Cell Penetrating Domain (CPD)
In some aspects, the shuttle agents of the present disclosure may comprise a Cell Penetrating Domain (CPD). As used herein, the expression "cell penetrating domain" refers to an amino acid sequence that confers the ability of a CPD-containing macromolecule (e.g., peptide or protein) to transduce into a cell.
In some embodiments, the CPD may be (or may be derived from) a cell penetrating peptide or a protein transduction domain of a cell penetrating peptide. The cell penetrating peptide can be used as a carrier for successful intracellular delivery of a variety of cargo (e.g., polynucleotides, polypeptides, small molecule compounds, or other macromolecules/compounds that are otherwise impermeable to the membrane). Cell penetrating peptides typically include short peptides rich in basic amino acids that mediate their internalization into cells upon fusion (or otherwise operative attachment) to macromolecules (Shaw, catchpole et al, 2008). The first cell penetrating peptide was identified by analysis of the cell penetrating capacity of the HIV-1 transcriptional cis-activator (Tat) protein (Green and Loewenstein 1988, vitamins, brodin et al 1997). Such proteins contain a short hydrophilic amino acid sequence called "TAT" which facilitates their insertion into the plasma membrane and the formation of pores. From this discovery, a variety of other cell penetrating peptides have been described. In this regard, in some embodiments, the CPD may be a cell penetrating peptide as set forth in table II or a variant thereof having cell penetrating peptide activity.
Table II: examples of cell penetrating peptides
Without being bound by theory, it is believed that the cell penetrating peptide interacts with the cytoplasmic membrane and then spans by pinocytosis or endocytosis. In the case of TAT peptides, their hydrophilic nature and charge are thought to promote their insertion into the plasma membrane and formation of pores (Herce and Garcia 2007). The alpha helical motif within hydrophobic peptides such as SP is also thought to form pores within the plasma membrane (Veach, liu et al, 2004).
In some embodiments, the shuttle agent of the present description may comprise one or more CPDs or one or more types of CPDs. More particularly, it may comprise at least 2, at least 3, at least 4 or at least 5 or more CPDs. In some embodiments, the shuttle agent may comprise between 1 and 10 CPDs, between 1 and 6 CPDs, between 1 and 5 CPDs, between 1 and 4 CPDs, between 1 and 3 CPDs, and the like.
In some embodiments, CPD may be TAT having the amino acid sequence of SEQ ID NO:17 of WO/2016/161516, or a variant thereof having 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% or 95% identity to SEQ ID NO:17 of WO/2016/161516 and having cell penetrating activity; or a transmembrane peptide having the amino acid sequence of SEQ ID NO:18 of WO/2016/161516, or a variant thereof having 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% or 95% identity and having cell penetrating activity with SEQ ID NO:18 of WO/2016/161516.
In some embodiments, the CPD may be PTD4 having the amino acid sequence of SEQ ID NO:65 of WO/2016/161516, or a variant thereof having 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% or 95% identity to SEQ ID NO:65 of WO/2016/161516.
In some embodiments, the order or position of CPD relative to other domains (ELD, histidine-rich domain) within the shuttle agent of the present description may vary, so long as the transduction ability of the shuttle agent is maintained.
In some embodiments, the CPD may be a variant or fragment of any of those listed in table II and having cell penetrating activity. In some embodiments, the CPD may comprise or consist of an amino acid sequence of any of SEQ ID NOs 16-27 or 65 of WO/2016/161516, or a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 90%, 91%, 92%, 93%, 94% or 95% identical to any of SEQ ID NOs 16-27 or 65 of WO/2016/161516 and has cell penetrating activity.
In some embodiments, the shuttle agent of the present description does not comprise any of the amino acid sequences of SEQ ID NOS: 16-27 or 65 of WO/2016/161516.
Methods, kits, uses, compositions and cells
In some embodiments, the present description relates to methods for delivering non-anionic polynucleotide analog cargo from the extracellular space to the cytosol and/or nucleus of a target eukaryotic cell. The method comprises contacting a target eukaryotic cell with cargo in the presence of a concentration of a shuttle agent sufficient to increase transduction efficiency of the cargo compared to the absence of the shuttle agent. In some embodiments, contacting a target eukaryotic cell with cargo in the presence of the shuttle agent results in an increase in transduction efficiency of the non-anionic polynucleotide analog cargo of at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold as compared to the absence of the shuttle agent.
In some embodiments, the present description relates to methods for increasing transduction efficiency of non-anionic polynucleotide analog cargo into the cytosol and/or nucleus of a target eukaryotic cell. As used herein, the expression "increase transduction efficiency" refers to the ability of the shuttle agents of the present description to improve the percentage or proportion of a target cell population to which a cargo of interest (e.g., a non-anionic polynucleotide analog cargo) is delivered intracellularly. Immunofluorescence microscopy, flow cytometry, and other suitable methods may be used to assess cargo transduction efficiency. In some embodiments, the shuttle agents of the present disclosure may achieve a transduction efficiency of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%, e.g., as measured by immunofluorescence microscopy, flow cytometry, FACS, and other suitable methods. In some embodiments, the shuttle agent of the present disclosure may achieve one of the transduction efficiencies described above and at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% cell viability, e.g., as measured by the assay described in example 3.3a of WO/2018/068135 or by another suitable assay known in the art.
In addition to increasing the transduction efficiency of target cells, the shuttle agents of the present disclosure may also facilitate delivery of cargo of interest (e.g., non-anionic polynucleotide analog cargo) into the cytosol and/or nucleus of target cells. In this regard, the use of peptides to efficiently deliver extracellular cargo to the cytosol and/or nucleus of a target cell can be challenging because cargo tends to become trapped in the endosome within the cell after crossing the plasma membrane, which can limit its intracellular availability and can lead to its eventual metabolic degradation. For example, the use of protein transduction domains from HIV-1Tat proteins has been reported to result in substantial sequestration of cargo into intracellular vesicles. In some aspects, the shuttle agents of the present description may facilitate the ability of endosomal captured cargo to escape from the endosome and enter the cytoplasmic compartment. In this regard, the expression "cytosol" in the phrase "increasing the transduction efficiency of non-anionic polynucleotide analogue cargo into the cytosol" is intended to mean that the shuttle agent of the present specification allows intracellular delivery of the cargo of interest to evade endosomal capture and into the cytosol and/or the nuclear compartment. After the cargo of interest enters the cytosol, it is free to bind its intracellular targets (e.g., in the cytosol, nucleus, nucleolus, mitochondria, peroxisomes). In some embodiments, the expression "to cytosol" is therefore intended to encompass not only cytosolic delivery, but also delivery to other subcellular compartments that first require cargo to enter the cytosolic compartment.
In some embodiments, the methods of the present description are in vitro methods (e.g., such as for therapeutic and/or diagnostic purposes). In other embodiments, the methods of the present description are in vivo methods (e.g., such as for therapeutic and/or diagnostic purposes). In some embodiments, the methods of the present description include topical, enteral/gastrointestinal (e.g., oral) or parenteral administration of non-anionic polynucleotide analog cargo and synthetic peptide shuttle agents. In some embodiments, described herein are compositions formulated for topical, enteral/gastrointestinal (e.g., oral) or parenteral administration of non-anionic polynucleotide analog cargo and synthetic peptide shuttle agents.
In some embodiments, the methods of the present disclosure may include contacting a target eukaryotic cell with a shuttle agent or composition as defined herein and a non-anionic polynucleotide analog cargo. In some embodiments, the shuttle agent or composition may be pre-incubated with cargo to form a mixture, and then the target eukaryotic cells are exposed to the mixture. In some embodiments, the type of shuttle agent may be selected based on the characteristics and/or physicochemical properties of the cargo to be delivered intracellularly. In other embodiments, the type of shuttle agent may be selected to take into account the characteristics and/or physicochemical properties, cell type, tissue type, etc. of the cargo to be delivered intracellularly.
In some embodiments, the method may comprise treating the target cells with the shuttle agent or composition multiple times (e.g., 1, 2, 3, 4 or more times per day, and/or on a predetermined schedule). In such cases, a lower concentration of shuttle agent or composition may be desirable (e.g., to reduce toxicity). In some embodiments, the cells may be suspension cells or adherent cells. In some embodiments, one of skill in the art will be able to use different combinations of shuttle agents, domains, uses, and methods to tailor the teachings of the present disclosure to the specific needs of delivering non-anionic polynucleotide analog cargo to specific cells having the desired viability.
In some embodiments, the methods of the present description may be applied to methods of intracellular delivery of non-anionic polynucleotide analogs to cells in vivo. Such methods may be accomplished by parenteral administration or direct injection into a tissue, organ or system.
In some aspects, the compositions or synthetic peptide shuttling agents of the present disclosure may be used in an in vitro or in vivo method of increasing transduction efficiency of non-anionic polynucleotide analog cargo (e.g., targeted therapeutic or biologically relevant RNA molecules) into a target eukaryotic cell, wherein the synthetic peptide shuttling agent or synthetic peptide shuttling agent variant is used at a concentration or is formulated for use at a concentration sufficient to increase transduction efficiency and cytosol and/or nuclear delivery of cargo into the target eukaryotic cell as compared to the absence of the synthetic peptide shuttling agent or synthetic peptide shuttling agent variant.
In some embodiments, the compositions or synthetic peptide shuttles of the present disclosure can be used in therapy, wherein the synthetic peptide shuttles or synthetic peptide shuttles variants transduce a therapeutically relevant non-anionic polynucleotide analog cargo into the cytosol and/or nucleus of a target eukaryotic cell, wherein the synthetic peptide shuttles or synthetic peptide shuttles variants are used at a concentration (or are formulated for use at a concentration) sufficient to increase the transduction efficiency of the cargo into the target eukaryotic cell as compared to the absence of the synthetic peptide shuttles.
In some aspects, described herein are compositions for transduction of non-anionic polynucleotide analog cargo into a target eukaryotic cell, the compositions comprising a synthetic peptide shuttling agent formulated with a pharmaceutically suitable excipient, wherein the concentration of synthetic peptide shuttling agent in the composition is sufficient to increase transduction efficiency and cytosol and/or nuclear delivery of cargo into the target eukaryotic cell after administration compared to the absence of the synthetic peptide shuttling agent. In some embodiments, the composition further comprises cargo. In some embodiments, the composition may be mixed with cargo prior to administration or therapeutic use.
In some aspects, described herein are compositions for use in therapy comprising a synthetic peptide shuttling agent formulated with a non-anionic polynucleotide analog cargo to be transduced into a target eukaryotic cell by the synthetic peptide shuttling agent, wherein the concentration of synthetic peptide shuttling agent in the composition is sufficient to increase transduction efficiency and cytosol and/or nuclear delivery of cargo into the target eukaryotic cell after administration compared to the absence of the synthetic peptide shuttling agent.
In some aspects, described herein are compositions comprising a non-anionic polynucleotide analogue cargo for intracellular delivery and a synthetic peptide shuttling agent independent of or not covalently linked to the non-anionic polynucleotide analogue cargo, the synthetic peptide shuttling agent being a peptide comprising an amphipathic α -helical motif having both a positively charged hydrophilic outer face and a hydrophobic outer face, wherein synthetic peptide shuttling agent increases cytosolic/nuclear delivery of the non-anionic polynucleotide analogue cargo in eukaryotic cells compared to the absence of the synthetic peptide shuttling agent. In some embodiments, the compositions and/or shuttle agents described herein do not contain an organic solvent (e.g., DMSO) or do not contain concentrations of an organic solvent unsuitable for therapeutic or human use. In some embodiments, the shuttle agents described herein are advantageously designed with consideration of water solubility, thereby eliminating the need for the use of organic solvents.
In some embodiments, the shuttle agent or composition and the non-anionic polynucleotide analog cargo may be exposed to target cells in the presence or absence of serum. In some embodiments, the methods may be suitable for clinical or therapeutic use.
In some embodiments, the present description relates to kits for delivering non-anionic polynucleotide analog cargo from an extracellular space to the cytosol and/or nucleus of a target eukaryotic cell. In some embodiments, the present description relates to kits for increasing transduction efficiency of non-anionic polynucleotide analog cargo into the cytosol of a target eukaryotic cell. The kit may comprise a shuttle agent or composition as defined herein, or a suitable container.
In some embodiments, the target eukaryotic cell may be an animal cell, a mammalian cell, or a human cell. In some embodiments, the target eukaryotic cell may be a stem cell (e.g., embryonic stem cell, pluripotent stem cell, induced pluripotent stem cell, neural stem cell, mesenchymal stem cell, hematopoietic stem cell, peripheral blood stem cell), primary cell (e.g., myoblast, fibroblast), immune cell (e.g., NK cell, T cell, dendritic cell, antigen presenting cell), epithelial cell, skin cell, gastrointestinal tract cell, mucosal cell, or lung (lung) cell. In some embodiments, the target cells include those cells that have a cellular mechanism for endocytosis (i.e., production of endosomes).
In some embodiments, the present description relates to isolated cells comprising a synthetic peptide shuttling agent as defined herein. In some embodiments, the cell may be a pluripotent stem cell. It will be appreciated that cells that are generally resistant to or unsuitable for DNA transfection may be candidates for the synthetic peptide shuttle agents of the present description.
In some embodiments, the present description relates to the compositions or methods described herein, wherein the non-anionic polynucleotide analog cargo is a non-anionic antisense oligonucleotide of a gene targeting a hedgehog pathway. In some embodiments, the non-anionic antisense oligonucleotide targets Gli1 for knockdown. In some embodiments, the non-anionic antisense oligonucleotide hybridizes to a polynucleotide sequence of any one of SEQ ID NOS: 365-368 (e.g., when in the cytosol or under cytosolic conditions). In some embodiments, the non-anionic antisense oligonucleotides described herein comprise sequences that hybridize to any of SEQ ID NOS 365-368. In some embodiments, the present description relates to a composition or method described herein, wherein the composition or method is for treating gollin's syndrome and/or basal cell carcinoma.
Examples
Example 1: materials and methods
All materials and methods not described or specified herein are generally carried out as in WO/2018/068135, CA 3,040,645 and/or WO/2020/210916.
Materials and reagents
Cell lines and culture conditions
The cells were cultured according to the manufacturer's instructions.
Phosphoric acid diamide morpholino oligomer transduction protocol
1mM of a diamide morpholino phosphate oligomer labeled with the fluorophore FITC (PMO-FITC) was prepared in sterile water.
HeLa cells were plated in 96-well dishes (20 000 cells/well) the day prior to the experiment. Each delivery mixture containing synthetic peptide shuttle (7.5, 10 or 20. Mu.M) and PMO-FITC (6. Mu.M) was prepared and made up to 50. Mu.L with RPMI-1640 medium. Cells were washed once with PBS and shuttle/PMO-FITC or PMO-FITC alone was added to the cells for 5 minutes. mu.L of DMEM containing 10% FBS was then added to the mixture and removed. Cells were washed once with PBS and incubated in DMEM containing 10% FBS. After 2 hours of incubation the cells were analyzed by flow cytometry.
Antisense GFP-PMO transduction protocols in GFPd reporter HeLa cells
Stock solutions of cargo were prepared as follows: PMO stock (1 mM in water); siRNA stock (in 60mM KCl, 6mM HEPES-pH 7.5 and 0.2mM MgCl) 2 100 μm) in the middle).
And (3) delivery. HeLa-plex-TetO-GFPd cells were plated in 96-well dishes (20 000 cells/well) one day prior to the experiment. Each delivery mixture containing synthetic peptide shuttle (7.5. Mu.M) and PMO (0.1 or 10. Mu.M) was prepared and completed to 50. Mu.L with RPMI-1640 medium. Cells were washed once with PBS and shuttle/PMO or PMO was added only to cells for 5 minutes. mu.L of DMEM containing 10% FBS was then added to the mixture and removed. Cells were washed once with PBS and incubated in DMEM containing 10% FBS. After 5 hours of incubation the cells were analyzed by flow cytometry.
And (5) transfection. HeLa-plex-TetO-GFPd cells were plated in 96-well dishes (20 000 cells/well) one day prior to the experiment. Lipofectamine was used according to the manufacturer's instructions TM RNAiMax reagent transfected siRNA (2.5 pmol). Lipofectamine TM RNAiMax was diluted in Opti-MEM (0.3. Mu.L in 25. Mu.L). The siRNA stock was first diluted at 10. Mu.M in RNase-free water and then 2.5pmol (0.25. Mu.L) was added to 25. Mu.L Opti-MEM. Diluting Lipofectamine TM RNAiMax was mixed with diluted siRNA (50 nM final concentration) and incubated for 5 min at room temperature. Cells were washed once with PBS and 100 μl DMEM containing 10% FBS was added to the cells. Will be in Lipofectamine TM The siRNA diluted in RNAiMax is added to the cells. After 24h, the medium was replaced with 100. Mu.L of fresh DMEM containing 10% FBS. Cells were analyzed by flow cytometry 48 hours after transfection.
Antisense PMO transduction protocol to knock down Gli1 expression in DU145 cells
The day before the experiment, DU145 cells were trypsinized and plated in 24-well dishes (500 000 cells/well). Each delivery mix containing synthetic peptide shuttle agent alone (5. Mu.M) and PMO-FITC (6. Mu.M) or together with antisense PMO (6. Mu.M) designed to knock down target protein expression was prepared and completed to 1mL with normal RPMI-1640 medium. Cells were washed once with PBS and the delivery mixture was added to the cells for 5 minutes. 2mL of RPMI-1640 containing 10% FBS was then added to the mixture and removed. Untreated cells were incubated with RPMI-1640 only. Cells were incubated in fresh RPMI-1640 with 10% FBS. After 48 hours, the medium was removed and the cells were washed once with PBS prior to trypsin digestion. Cells were harvested, collected by centrifugation, washed, and resuspended in PBS. PMO-FITC positive and PMO-FITC negative cells were then sorted and collected by FACS (BD FACS Aria Fusion). FITC-positive and FITC-negative cell samples were collected by centrifugation and resuspended in 50. Mu.L of protein extraction RIPA buffer (150 mM NaCl, 1% Nonidet TM P-40, 0.1% SDS, 0.5% sodium deoxycholate, 25mM Tris). The total protein concentration was measured with BCA protein assay kit. For all conditions, 10. Mu.g of protein was prepared with 4 XLaemmeli and RIPA buffer in a final volume of 40. Mu.L. The protein samples were then heated at 90℃and separated by 8% SDS-PAGE gel. The proteins were then transferred overnight (25 volts) onto a 0.2 μm PVDF membrane. Using 5% bovine serum albumin, tris buffer saline, 0.1%The membrane was blocked with 20 solution (5% BSA/TBS-T) for 1 hour. After blocking, the membranes were incubated with a 1:1000 solution of 5% BSA/TBS-T containing primary anti-alpha-actin (D6F 6) antibody for 1 hour. The alpha-actin present in the cell lysates was used as a load control. Subsequently, the membranes were incubated overnight with a 5% BSA/TBS-T solution containing anti-Gli 1 primary antibody diluted 1:500. The membrane was then subjected to 1 hour incubation at a 1:5000 dilution of HRP conjugated goat anti-rabbit secondary 5% BSA/TBS-T solution. Using Clarity TM Western ECL substrate and Chemidoc TM The XRS instrument performs chemiluminescent detection. Using ImageJ TM Software evaluates Gli1 and alpha-actin densitometry.
Propidium iodide or GFP-NLS transduction protocols
HeLa cells were plated in 96-well dishes (20 000 cells/well) the day prior to the experiment. Each delivery mixture containing synthetic peptide shuttle (10 μm) and Propidium Iodide (PI) (10 μg/mL) or GFP-NLS (10 μm) was prepared and completed to 50 μl with Phosphate Buffered Saline (PBS) for PI or RPMI-1640 medium for GFP-NLS. Cells were washed once and shuttle/PI or shuttle/GFP-NLS was added to cells for 1 min (PI) or 5 min (GFP-NLS). mu.L of DMEM containing 10% FBS was then added to the mixture and removed. Cells were washed once with PBS and incubated in DMEM containing 10% FBS. After 2 hours of incubation the cells were analyzed by flow cytometry. Cells were analyzed 1 hour after PI or GFP-NLS treatment.
Example 2: synthetic peptide shuttle agent: novel class of intracellular delivery peptides
Synthetic peptides, known as shuttles, represent a new class of intracellular delivery peptides that have the ability to rapidly transduce polypeptide cargo into the cytosol/nuclear compartment of eukaryotic cells. In contrast to traditional cell penetrating peptide-based intracellular delivery strategies, synthetic peptide shuttles are not covalently linked to their polypeptide cargo. In fact, covalent attachment of shuttle agents to their cargo in a non-cleavable manner often has a negative impact on their transduction activity.
A first generation synthetic peptide shuttle agent is described in WO/2016/161516 and consists of a multi-domain based peptide having an Endosomal Leakage Domain (ELD) operably linked to a Cell Penetrating Domain (CPD), and optionally further comprising one or more histidine-rich domains. Although it was originally believed that shuttle-mediated transduction of cargo occurred through a mechanism similar to conventional cell penetrating peptides, the rate and efficiency of cargo delivery into the cytosol/nucleus compartment suggests a strong contribution from a more direct delivery mechanism across the plasma membrane without the need for complete endosomal formation (Del' guide et al 2018). Thus, using the first generation shuttle agents as a starting point, large-scale iterative design and screening projects were performed to optimize the shuttle agents to transduce polypeptide cargo quickly and efficiently while reducing cytotoxicity. The project involves manual and computer-aided design/modeling of nearly 11,000 synthetic peptides, and the synthesis and testing of hundreds of different peptides for their ability to rapidly and efficiently transduce a variety of polypeptide cargo in a variety of cells and tissues. Rather than consider the shuttle agent as a fusion of known cell penetrating peptides (CPDs) and endosomolytic peptides (ELDs) from the literature, each peptide is fully contemplated based on its predicted three-dimensional structure and physicochemical properties. The design and screening procedure ultimately resulted in a second generation synthetic peptide shuttle agent defined by a rationally designed set of 15 parameters that control the shuttle agent described in WO/2018/068135, which has improved transduction/toxicity characteristics of polypeptide cargo compared to the first generation shuttle agent. These second generation synthetic peptide shuttles were designed and empirically screened for rapid transduction of polypeptide cargo (i.e., typically within 5 minutes) and thus were designed primarily to lack prototype CPD.
Example 3: synthetic peptide shuttles deliver naked DNA/RNA cargo inefficiently into the cytosol/nucleus compartment
For decades, cell Penetrating Peptides (CPPs) have been used for transfection strategies for intracellular delivery of DNA/RNA. Delivery of polynucleotides using CPPs can be categorized into two classes, wherein CPPs are covalently or electrostatically bound to their polynucleotide cargo. The complexity of the synthesis of the former is a significant obstacle, whereas the latter is relatively simple in view of the cationic nature of CPPs and the negatively charged phosphate backbone of DNA/RNA. Thus, during the screening of first generation synthetic peptide shuttles, experiments were conducted to determine if the shuttles were able to efficiently transduce plasmid DNA cargo into the nucleus for gene expression. The results of these transfection experiments are reported in example 7.2 of WO/2016/161516, where the first-generation shuttle CM18-TAT-Cys was indeed able to deliver the fluorescently labeled plasmid DNA encoding GFP in cells. However, GFP expression was only detected in 0.1% of the cells (see table 7.1 of WO/2016/161516), which strongly suggests that internalized plasmid DNA was still captured in endosomes and was unable to enter the cytosol/nuclear compartment. Example 7.3 of WO/2018/068135 reexamined the ability of a variety of first and second generation synthetic peptide shuttles to successfully transfect cells with GFP encoding plasmids. Results were similar-GFP expression was detected in less than 1% of cells for all shuttle agents (see table 7.2 of WO/2018/068135). These results indicate that synthetic peptide shuttles are not suitable for transduction of DNA/RNA into the cytosol/nucleus of eukaryotic cells.
Several strategies have been performed to attempt to deliver DNA/RNA cargo into the cytosol/nucleus compartment, but with no success. Interestingly, in experiments attempting to co-transduce both GFP and polynucleotide cargo, the presence of the polynucleotide was observed to reduce the transduction efficiency of GFP cargo in a concentration-dependent manner. Given that the inhibitory effect of polynucleotides may be due to negatively charged phosphate backbones, we attempted to coat polynucleotides with positively charged small molecules to neutralize negative charge prior to transduction. Attempted positively charged small molecules include 1, 3-diaminoguanidine monohydrochloride; 3, 5-diamino-1, 2, 4-triazole; guanidine hydrochloride; and L-arginine amide dihydrochloride at a concentration ranging from 100nM to 10mM. However, these strategies fail to significantly improve cytosolic/nuclear delivery of DNA/RNA cargo, where endosomal capture remains problematic, potentially suggesting that shuttle-mediated transduction requires more than just charge neutralization.
Example 4: synthetic peptide shuttles capable of intracellular delivery of FITC-labeled charge-neutral polynucleotide analogs
Diamide morpholino oligomer (PMO) is a short single stranded polynucleotide analog that can be used as an antisense oligonucleotide for modifying gene expression by steric hindrance. Their molecular structure contains DNA/RNA nucleobases attached to the backbone of a methylenemorpholine ring linked via a diamide phosphate group. Because of its charge-neutral structure, many of the intracellular delivery systems developed for DNA/RNA (e.g., cationic lipids; electrostatic coupling to cationic cell penetrating peptides) are not suitable for intracellular delivery of PMO. Thus, modified forms of PMO have been developed for intracellular delivery, including covalent attachment of PMO to eight guanidinium head groups (Vivo-morphino) or cell Penetrating Peptides (PPMO). However, besides the direct injection strategy, few reports indicate that cytosol delivery of unmodified PMOs capable of modifying gene expression is successful.
To explore whether synthetic peptide shuttles were capable of intracellular delivery of PMO, heLa cells were exposed to normal RPMI medium containing 6. Mu.M of 25 mer PMO molecules covalently labeled with fluorophores ("PMO-FITC"; SEQ ID NO: 348) in the presence of 7.5, 10, or 20. Mu.M representative members of first and second generation synthetic peptide shuttles for 5 minutes. The results are shown in fig. 1, where the average cell viability ("average viability") and "average% of pmo+ cells" (number of PMO-FITC cargo positive living cells) were evaluated by flow cytometry, as described in WO/2018/068135. The "average delivery score" provides a further indication of the total amount of cargo delivered (PMO-FITC) per cell in all cargo positive cells and is calculated by: the average fluorescence intensity measured for the live PMO-fitc+ cells (of at least duplicate samples) was multiplied by the average percentage of live PMO-fitc+ cells divided by 100,000. Finally, the "delivery-viability score" for each peptide was calculated as the average viability times the average delivery score times 10, enabling the shuttle agent to be ranked according to its transduction activity and toxicity. Rows in fig. 1 are ranked from lowest to highest according to their delivery-vitality scores.
As negative controls, "cargo only" (cells incubated with PMO-FITC in the absence of peptide) and "FSD10 disorder" (control peptide having the same amino acid composition as shuttle FSD10, except that the primary amino acid sequence is "scrambled" to disrupt the cationic amphiphilic structure common to all shuttles) were included. The results in fig. 1 show that the second generation shuttle agent designed to transduce protein cargo-both in terms of transduction efficiency (average% pmo+ cells) and in terms of average amount of cargo delivered to each cell (delivery score) -was generally superior to the first generation shuttle agent. The latter is particularly advantageous for gene expression modification purposes, given that the effect of PMO and other antisense oligonucleotide analogs depends on intracellular concentration. The first generation shuttle agents CM 18-penetrating protein-cys, CM18-TAT and His-CM18-PTD4 included in the experiments exhibited higher toxicity (i.e. average viability below 50%) at the lowest concentration tested (7.5 μm) and were therefore excluded from fig. 1. This result is unexpected because such first generation shuttle agents have been used at higher concentrations to deliver GFP-NLS in the same HeLa cells without a comparable negative impact on viability. The increase in toxicity cannot be attributed solely to PMO-FITC cargo toxicity, as many other shuttles tested showed much higher average% and average PMO delivery scores for pmo+ cells without the same level of toxicity. These results indicate that toxicity may be due to interactions between each shuttle agent and PMO-FITC cargo, and that "matching" of shuttle agents/cargo may be considered for toxicity and transduction activity purposes.
Taken together, the results in fig. 1 show that synthetic peptide shuttles are capable of intracellular delivery of PMO-FITC cargo, and that second generation shuttles are generally superior to first generation shuttles in terms of transduction efficiency, amount of cargo delivered/cell, and toxicity.
Example 5: closing devicePeptide-forming shuttle FSD10 transduces antisense PMO into the cytosol, enabling the expression in HeLa cells
Mid-knock down GFP gene expression
Endosomal trapping was found to be problematic for shuttling agent mediated transduction of naked DNA/RNA cargo (example 3). The flow cytometry results presented in example 4 and FIG. 1 suggest that cells were positive for PMO-FITC cargo, whether the cargo was cytosol/nucleus or endosome captured. In addition, recent reports from other teams indicate that hydrophobic fluorophores (e.g., tetramethyl rhodamine) may interact with lipid bilayers and enhance membrane instability (Brock et al, 2018). Thus, experiments were conducted to demonstrate that shuttle agents can deliver unlabeled PMO cargo in a biologically active form into the cytosol/nucleus compartment to knock down expression of target genes.
A "HeLa plex TetO GFPd" cell line was created that consisted of HeLa cells stably expressing GFP variant ("GFPd") engineered to have a short half-life of about 2 hours. HeLa plex TetO GFPd cells were exposed to normal RPMI medium containing varying concentrations of antisense PMO molecules designed to knock down GFPd expression ("PMO-GFP"; SEQ ID NO: 345) or off-target antisense PMO molecules targeting GLI1 expression ("PMO-Gli 1"; SEQ ID NO: 346) and with or without synthetic peptide shuttling agent for 5 minutes. As a further control, siRNAs with the same sequence as the antisense ("siRNA-GFP"; SEQ ID NO: 349) and non-specific ("siRNA-Gli 1"; SEQ ID NO: 350) PMO molecules were included and passed through a commercial cationic lipid-based delivery system (Lipofectamine) TM RNAiMax) delivery. Following transduction/transfection, cells were washed, cultured in growth medium, and then analyzed by flow cytometry to evaluate the effect on GFPd expression at appropriate times to observe knockdown (5 hours for PMO treatment or 48 hours for siRNA treatment).
As shown in fig. 2, the baseline of untreated HeLa plex TetO GFPd cells was 65% average gfp+ cells, and exposure of the cells to PMO cargo alone (no shuttle agent) resulted in no change in this regard. Interestingly, exposure of cells to 10. Mu.M PMO-GFP cargo in the presence of 7.5. Mu.M shuttle FSD10 resulted in a reduction of the percentage of GFP+ cells to 34%. The effect was specific for PMO-GFP cargo, as no effect on GFPd expression was observed in the PMO-non-specific cargo of the negative control. Finally, the effect was dose dependent in that knockdown of GFPd expression was lost by lowering PMO-GFP cargo concentration to 0.1 μm.
Example 6: synthetic peptide shuttling agent FSD250 transduces antisense PMO into the cytosol, enabling the refinement of DU145
Intracellular knockout of Gli1 expression
Human DU145 cells were exposed to normal RPMI medium containing 6. Mu.M of an antisense PMO molecule designed to knock down Gli1 protein expression ("PMO-Gli 1"; SEQ ID NO: 346) or an antisense PMO molecule designed to knock down Wnt1 protein expression ("PMO-Wnt 1"; SEQ ID NO: 347) in the presence of 5. Mu.M synthetic peptide shuttle FSD250 for 5 min. The tracer PMO-FITC molecule (6 μm) was also included under both conditions to enable Fluorescence Activated Cell Sorting (FACS) of transduced cells from non-transduced cells in the same cell population. At 48 hours post transduction, cells were analyzed by flow cytometry, and then isolated by FACS into a FITC positive population and a FITC negative population.
For PMO-Gli/PMO-FITC transduction, the average% of PMO-FITC+ cells was 37%, and viability was 95.8%. For PMO-Wnt1/PMO-FITC transduction, the average% of PMO-FITC+ cells was 36.8%, and viability was 75.7%. For untreated cells, the average% of PMO-fitc+ cells was 0.6% and viability was 91.3%.
Fitc+ and FITC-cell populations were then lysed, resolved by SDS-PAGE, and western blot analysis was performed using anti-Gli 1 polyclonal antibodies, anti-actin polyclonal antibodies as loading controls, and appropriate enzyme conjugated secondary antibodies. The results are shown in fig. 3, including densitometry scan values for each band. FIG. 3 clearly shows that intracellular delivery of antisense PMO-Wnt1 and antisense PMO-Gli1 by shuttling agent in transduced cells ("FITC+ cells") knockdown the expression of Gli1 protein compared to non-transduced cells ("FITC-cells"). As expected, the Gli1 knock-down effect of directly knocking down Gli1 mRNA with PMO-Gli1 was stronger than the indirect knock-down of Gli1 by knocking down Wnt1 mRNA (which is part of the same signaling pathway).
Example 7: synthetic peptide shuttling agent FSD250 transduces antisense PMO into the cytosol, enabling the refinement of DU145
Intracellular knockout of Gli1 expression
Human DU145 cells were exposed to normal RPMI medium containing 6. Mu.M PMO-Gli1 (SEQ ID NO: 346) or PMO-GFP (SEQ ID NO: 345) for 5 min in the presence of 3.75. Mu.M synthetic peptide shuttle FSD 250. The tracer PMO-FITC molecule was also included under both conditions (and separately as an additional negative control) to enable separation of transduced cells from non-transduced cells by FACS. At 48 hours post transduction, cells were separated by FACS into FITC positive and FITC negative populations.
For PMO-FITC-only transduction, the average% of PMO-FITC+ cells was 44.1%, and viability was 77.7%. For PMO-Gli1/PMO-FITC transduction, the average% of PMO-FITC+ cells was 36.0%, and viability was 61.1%. For PMO-GFP/PMO-FITC transduction, the average% PMO-FITC+ cells was 48.9%, and viability was 77.0%. For untreated cells, the average% of PMO-fitc+ cells was 0.1% and viability was 84.5%.
Fitc+ and FITC-cell populations were then lysed, resolved by SDS-PAGE, and western blot analysis was performed using anti-Gli 1 polyclonal antibodies, anti-actin polyclonal antibodies as loading controls, and appropriate enzyme conjugated secondary antibodies. The results are shown in fig. 4, including the relative densitometry scan of each band normalized to its corresponding actin load control band. FIG. 4 clearly shows that the Gli1 protein expression was knockdown by shuttle-mediated intracellular delivery of antisense PMO-Gli1, whereas cells transduced with the tracer PMO-FITC alone or with PMO-GFP were not.
Example 8: large-scale screening of candidate peptide shuttles for Propidium Iodide (PI) and GFP-NLS transduction Activity
A proprietary library of over 300 candidate peptide shuttles was screened in parallel for both propidium iodide (PI; small molecule cargo) and GFP-NLS transduction activity in HeLa cells using flow cytometry as generally described in example 1. PI is used as cargo because it exhibits 20 to 30 fold enhanced fluorescence and a detectable shift in the maximum excitation/emission spectrum only after binding to genomic DNA-this property makes it particularly useful for distinguishing endosomal captured cargo from endosomal escape cargo that can enter the cytosol/nuclear compartment. Thus, both intracellular delivery and endosomal escape can be measured by flow cytometry, since any PI that remains trapped in the endosome does not reach the nucleus and exhibits neither enhanced fluorescence nor spectral shift.
Since a large number of peptides were screened, negative controls were performed in parallel for each experimental batch, and included "no-treatment" (NT) controls in which cells were not exposed to shuttle peptide or cargo, and "cargo-only" controls in which cells were exposed to cargo in the absence of shuttle agent. The results are shown in fig. 5, where "transduction efficiency" refers to the percentage of all living cells positive for cargo (PI or GFP-NLS). The "average delivery score" provides a further indication of the total amount of cargo delivered per cell in all cargo positive cells. The average PI or GFP-NLS delivery scores were calculated by: the measured average fluorescence intensity (of at least duplicate samples) for either live pi+ or gfp+ cells is multiplied by the average percentage of live pi+ or gfp+ cells divided by 100,000 (for GFP delivery) or divided by 10,000 (for PI delivery). The average delivery scores for PI and GFP-NLS for each candidate shuttle agent were then normalized by dividing by the average delivery score for the "cargo only" negative control performed in parallel for each experimental batch. Thus, "normalized average delivery score" in fig. 5 represents the fold increase in average delivery score relative to the "cargo only" negative control.
The observed batch-to-batch variation for the negative control was relatively small for GFP-NLS but significantly higher when PI was used as cargo. For example, the transduction efficiency of the "cargo-only" negative control varied from 0.4% to 1.3% for GFP-NLS and from 0.9% to 6.3% for PI. Furthermore, the transduction efficiency (e.g., FSD174 disorder; data not shown) of several negative control peptides tested in parallel (i.e., peptides known to have low or no GFP transduction activity) sometimes resulted in lower transduction efficiency for PI (but not for GFP-NLS) than the "cargo-only" negative control, in some cases up to 5% lower transduction efficiency, possibly due to non-specific interactions between PI and peptide. This was not observed for GFP-NLS transduction experiments. The foregoing suggests that the shuttle transduction efficiency for at least PI may be more suitable for comparison with the transduction efficiency of the negative control peptide than with the "cargo only" condition.
Among the candidate peptide shuttles having an average PI transduction efficiency of at least 20% in fig. 5 are peptides having a length of less than 20 residues: FSD390 (17 aa), FSD367 (19 aa) and FSD366 (18 aa). Also included among candidate peptide shuttles having an average PI transduction efficiency of at least 20% are peptides comprising non-physiological amino acid analogs (e.g., FSD435, which corresponds to FSD395, except that lysine residue (K) is replaced with an L-2, 4-diaminobutyric acid residue) or chemical modifications (e.g., FSD438, which corresponds to FSD10, except that there is an N-terminal octanoic acid modification; FSD436, which corresponds to FSD222, except that phenylalanine residue (F) is replaced with a (2-naphthyl) -L-alanine residue; FSD171, which corresponds to FSD168, except that there is an N-terminal acetyl and C-terminal cysteamide group). These results demonstrate the robustness of peptide shuttle platform technology to withstand the use of non-physiological amino acids or analogs thereof in place of physiological amino acids and/or the use of chemical modifications.
Additional screening assays were performed using further shuttle agents as shown in figure 6. Among these results, there are several active shuttles that were truncations of longer shuttles previously shown to have transduction activity: FSD10-15 (15 aa) and FSD418-12-2 (12 aa), FSD418 (15 aa), CM18 (18 aa) and penetratin (16 aa).
Example 9: synthetic peptide shuttling agents efficiently transduce PMO and PNA in HeLa cells
HeLa cells were transduced with 10. Mu.M PMO-FITC or fluorescent-labeled Peptide Nucleic Acid (PNA) (PNA TelC-Alexa 488; catalog number F1004; PNA BIO Inc.), as generally described in the transduction protocol described in example 1 with some modifications. The shuttle peptide used was FSD250 (5 μm) and the cells were contacted with cargo and shuttle for two minutes and analyzed by flow cytometry after 1 hour incubation. In addition, PNA was re-dissolved in water instead of Dimethylformamide (DMF) recommended by the manufacturer, since inclusion of DMF in the medium resulted in cell viability below 50%. The cargo transduction results in fig. 7A demonstrate that FSD250 greatly increases intracellular delivery of both PMO ("fsd250+pmo" delivery) and PNA ("fsd250+pna") cargo compared to the absence of shuttle agents ("PMO only" and "PNA only"; "NT" = untreated cells). Cell viability remained high under all test conditions as shown in figure 7B. Parallel experiments using FSD250 in an attempt to transduce fluorescent-labeled siRNA as cargo resulted in only 5% intracellular delivery (data not shown). These results indicate that synthetic peptide shuttles can transduce other non-anionic polynucleotide analogs, such as PNA, in addition to PMO, rapidly and efficiently.
Example 10: effects of naked DNA/RNA on shuttle-mediated PMO cargo transduction
While naked DNA/RNA cargo itself is shown to be a bad cargo for synthetic peptide shuttles (examples 3 and 9), this example evaluates their potential dominant negative impact on shuttles-mediated PMO cargo transduction in trans. Briefly, RH-30 cells (150,000 cells/well in 24-well dishes) were contacted with a delivery mixture of 6. Mu.M PMO-FITC and 5. Mu.M synthetic peptide shuttle FSD250 in RPMI in the presence of increasing amounts of DNA oligonucleotides or labeled sgRNA in the medium for 2 minutes. Cells were then washed, incubated in growth medium, and then collected after 1h for flow cytometry analysis. The results in FIG. 8 show that reduced PMO-FITC transduction efficiency was observed in the presence of 1.5 μg of DNA oligonucleotide (3 μg/mL) (FIG. 8A) and 2 μg of sgRNA (4 μg/mL) (FIG. 8B). Cell viability remained above 75% under all test conditions.
Example 11: comparison of first and second Generation synthetic peptide shuttles to transduce PMO cargo
In general, second generation synthetic peptide shuttles exhibit higher cargo transduction efficiencies than the first generation shuttles. This example compares PMO transduction activity of a first generation shuttle agent comprising prototype CPD with two rationally designed second generation synthetic peptide shuttle agents. Briefly, RH-30 cells (20,000 cells/well in 96-well dishes) were contacted with a delivery mixture of 6. Mu.M PMO-FITC and increasing concentrations of the first generation shuttle His-CM18-PTD4 or CPD (FSD 250 and FSD 10) of two second generation synthetic peptide shuttles lacking CPD in RPMI for 2 minutes. Cells were washed, incubated in complete medium, and then after 1h cells were collected for flow cytometry analysis. PMO-FITC transduction efficiencies are shown in fig. 9A, and cell viability is shown in fig. 9B. The results in FIG. 9A show that FSD250 and FSD10 produce higher transduction efficiencies for PMO-FITC cargo than His-CM18-PTD 4.
Example 12: comparison of synthetic peptide shuttling agent mediated PMO transduction with self-internalizing VivoPMO
Gli1 knockdown experiments were generally performed as described in example 6 to compare shuttle-mediated transduction of unmodified PMO with commercially available internalized Vivo-Morpholino (VivoPMO), a PMO chemically modified with terminal octaguanidinium dendrimers to promote entry into cells. Briefly, RH-30 cells were contacted with a delivery mixture of 6. Mu.M cargo (PMO-Gli 1 or VivoPMO-Gli 1) in RPMI for 2 minutes in the presence or absence of 5. Mu.M synthetic peptide shuttle FSD 250. Cells were then washed, incubated in complete medium, and then collected after 24h to analyze Gli1 protein expression by western blot collection using anti-Gli 1 polyclonal abs (Abcam Ab273018, 150 kDa), anti-GAPDH abs (Abcam Ab181602, 37 kDa), and anti-rabbit HRP secondary abs. Western blot results are shown in fig. 10A, and corresponding densitometry scan analysis is shown in fig. 10B. The Gli1 protein knockdown was only observed in cells treated with FSD250 and PMO-Gli1 cargo. The lack of Gli1 knockdown observed in cells exposed to VivoPMO-Gli1 alone suggests that the 2 minute incubation time in fig. 10 is insufficient to self-internalize VivoPMO. In fact, this is consistent with the manufacturer's recommended 2-4 hour incubation time for the VivoPMO to achieve adequate self-internalization by endocytosis. Regardless, the results in fig. 10 highlight the tremendous difference in internalization kinetics between slow endocytosis-dependent intracellular delivery of VivoPMO and the rapid and efficient PMO transduction observed with synthetic peptide shuttles.
Example 13: ratio of synthetic peptide shuttling agent mediated PMO transduction to Endoporter mediated intracellular PMO delivery
Compared with
PMO cargo delivery experiments were performed in HeLa cells to directly compare synthetic peptide shuttles-mediated PMO transduction with Endoporter-mediated intracellular PMO delivery. For shuttle-mediated transduction, heLa cells were exposed to 10 μm PMO-FITC in RPMI for 5 min in the presence of 2.5, 5, 7.5 or 10 μm second generation shuttle FSD 396. For Endoporter mediated delivery, endoporters are commercially available at 2.5, 5, 7.5 or 10 μm in growth medium TM HeLa cells were exposed to 10. Mu.M PMO-FITC in the presence of peptide (GeneTools, LLC) for a minimum incubation time of at least 24 hours as recommended by the manufacturer. After the washing step, intracellular PMO-FITC fluorescence was observed by immunofluorescence microscopy, the delivery results were compared, and the results are shown in FIG. 11. HeLa cells (FIG. 11B) exposed to PMO-FITC cargo only exhibited only slight intracellular delivery after 48h compared to untreated cells (FIG. 11A). Although FSD396 induced robust intracellular delivery of PMO-FITC after 5 minutes of incubation (FIG. 11C), comparable levels of intracellular PMO-FITC were reached only after 48 hours of incubation with the Endoporter peptide (FIG. 11D). Furthermore, the Endoporter treated cells exhibited undesired morphological changes that were not observed in untreated cells (fig. 11A), in cells treated with cargo only (fig. 11B), or in cells treated with shuttle agents (fig. 11C). Large PMO-FITC aggregates were also observed in the Endoporter treated cells, which cannot be removed from the cells by the washing step.
Example 14: gli1 knockdown triggers increased apoptosis in BCC cell lines, but in normal human skin cell lines
Will not in (3)
Gollin syndrome, also known as nevus basal cell carcinoma syndrome or Basal Cell Carcinoma Nevus Syndrome (BCCNS), is a genetic disease associated with aberrant hedgehog (Hh) pathway signaling, leading to frequent growth of Basal Cell Carcinoma (BCC) on the face, hands, back and neck. Patients with gollin syndrome may develop up to 30 lesions each year that originate from the basal cell layer of the skin between the epidermis and dermis. Gollin patients have a genetic mutation that results in constitutive activation of the Hh pathway.
Gli1 is a transcription factor responsible for expressing determinants of the Hh pathway and thus can be considered as a major regulator of Hh signaling. The effect of Gli1 knockdown on two human skin cell lines was evaluated: a skin epithelial-like cell line derived from normal human skin (NCTC-2544) and a human basal cell carcinoma cell line (UW-BCC 1). Normal-derived NCTC-2544 cells and BCC-derived UW-BCC1 cells were exposed to self-internalizing VivoPMO-Gli1 (15 μm) for 24 or 48h in complete cell culture medium. After 48h, about 60% knockdown of Gli1 protein expression was observed by western blotting. In parallel, the percentage of apoptosis was measured by flow cytometry with fluorescence labelled annexin-V. Interestingly, treatment with VivoPMO-Gli1 resulted in 68% -72% apoptotic UW-BCC1 cells after 48h, compared to only 11% apoptotic cells treated with negative control VivoPMO. In contrast, treatment with VivoPMO-Gli1 resulted in only 3% -6% of apoptotic NCTC-2544 cells after 48h, and only 2% of apoptosis in cells treated with negative control VivoPMO. These results support the knockdown of Gli1 by intracellular delivery of Gli 1-specific PMOs to treat basal cell carcinoma.
Example 15: design, synthesis and shuttle-mediated transduction of PMO for Gli1 knockdown
Four different PMOs were designed and synthesized, targeting different regions proximal to the 5' untranslated region or start codon of the human Gli1 gene. Four PMOs were synthesized in ascending order of their distance from Gli1 start codon: PMO-Gli1_Opt (bound to SEQ ID NO:365 and spanning the Gli1 start codon); PMO-Gli1_Opt1 (binds to SEQ ID NO: 366); PMO-Gli1_Opt2 (bound to SEQ ID NO: 367); and PMO-Gli1_Opt3 (binding to SEQ ID NO: 368). RH-30 cells were transduced with each of the four PMO cargoes (6. Mu.M) or with negative control PMO-FITC with shuttle FSD250 (6. Mu.M) as described in example 12. The overall transduction efficiency in the transduction experiments was about 80% as estimated by flow cytometry of cells transduced with PMO-FITC control cargo. Cells were harvested 24 hours after delivery and knockdown of Gli1 protein expression was assessed by western blotting (fig. 12). When transduced with a shuttle agent, all four PMOs knocked down the expression of Gli1 protein, but minimal knockdown was observed in the absence of the shuttle agent. Densitometry analysis of Gli1 and control actin bands revealed that four PMOs knocked down Gli1 expression to 27% -45% of untreated cells (fig. 12). Transduction experiments were repeated with increasing concentrations of PMO-gli1_opt, and Gli1 knockdown was found to be dose dependent: 0.325, 0.75, 1.5, 3 and 6. Mu.M PMO-Gli1_Opt knockdown Gli1 levels to 48%, 43%, 34%, 33% and 22% of untreated cells, respectively.
Example 16: shuttle-mediated use for Gli1 knockdown in basal cells of patient-derived tumor explants
Transduction of PMO
Freshly obtained basal cell carcinoma tumors after Mohs-type surgery were incubated in complete DMEM medium on a silk screen with air-exposed surface. To allow delivery of cargo to epidermis and dermis, tumors were washed with PBS 1X and washed with Pantec PLEASE TM The laser permeabilizes the stratum corneum of the explant according to the following parameters: the density of the holes is 2.5%, the array size is 14mm, the hole depth is 20 μm (4.9J/cm) 2 0.8W). The explants were then split in half, with one half treated with PBS 1x-2% hydroxyethylcellulose solution containing 25. Mu.M PMO-Gli1-Cy5 and 40. Mu.M FSD250, and the other half treated with the same solution containing PMO-Gli1-Cy5 alone (no shuttle agent; control). After treatment, the tumors were incubated at 37 ℃ for 4 hours and fixed with 4% Paraformaldehyde (PFA), then treated with 30% sucrose and frozen at OCT (optimal cutting temperature). Transfer 10 μm sections onto coverslips and use ProLong TM The Diamond treatment was used for fluorescence microscopy analysis. As shown in FIG. 13, the treatment with PMO-Gli1-Cy5 aloneIncreased fluorescence levels, especially at the epidermis level, were observed in half of the tumors treated with PMO-Gli1-cy5 and shuttle combination. In addition, after tumor incubation, dermal and epidermal cells were isolated by enzymatic treatment with thermolysin and trypsin, and at 37 ℃ at 5% CO 2 Further culturing. After fixation and labelling of isolated dermal and epidermal cells with anti-Gli 1-Alexa 647 antibodies, the level of Gli1 protein was measured by cytofluorometry at different incubation times. Between 24 and 48 hours of incubation after tumor treatment, gli1 protein levels were significantly reduced (50%) in cultured cells treated with PMO-Gli1 and shuttle combination as measured by flow cytometry (data not shown).
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SEQUENCE LISTING
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<210> 16
<211> 42
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD19
<400> 16
His His His His His His Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys
1 5 10 15
Thr Trp Thr Gln Gly Arg Arg Leu Lys Ala Lys Ser Ala Gln Ala Ser
20 25 30
Thr Arg Gln Ala His His His His His His
35 40
<210> 17
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD21
<400> 17
His His His His His His Phe Leu Lys Ile Trp Ser Arg Leu Ile Lys
1 5 10 15
Ile Trp Thr Gln Gly Arg Arg Lys Gly Ala Gln Ala Ala Phe Arg
20 25 30
<210> 18
<211> 37
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD23
<400> 18
His His His His His His Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys
1 5 10 15
Glu Trp Thr Gln Gly Arg Arg Leu Glu Ala Lys Arg Ala Glu Ala His
20 25 30
His His His His His
35
<210> 19
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD120
<400> 19
His His His His His His Phe Leu Lys Ile Trp Ser Arg Leu Ile Lys
1 5 10 15
Ile Trp Thr Gln Gly Leu Arg Lys Gly Ala Gln Ala Ala Lys Arg
20 25 30
<210> 20
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD127
<400> 20
His His His His His His Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys
1 5 10 15
Gly Trp Thr Gln Gly Trp Arg Thr Ile Ala Gln Ala Leu Gly
20 25 30
<210> 21
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD129
<400> 21
Phe Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Arg Arg Lys Gly Ala Gln Ala Ala Phe Arg
20 25 30
<210> 22
<211> 37
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD131
<400> 22
His His His His His His Phe Leu Lys Ile Trp Ser Arg Leu Ile Lys
1 5 10 15
Ile Trp Thr Gln Gly Arg Arg Lys Gly Ala Gln Ala Ala Phe Arg His
20 25 30
His His His His His
35
<210> 23
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD134
<400> 23
Leu Ile Arg Lys Trp Ile His Leu Ile His Ser Trp Phe Gln Asn Leu
1 5 10 15
Arg Arg Leu Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg Ala
20 25 30
<210> 24
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD146
<400> 24
Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Thr Gln Gly Gly
1 5 10 15
Ser Pro Pro Pro Ser Gly Arg Arg Leu Lys Ala Lys Arg Ala Lys Ala
20 25 30
<210> 25
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD155
<400> 25
Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Thr Gln Gly Gly
1 5 10 15
Ser Gly Glu Gly Ser Gly Arg Arg Leu Lys Ala Lys Arg Ala Lys Ala
20 25 30
<210> 26
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD156
<400> 26
Trp Ile Arg Leu Phe Thr Lys Leu Trp Arg Ile Phe Gln Gln Gly Lys
1 5 10 15
Arg Ile Lys Ala Lys Arg Ala
20
<210> 27
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD157
<400> 27
Trp Ile Arg Leu Phe Thr Lys Leu Trp Arg Ile Phe Gln Gln Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Lys Arg Ile Lys Ala Lys Arg Ala
20 25
<210> 28
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD159
<400> 28
Trp Ile Arg Leu Phe Thr Lys Leu Trp Arg Ile Phe Arg Gln Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Lys Arg Ile Lys Ala Lys Ala Ala
20 25
<210> 29
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD162
<400> 29
Ile Leu Lys Leu Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Lys Lys Ala Gln Ala Ala Lys Arg
20 25
<210> 30
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD168
<400> 30
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Ala Arg Ala Gln Ala Arg
20 25
<210> 31
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD173
<400> 31
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Ala Arg Ala Ala Arg Gln Ala Arg
20 25
<210> 32
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD174
<400> 32
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala
20 25 30
Arg
<210> 33
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD194
<400> 33
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Arg Arg Leu Lys Ala Lys Arg Ala Lys Ala
20 25 30
<210> 34
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD220
<400> 34
Trp Ala Arg Ala Phe Ala Lys Ala Trp Arg Ile Phe Gln Gln Gly Lys
1 5 10 15
Arg Ile Lys Ala Lys Arg Ala
20
<210> 35
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD250
<400> 35
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 36
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD250D
<220>
<221> MISC_FEATURE
<222> (1)..(30)
<223> All D-amino acids
<400> 36
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 37
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD253
<400> 37
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Arg Gly Gly Arg Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 38
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD258
<400> 38
Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25
<210> 39
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD262
<400> 39
Lys Trp Lys Leu Leu Arg Leu Trp Ser Arg Leu Leu Arg Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 40
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD263
<400> 40
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Ala Arg Gln Ala Arg
20 25 30
<210> 41
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD264
<400> 41
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Ala Arg Ala Ala Arg
20 25 30
<210> 42
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD265
<400> 42
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Ala Ala Arg Gln Ala Arg
20 25 30
<210> 43
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD268
<400> 43
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 44
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD286
<400> 44
Lys Trp Lys Leu Leu Arg Ala Leu Ala Arg Leu Leu Lys Leu Ala Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Arg Arg Leu Gly Ala Arg Ala Gln Ala
20 25 30
Arg
<210> 45
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD271
<400> 45
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Arg
1 5 10 15
Gly Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 46
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD272
<400> 46
Lys Trp Lys Leu Ala Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 47
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD273
<400> 47
Lys Trp Lys Leu Leu Arg Ala Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 48
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD276
<400> 48
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Arg Ala Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 49
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD268 Cyclic Amide
<220>
<221> MISC_FEATURE
<222> (1)..(32)
<223> Cyclic peptide: covalent link between K1 and R32
<400> 49
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 50
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD268 Disulfide
<220>
<221> MISC_FEATURE
<222> (1)..(32)
<223> Cyclic peptide: disulfiude bond between C1 and C34
<400> 50
Cys Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp
1 5 10 15
Gly Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala
20 25 30
Arg Cys
<210> 51
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD10 Scarmble
<400> 51
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Tyr Ala Arg Ala Leu Arg Arg Gln Ala Arg
20 25 30
Thr Gly
<210> 52
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD268 Scramble
<400> 52
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 53
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD174 Scramble
<400> 53
Leu Gly Arg Ser Gly Arg Ile Lys Ile Gly Gly Trp Ser Ala Leu Ala
1 5 10 15
Ser Arg Ala Arg Gln Ala Arg Gly Leu Lys Ile Trp Thr Gln Gly Arg
20 25 30
Leu
<210> 54
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> FSN3
<400> 54
His His His His His His Gln Phe Leu Cys Phe Trp Leu Asn Lys Met
1 5 10 15
Gly Lys His Asn Thr Val Trp His Gly Arg His Leu Lys Cys His Lys
20 25 30
Arg Gly Lys Gly
35
<210> 55
<211> 35
<212> PRT
<213> Artificial Sequence
<220>
<223> FSN4
<400> 55
His His His His His His Leu Leu Tyr Leu Trp Arg Arg Leu Leu Lys
1 5 10 15
Phe Trp Cys Ala Gly Arg Arg Val Tyr Ala Lys Cys Ala Lys Ala Tyr
20 25 30
Gly Cys Phe
35
<210> 56
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSN7
<400> 56
Leu Ile Lys Leu Trp Ser Arg Phe Ile Lys Phe Trp Thr Gln Gly Arg
1 5 10 15
Arg Ile Lys Ala Lys Leu Ala Arg Ala Gly Gln Ser Trp Phe Gly
20 25 30
<210> 57
<211> 19
<212> PRT
<213> Artificial Sequence
<220>
<223> FSN8
<400> 57
His His His His His His Phe Arg Lys Leu Trp Leu Ala Ile Val Arg
1 5 10 15
Ala Lys Lys
<210> 58
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD117
<400> 58
His His His His His His Phe Leu Lys Phe Trp Ser Arg Leu Phe Lys
1 5 10 15
Phe Trp Thr Gln Gly Arg Arg Lys Gly Ala Gln Ala Ala Phe Arg
20 25 30
<210> 59
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD118
<400> 59
His His His His His His Ile Leu Lys Ile Trp Ser Arg Leu Ile Lys
1 5 10 15
Ile Trp Thr Gln Gly Arg Arg Lys Gly Ala Gln Ala Ala Ile Arg
20 25 30
<210> 60
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD119
<400> 60
His His His His His His Phe Leu Lys Ile Trp Ser Arg Ala Leu Ile
1 5 10 15
Lys Ile Trp Thr Gln Gly Leu Arg Lys Gly Ala Gln Ala Ala Lys Arg
20 25 30
<210> 61
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD121
<400> 61
His His His His His His Val Leu Lys Ile Trp Ser Arg Leu Ile Lys
1 5 10 15
Ile Trp Thr Gln Gly Arg Arg Lys Gly Ala Gln Ala Ala Val Arg
20 25 30
<210> 62
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD122
<400> 62
His His His His His His Phe Leu Lys Val Trp Ser Arg Leu Val Lys
1 5 10 15
Val Trp Thr Gln Gly Arg Arg Lys Gly Ala Gln Ala Ala Phe Arg
20 25 30
<210> 63
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD123
<400> 63
His His His His His His Val Leu Lys Val Trp Ser Arg Leu Val Lys
1 5 10 15
Val Trp Thr Gln Gly Arg Arg Lys Gly Ala Gln Ala Ala Val Arg
20 25 30
<210> 64
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD124
<400> 64
His His His His His His Phe Leu Lys Ile Trp Gln Arg Leu Ile Lys
1 5 10 15
Ile Trp Gln Gln Gly Arg Arg Lys Gly Ala Gln Ala Ala Phe Arg
20 25 30
<210> 65
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD125
<400> 65
His His His His His His Phe Leu Lys Ile Trp Asn Arg Leu Ile Lys
1 5 10 15
Ile Trp Asn Asn Gly Arg Arg Lys Gly Ala Asn Ala Ala Phe Arg
20 25 30
<210> 66
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD126
<400> 66
His His His His His His Phe Leu Lys Ile Trp Ser Arg Leu Ile Lys
1 5 10 15
Ile Trp Thr Gln Gly Trp Arg Thr Gly Ala Gln Ala Gly Phe
20 25 30
<210> 67
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD127
<400> 67
His His His His His His Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys
1 5 10 15
Gly Trp Thr Gln Gly Trp Arg Thr Ile Ala Gln Ala Leu Gly
20 25 30
<210> 68
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD128
<400> 68
His His His His His His Phe Leu Lys Ile Trp Ser Arg Leu Ile Lys
1 5 10 15
Ile Trp Pro Gln Pro Arg Arg Lys Gly Ala Gln Ala Ala Phe Arg
20 25 30
<210> 69
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD130
<400> 69
Leu Ile Lys Ile Trp Thr Gln Phe Leu Lys Ile Trp Ser Arg Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Arg Arg Leu Gly Ala Arg Ala Gln Ala Arg
20 25 30
<210> 70
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD132
<400> 70
His His His His His His Arg Phe Ala Ala Gln Ala Gly Lys Arg Arg
1 5 10 15
Gly Gln Thr Trp Ile Lys Ile Leu Arg Ser Trp Ile Lys Leu Phe
20 25 30
<210> 71
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD133
<400> 71
His His His His Phe Leu His His Ser Trp Ile Lys Lys Ile Leu Arg
1 5 10 15
Thr Trp Ile Arg Arg Gly Gln Gln Ala Gly Lys Phe Ala Ala Arg
20 25 30
<210> 72
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD135
<400> 72
Leu Ile Arg Lys Trp Ile His Leu Ile His Ser Trp Phe Gln Asn Leu
1 5 10 15
Arg Arg Leu Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25
<210> 73
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD137
<400> 73
Leu Leu Arg Lys Trp Ser His Leu Leu His Ile Trp Gly Gly Ser Gly
1 5 10 15
Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25
<210> 74
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD138
<400> 74
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Ile Phe Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Arg Arg Leu Lys Ala Lys Arg Ala Lys
20 25 30
Ala
<210> 75
<211> 40
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD139
<400> 75
His His His His His His Leu Ile Arg Leu Trp Ser His Leu Ile His
1 5 10 15
Ile Trp Phe Gln Asn Arg Arg Leu Lys Trp Lys Lys Lys Tyr Ala Arg
20 25 30
Ala Ala Ala Arg Gln Ala Arg Ala
35 40
<210> 76
<211> 46
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD140
<400> 76
His His His His His His Leu Ile Arg Leu Trp Ser His Leu Ile His
1 5 10 15
Ile Trp Phe Gln Asn Arg Arg Leu Lys Trp Lys Lys Lys Tyr Ala Arg
20 25 30
Ala Ala Ala Arg Gln Ala Arg Ala His His His His His His
35 40 45
<210> 77
<211> 41
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD141
<400> 77
Leu Ile Arg Leu Trp Ser His Leu Ile His Ile Trp Phe Gln Asn Arg
1 5 10 15
Arg Leu Lys Trp Lys Lys Lys Gly Gly Ser Gly Gly Gly Ser Tyr Ala
20 25 30
Arg Ala Ala Ala Arg Gln Ala Arg Ala
35 40
<210> 78
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD142
<400> 78
Phe Leu Lys Ile Trp Ser His Leu Ile His Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Lys Ala Lys Arg Ala Lys Ala
20 25
<210> 79
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD143
<400> 79
Leu Ile Arg Lys Trp Ile His Leu Ile His Ser Trp Phe Gln Gly Arg
1 5 10 15
Arg Leu Gly Ala Arg Ala
20
<210> 80
<211> 49
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD144
<400> 80
His His His His His His Lys Lys Ala Leu Leu Ala His Ala Leu His
1 5 10 15
Leu Leu Ala Leu Leu Ala Leu His Leu Ala His Ala Leu Lys Lys Ala
20 25 30
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg His His His His His
35 40 45
His
<210> 81
<211> 52
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD145
<400> 81
His His His His His His Lys Lys His Leu Leu Ala His Ala Leu His
1 5 10 15
Leu Leu Ala Leu Leu Ala Leu His Leu Ala His Ala Leu Ala His Leu
20 25 30
Lys Lys Ala Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg His His
35 40 45
His His His His
50
<210> 82
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD147
<400> 82
Leu Leu Lys Leu Trp Thr Gln Leu Leu Lys Leu Trp Ser Arg Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Arg Arg Leu Lys Ala Lys Arg Ala Lys Ala
20 25 30
<210> 83
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD148
<400> 83
His His His His His His Met Val Thr Val Leu Phe Arg Arg Leu Arg
1 5 10 15
Ile Arg Arg Ala Cys Gly Pro Pro Arg Val Arg Val
20 25
<210> 84
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD149
<400> 84
His His His His His His Met Val Arg Val Leu Thr Arg Phe Leu Arg
1 5 10 15
Ile Gly Ala Arg Cys Arg Arg Pro Pro Val Val Arg
20 25
<210> 85
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD150
<400> 85
His His His His His His Trp Ile Thr Trp Leu Phe Lys Arg Leu Lys
1 5 10 15
Ile Arg Arg Ala Ala Gly Gln Ser Lys Phe Arg Ile Ala Gly
20 25 30
<210> 86
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD151
<400> 86
His His His His His His Trp Ile Thr Trp Leu Arg Lys Ile Leu Lys
1 5 10 15
Arg Phe Arg Lys Ala Ala Gln Ser Gly Phe Arg Ile Ala Gly
20 25 30
<210> 87
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD152
<400> 87
His His His His His His Trp Ile Thr Trp Leu Arg Lys Ile Leu Lys
1 5 10 15
Arg Phe Gly Lys Ala Ala Gln Ser Gly Phe Arg Ile Ala Arg
20 25 30
<210> 88
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD153
<400> 88
His His His His His His Trp Ile Thr Trp Leu Arg Lys Ile Leu Lys
1 5 10 15
Arg Leu Gly Gly Ala Ala Gln Ser Ile Ile Thr Gly Gly Gln
20 25 30
<210> 89
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD154
<400> 89
His His His His His His Trp Ile Thr Trp Leu Phe Lys Arg Leu Lys
1 5 10 15
Ile Arg Arg Ala Ala Gly Gly Ser Gly Gly Gly Ser Gln Ser Lys Phe
20 25 30
Arg Ile Ala Gly
35
<210> 90
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD158
<400> 90
Trp Ile Arg Leu Phe Thr Lys Leu Trp Arg Ile Phe Arg Gln Gly Lys
1 5 10 15
Arg Ile Lys Ala Lys Ala Ala
20
<210> 91
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD160
<400> 91
Ile Leu Lys Leu Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Ala Gln Ala Ala Leu Arg
20 25
<210> 92
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD161
<400> 92
Ile Leu Lys Leu Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Arg Arg Leu Gly Ala Gln Ala Ala Leu Arg
20 25 30
<210> 93
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD163
<400> 93
Ile Leu Lys Leu Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Arg Arg Lys Lys Ala Gln Ala Ala Lys Arg
20 25 30
<210> 94
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD164
<400> 94
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Ala Arg Ala Ala Arg Ala
20 25
<210> 95
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD165
<400> 95
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Arg Arg Lys Lys Ala Arg Ala Ala Arg Ala
20 25 30
<210> 96
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD166
<400> 96
Leu Leu Lys Leu Trp Ser Arg Leu Ile Lys Ile Trp Thr Lys Gly Arg
1 5 10 15
Arg Lys Lys Ala Arg Ala Ala Gln Ala Arg
20 25
<210> 97
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD167
<400> 97
Leu Leu Lys Leu Trp Ser Arg Leu Ile Lys Ile Trp Thr Lys Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Arg Arg Lys Lys Ala Arg Ala Ala Gln Ala Arg
20 25 30
<210> 98
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD169
<400> 98
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Arg Arg Leu Gly Ala Arg Ala Gln Ala Arg
20 25 30
<210> 99
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD170
<400> 99
Leu Ile Lys Ile Trp Thr Gln Leu Leu Lys Ile Trp Ser Arg Gly Arg
1 5 10 15
Arg Leu Gly Ala Arg Ala Gln Ala Arg
20 25
<210> 100
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD171
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> Acetyl
<220>
<221> MISC_FEATURE
<222> (25)..(25)
<223> Amide
<220>
<221> MISC_FEATURE
<222> (25)..(25)
<223> Cysteamide
<400> 100
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Ala Arg Ala Gln Ala Arg
20 25
<210> 101
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD172
<400> 101
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Gly Ser Gly Gly Gly Ser Ala Arg Ala Gln Ala Arg
20 25 30
<210> 102
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD175
<400> 102
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25
<210> 103
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD176
<400> 103
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Gly Ser Gly Gly Gly Ser Gly Gly Ser Ala Arg Ala Ala
20 25 30
Arg Gln Ala Arg
35
<210> 104
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD177
<400> 104
Lys Leu Lys Ile Trp Ser Arg Leu Ile Arg Lys Trp Thr Lys Gly Leu
1 5 10 15
Arg Leu Gly Ala Gln Ala Gln Ala Arg
20 25
<210> 105
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD178
<400> 105
Lys Leu Lys Ile Trp Ser Arg Leu Ile Arg Lys Trp Thr Lys Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Leu Arg Leu Gly Ala Gln Ala Gln Ala Arg
20 25 30
<210> 106
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD179
<400> 106
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Gly Arg Glu Ser Arg Lys Pro Arg Lys Ser Arg Gln
20 25
<210> 107
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD180
<400> 107
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Arg Gly Arg Glu Ser Arg Lys Pro Arg Lys Ser
20 25 30
Arg Gln
<210> 108
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD181
<400> 108
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Leu
1 5 10 15
Gly Leu Leu Val Leu Arg Val Arg Ala Gly Lys Arg
20 25
<210> 109
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD182
<400> 109
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Leu Gly Leu Leu Val Leu Arg Val Arg Ala Gly
20 25 30
Lys Arg
<210> 110
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD183
<400> 110
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Ala Arg Ala
20
<210> 111
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD184
<400> 111
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Ala Arg Ala Ala Arg
20
<210> 112
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD185
<400> 112
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Ala Arg Ala Ala Arg Gln
20 25
<210> 113
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD186
<400> 113
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Gly Leu Glu Ala Arg Ala Pro Arg Lys Ala Arg
20 25
<210> 114
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD187
<400> 114
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Ala Arg Lys Pro Arg Lys Ser Arg Gln
20 25
<210> 115
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD188
<400> 115
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Gly Arg Glu Ser Arg Ala Ala Arg Gln Ala Arg
20 25
<210> 116
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD189
<400> 116
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Arg Ala Gln Arg Ala Gln Arg Ala
20 25
<210> 117
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD190
<400> 117
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Ala Gln Arg Ala Gln Arg Ala
20
<210> 118
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD191
<400> 118
His His His His His His Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys
1 5 10 15
Ile Trp Thr Gln Gly Thr Arg Ser Lys Arg Ala Gly Leu Gln Phe Pro
20 25 30
<210> 119
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD192
<400> 119
His His His His His His Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys
1 5 10 15
Ile Trp Thr Gln Gly Val Gly Arg Val His Arg Leu Leu Arg Lys
20 25 30
<210> 120
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD193
<400> 120
Lys Trp Lys Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Arg
1 5 10 15
Arg Leu Gly Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala
20 25 30
Arg
<210> 121
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD195
<400> 121
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Lys Ala Arg Ala Gln Ala Arg
20 25
<210> 122
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD196
<400> 122
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Ala Arg Ala Ala Ala Arg
20 25
<210> 123
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD197
<400> 123
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Lys Ala Arg Ala Ala Ala Arg
20 25
<210> 124
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD198
<400> 124
Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Gly Ser Gly Gly Gly
1 5 10 15
Ser Arg Arg Lys Gly Ala Gln Ala Ala Phe Arg
20 25
<210> 125
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD199
<400> 125
Trp Ser Arg Leu Ile Thr Lys Ile Trp Arg Ile Phe Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Ala Arg Ala Ala
20
<210> 126
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD200
<400> 126
Trp Ser Arg Leu Ile Thr Lys Ile Trp Arg Ile Phe Thr Gln Gly Arg
1 5 10 15
Arg Leu Lys Ala Arg Ala Ala
20
<210> 127
<211> 19
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD201
<400> 127
Trp Ser Arg Leu Ile Lys Leu Trp Thr Gln Gly Arg Arg Leu Lys Ala
1 5 10 15
Arg Ala Ala
<210> 128
<211> 19
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD202
<400> 128
Trp Ile Arg Leu Phe Lys Leu Trp Gln Gln Gly Lys Arg Ile Lys Ala
1 5 10 15
Lys Arg Ala
<210> 129
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD203
<400> 129
Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg Arg Leu Gly Ala
1 5 10 15
Arg Ala Gln Ala Arg
20
<210> 130
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD204
<400> 130
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Ala Arg
20
<210> 131
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD205
<400> 131
Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg Arg Leu Gly Ala
1 5 10 15
Arg
<210> 132
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD206
<400> 132
Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg Arg Leu
1 5 10 15
Gly Ala Arg Ala Gln
20
<210> 133
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD207
<400> 133
Leu Ala Lys Ala Trp Ala Arg Ala Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Ala Arg Ala Gln Ala Arg
20 25
<210> 134
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD208
<400> 134
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Arg Arg Leu Gly Ala Arg Ala Gln Ala Arg
20 25 30
<210> 135
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD209
<400> 135
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Tyr Ala Arg Ala Leu Arg Arg Gln Ala Arg Thr
20 25 30
Gly
<210> 136
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD210
<400> 136
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Arg Arg Leu Lys Ala Lys Arg Ala Lys Ala
20 25 30
<210> 137
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD211
<400> 137
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Tyr Ala Arg Ala Leu Arg Arg Gln Ala Arg
20 25 30
Thr Gly
<210> 138
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD212
<400> 138
Trp Ser Arg Leu Leu Lys Leu Trp Gly Gly Ser Gly Gly Gly Ser Arg
1 5 10 15
Arg Leu Lys Ala Lys Arg Ala Lys Ala
20 25
<210> 139
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD213
<400> 139
Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly Gly Ser Gly
1 5 10 15
Gly Gly Ser Arg Arg Leu Lys Ala Lys
20 25
<210> 140
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD214
<400> 140
Trp Ser Arg Leu Leu Lys Leu Trp Gly Gly Ser Gly Gly Gly Ser Arg
1 5 10 15
Arg Leu Lys Ala Lys
20
<210> 141
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD215
<400> 141
Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly Gly Ser Gly Gly Gly
1 5 10 15
Ser Arg Arg Leu Lys Ala Lys Arg Ala
20 25
<210> 142
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD216
<400> 142
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Gly Arg Ser Arg Lys Pro Arg Lys Ser Arg Gln
20 25
<210> 143
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD217
<400> 143
Lys Trp Lys Leu Lys Leu Trp Arg Leu Lys Gly Gly Ser Gly Gly Gly
1 5 10 15
Ser Arg Arg Ala Lys Ala
20
<210> 144
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD218
<400> 144
Lys Trp Lys Leu Lys Leu Trp Arg Leu Lys Ser Arg Leu Lys Leu Trp
1 5 10 15
Arg Leu Lys Gly Gly Ser Gly Gly Gly Ser Arg Arg Ala Lys Ala
20 25 30
<210> 145
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD219
<400> 145
Trp Ile Arg Leu Trp Thr His Leu Trp His Ile Trp Gln Gln Gly Lys
1 5 10 15
Arg Ile Lys Ala Lys Arg Ala
20
<210> 146
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD221
<400> 146
Trp Lys Leu Ile Arg Leu Phe Thr Arg Leu Ile Lys Ile Trp Gly Gln
1 5 10 15
Arg Arg Leu Lys Ala Lys Arg Ala
20
<210> 147
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD222
<400> 147
Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Ile Phe Gly Gln Arg Arg
1 5 10 15
Leu Lys Ala Lys Arg Ala
20
<210> 148
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD223
<400> 148
Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Ile Phe Gln Gly Gly Ser
1 5 10 15
Gly Gly Gly Ser Arg Arg Leu Lys Ala Lys Arg Ala
20 25
<210> 149
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD224
<400> 149
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Ile Phe Gly
1 5 10 15
Gln Arg Arg Leu Lys Ala Lys Arg Ala
20 25
<210> 150
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD225
<400> 150
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Ile Phe Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Arg Arg Leu Lys Ala Lys Arg Ala
20 25 30
<210> 151
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD226
<400> 151
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Ile Phe Gly
1 5 10 15
Gln Arg Arg Leu Gly Ala Arg Ala Gln Ala Arg
20 25
<210> 152
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD227
<400> 152
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Ile Phe Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Arg Arg Leu Gly Ala Arg Ala Gln Ala
20 25 30
Arg
<210> 153
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD228
<400> 153
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Ile Phe Gly
1 5 10 15
Gln Arg Arg Leu Lys Ala Lys Arg Ala Lys Ala
20 25
<210> 154
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD229
<400> 154
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Ile Phe Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Arg Arg Leu Lys Ala Lys Arg Ala Lys
20 25 30
Ala
<210> 155
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD230
<400> 155
Lys Trp Lys Leu Ala Lys Ala Trp Ala Arg Ala Leu Lys Leu Trp Gly
1 5 10 15
Arg Arg Leu Gly Ala Arg Ala Gln Ala Arg
20 25
<210> 156
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD231
<400> 156
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Arg Arg Leu Lys Ala Lys Arg Ala Leu Lys
20 25 30
Ala
<210> 157
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD232
<400> 157
Lys Trp Lys Trp Ala Arg Ala Trp Ala Arg Ala Trp Lys Lys Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Arg Arg Leu Gly Ala Arg Ala Gln Ala Arg
20 25 30
<210> 158
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD233
<400> 158
Lys Leu Lys Leu Ala Arg Ala Leu Ala Arg Ala Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Arg Arg Leu Gly Ala Arg Ala Gln Ala Arg
20 25 30
<210> 159
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD234
<400> 159
Lys Ile Lys Ile Ala Arg Ala Ile Ala Arg Ala Ile Lys Lys Ile Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Arg Arg Leu Gly Ala Arg Ala Gln Ala Arg
20 25 30
<210> 160
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD235
<400> 160
Lys Phe Lys Phe Ala Arg Ala Phe Ala Arg Ala Phe Lys Lys Phe Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Arg Arg Leu Gly Ala Arg Ala Gln Ala Arg
20 25 30
<210> 161
<211> 39
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD236
<400> 161
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Ser
1 5 10 15
Arg Leu Leu Lys Leu Trp Gly Gly Ser Gly Gly Gly Ser Arg Arg Leu
20 25 30
Gly Ala Arg Ala Gln Ala Arg
35
<210> 162
<211> 39
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD237
<400> 162
Lys Trp Lys Leu Leu Lys Leu Trp Thr Gln Leu Leu Lys Leu Trp Thr
1 5 10 15
Gln Leu Leu Lys Leu Trp Gly Gly Ser Gly Gly Gly Ser Arg Arg Leu
20 25 30
Gly Ala Arg Ala Gln Ala Arg
35
<210> 163
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD238
<400> 163
Lys Trp Lys Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 164
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD239
<400> 164
Lys Trp Lys Leu Leu Lys Ile Trp Thr Gln Leu Ile Lys Ile Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Gln Ala Arg Gln Ala Arg
20 25 30
<210> 165
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD240
<400> 165
Lys Trp Lys Ala Leu Leu Ala Leu Ala Leu His Leu Ala His Leu Ala
1 5 10 15
Leu His Leu Lys Lys Ala Gly Arg Arg Lys Gly Ala Gln Ala Ala Phe
20 25 30
Arg
<210> 166
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD241
<400> 166
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg
20 25 30
Ala
<210> 167
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD243
<400> 167
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Gly Ser Gly Gly Gly Ser Tyr Ala Arg Ala Ala Ala Arg
20 25 30
Gln Ala Arg Ala
35
<210> 168
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD244
<400> 168
Lys Trp Lys Leu Ala Lys Ala Trp Ala Arg Ala Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Tyr Ala Arg Ala Ala Ala Arg Lys Ala Lys
20 25 30
Arg Ala
<210> 169
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD246
<400> 169
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Tyr Ala Arg Ala Ala Ala Arg Lys Ala Lys
20 25 30
Arg Ala
<210> 170
<211> 37
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD247
<400> 170
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Gly Ser Gly Gly Gly Ser Tyr Ala Arg Ala Ala Ala Arg
20 25 30
Lys Ala Lys Arg Ala
35
<210> 171
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD248
<400> 171
Lys Trp Lys Leu Ala Lys Ala Trp Ala Arg Ala Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 172
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD250 Scramble
<400> 172
Arg Gly Lys Leu Trp Ser Leu Ser Lys Leu Lys Gly Trp Gly Gly Ala
1 5 10 15
Arg Ala Ser Lys Ala Gln Leu Ala Arg Leu Gly Leu Trp Arg
20 25 30
<210> 173
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD250E
<400> 173
Lys Trp Lys Leu Leu Glu Leu Trp Ser Glu Leu Leu Glu Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 174
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD251
<400> 174
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Glu Ala Ala Glu Gln Ala Glu
20 25 30
<210> 175
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD254
<400> 175
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Arg Gly Gly Arg Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 176
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD255
<400> 176
Lys Trp Lys Leu Leu Lys Leu Trp Gly Gly Ser Arg Leu Leu Lys Leu
1 5 10 15
Trp Gly Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 177
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD256
<400> 177
Lys Trp Lys Leu Leu Lys Leu Gly Arg Trp Ser Arg Leu Gly Leu Lys
1 5 10 15
Leu Trp Gly Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala
20 25 30
Arg
<210> 178
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD257
<400> 178
Lys Trp Lys Leu Leu Lys Leu Trp Ala Ala Ser Arg Leu Leu Lys Leu
1 5 10 15
Trp Gly Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 179
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD259
<400> 179
Lys Trp Lys Leu Leu Lys Leu Ala Arg Trp Ser Arg Leu Ala Leu Lys
1 5 10 15
Leu Trp Gly Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala
20 25 30
Arg
<210> 180
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD260
<400> 180
Arg Trp Arg Leu Leu Arg Leu Trp Ser Arg Leu Leu Arg Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 181
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD261
<400> 181
Gly Gly Ser Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 182
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD266
<400> 182
Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly Gly Ser Gly
1 5 10 15
Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25
<210> 183
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD267
<400> 183
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Tyr Ala Arg Ala Ala Arg Tyr Ala Arg
20 25 30
<210> 184
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD269
<400> 184
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Tyr Ala Arg Ala Tyr Ala Arg Tyr Ala Arg
20 25 30
<210> 185
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD270
<400> 185
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Ala Ala Ala Glu Lys
20 25
<210> 186
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD274
<400> 186
Lys Trp Lys Leu Ala Arg Ala Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 187
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD275
<400> 187
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Ala Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 188
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD276
<400> 188
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Arg Ala Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 189
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD277
<400> 189
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Ala Arg Ala Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 190
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD278
<400> 190
Lys Trp Lys Leu Ala Arg Ala Trp Ser Arg Leu Ala Arg Ala Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 191
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD279
<400> 191
Lys Trp Lys Leu Ala Arg Ala Leu Ala Arg Ala Trp Ser Arg Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25
<210> 192
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD280
<400> 192
Lys Trp Lys Leu Leu Lys Leu Trp Lys Arg Leu Leu Lys Lys Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 193
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD281
<400> 193
Lys Trp Ser Leu Leu Lys Leu Trp Ser Ala Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 194
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD282
<400> 194
Lys Trp Lys Leu Trp Lys Leu Leu Ser Arg Leu Trp Lys Leu Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 195
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD283
<400> 195
Lys Trp Lys Leu Ala Arg Lys Phe Lys Arg Ala Ile Lys Lys Phe Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 196
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD284
<400> 196
Lys Trp Ala Leu Ala Arg Ala Phe Ala Arg Ala Ile Ala Ile Phe Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 197
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD285
<400> 197
Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Ile Phe Gly Gly Ser Gly
1 5 10 15
Gly Gly Ser Gln Arg Arg Leu Gly Ala Arg Ala Gln Ala Arg
20 25 30
<210> 198
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD287
<400> 198
Lys Trp Lys Leu Leu Arg Ala Leu Ala Arg Leu Leu Lys Ala Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Arg Arg Leu Gly Ala Arg Ala Gln Ala
20 25 30
Arg
<210> 199
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD288
<400> 199
Lys Trp Lys Leu Leu Lys Trp Trp Ser Arg Leu Leu Lys Trp Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 200
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD289
<400> 200
Lys Trp Lys Leu Leu Lys Phe Trp Ser Arg Leu Leu Lys Phe Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 201
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD290
<400> 201
Lys Trp Lys Leu Leu Lys Leu Tyr Ser Arg Leu Leu Lys Leu Tyr Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 202
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD291
<400> 202
Lys Trp Lys Leu Leu Lys Leu Phe Ser Arg Leu Leu Lys Leu Phe Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 203
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD292
<400> 203
Lys Trp Lys Leu Leu Ser Leu Trp Ser Ser Leu Leu Ser Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 204
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD293
<400> 204
Lys Trp Lys Leu Leu Ser Leu Trp Ser Arg Leu Leu Ser Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 205
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD2294
<400> 205
Lys Trp Lys Leu Leu Lys Leu Trp Ser Ser Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 206
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD295
<400> 206
Lys Trp Lys Leu Leu Lys Leu Trp Ser Leu Leu Lys Leu Trp Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 207
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD296
<400> 207
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gln
1 5 10 15
Gln Gly Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln
20 25 30
Ala Arg
<210> 208
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD297
<400> 208
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Asn
1 5 10 15
Asn Gly Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln
20 25 30
Ala Arg
<210> 209
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD298
<400> 209
Ser Trp Ser Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 210
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD299
<400> 210
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Ile
1 5 10 15
Lys Ile Phe Gly Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 211
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD300
<400> 211
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Trp
1 5 10 15
Arg Ile Phe Gly Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 212
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD301
<400> 212
Gly Gly Ser Gly Gly Gly Ser Lys Trp Lys Leu Leu Lys Leu Trp Ser
1 5 10 15
Arg Leu Leu Lys Leu Trp Gly Gly Ser Gly Gly Gly Ser
20 25
<210> 213
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD302
<400> 213
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Gly Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25
<210> 214
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD303
<400> 214
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25
<210> 215
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD304
<400> 215
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gln
1 5 10 15
Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25
<210> 216
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD305
<400> 216
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Gly Gly Gly Gly Gly Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 217
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD306
<400> 217
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Gln Ala Arg
20 25
<210> 218
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD307
<400> 218
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg
20 25
<210> 219
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD308
<400> 219
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gly Ala Arg Ala Gly Ala Arg Gly Ala Arg
20 25 30
<210> 220
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD309
<400> 220
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Gly Ala Gln Ala Gly Gln Ala Gly
20 25 30
<210> 221
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD310
<400> 221
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Gly Arg Gly Gln Gly Arg Gln Gly Arg
20 25 30
<210> 222
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD311
<400> 222
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Arg Gly Gly Arg Gly Gly Gly Arg
20 25 30
<210> 223
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD312
<400> 223
Trp Ile Arg Leu Phe Thr Lys Leu Trp Ile Phe Gln Gln Gly Gly Ser
1 5 10 15
Gly Gly Gly Ser Lys Arg Ile Lys Ala Lys Arg Ala
20 25
<210> 224
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD313
<400> 224
Trp Ile Arg Leu Phe Ser Arg Leu Trp Arg Ile Phe Gln Gln Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Lys Arg Ile Lys Ala Lys Arg Ala
20 25
<210> 225
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD314
<400> 225
Lys Trp Lys Trp Ile Arg Leu Phe Ser Arg Leu Trp Arg Ile Phe Gln
1 5 10 15
Gln Gly Gly Ser Gly Gly Gly Ser Lys Arg Ile Lys Ala Lys Arg Ala
20 25 30
<210> 226
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD315
<400> 226
Trp Ile Arg Leu Phe Ser Arg Leu Trp Arg Ile Phe Gln Gln Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 227
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD316
<400> 227
Lys Trp Lys Trp Ile Arg Leu Phe Ser Arg Leu Trp Arg Ile Phe Gln
1 5 10 15
Gln Gly Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln
20 25 30
Ala Arg
<210> 228
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD317
<400> 228
Trp Ile Arg Leu Phe Thr Lys Leu Trp Gln Ile Phe Gln Gln Gly Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 229
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD318
<400> 229
Trp Ile Arg Leu Phe Thr Lys Leu Trp Arg Ile Phe Gln Gln Gly Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 230
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD319
<400> 230
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Ala Ala Ala Gln Lys
20 25
<210> 231
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD320
<400> 231
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Ala Ala Ala Gln Gln
20 25
<210> 232
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD321
<400> 232
Lys Trp Lys Leu Ala Lys Ala Trp Ser Arg Ala Ile Lys Ile Trp Gly
1 5 10 15
Ala Arg Ala Gln Ala Arg Gln Ala
20
<210> 233
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD322
<400> 233
Lys Trp Lys Leu Ala Lys Ala Trp Ser Arg Ala Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Gln Ala Arg Gln Ala
20 25 30
<210> 234
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD323
<400> 234
Trp Ile Arg Leu Phe Thr Arg Leu Ile Lys Ile Trp Gly Gln Arg Arg
1 5 10 15
Leu Lys Ala Lys Arg Ala
20
<210> 235
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD324
<400> 235
Trp Ala Arg Ala Phe Ala Arg Ala Trp Arg Ile Phe Gln Gln Arg Arg
1 5 10 15
Leu Lys Ala Lys Arg Ala
20
<210> 236
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD325
<400> 236
Trp Ala Arg Ala Phe Ala Arg Ala Trp Arg Ile Phe Gln Gln Arg Arg
1 5 10 15
Leu Ala Arg Ala Ala Arg Gln Ala Arg
20 25
<210> 237
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD326
<400> 237
Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Ile Phe Gly Gln Ala Arg
1 5 10 15
Ala Gln Ala Arg Gln Ala Arg
20
<210> 238
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD327
<400> 238
Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Ile Phe Gly Arg Arg Leu
1 5 10 15
Lys Ala Lys Arg Ala Lys Ala
20
<210> 239
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD328
<400> 239
Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Ile Phe Gly Arg Arg Leu
1 5 10 15
Gly Ala Arg Ala Gln Ala Arg
20
<210> 240
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD330
<400> 240
Leu Ala Arg Ala Phe Ala Arg Ala Leu Leu Lys Leu Trp Gly Gln Arg
1 5 10 15
Arg Leu Lys Ala Lys Arg Ala
20
<210> 241
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD331
<400> 241
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Gly Gln Arg Arg Leu
1 5 10 15
Lys Ala Lys Arg Ala
20
<210> 242
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD332
<400> 242
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Gly Arg Arg Leu Gly
1 5 10 15
Ala Arg Ala Gln Ala Arg
20
<210> 243
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD333
<400> 243
Lys Trp Lys Leu Leu Arg Leu Leu Leu Arg Leu Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 244
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD334
<400> 244
Lys Trp Lys Leu Leu Arg Trp Leu Trp Arg Leu Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 245
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD335
<400> 245
Lys Trp Lys Leu Ala Arg Leu Leu Leu Arg Ala Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 246
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD336
<400> 246
Lys Trp Lys Leu Leu Arg Leu Phe Leu Arg Leu Phe Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 247
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD337
<400> 247
Lys Trp Lys Leu Ala Arg Trp Leu Trp Arg Ala Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 248
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD338
<400> 248
Lys Trp Lys Leu Leu Arg Trp Phe Trp Arg Leu Phe Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 249
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD339
<400> 249
Lys Trp Lys Leu Ala Arg Leu Phe Leu Arg Ala Phe Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 250
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD340
<400> 250
Lys Trp Lys Leu Ala Arg Trp Phe Trp Arg Ala Phe Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 251
<211> 37
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD341
<400> 251
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly Arg
1 5 10 15
Arg Leu Gly Gly Ser Gly Gly Gly Ser Tyr Ala Arg Ala Leu Arg Arg
20 25 30
Gln Ala Arg Thr Gly
35
<210> 252
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD342
<400> 252
Lys Trp Lys Leu Ala Arg Trp Phe Trp Arg Ala Phe Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Tyr Ala Arg Ala Leu Arg Arg Gln Ala Arg
20 25 30
Thr Gly
<210> 253
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD343
<400> 253
Lys Trp Lys Leu Leu Gln Leu Trp Ser Arg Leu Leu Gln Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 254
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD344
<400> 254
Gln Trp Gln Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 255
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD345
<400> 255
Lys Leu Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 256
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD346
<400> 256
Lys Phe Lys Leu Leu Lys Leu Phe Ser Arg Leu Leu Lys Leu Phe Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 257
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD347
<400> 257
Lys Trp Lys Leu Leu Lys Leu Leu Ser Arg Leu Leu Lys Leu Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 258
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD348
<400> 258
Lys Trp Lys Leu Leu Lys Leu Leu Ser Arg Leu Leu Lys Leu Leu Gly
1 5 10 15
Gly Gly Gly Gly Gly Gly Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 259
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD349
<400> 259
Lys Trp Lys Trp Leu Lys Leu Trp Ser Arg Leu Trp Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 260
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD350
<400> 260
Lys Trp Lys Leu Leu Lys Phe Trp Ser Arg Leu Leu Lys Phe Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 261
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD351
<400> 261
Lys Trp Lys Leu Leu Lys Leu Phe Ser Arg Leu Phe Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 262
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD352
<400> 262
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Ile Lys Ile Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 263
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD353
<400> 263
Lys Trp Lys Leu Leu Lys Leu Gln Ser Arg Leu Leu Lys Leu Gln Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 264
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD354
<400> 264
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Gly Arg
20 25
<210> 265
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD355
<400> 265
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gly Ala Arg
20 25
<210> 266
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD356
<400> 266
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Gly
20 25
<210> 267
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD357
<400> 267
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Arg Arg Arg
20 25
<210> 268
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD358
<400> 268
Lys Trp Lys Leu Leu His Leu Trp Ser Arg Leu Leu His Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 269
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD359
<400> 269
Lys Trp Lys Leu Leu Lys Leu Trp Ser Lys Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Gly Gly Gly Gly Gly Ala Lys Ala Ala Lys Gln Ala Lys
20 25 30
<210> 270
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD360
<400> 270
Arg Trp Arg Leu Leu Arg Leu Trp Ser Arg Leu Leu Arg Leu Trp Gly
1 5 10 15
Gly Gly Gly Gly Gly Gly Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 271
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD361
<400> 271
Leu Leu Lys Leu Trp Ser Lys Leu Leu Lys Leu Trp Gly Gly Gly Gly
1 5 10 15
Gly Gly Gly Ala Lys Ala Ala Lys Gln Ala Lys
20 25
<210> 272
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD362
<400> 272
Leu Leu Arg Leu Trp Ser Arg Leu Leu Arg Leu Trp Gly Gly Gly Gly
1 5 10 15
Gly Gly Gly Ala Arg Ala Ala Arg Gln Ala Arg
20 25
<210> 273
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD363
<400> 273
Leu Leu Lys Leu Trp Ser Lys Leu Leu Lys Leu Trp Gly Gly Gly Ala
1 5 10 15
Lys Ala Ala Lys Gln Ala Lys
20
<210> 274
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD364
<400> 274
Leu Leu Arg Leu Trp Ser Arg Leu Leu Arg Leu Trp Gly Gly Gly Ala
1 5 10 15
Arg Ala Ala Arg Gln Ala Arg
20
<210> 275
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD365
<400> 275
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Gly Gln Ala Arg
20
<210> 276
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD366
<400> 276
Lys Trp Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly Gly Gly Gln
1 5 10 15
Ala Arg
<210> 277
<211> 19
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD367
<400> 277
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Gly Gly Gly
1 5 10 15
Gln Ala Arg
<210> 278
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD368
<400> 278
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Ser Arg Leu Leu Lys
1 5 10 15
Leu Trp Gly Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala
20 25 30
Arg
<210> 279
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD369
<400> 279
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 280
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD370
<400> 280
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 281
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD371
<400> 281
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gln
1 5 10 15
Gln Gly Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 282
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD372
<400> 282
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Lys Leu Asn
1 5 10 15
Asn Gly Gly Ser Gly Gly Gly Ser Tyr Ala Arg Ala Leu Arg Arg Gln
20 25 30
Ala Arg Thr Gly
35
<210> 283
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD373
<400> 283
Gly Gly Ser Gly Gly Gly Ser Leu Leu Lys Leu Trp Ser Arg Leu Leu
1 5 10 15
Lys Leu Trp Gly Gly Ser Gly Gly Gly Ser
20 25
<210> 284
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD374
<400> 284
Gly Gly Ser Gly Gly Gly Ser Leu Leu Lys Ile Trp Ser Arg Leu Ile
1 5 10 15
Lys Ile Trp Thr Gln Gly Arg Arg Leu Gly Gly Ser Gly Gly Gly Ser
20 25 30
<210> 285
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD375
<400> 285
Gly Gly Ser Gly Gly Gly Ser Lys Trp Lys Leu Ala Arg Ala Phe Ala
1 5 10 15
Arg Ala Ile Lys Lys Leu Gly Gly Ser Gly Gly Gly Ser
20 25
<210> 286
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD376
<400> 286
Gly Gly Ser Gly Gly Gly Ser Leu Ala Arg Ala Phe Ala Arg Ala Ile
1 5 10 15
Lys Ile Phe Gly Gly Ser Gly Gly Gly Ser
20 25
<210> 287
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD377
<400> 287
Gly Gly Gly Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys
1 5 10 15
Leu Trp Gly Gly Gly
20
<210> 288
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD378
<400> 288
Gly Gly Ser Gly Gly Gly Ser Lys Trp Lys Trp Ile Arg Leu Phe Ser
1 5 10 15
Arg Trp Ile Arg Leu Phe Gly Gly Ser Gly Gly Gly Ser
20 25
<210> 289
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD379
<400> 289
Lys Trp Lys Leu Ser Lys Leu Trp Ser Lys Leu Ser Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 290
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD381
<400> 290
Leu Leu Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Leu Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25 30
<210> 291
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD382
<400> 291
Leu Leu Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Leu Leu Gly
1 5 10 15
Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25
<210> 292
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD383
<400> 292
Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys Leu Leu Gly Gly Ser Gly
1 5 10 15
Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25
<210> 293
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD384
<400> 293
Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Leu Leu Gly Gln Ala Arg
1 5 10 15
Ala Gln Ala Arg Gln Ala Arg
20
<210> 294
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD385
<400> 294
Leu Leu Lys Leu Leu Lys Leu Leu Leu Lys Leu Leu Gly Gly Ser Gly
1 5 10 15
Gly Gly Ser Gln Ala Arg Ala Gln Ala Arg Gln Ala Arg
20 25
<210> 295
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD386
<400> 295
Leu Leu Lys Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys Leu Leu Gly
1 5 10 15
Gly Gly Gly Lys Gly Gly Gly Lys Gly Gly Lys
20 25
<210> 296
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD387
<400> 296
Gln Leu Gln Leu Leu Arg Leu Leu Leu Arg Leu Leu Lys Lys Leu Gln
1 5 10 15
Leu Gln
<210> 297
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD388
<400> 297
Lys Trp Lys Leu Ala Arg Ala Phe Ser Arg Ala Ile Lys Leu Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Tyr Ala Arg Ala Leu Arg Arg Gln Ala Arg
20 25 30
Thr Gly
<210> 298
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD389
<400> 298
Lys Trp Lys Leu Ala Lys Ala Phe Ser Lys Ala Ile Lys Leu Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Tyr Ala Lys Ala Leu Lys Lys Gln Ala Lys
20 25 30
Thr Gly
<210> 299
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD390
<400> 299
Lys Trp Lys Leu Trp Ser Lys Leu Leu Lys Leu Trp Ser Lys Leu Trp
1 5 10 15
Lys
<210> 300
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD391
<400> 300
Gly Gly Lys Gly Gly Lys Gly Gly Lys Trp Lys Leu Leu Lys Leu Trp
1 5 10 15
Ser Arg Leu Leu Lys Leu Trp Gly Gly Lys Gly Gly Lys Gly Gly
20 25 30
<210> 301
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD392
<400> 301
Gly Gly Trp Gly Gly Trp Gly Gly Lys Trp Lys Leu Leu Lys Leu Trp
1 5 10 15
Ser Arg Leu Leu Lys Leu Trp Gly Gly Trp Gly Gly Trp Gly Gly
20 25 30
<210> 302
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD393
<400> 302
Arg Ala Gln Arg Ala Ala Arg Ala Ser Gly Gly Gly Ser Gly Gly Trp
1 5 10 15
Leu Lys Leu Leu Arg Ser Trp Leu Lys Leu Leu Lys Trp Lys
20 25 30
<210> 303
<211> 40
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD394
<400> 303
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Ile Phe Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gly Gly Gly Lys Trp Lys Leu Ala Arg Ala
20 25 30
Phe Ala Arg Ala Ile Lys Ile Phe
35 40
<210> 304
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD395
<400> 304
Lys Leu Lys Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Lys Ala Gln Ala Lys Gln Ala Lys
20 25 30
<210> 305
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD396
<400> 305
Lys Leu Lys Leu Ala Lys Leu Leu Leu Lys Ala Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Lys Ala Gln Ala Lys Gln Ala Lys
20 25 30
<210> 306
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD397
<400> 306
Lys Leu Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Lys Ala Gln Ala Lys Gln Ala Lys
20 25 30
<210> 307
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD398
<400> 307
Lys Leu Lys Leu Leu Lys Ala Leu Ala Lys Leu Leu Lys Lys Ala Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Lys Ala Gln Ala Lys Gln Ala Lys
20 25 30
<210> 308
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD399
<400> 308
Lys Leu Lys Leu Ala Lys Ala Leu Leu Lys Ala Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Lys Ala Gln Ala Lys Gln Ala Lys
20 25 30
<210> 309
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD400
<400> 309
Lys Leu Lys Ala Ala Lys Ala Leu Ala Lys Ala Leu Lys Ala Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Lys Ala Gln Ala Lys Gln Ala Lys
20 25 30
<210> 310
<211> 37
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD401
<400> 310
Gly Gly Ser Gly Gly Gly Ser Lys Trp Lys Leu Leu Lys Leu Trp Ser
1 5 10 15
Arg Leu Leu Lys Leu Trp Gly Gly Ser Gly Gly Gly Ser Ala Arg Ala
20 25 30
Ala Arg Gln Ala Arg
35
<210> 311
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD402
<400> 311
Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys Lys Leu Gly Gly Ser Gly
1 5 10 15
Gly Gly Ser Gln Ala Lys Ala Gln Ala Lys Gln Ala Lys
20 25
<210> 312
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD403
<400> 312
Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Lys Leu Gly Gly Ser Gly
1 5 10 15
Gly Gly Ser Gln Ala Lys Ala Gln Ala Lys Gln Ala Lys
20 25
<210> 313
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD404
<400> 313
Lys Leu Lys Leu Leu Leu Lys Leu Leu Lys Lys Leu Gly Gly Ser Gly
1 5 10 15
Gly Gly Ser Gln Ala Lys Ala Gln Ala Lys Gln Ala Lys
20 25
<210> 314
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD406
<400> 314
Lys Leu Lys Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Lys Ala Gln Ala Lys Gln Ala
20 25 30
<210> 315
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD407
<400> 315
Lys Leu Lys Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Lys Ala Ala Lys Gln Ala Lys
20 25 30
<210> 316
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD408
<400> 316
Lys Leu Lys Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Gly
20 25
<210> 317
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD409
<400> 317
Lys Leu Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Gly
20 25
<210> 318
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD410
<400> 318
Lys Leu Lys Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Leu Ala Lys Ala Leu Ala Lys Leu Ala Lys
20 25 30
<210> 319
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD411
<400> 319
Lys Leu Lys Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Lys Ala Leu Ala Lys Gln Ala Lys
20 25 30
<210> 320
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD412
<400> 320
Lys Leu Lys Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Leu Ala Gly
20 25
<210> 321
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD413
<400> 321
Lys Leu Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Lys Ala Leu Ala Lys Gln Ala Lys
20 25 30
<210> 322
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD414
<400> 322
Leu Leu Lys Lys Leu Leu His Leu Leu His Ser Leu Leu Gln Asn Leu
1 5 10 15
Lys Lys Leu Gly Gly Ser Gly Gly Gly Ser Gln Ala Lys Ala Gln Ala
20 25 30
Lys Gln Ala Lys
35
<210> 323
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD415
<400> 323
Leu Ile Arg Lys Trp Ile His Leu Ile His Ser Trp Phe Gln Asn Leu
1 5 10 15
Arg Arg Leu Gly Gly Ser Gly Gly Gly Ser Gln Ala Lys Ala Gln Ala
20 25 30
Lys Gln Ala Lys
35
<210> 324
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD416
<400> 324
Gly Gly Ser Gly Gly Gly Ser Lys Trp Lys Leu Ala Lys Ala Trp Ser
1 5 10 15
Arg Ala Leu Lys Leu Trp Gly Gly Ser Gly Gly Gly Ser
20 25
<210> 325
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD417
<400> 325
Gly Gly Ser Gly Gly Gly Ser Leu Ala Lys Ala Trp Ser Arg Ala Leu
1 5 10 15
Lys Leu Trp Gly Gly Ser Gly Gly Gly Ser
20 25
<210> 326
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD418
<400> 326
Gly Gly Ser Gly Gly Gly Ser Lys Leu Lys Leu Leu Lys Leu Leu Leu
1 5 10 15
Lys Leu Leu Lys Lys Leu Gly Gly Ser Gly Gly Gly Ser
20 25
<210> 327
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD419
<400> 327
Gly Gly Ser Gly Gly Gly Ser Lys Leu Lys Leu Ala Lys Ala Leu Ala
1 5 10 15
Lys Ala Leu Lys Lys Leu Gly Gly Ser Gly Gly Gly Ser
20 25
<210> 328
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD421
<400> 328
Gly Gly Ser Gly Gly Gly Ser Leu Leu Lys Lys Leu Leu His Leu Leu
1 5 10 15
His Ser Leu Leu Gln Asn Leu Lys Lys Leu Gly Gly Ser Gly Gly Gly
20 25 30
Ser
<210> 329
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD422
<400> 329
His His His His His His Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg
1 5 10 15
Ala Ile Lys Lys Leu His His His His His His
20 25
<210> 330
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD423
<400> 330
His His His His His His Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys
1 5 10 15
Ile Phe His His His His His His
20
<210> 331
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD424
<400> 331
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gly Gly Ser Gly Gly Gly Ser
20 25
<210> 332
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD425
<400> 332
Lys Leu Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Leu Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Lys Ala Gln Ala Lys Gln Ala Lys
20 25 30
<210> 333
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD426
<400> 333
Lys Leu Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Lys Lys Leu Lys Ala Lys Lys Ala Leu Lys
20 25 30
Ala
<210> 334
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD427
<400> 334
Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Lys Leu Gly Gly Ser Gly
1 5 10 15
Gly Gly Ser Lys Lys Leu Lys Ala Lys Lys Ala Leu Lys Ala
20 25 30
<210> 335
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD428
<400> 335
Lys Leu Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Lys Lys Leu Lys Ala Lys Lys Ala
20 25 30
<210> 336
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD429
<400> 336
Lys Leu Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Lys Lys Leu Lys Ala Lys
20 25
<210> 337
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD430
<400> 337
Lys Leu Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Leu Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Lys Lys Leu Lys Ala Lys Leu Ala Leu Lys
20 25 30
Ala
<210> 338
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD431
<400> 338
Lys Trp Lys Leu Ala Lys Ala Phe Ala Lys Ala Ile Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Tyr Ala Lys Ala Leu Lys Lys Gln Ala Lys
20 25 30
Thr Gly
<210> 339
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD432
<400> 339
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Lys Ala Gln Ala Lys Gln Ala Lys
20 25 30
<210> 340
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD433
<400> 340
Lys Leu Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Tyr Ala Arg Ala Leu Arg Arg Gln Ala Arg
20 25 30
Thr Gly
<210> 341
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD434
<400> 341
Lys Trp Lys Leu Ala Lys Ala Phe Ala Lys Ala Ile Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gly Gly Lys Gly Gly Lys Lys Gln Gly Lys
20 25 30
Thr Gly
<210> 342
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD435
<220>
<221> MISC_FEATURE
<222> (1)..(32)
<223> Xaa is L-2,4-diaminobutyric acid
<400> 342
Xaa Leu Xaa Leu Leu Xaa Leu Leu Leu Xaa Leu Leu Xaa Xaa Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Gln Ala Xaa Ala Gln Ala Xaa Gln Ala Xaa
20 25 30
<210> 343
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD436
<220>
<221> MISC_FEATURE
<222> (1)..(22)
<223> Xaa is (2-naphthyl)-L-alanine
<400> 343
Leu Ala Arg Ala Xaa Ala Arg Ala Ile Lys Ile Xaa Gly Gln Arg Arg
1 5 10 15
Leu Lys Ala Lys Arg Ala
20
<210> 344
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD438
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> N-ter octanoic acid
<400> 344
Lys Trp Lys Leu Ala Arg Ala Phe Ala Arg Ala Ile Lys Lys Leu Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Tyr Ala Arg Ala Leu Arg Arg Gln Ala Arg
20 25 30
Thr Gly
<210> 345
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> PMO-GFP
<400> 345
acagctcctc gcccttgctc accat 25
<210> 346
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> PMO-Gli1
<400> 346
gtcatcgagt tgaacatggc gtctc 25
<210> 347
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> PMO-Wnt1
<400> 347
gcaacagcgc ccagagcccc atg 23
<210> 348
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> PMO-FITC
<400> 348
cctcttacct cagttacaat ttata 25
<210> 349
<211> 25
<212> RNA
<213> Artificial Sequence
<220>
<223> siRNA-GFP
<400> 349
acagcuccuc gcccuugcuc accau 25
<210> 350
<211> 21
<212> RNA
<213> Artificial Sequence
<220>
<223> siRNA-Gli1
<400> 350
aacuccacag gcauacagga u 21
<210> 351
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> PTD4
<400> 351
Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg Ala
1 5 10
<210> 352
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> TAT
<400> 352
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
1 5 10
<210> 353
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> CM18
<400> 353
Lys Trp Lys Leu Phe Lys Lys Ile Gly Ala Val Leu Lys Val Leu Thr
1 5 10 15
Thr Gly
<210> 354
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> KALA
<400> 354
Trp Glu Ala Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Ala Lys His
1 5 10 15
Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Ala Cys Glu Ala
20 25 30
<210> 355
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> Penetratin
<400> 355
Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys
1 5 10 15
<210> 356
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD92
<400> 356
Leu Leu Lys Ile Trp Ser Arg Leu Ile Lys Ile Trp Thr Gln Gly
1 5 10 15
<210> 357
<211> 19
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD418-19
<400> 357
Lys Leu Lys Leu Leu Lys Leu Leu Leu Lys Leu Leu Leu Lys Leu Leu
1 5 10 15
Lys Lys Leu
<210> 358
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD418-12-2
<400> 358
Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys Lys Leu
1 5 10
<210> 359
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD418-15
<400> 359
Lys Leu Lys Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys Lys Leu
1 5 10 15
<210> 360
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD440
<400> 360
Gly Gly Ser Gly Gly Gly Ser Lys Trp Lys Leu Phe Lys Lys Ile Gly
1 5 10 15
Ala Val Leu Lys Val Leu Thr Thr Gly Gly Gly Ser Gly Gly Gly Ser
20 25 30
<210> 361
<211> 40
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD441
<400> 361
Gly Gly Ser Gly Gly Gly Ser Lys Lys Ala Leu Leu Ala Leu Ala Leu
1 5 10 15
His His Leu Ala His Leu Ala Leu His Leu Ala Leu Ala Leu Lys Lys
20 25 30
Ala Gly Gly Ser Gly Gly Gly Ser
35 40
<210> 362
<211> 44
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD443
<400> 362
Gly Gly Ser Gly Gly Gly Ser Trp Glu Ala Lys Leu Ala Lys Ala Leu
1 5 10 15
Ala Lys Ala Leu Ala Lys His Leu Ala Lys Ala Leu Ala Lys Ala Leu
20 25 30
Lys Ala Cys Glu Ala Gly Gly Ser Gly Gly Gly Ser
35 40
<210> 363
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD445
<400> 363
Gly Trp Gly Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser Ala Arg Ala Ala Arg Gln Ala Arg
20 25 30
<210> 364
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> FSD446
<400> 364
Lys Trp Lys Leu Leu Lys Leu Trp Ser Arg Leu Leu Lys Leu Trp Gly
1 5 10 15
Gly Ser Gly Gly Gly Ser His Thr Ser Asp Gln Thr Asn
20 25
<210> 365
<211> 25
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<222> (9)..(11)
<223> Start codon
<400> 365
gagacgccat gttcaactcg atgac 25
<210> 366
<211> 25
<212> DNA
<213> Homo sapiens
<400> 366
aagtttgcgc ttctcgcggg tggtc 25
<210> 367
<211> 25
<212> DNA
<213> Homo sapiens
<400> 367
gaaatagaag ggaggtgagg ggcga 25
<210> 368
<211> 24
<212> DNA
<213> Homo sapiens
<400> 368
catcctccag aacggcaaga ggga 24
Claims (34)
1. A composition comprising a non-anionic polynucleotide analogue cargo for intracellular delivery and a synthetic peptide shuttle agent independent of or not covalently linked to the non-anionic polynucleotide analogue cargo, the synthetic peptide shuttle agent being a peptide comprising an amphiphilic α -helical motif having both a positively charged hydrophilic outer face and a hydrophobic outer face, wherein synthetic peptide shuttle agent increases cytosolic/nuclear delivery of the non-anionic polynucleotide analogue cargo in eukaryotic cells compared to the absence of the synthetic peptide shuttle agent.
2. The composition of claim 1, wherein the non-anionic polynucleotide analog cargo is a charge neutral or cationic Antisense Synthetic Oligonucleotide (ASO).
3. The composition of claim 1 or 2, wherein the non-anionic polynucleotide analog cargo is:
(a) Charge neutral polynucleotide analog cargo having a phosphodiamide backbone, an amide (e.g., peptide) backbone, a methylphosphonate backbone, a neutral phosphotriester backbone, a sulfone backbone, or a triazole backbone; or (b)
(b) Cationic polynucleotide analog cargo having an aminoalkylated phosphoramidate backbone, a guanidinium backbone, an S-methyl thiourea backbone, or a Nucleotidyl Amino Acid (NAA) backbone.
4. The composition of any one of claims 1 to 3, wherein the non-anionic polynucleotide analog cargo is a Phosphodiamide Morpholino Oligomer (PMO), a Peptide Nucleic Acid (PNA), a methylphosphonate oligomer, or a short interfering ribonucleic acid neutral oligonucleotide (siRNN).
5. The composition of any one of claims 1 to 4, wherein the non-anionic polynucleotide analog cargo is 5 to 50 mers, 5 to 75 mers, or 5 to 100 mers.
6. The composition of any one of claims 1 to 5, wherein the non-anionic polynucleotide analog cargo is not covalently linked to a cell penetrating peptide, octaguanidine dendrimer, or other intracellular delivery moiety.
7. The composition of any one of claims 1 to 6, wherein the shuttle agent is:
(1) A peptide of at least 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length comprising
(2) Amphipathic alpha-helical motifs with
(3) A positively charged hydrophilic outer face and a hydrophobic outer face,
wherein at least five of the following parameters (4) to (15) are observed:
(4) The hydrophobic outer face comprises a highly hydrophobic core consisting of spatially adjacent L, I, F, V, W and/or M amino acids, which account for 12% to 50% of the amino acids of the peptide, based on an open cylindrical representation of an a-helix of 3.6 residues per turn;
(5) The peptide has a hydrophobic moment (μ) of 3.5 to 11;
(6) The peptide has a predicted net charge of at least +3 or +4 at physiological pH;
(7) The peptide has an isoelectric point (pI) of 8 to 13;
(8) The peptide consists of 35% to 65% of any combination of the following amino acids: A. c, G, I, L, M, F, P, W, Y and V;
(9) The peptide consists of any combination of 0% to 30% of the following amino acids: n, Q, S and T;
(10) The peptide consists of 35% to 85% of any combination of the following amino acids: A. l, K or R;
(11) The peptide consists of any combination of 15% to 45% of the following amino acids: a and L, provided that at least 5% L is present in the peptide;
(12) The peptide consists of any combination of 20% to 45% of the following amino acids: k and R;
(13) The peptide consists of any combination of 0% to 10% of the following amino acids: d and E;
(14) The difference between the percentage of a and L residues in the peptide (a+l%) and the percentage of K and R residues in the peptide (k+r) is less than or equal to 10%; and
(15) The peptide consists of any combination of 10% to 45% of the following amino acids: q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T and H.
8. The composition of claim 7, wherein:
(a) The shuttle agent complies with at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or complies with all of parameters (4) to (15);
(b) The shuttle agent is a peptide having a minimum length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids and a maximum length of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids;
(c) The amphiphilic α -helical motif has a hydrophobic moment (μ) between the lower limit of 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and the upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9 or 11.0;
(d) The amphiphilic α -helical motif comprises a positively charged hydrophilic outer face comprising, based on an α -helix with a rotation angle of 100 degrees between consecutive amino acids and/or an α -helix with 3.6 residues per turn: (i) At least two, three or four adjacent positively charged K and/or R residues upon helical projection; and/or (ii) a segment comprising six adjacent residues of three to five K and/or R residues upon helical projection;
(e) The amphiphilic α -helical motif comprises a hydrophobic outer face comprising, based on an α -helix with a rotation angle of 100 degrees between consecutive amino acids and/or an α -helix with 3.6 residues per turn: (i) at least two adjacent L residues upon helical projection; and/or (ii) a segment comprising ten adjacent residues of at least five hydrophobic residues selected from L, I, F, V, W and M upon helical projection;
(f) The hydrophobic outer face comprises a highly hydrophobic core consisting of spatially adjacent L, I, F, V, W and/or M amino acids, the amino acids comprising from 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5% or 20% to 25%, 30%, 35%, 40% or 45% of the amino acids of the shuttle agent;
(g) The shuttle agent has a hydrophobic moment (μ) between a lower limit of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and an upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4 or 10.5;
(h) The shuttle agent has a predicted net charge between +3, +4, +5, +6, +7, +8, +9 to +10, +11, +12, +13, +14 or +15;
(i) The shuttle agent has a predicted pI of 10 to 13; or (b)
(j) Any combination of (a) to (i).
9. The composition of claim 7 or 8, wherein the shuttle agent complies with at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all of the following parameters:
(8) The shuttle agent consists of 36% to 64%, 37% to 63%, 38% to 62%, 39% to 61%, or 40% to 60% of any combination of the following amino acids: A. c, G, I, L, M, F, P, W, Y and V;
(9) The shuttle agent consists of 1% to 29%, 2% to 28%, 3% to 27%, 4% to 26%, 5% to 25%, 6% to 24%, 7% to 23%, 8% to 22%, 9% to 21%, or 10% to 20% of any combination of the following amino acids: n, Q, S and T;
(10) The shuttle agent consists of 36% to 80%, 37% to 75%, 38% to 70%, 39% to 65%, or 40% to 60% of any combination of the following amino acids: A. l, K or R;
(11) The shuttle agent consists of 15% to 40%, 20% to 35%, or 20% to 30% of any combination of the following amino acids: a and L;
(12) The shuttle agent consists of 20% to 40%, 20% to 35%, or 20% to 30% of any combination of the following amino acids: k and R;
(13) The shuttle agent consists of any combination of 5% to 10% of the following amino acids: d and E;
(14) The difference between the percentage of a and L residues in the shuttle agent (a+l%) and the percentage of K and R residues in the shuttle agent (k+r) is less than or equal to 9%, 8%, 7%, 6% or 5%; and
(15) The shuttle agent consists of 15% to 40%, 20% to 35%, or 20% to 30% of any combination of the following amino acids: q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T and H.
10. The composition of any one of claims 1 to 9, wherein the shuttle agent comprises a histidine-rich domain, optionally wherein the histidine-rich domain:
(i) Positioning toward the N-terminus and/or C-terminus of the shuttle agent;
(ii) Is an extension of at least 3, at least 4, at least 5 or at least 6 amino acids comprising at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% histidine residues; and/or comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 consecutive histidine residues; or (b)
(iii) Both (i) and (ii).
11. The composition of any one of claims 1 to 10, wherein the shuttle agent comprises a flexible linker domain that is rich in serine and/or glycine residues (e.g., separates the N-terminal and C-terminal segments of the shuttle agent; or is positioned N-terminal and/or C-terminal to a central amphiphilic cationic alpha-helical domain).
12. The composition of any one of claims 1 to 11, wherein the shuttle agent comprises or consists of the amino acid sequence:
(a) [ X1] - [ X2] - [ linker ] - [ X3] - [ X4] (formula 1);
(b) [ X1] - [ X2] - [ linker ] - [ X4] - [ X3] (formula 2);
(c) [ X2] - [ X1] - [ linker ] - [ X3] - [ X4] (formula 3);
(d) [ X2] - [ X1] - [ linker ] - [ X4] - [ X3] (formula 4);
(e) [ X3] - [ X4] - [ linker ] - [ X1] - [ X2] (formula 5);
(f) [ X3] - [ X4] - [ linker ] - [ X2] - [ X1] (formula 6);
(g) [ X4] - [ X3] - [ linker ] - [ X1] - [ X2] (formula 7); or (b)
(h) [ X4] - [ X3] - [ linker ] - [ X2] - [ X1] (formula 8),
(i) [ linker ] - [ X1] - [ X2] - [ linker ] (formula 9);
(j) [ linker ] - [ X2] - [ X1] - [ linker ] (formula 10);
(k) [ X1] - [ X2] - [ linker ] (formula 11);
(l) [ X2] - [ X1] - [ linker ] (formula 12);
(m) [ linker ] - [ X1] - [ X2] (formula 13);
(n) [ linker ] - [ X2] - [ X1] (formula 14);
(o) [ X1] - [ X2] (formula 15); or (b)
(p) [ X2] - [ X1] (formula 16),
wherein:
[ X1] is selected from: 2[ phi ] -1 < + > -2[ phi ] -1[ zeta ] -1 < + > -;2[ phi ] -1 < + > -2[ phi ] -2 < + > -;1 < + > -1[ phi ] -1 < + > -2[ phi ] -1[ zeta ] -1 < + > -; and 1 < + > -2 < + >;
[ X2] is selected from: -2[ phi ] -1+ ] -2[ phi ] -2[ zeta ] -; -2[ phi ] -1 < + > -2[ phi ] -2 < + > -; -2[ phi ] -1 < + > -1[ ζ ] -; -2[ phi ] -1 + ] -2[ phi ] -1[ zeta ] -1 + ]; -2[ phi ] -2 < + > -1[ phi ] -2 < + > -; -2[ phi ] -2 < + > -1[ phi ] -2[ zeta ] -; -2[ phi ] -2 < + > -1[ phi ] -1 < + > -1[ ζ ] -; and-2 [ phi ] -2 < + > -1[ phi ] -1[ zeta ] -1 < + > -;
[ X3] is selected from: -4 < + > -A-; ext> -ext> 3ext> <ext> +ext> >ext> -ext> Gext> -ext> Aext> -ext>;ext> -3 < + > -A-A-; -2 < + > -1 < + > -A-; ext> -ext> 2ext> <ext> +ext> >ext> -ext> 1ext> [ext> phiext> ]ext> -ext> Gext> -ext> Aext> -ext>;ext> -2 < + > -1[ phi ] -A-A-; or-2 < + > -A-1 < + > -A; ext> -ext> 2ext> <ext> +ext> >ext> -ext> Aext> -ext> Gext> -ext> Aext>;ext> -2 < + > -A-A-A-; -1[ phi ] -3 < + > -A-; ext> -ext> 1ext> [ext> phiext> ]ext> -ext> 2+ext> ]ext> -ext> Gext> -ext> Aext> -ext>;ext> -1[ phi ] -2 < + > -A-A-; -1[ phi ] -1[ phi+ ] -A; ext> -ext> 1ext> [ext> phiext> ]ext> -ext> 1ext> +ext> ]ext> -ext> 1ext> [ext> phiext> ]ext> -ext> Gext> -ext> Aext>;ext> -1[ phi ] -1 + ] -1[ phi ] -A-A; -1[ phi ] -1 < + > -A; ext> -ext> 1ext> [ext> phiext> ]ext> -ext> 1+ext> ]ext> -ext> Aext> -ext> Gext> -ext> Aext>;ext> -1[ phi ] -1 < + > -A-A-A; -A-1 < + > -A; ext> -ext> Aext> -ext> 1ext> <ext> +ext> >ext> -ext> Aext> -ext> Gext> -ext> Aext>;ext> and-A-1 < + > -A-A-A;
[ X4] is selected from: -1[ ζ ] -2A-1+ ] -A; -1[ zeta ] -2A-2+ ]; -1 < + > -2A-1 < + > -A; -1[ zeta ] -2A-1 < + > -1[ zeta ] -A-1 < + >; -1[ zeta ] -A-1+ ]; -2 < + > -A-2 < + >; -2 < + > -A-1 < + > -A; -2 < + > -A-1 < + > -1[ ζ ] -A-1 < + >; -2 < + > -1[ ζ ] -A-1 < + >; -1 < + > -1[ ζ ] -A-1 < + > -A; -1 < + > -1[ ζ ] -A-2 < + >; -1 < + > -1[ zeta ] -A-1 < + >; -1 < + > -2[ ζ ] -A-1 < + >; -1 < + > -2 < + >; -1 < + > -2[ ζ ] -1 < + > -A; -1 < + > -2[ zeta ] -1 < + > -1[ zeta ] -A-1 < + >; -1 < + > -2[ zeta ] -1[ zeta ] -A-1 < + >; -3[ zeta ] -2+ ]; -3[ ζ ] -1+ ] -A; -3[ zeta ] -1 < + > -1[ zeta ] -A-1 < + >; -1[ ζ ] -2A-1+ ] -A; -1[ zeta ] -2A-2+ ]; -1[ zeta ] -2A-1 < + > -1[ zeta ] -A-1 < + >; -2 < + > -A-1 < + > -A; -2 < + > -1[ ζ ] -1 < + > -A; -1 < + > -1[ ζ ] -A-1 < + > -A; -1 < + > -2A-1 < + > -1[ zeta ] -A-1 < + >; and-1 [ zeta ] -A-1+ ]; and is also provided with
[ linker ] is selected from: -Gn-; -Sn-; - (GnSn) n-; - (GnSn) nGn-; - (GnSn) nSn-; - (GnSn) nGn- (GnSn) n-; and- (GnSn) nSn (GnSn) n-;
wherein:
[ phi ] is an amino acid which is: leu, phe, trp, ile, met, tyr or Val, preferably Leu, phe, trp or Ile;
the [ + ] is an amino acid which is: lys or Arg;
[ ζ ] is an amino acid, which is: gln, asn, thr or Ser;
a is the amino acid Ala;
g is amino acid Gly;
s is the amino acid Ser; and is also provided with
n is an integer from 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, or 1 to 3.
13. The composition of any one of claims 1 to 12, wherein the shuttle agent comprises or consists of:
(i) The amino acid sequence of any of SEQ ID NOs 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311, 313 to 324, 326 to 332, 338 to 342, 344, or 353 to 364;
(ii) Amino acid sequences that differ from any of SEQ ID NOs 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311, 313 to 324, 326 to 332, 338 to 342, 344, or 353 to 364 by NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., do not include any linker domains);
(iii) An amino acid sequence that is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (e.g., calculated without any linker domain) to any of SEQ ID NOs 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311 to 324, 326 to 332, 338 to 342, 344, or 353 to 364;
(iv) Amino acid sequence differing from any of SEQ ID NOs 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311, 313 to 324, 326 to 332, 338 to 342, 344, or 353 to 364 by only conservative amino acid substitutions (e.g., by NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, preferably not including any linker domain), wherein each conservative amino acid substitution is selected from amino acids within the same amino acid class, the amino acids being: aliphatic: G. a, V, L and I; hydroxyl-containing or sulfur/selenium: s, C, U, T and M; aromatic: F. y and W; alkaline: H. k and R; acidic and its amides: D. e, N and Q; or (b)
(v) Any combination of (i) to (iv).
14. The composition of any one of claims 1 to 13, wherein the shuttle agent comprises or consists of a variant of the synthetic peptide shuttle agent that is identical to the synthetic peptide shuttle agent as defined in any one of claims 1 or 7 to 13, except that at least one amino acid is replaced with a corresponding synthetic amino acid having a side chain with physiochemical properties (e.g., structure, hydrophobicity, or charge) similar to that of the replaced amino acid, wherein the variant increases cytosolic/nuclear delivery of the non-anionic polynucleotide analogue cargo in eukaryotic cells compared to the absence of the synthetic peptide shuttle agent, preferably wherein the synthetic amino acid replacement:
(a) Substituting a basic amino acid with any one of: α -aminoglycine, α, γ -diaminobutyric acid, ornithine, α, β -diaminopropionic acid, 2, 6-diamino-4-hexynoic acid, β - (1-piperazinyl) -alanine, 4, 5-dehydro-lysine, δ -hydroxylysine, ω -dimethylarginine, homoarginine, ω' -dimethylarginine, ω -methylarginine, β - (2-quinolinyl) -alanine, 4-aminopiperidine-4-carboxylic acid, α -methylhistidine, 2, 5-diiodohistidine, 1-methylhistidine, 3-methylhistidine, spinacin, 4-aminophenylalanine, 3-aminotyrosine, β - (2-pyridinyl) -alanine or β - (3-pyridinyl) -alanine;
(b) Substitution of a nonpolar (hydrophobic) amino acid with any of the following: dehydro-alanine, beta-fluoroalanine, beta-chloroalanine, beta-iodoalanine, alpha-aminobutyric acid, alpha-aminoisobutyric acid, beta-cyclopropylalanine, azetidine-2-carboxylic acid, alpha-allylglycine, propargylglycine, t-butylalanine, beta- (2-thiazolyl) -alanine, thioproline, 3, 4-dehydroproline, t-butylglycine, beta-cyclopentylalanine, beta-cyclohexylalanine, alpha-methylproline, norvaline, alpha-methylvaline, penicillamine, beta, beta-dicyclohexylalanine, 4-fluoroproline, 1-aminocyclopentanecarboxylic acid, piperidinecarboxylic acid, 4, 5-dehydroleucine, allo-isoleucine, norleucine, alpha-methylleucine, cyclohexylglycine, cis-octahydroindole-2-carboxylic acid, beta- (2-thienyl) -alanine, phenylglycine, alpha-methylphenylalanine, homophenylalanine, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, beta- (3-benzothienyl) -alanine, 4-nitrophenylalanine, 4-bromophenylalanine, 4-tert-butylphenylalanine, alpha-methyltryptophan, beta- (2-naphthyl) -alanine, beta- (1-naphthyl) -alanine, 4-iodophenylalanine, 3-fluorophenylalanine, 4-methyltryptophan, 4-chlorophenylalanine, 3, 4-dichloro-phenylalanine, 2, 6-difluoro-phenylalanine, n-in-methyltryptophan, 1,2,3, 4-tetrahydronor Ha Erman-3-carboxylic acid, β -diphenylalanine, 4-methylphenylalanine, 4-phenylphenylalanine, 2,3,4,5, 6-pentafluoro-phenylalanine or 4-benzoylphenylalanine;
(c) Substituting a polar uncharged amino acid with any of the following: beta-cyanoalanine, beta-ureidoalanine, homocysteine, allothreonine, pyroglutamic acid, 2-oxothiazolidine-4-carboxylic acid, citrulline, thiocitrulline, homoccitrulline, hydroxyproline, 3, 4-dihydroxyphenylalanine, beta- (1, 2, 4-triazol-1-yl) -alanine, 2-mercaptohistidine, beta- (3, 4-dihydroxyphenyl) -serine, beta- (2-thienyl) -serine, 4-azidophenalanine, 4-cyanophenylalanine, 3-hydroxymethyltyrosine, 3-iodotyrosine, 3-nitrotyrosine, 3, 5-dinitrotyrosine, 3, 5-dibromotyrosine, 3, 5-diiodotyrosine, 7-hydroxy-1, 2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, 5-hydroxytryptophan, thyronine, beta- (7-methoxycoumarin-4-yl) -alanine or 4- (7-hydroxy-4-coumarin) -aminobutyric acid; and/or
(d) Replacing an acidic amino acid with any one of: gamma-hydroxy glutamic acid, gamma-methylene glutamic acid, gamma-carboxy glutamic acid, alpha-amino adipic acid, 2-amino pimelic acid, alpha-amino suberic acid, 4-carboxy phenylalanine, sulfoalanine, 4-phosphonophenylalanine or 4-sulfomethyl phenylalanine.
15. The composition of any one of claims 1 to 14, wherein the shuttle agent does not comprise a Cell Penetrating Domain (CPD), a Cell Penetrating Peptide (CPP), or a Protein Transduction Domain (PTD).
16. The composition of any one of claims 1 to 14, wherein the shuttle agent does not comprise CPD fused to an Endosomal Leakage Domain (ELD).
17. The composition of any one of claims 1 to 14, wherein the shuttle agent comprises an Endosomal Leakage Domain (ELD) and/or a Cell Penetration Domain (CPD).
18. The composition of any one of claims 15 to 17, wherein:
(i) The ELD is or is derived from: endosomally lytic peptides; antimicrobial peptides (AMPs); linear cationic alpha-helical antimicrobial peptides; cecropin-a/melittin hybrid (CM) peptide; a pH dependent membrane active peptide (PAMP); a peptide amphiphile; a peptide derived from the N-terminus of the HA2 subunit of influenza Hemagglutinin (HA); CM18; diphtheria toxin T Domain (DT); GALA; PEA; INF-7; LAH4; HGP; h5WYG; HA2; EB1; VSVG; a pseudomonas toxin; melittin; KALA; JST-1; c (LLKK) 3 C;G(LLKK) 3 G, G; or any combination thereof;
(ii) The CPD is or is derived from: a cell penetrating peptide or a protein transduction domain from a cell penetrating peptide; TAT; PTD4; a penetrating protein; pVEC; m918; pep-1; pep-2; xentry; an arginine extension; a transporter; synB1; synB3; or any combination thereof; or (b)
(iii) Both (i) and (ii).
19. The composition of any one of claims 1 to 18, wherein the shuttle agent is a cyclic peptide and/or comprises one or more D-amino acids.
20. The composition of any one of claims 1 to 19, wherein the shuttle agent increases the transduction efficiency and/or total amount of non-anionic polynucleotide analog cargo delivered intracellularly in the eukaryotic cell by at least a factor of 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 relative to a corresponding negative control lacking the shuttle agent.
21. The composition of any one of claims 1-20, wherein the shuttle agent further comprises a chemical modification to one or more amino acids, wherein the chemical modification does not disrupt the transduction activity of the synthetic peptide shuttle agent.
22. The composition of claim 21, wherein the chemical modification is at the N-and/or C-terminus of the shuttle agent.
23. The composition of claim 21 or 22, wherein the chemical modification is addition of an acetyl group (e.g., an N-terminal acetyl group), a cysteamine group (e.g., a C-terminal cysteamine group), or a fatty acid (e.g., a C4-C16 fatty acid, preferably N-terminal).
24. The composition of any one of claims 1 to 23, wherein the concentration of the non-anionic polynucleotide analogue cargo and/or the synthetic peptide shuttling agent in the composition is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 μΜ.
25. The composition according to any one of claims 1 to 24:
(i) For modulating gene expression in said eukaryotic cell (e.g., gene expression knockdown, modified splicing (e.g., exon skipping), modification of miRNA activity and/or maturation, translation frame shift induction, RNA editing interference, or modification of poly a tailing);
(ii) For use in therapy, wherein the non-anionic polynucleotide analog cargo modulates gene expression of a therapeutically relevant target RNA in the eukaryotic cell;
(iii) As antiviral agents, wherein the non-anionic polynucleotide analog cargo modulates gene expression of viral genes required for virulence and/or pathogenicity;
(iv) For delivering non-therapeutic non-anionic polynucleotide analog cargo as a diagnostic agent;
(v) For the manufacture of a medicament or diagnostic agent (e.g., formulated for topical, enteral/parenteral (e.g., oral) or parenteral administration);
(vi) For treating cancer (e.g., skin cancer, basal cell carcinoma, nevus basal cell carcinoma syndrome), inflammation or inflammation-related diseases (e.g., psoriasis, atopic dermatitis, ulcerative colitis, urticaria, dry eye, dry or wet age-related macular degeneration, digital ulcers, keratosis, idiopathic pulmonary fibrosis), pain (e.g., chronic or acute), or diseases affecting the lung (e.g., cystic fibrosis, asthma, chronic Obstructive Pulmonary Disease (COPD), or idiopathic pulmonary fibrosis); or (b)
(vii) Any combination of (i) to (vi).
26. The composition of any one of claims 1 to 24 or the composition for the use of claim 25, wherein the eukaryotic cell is an animal cell, a mammalian cell, a human cell, a stem cell, a primary cell, an immune cell, a T cell, an NK cell, a dendritic cell, an epithelial cell, a skin cell, a gastrointestinal tract cell, a lung cell, or an ocular cell.
27. A method for modifying gene expression in a eukaryotic cell, the method comprising:
(a) Providing a non-anionic polynucleotide analogue cargo for intracellular delivery, said non-anionic polynucleotide analogue cargo being designed to hybridise to a RNA of interest in said eukaryotic cell;
(b) Providing a synthetic peptide shuttle agent that is independent of or not covalently linked to the non-anionic polynucleotide analogue cargo;
(c) Contacting the eukaryotic cell with the non-anionic polynucleotide analogue cargo in the presence of the synthetic peptide shuttle agent at a concentration sufficient to increase transduction efficiency and/or cytosol/nucleus delivery of the non-anionic polynucleotide analogue cargo as compared to the absence of the synthetic peptide shuttle agent, wherein the non-anionic polynucleotide analogue cargo hybridizes to the RNA of interest following cytosol/nucleus delivery, thereby effecting modification of gene expression.
28. The method of claim 27, which is an in vitro method (e.g., for therapeutic and/or diagnostic purposes).
29. The method of claim 27, which is an in vivo method (e.g., for therapeutic and/or diagnostic purposes).
30. The method of any one of claims 27 to 29, wherein:
(i) The non-anionic polynucleotide analogue cargo is as defined in any one of claims 1 to 6;
(ii) The synthetic peptide shuttle agent is as defined in any one of claims 1 or 7 to 23;
(iii) Contacting the eukaryotic cell with a concentration of a non-anionic polynucleotide analogue cargo and/or synthetic peptide shuttle agent as defined in claim 24;
(iv) The method is for use as defined in claim 25;
(v) The eukaryotic cell is as defined in claim 26; or (b)
(vi) Any combination of (i) to (v).
31. The composition of any one of claims 1 to 26 or the method of any one of claims 27 to 30, wherein the non-anionic polynucleotide analog cargo is a non-anionic antisense oligonucleotide targeting a gene of a hedgehog pathway.
32. The composition of claim 31 or method of claim 31, wherein the non-anionic antisense oligonucleotide targets Gli1 for knockdown.
33. The composition of claim 32 or the method of claim 32, wherein the non-anionic antisense oligonucleotide hybridizes to a polynucleotide sequence of any one of SEQ ID NOs 365-368.
34. The composition of any one of claims 31 to 33 or the method of any one of claims 31 to 33, wherein the composition or method is for treating gollin syndrome and/or basal cell carcinoma.
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PCT/CA2021/051458 WO2022077121A1 (en) | 2020-10-18 | 2021-10-18 | Peptide-based transduction of non-anionic polynucleotide analogs for gene expression modulation |
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