Intracellular delivery of gene-editing enzymes and uses thereof

Abstract

Described herein are peptides including a gene editing polypeptide having at least 90% sequence similarity to SEQ ID NOs: 141, 148-151, or 236-251 linked to a membrane translocation domain having one or more cell penetrating peptide motifs, where at least one cell penetrating peptide motif is from 3 to 10 amino acid residues in length and has at least three arginine and / or lysine residues; or where at least one cell penetrating peptide motif is from 3 to 10 amino acid residues in length and has at least two arginine and / or lysine residues and at least one other cell penetrating peptide motif is from 2 to 8 amino acid residues in length and has at least two hydrophobic residues.

Description

[0001] INTRACELLULAR DELIVERY OF GENE-EDITING

[0002] ENZYMES AND USES THEREOF

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under Grant No. GM122459 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] CROSS-REFERENCE TO RELATED APPLICATION

[0006] This application claims priority to, and the benefit of U.S. Provisional Application 63 / 603,930. filed on November 29. 2023 and U.S. Provisional Application No. 63 / 697,761. filed on September 23, 2024 the contents of which are hereby incorporated in their entirety.

[0007] REFERENCE TO SEQUENCE LISTING

[0008] The Sequence Listing submitted November 29, 2024, as a text filed named ‘T03361- 606WOl_ST26.xmU created November 28, 2024, and having a file size of 633.768 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

[0009] BACKGROUND

[0010] Gene editing has the potential to revolutionize the treatment of previously incurable human diseases (H. Li, et al., Signal Transduct Target Ther, vol. 5, no. 1, p. 1, Jan 03 2020) and the generation of genetically edited plants (Wada, N., et al.. BMC Plant Biol 20, 234 (2020)). Four different gene-editing systems have been developed, including meganucleases (G. Silva et al., Curr Gene Ther, vol. 11, no. 1, pp. 11-27, Feb 2011), zinc finger nucleases (T. Gaj, , et al., Trends Biotechnol, vol. 31, no. 7, pp. 397-405, Jul 2013), transcription activator-like effector nucleases (TALENs) (J. K. Joung, et al., Nat Rev Mol Cell Biol. vol. 14, no. 1, pp. 49-55, Jan 2013). and clustered regularly interspaced short palindromic repeats (CRISPR)ZCRISPR associated (Cas) systems (J. A. Doudna, et al.. Science, vol. 346, no. 6213, p. 1258096, Nov 28 2014). Among these methods, the CRISPR / Cas9 system has been most widely used due to its simplicity and versatility . Cas9 is an endonuclease derived from Streptococcus pyogenes. Cas9 binds to a guide RNA (gRNA) containing an ~20-nucleotide sequence designed to match the target genes and the resulting ribonucleoprotein (RNP) targets the DNA substrate containing the complementary sequence and generates site-specific double-strand breaks (DSBs) (Jinek, M., et al., (2012). Science 337(6096), 816-821; Cong, L., et al.. (2013). Science 339, 819-823; Mali, P., et al., (2013). Science (New York, N.Y.), 339(6121), 823-826). The DSBs are subsequently repaired through non-homologous endjoining (NHEJ) or homology-directed repair (HDR) in cells. Catalytically inactive Cas9 mutants have also been fused to adenosine or cytosine deaminase to site-specifically edit DNA sequences without generating DNA strand breaks (Komor AC, et al.. Nature. 2016;533(7603):420-4; Nishida K, et al., Science. 2016;353(6305):aaf8729; Gaudelli NM, et al., Nature. 2017;551(7681):464- 71). In addition, CRISPR-Cas systems have been developed to site-specifically edit mRNAs instead of DNAs (Abudayyeh, O., et al., Nature 550. 280-284 (2017); Cox. D. B. T„ et al., (2017). Science (New York, N.Y.), 358(6366), 1019-1027; East-Seletsky, A., et al., Molecular cell, 66(3), 373-383. e3; Knott, G. J., et al.. (2017). Nature structural & molecular biology, 24(10), 825-833).

[0011] For therapeutic applications of gene-editing systems, intracellular delivery of the proteins (or ribonucleoproteins) remains a key challenge. Several delivery’ platforms have been explored but each has significant limitations. Viral-based delivery platforms can provide sustained expression of the gene-editing proteins but are associated with safety concerns (including risks of integration into the host genome and existing immunity’ against the viral vectors) and limited in the size of the DNA fragment that can be packaged efficiently (Ran, F„ et al., Nature 520, 186-191 (2015); Wang, D„ et al., (2020). Cell, 181(1), 136-150). Sustained expression of a gene-editing enzyme in cells also increases the chance of off-target cleavage. Non-viral methods such as physical methods (e.g., electroporation), liposomes, nanoparticles, and cell-penetrating peptides (CPPs) result in the transient presence of nucleases in cells, thus reducing off-target effects and immune responses (Lino. C. A., et al., (2018). Drug delivery, 25(1), 1234-1257; Li, J., et al., (2021). Advanced drug delivery reviews, 168, 99-117; Sinclair, F., etal. DrugDeliv. and Transl. Res. 13, 1500-1519 (2023)). However, the non-viral methods generally have low cytosolic delivery efficiency due to inefficient endosomal escape or are incompatible with in vivo applications (in the case of electroporation). More recently. Tian et al. utilized detoxified bacterial toxins such as diphtheria toxin (DT) and botulinum neurotoxin (BoNT)-like toxin (BoNT / X) as carriers to deliver large protein cargoes including Casl3a, CasRx, Cas9, and Cre recombinase into cells in a receptor-dependent manner (Tian, S., et al., (2022). Cell reports, 38(10), 110476). This method is, however, limited to cells that express proper receptors for the toxins and failed to deliver ribonucleoproteins. Furthermore, most people are already immunized against the DT, limiting the potential use of DT-based delivery.

[0012] Accordingly, there is a need for an effective delivery composition for gene-editing enzymes. The compositions and methods disclosed herein address these and other needs.

[0013] SUMMARY

[0014] Provided herein are peptides, including: a gene editing polypeptide having at least 90% sequence similarity to SEQ ID NOs: 141, 148-151, or 236-251 linked to a membrane translocation domain having one or more cell penetrating peptide motifs, where at least one cell penetrating peptide motif is from 3 to 10 amino acid residues in length and has at least three arginine and / or lysine residues; or where at least one cell penetrating peptide motif is from 3 to 10 amino acid residues in length and has at least two arginine and / or lysine residues and at least one other cell penetrating peptide motif is from 2 to 8 amino acid residues in length and has at least two hydrophobic residues.

[0015] In some embodiments, the gene editing polypeptide having at least 90% sequence similarity to SEQ ID NOs: 141. 148-151, or 236-251 is linked to the membrane translocation domain at a N-terminus or C-terminus of the membrane translocation domain, or at a side chain within the membrane translocation domain. In some embodiments, the gene editing polypeptide having at least 90% sequence similarity to SEQ ID Nos: 141, 148-151, or 236- 251 is linked to the membrane translocation domain at the C-terminus of the membrane translocation domain.

[0016] In some embodiments, the membrane translocation domain is human fibronectin type III. In some embodiments, the human fibronectin type III has 90% sequence similarity with SEQ ID NO: 118. In some embodiments, the membrane translocation domain is human fibronectin type III having BC, DE, CD, and FG loops and one or more of the BC, DE. CD, or FG loops have cell penetrating peptide motifs. In some embodiments, the membrane translocation domain is human fibronectin type III having BC, DE, CD, and FG loops and two of the BC, DE, CD, or FG loops have cell penetrating peptide motifs. In some embodiments, the membrane translocation domain is human fibronectin type III having BC, DE, CD, and FG loops and the BC and either the DE, CD. or FG loops have cell penetrating peptide motifs. In some embodiments, the membrane translocation domain is human fibronectin type III having BC, DE, CD, and FG loops and the BC and FG loops have cell penetrating peptide motifs. In some embodiments, the cell penetrating peptide motif in the BC loop has from 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues and the cell penetrating peptide motif in the FG loop has from 3 to 10 amino acid residues and has at least two adjacent arginine and / or lysine residues. In some embodiments, the cell penetrating peptide motif in the FG loop has from 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues and the cell penetrating peptide motif in the BC loop has from 3 to 10 amino acid residues and has at least two adjacent arginine and / or lysine residues.

[0017] In some embodiments, the cell penetrating motif has from 3 to 10 adjacent arginine residues. In some embodiments, a second cell penetrating peptide motif is present and is WW, FF, WF, FW, WWW, FFF, WFW, FWF, WWF, WFF, FWW, FFW, WYW, WWH, YWW, or WYH. In some embodiments, the cell penetrating peptide motif can include RRRWWW (SEQ ID NO: 104), WWWRRR (SEQ ID NO: 105), TGRRRRWWWSKPI (SEQ ID NO: 1 11); APWWWRRRRYY (SEQ ID NO: 112); GGRRRRWWWVQE (SEQ ID NO: 113); APAWYWRYY (SEQ ID NO: 114); TGRRRRSKPI (SEQ ID NO: 115); APARRRRYY (SEQ ID NO: 116); TGWYWRSKPI (SEQ ID NO: 117); SEQ ID NO: 153, SEQ ID NO: 154. SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, or SEQ ID NO: 162. In some embodiments, the cell penetrating peptide motif is APAWYWRYY (SEQ ID NO: 114) and TGRRRRSKPI (SEQ ID NO: 115).

[0018] In some embodiments, the membrane translocation domain comprises SEQ ID NO.s: 119, 120. 121, 122, 123, 124, 125. 135, 136. 137, 138, or 139. In some embodiments, the membrane translocation domain is SEQ ID NO: 122.

[0019] In some embodiments, the peptide can further include a cargo moiety. In some embodiments, the peptide has at least 90% sequence similarity to SEQ ID NO: 140.

[0020] Described herein are also pharmaceutical composition including an effective amount of the peptide described herein and a pharmaceutically acceptable carrier.

[0021] Described herein are also methods for treating or preventing cancer, the method including administering to a subject in need thereof an effective amount of the pharmaceutical composition described herein, or an effective amount of a peptide described herein.

[0022] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims. DESCRIPTION OF DRAWINGS

[0023] FIG. 1 shows a predicted 3D structure of MTD4-Cas9 by Phyre2. The his-tag, the CPP motifs in MTD4, and the GGS3 linker are labeled the structure.

[0024] FIGs. 2A-2C show images of bacterial expression and purification of MTD4-Cas9. FIG. 2A) SDS-PAGE showing the different ammonium sulfate fractions. CL, crude cell lysate; S, soluble fraction; P. precipitated fraction. FIG. 2B) SDS-PAGE showing the different fractions from the Ni-NTA column. Inj. redissolved protein solution of the 60% ammonium sulfate fraction from a), which was loaded onto the Ni-NTA column; FT, flow through. FIG. 2C) SDS-PAGE showing the purified MTD4-Cas9 (with different amounts loaded into different lanes). L, molecular weight markers.

[0025] FIGs. 3 A and 3B show the effect of MTD4. PTEN. and MTD4-PTEN on the viability of MDA-MB-468 cells (n = 3). Western blot showing the effect of PTEN (100 nM) and MTD4-PTEN (3-100 nM) on the phosphorylation of Akt.

[0026] FIG. 4 shows representative thermal denaturation profiles of FN3 and MTD4 as monitored by high-throughput thermal scanning (HTTS). The HTTS method employs a hydrophobic dye (SYPRO™ Orange), which fluoresces upon binding to molten globules and thermal denaturation intermediates. The melting temperature (Tm) of a protein is defined as the temperature at which half of the protein is in the unfolded state. The Tm values reported represent the mean ± SD of three independent experiments (n = 3).

[0027] FIGs 5A-5D. Serum stability of MTD4 and MTD4s. (Fig. 5A) Amino acid sequence of MTD4, with the proteolytic cleavage sites indicated by arrows. (Fig. 5B) Amino acid sequence of MTD4s. (Fig. 5C) Coomassie blue-stained SDS-PAGE gel showing the timedependent proteolysis of MTD4 (13.2 kD) into a fragment(s) of ~12 kD (11 / 2 ~3 h). (Fig. 5D) SDS-PAGE gel showing MTD4s (12.6 kDa) after varying periods of incubation in human serum. No significant cleavage was observed after 24 h of incubation (11 / 2 >24 h). M, molecular weight markers; NS, no serum (MTD4s only).

[0028] Like reference symbols in the various drawings indicate like elements.

[0029] DETAILED DESCRIPTION

[0030] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. Definitions

[0031] To facilitate understanding of the disclosure set forth herein, a number of terms are defined below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary' skill in the art to which this disclosure belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0032] As used in this specification and the following claims, the terms "‘comprise’7(as well as forms, derivatives, or variations thereof, such as “comprising” and “comprises”) and “include” (as well as forms, derivatives, or variations thereof, such as “including” and “includes”) are inclusive (i.e., open-ended) and do not exclude additional elements or steps. For example, the terms "comprise" and / or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Other than where noted, all numbers expressing quantities of ingredients, reaction conditions, geometries, dimensions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary' rounding approaches.

[0033] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. By “about” is meant within 5% of the value, e.g., within 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Arange may be construed to include the start and the end of the range. For example, a range of 10% to 20% (i.e., range of 10%-20%) can includes 10% and also includes 20%, and includes percentages in between 10% and 20%, unless explicitly stated otherwise herein.

[0034] As used herein, the terms "may." "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0035] It is understood that when combinations, subsets, groups, etc. of elements are disclosed (e.g., combinations of components in a composition, or combinations of steps in a method), that while specific reference of each of the various individual and collective combinations and permutations of these elements may not be explicitly disclosed, each is specifically contemplated and described herein.

[0036] “Administration" to a subject includes any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra- articular, intra-synovial, intrastemal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like. "Concurrent administration", "administration in combination", "simultaneous administration" or "administered simultaneously" as used herein, means that the compounds are administered at the same point in time or essentially immediately following one another. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time. "Systemic administration" refers to the introducing or delivering to a subj ect an agent via a route which introduces or delivers the agent to extensive areas of the subject's body (e.g. greater than 50% of the body), for example through entrance into the circulatory or lymph systems. By contrast, "local administration" refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject's body. Administration includes self-administration and the administration by another.

[0037] Throughout the present specification, the terms “about” and / or “approximately” maybe used in conjunction with numerical values and / or ranges. The term “about” is understood to mean those values near to a recited value, as well as the recited value. Throughout the present specification, numerical ranges are provided for certain quantities. It is to be understood that these ranges comprise all values and subranges therein. Thus, the range '‘from 50 to 80" includes all possible values therein (e.g., 50, 51, 52, 53, 54, 55, 56, etc.) and all possible ranges therein (e.g., 51-79, 52-78, 53-77, 54-76, 55-75, 60-70, etc.). Furthermore, all values within a given range may be an endpoint for the range encompassed thereby (e.g., the range 50-80 includes the ranges with endpoints such as 55- 80, 50-75. etc.).

[0038] The term "a" or “an’’ refers to one or more of that entity; for example, “a polypeptide conjugate” refers to one or more polypeptide conjugates or at least one polypeptide conjugate. As such, the terms “a” (or “an”), “one or more” and “at least one” are used interchangeably herein. In addition, reference to “a polypeptide conjugate” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the polypeptide conjugates is present, unless the context clearly requires that there is one and only one of the polypeptide conjugates.

[0039] As used herein, the term “adjacent” refers to two contiguous amino acids, which are connected by a covalent bond. “Adjacent” is also used interchangeably with “consecutive.”

[0040] The term “carrier” means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0041] As used herein, “therapeutically effective” refers to an amount of the polypeptide conjugate or the complex thereof of the present disclosure that can deliver an amount of a therapeutic nucleic acid which confers a therapeutic effect on a patient.

[0042] As used herein, “cell penetrating peptide” or “CPP” refers to any peptide which is capable of penetrating a cell membrane. As used herein, “cyclic cell penetrating peptide” or “cCPP” refers to any cyclic peptide which is capable of penetrating a cell membrane. Unless stated otherwise, CPP includes cCPP.

[0043] As used herein, “linker” or “L” refers to a moiety that covalently attaches two or more components of the polypeptide conjugates disclosed herein (e.g., a linker may covalently attach a CPP and a group that binds to a nucleic acid sequence by electrostatic interactions (i.e.. P). In some embodiments, the linker can be natural or non-natural amino acid or polypeptide. In other embodiments, the linker is a synthetic compound containing two or more appropriate functional groups suitable to bind, e.g., the CPP and, independently, P. In some embodiments, the linker is about 3 to about 100 (e.g., about 3 to about 20) atoms in linear length (not counting the branched atoms or substituents). In some embodiments, the linker provides about 1 A to about 400 A in distance of the two groups to which it connects.

[0044] As used herein, “polypeptide’’ refers to a string of at least two amino acids attached to one another by a peptide bond. There is no upper limit to the number of amino acids that can be included in a polypeptide. Further, polypeptides may include non-natural amino acids, amino acid analogs, or other synthetic molecules that are capable of integrating into a polypeptide.

[0045] As used herein, a “monomer” refers to an amino acid residue in a polypeptide. In some embodiments, an amino acid monomer is divalent. In other embodiments, an amino acid monomer may be trivalent if the monomer is further substituted. For example, a cysteine monomer can independently form peptide bonds at the N and C termini, and also form a disulfide bond.

[0046] As used herein, an “amino acid-analog” or “analog” (e.g., “arginine-analog”, “lysine- analog” or “histidine-analog”) refers to a variant of an amino acid that retains at least one function of the amino acid, such as the ability to bind an oligonucleotide through electrostatic interactions. Such variants may have an elongated or shorter side chain (e.g., by one or more -CH2- groups that retains the ability to bind an oligonucleotide through electrostatic interactions, or alternatively, the modification can improve the ability to bind an oligonucleotide through electrostatic interactions. For example, an arginine analog may include an additional methylene or ethylene between the backbone and guanidine / guanidinium group. Other examples include amino acids with one or more additional substituents (e.g., Me, Et, halogen, thiol, methoxy, ethoxy, Cl-haloalkyl, C2- haloalkyl, amine, guanidine, etc). The amino acid-analog can be monovalent, divalent, or trivalent.

[0047] Throughout the present specification, peptides and amino acid monomers are depicted as charge neutral species. It is to be understood that such species may bear a positive or negative charge depending on the conditions. For example, at pH 7, the N-terminus of an amino acid is protonated and bears a positive charge (-NH3 ). and the C-terminus of an amino acid is deprotonated and bears a negative charge (-CO2 ). Similarly, the side chains of certain amino acids may bear a positive or negative charge.

[0048] Each amino acid can be a natural or non-natural amino acid. The term “non-natural amino acid” refers to an organic compound that is a congener of a natural amino acid in that it has a structure similar to a natural amino acid so that it mimics the structure and reactivity of a natural amino acid. The non-natural amino acid can be a modified amino acid, and / or amino acid analog, that is not one of the 20 common naturally occurring amino acids or the rare natural amino acids selenocysteine or pyrrolysine. Non-natural amino acids can also be the D-isomer of the natural amino acids. Thus, as used herein, the term “amino acid” refers to natural and non-natural amino acids, and analogs and derivatives thereof. Examples of suitable amino acids include, but are not limited to, alanine, allosoleucine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, a derivative, or combinations thereof. Analogs of amino acids encompass that have a structural similar but not identical to an amino acid, e.g.. due to a modification to the side chain or backbone on said amino acid. Such modifications may increase the hydrophobicity of the side chain, including elongation of the side chain by one or more hydrocarbons, or increasing the the solvent accessible surface area (SASA as described herein) of an amino acid having an aromatic ring on its side chain, e.g., by conjugating a second aromatic ring or increasing the size of the aromatic ring. Derivatives of amino acids encompass natural and non-natural amino acids that have been modified (e.g., by susbstitution) to include a hydrophobic group as described herein. For example, a derivative of lysine includes lysine whose side chain has been substituted with alkylcarboxamidyl. These, and others, are listed in the Table 1 along with their abbreviations used herein.

[0049] Table 1. Amino Acid Abbreviations

[0050] * single letter abbreviations: when shown in capital letters herein it indicates the L-amino acid form, when shown in lower case herein it indicates the D-amino acid form. “Alkyl” or “alkyl group” refers to a fully saturated, straight or branched hydrocarbon chain radical having from one to twelve carbon atoms, and which is attached to the rest of the molecule by a single bond. Alkyls comprising any number of carbon atoms from 1 to 12 are included. An alkyl comprising up to 12 carbon atoms is a C1-C12 alkyd, an alkyl comprising up to 10 carbon atoms is a C1-C10 alkyl, an alkyl comprising up to 6 carbon atoms is a Ci-Cs alkyl and an alkyl comprising up to 5 carbon atoms is a C1-C5 alkyl. A C1-C5 alkyl includes Cs alkyls. C4 alkyls, C3 alkyls, C2 alkyls and Ci alkyl (z.e., methyl). A Ci-Cs alkyl includes all moieties described above for C1-C5 alkyls but also includes Cs alkyls. A C1-C10 alkyl includes all moieties described above for C1-C5 alkyls and Ci-Ce alkyls, but also includes C7, Cs, C9 and C10 alkyls. Similarly, a C1-C12 alkyl includes all the foregoing moieties. but also includes C11 and C12 alkyls. Non-limiting examples of C1-C12 alkyl include methyl, ethyl, n- propyl, z-propyl, sec-propyl, n-butyl, z-butyl, sec-butyl, / -butyl. n-pentyl, t-amyl, w-hexyl, n- heptyl, w-octyl, n-nonyl, w-decyl, w-undecyl, and w-dodecyl. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted.

[0051] “Alkylene” or “alkylene chain” refers to a fully saturated, straight or branched divalent hydrocarbon chain radical, having from one to forty carbon atoms. Non-limiting examples of C2-C40 alkylene include ethylene, propylene, n-butylene, pentylene, and the like. Unless stated otherwise specifically in the specification, an alky lene chain can be optionally substituted as described herein.

[0052] “Alkenyl” or “alkenyl group” refers to a straight or branched hydrocarbon chain radical having from two to twelve carbon atoms, and having one or more carbon-carbon double bonds. Each alkenyl group is attached to the rest of the molecule by a single bond. Alkenyl group comprising any number of carbon atoms from 2 to 12 are included. An alkenyl group comprising up to 12 carbon atoms is a C2-C12 alkenyl, an alkenyl comprising up to 10 carbon atoms is a C2-C10 alkenyl, an alkenyl group comprising up to 6 carbon atoms is a C2- Cs alkenyl and an alkenyl comprising up to 5 carbon atoms is a C2-C5 alkenyl. A C2-C5 alkenyl includes C5 alkenyls, C4 alkenyls, C3 alkenyls, and C2 alkenyls. A C2-C6 alkenyl includes all moieties described above for C2-C5 alkenyls but also includes Cs alkenyls. A C2-C10 alkenyl includes all moieties described above for C2-C5 alkenyls and C2-C6 alkenyls, but also includes C7, Cs, C9 and C10 alkenyls. Similarly, a C2-C12 alkenyl includes all the foregoing moieties, but also includes C11 and C12 alkenyls. Non-limiting examples of C2-C12 alkenyl include ethenyl (vinyl), 1 -propenyl, 2-propenyl (allyl), iso-propenyl, 2-methy 1-1 -propenyl, 1-butenyl,

[0053] 2-butenyl. 3-butenyl, 1 -pentenyl. 2-pentenyl, 3-pentenyl, 4-pentenyl, 1 -hexenyl, 2-hexenyl,

[0054] 3-hexenyL 4-hexenyl, 5-hexenyl, 1 -heptenyl, 2 -heptenyl, 3-heptenyL 4-heptenyL 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1- nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl. 8-nonenyl. 1- decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9- decenyl, 1 -undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7- undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1 -dodecenyl, 2-dodecenyl, 3- dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl. and 11 -dodecenyl. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted.

[0055] “Alkenylene” or “alkenylene chain” refers to a straight or branched divalent hydrocarbon chain radical, having from two to forty carbon atoms, and having one or more carbon-carbon double bonds. Non-limiting examples of C2-C40 alkenylene include ethenylene (-CH=CH-), propenylene, butenylene, and the like. Unless stated otherwise specifically in the specification, an alkenylene chain can be optionally substituted.

[0056] “Alkynyl” or “alkynyl group” refers to a straight or branched hydrocarbon chain radical having from two to twelve carbon atoms and having one or more carbon-carbon triple bonds. Each alkynyl group is attached to the rest of the molecule by a single bond. Alkynyl group comprising any number of carbon atoms from 2 to 12 are included. An alkynyl group comprising up to 12 carbon atoms is a C2-C12 alkynyl, an alkynyl comprising up to 10 carbon atoms is a C2-C10 alkynyl, an alkynyl group comprising up to 6 carbon atoms is a C2-C6 alkynyl and an alkynyl comprising up to 5 carbon atoms is a C2-C5 alkynyl. A C2-C5 alkynyl includes C5 alkynyls, C4 alkynyls, C3 alkynyls, and C2 alkynyls. A C2-C6 alkynyl includes all moieties described above for C2-C5 alkynyls but also includes Ce alkynyls. A C2-C10 alkynyl includes all moieties described above for C2-C5 alky nyls and C2-C6 alkynyls, but also includes C7, Cs, C9 and C10 alkynyls. Similarly, a C2-C12 alkynyl includes all the foregoing moieties, but also includes C11 and C12 alkynyls. Non-limiting examples of C2-C12 alkenyl include ethynyl, propynyl, butynyl, pentynyl and the like. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted.

[0057] “Alkynylene” or “alkynylene chain” refers to a straight or branched divalent hydrocarbon chain radical, having from two to forty carbon atoms, and having one or more carbon-carbon triple bonds. Non-limiting examples of C2-C40 alkynylene include ethynylene (-C=C-), propargylene and the like. Unless stated otherwise specifically in the specification, an alkynylene chain can be optionally substituted.

[0058] “Aryl” refers to a hydrocarbon ring system comprising hydrogen, 6 to 40 carbon atoms and at least one aromatic ring. For purposes of this disclosure, the aryl can be a monovalent or a divalent radical (not counting substituents), which can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, and which can include fused or bridged ring systems. Aryl radicals include, but are not limited to, radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as- indacene, s-indacenc. indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. In some embodiments, the aryl radical can be divalent when used as a linker or as a part of a linker. Unless stated otherwise specifically in the specification, an aryl group can be optionally substituted.

[0059] As used herein “aromatic” refers to an unsaturated cyclic molecule having 4n + 2 a electrons, wherein n is any integer. The term “non-aromatic” refers to any unsaturated cyclic molecule which does not fall within the definition of aromatic.

[0060] “Carbocyclyl,” “carbocyclic ring” or “carbocycle” refers to a rings structure, wherein the atoms which form the ring are each carbon. Carbocyclic rings can comprise from 3 to 20 carbon atoms in the ring. Carbocyclic rings include aryls and cycloalkyd and rings that are fully unsaturated, partially unsaturated, and fully saturated. In some embodiments, the carbocyclyl can be divalent when used as a linker or as a part of a linker. Unless stated otherwise specifically in the specification, a carbocyclyl group can be optionally substituted.

[0061] “Cycloalky 1” refers to a stable non-aromatic monocyclic or polycyclic fully saturated hydrocarbon radical having from 3 to 40 carbon atoms and at least one ring, wherein the ring consists solely of carbon and hydrogen atoms, which can include fused or bridged ring systems. For purposes of this disclosure, the cycloalkyl can be a monovalent or a divalent radical (not counting substituents). Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl radicals include, for example, adamantyl, norbomyl, decalinyl, 7,7-dimethyl-bicyclo[2.2. l]heptanyl, and the like. In some embodiments, the cycloalkyl radical can be divalent when used as a linker or as a part of a linker. Unless otherwise stated specifically in the specification, a cycloalkyd group can be optionally substituted.

[0062] “Cycloalkenyl” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical having from 3 to 40 carbon atoms, at least one ring having, and one or more carboncarbon double bonds, wherein the ring consists solely of carbon and hydrogen atoms, which can include fused or bridged ring systems. For purposes of this invention, the cycloalkenyl can be a monovalent or a divalent radical (not counting substituents). Monocyclic cycloalkenyl radicals include, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl, cycloctenyl, and the like. Polycyclic cycloalkenyl radicals include, for example, bicyclo[2.2. l]hept-2-enyl and the like. In some embodiments, the cycloalkenyl radical can be divalent when used as a linker or as a part of a linker. Unless otherwise stated specifically in the specification, a cycloalkenyl group can be optionally substituted.

[0063] “Cycloalkynyl” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical having from 3 to 40 carbon atoms, at least one ring, and one or more carbon-carbon triple bonds, wherein the ring consists solely of carbon and hydrogen atoms, which can include fused or bridged ring systems. For purposes of this invention, the cycloalkynyl can be a monovalent or a divalent radical (not counting substituents). Monocyclic cycloalkynyl radicals include, for example, cycloheptynyl, cyclooctynyl, and the like. In some embodiments, the cycloalky nyl radical can be divalent when used as a linker or as a part of a linker. Unless otherwise stated specifically in the specification, a cycloalkynyl group can be optionally substituted.

[0064] ‘'Heterocyclyl,” ‘'heterocyclic ring” or “heterocycle” refers to a stable 3- to 20-membered aromatic ring radical which consists of tw o to twelve carbon atoms and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. For purposes of this invention, the heterocyclyl radical can be a monovalent or a divalent radical (not counting substituents). Heterocyclycl or heterocyclic rings include heteroaryls as defined below. Unless stated otherwise specifically in the specification, the heterocyclyl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical can be optionally oxidized; the nitrogen atom can be optionally quatemized; and the heterocyclyl radical can be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl. isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyL 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl. and 1,1-dioxo-thiomorpholinyl. In some embodiments, the heterocyclyl radical can be divalent when used as a linker or as a part of a linker. Unless stated otherwise specifically in the specification, a heterocyclyl group can be optionally substituted.

[0065] “Heteroaryl” refers to a 5- to 20-membered ring system radical comprising hydrogen atoms, one to fourteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one aromatic ring. For purposes of this invention, the heteroaryl radical can be a monovalent or a divalent radical (not counting substituents) and can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl radical can be optionally oxidized; the nitrogen atom can be optionally quatemized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo |h|| 1.4] di oxepinyl, 1.4-benzodioxanyL benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[l,2-a]pyridinyl, carbazolyl. cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1- oxidopyrazinyl, 1 -oxidopyridazinyl, 1 -phenyl- UZ-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl. phthalazinyl, pteridinyl, purinyl. pyrrolyl, pyrazolyl. pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolmyl, quinoxalinyl, quinolmyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e. thienyl). In some embodiments, the heteroaryl radical can be divalent when used as a linker or as a part of a linker. Unless stated otherwise specifically in the specification, a heteroaryl group can be optionally substituted.

[0066] The term ‘’ether” used herein refers to a straight or branched divalent radical moiety - [(CH2)m-O-(CH2)n]z- wherein each of m, n, and z are independently selected from 1 to 40. Examples include, but are not limited to, polyethylene glycol. Unless stated otherwise specifically in the specification, the ether can be optionally substituted.

[0067] The term '‘substituted” used herein means any of the above groups (i.e., alkylene, alkenylene, alkynylene, aryl, carbocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, and / or ether) wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atom in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines. N-oxides, imides, and enamines; a silicon atom in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups. “Substituted” also means any of the above groups in which one or more hydrogen atoms are replaced by a higher-order bond (e.g., a double- or triple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, “substituted” includes any of the above groups in which one or more hydrogen atoms are replaced with -NRgRh, -NRgC(=O)Rh, -NRgC(=O)NRgRh, -NRgC(=O)ORh, -NRgSChRh, -OC(=O)NRgRh. -ORg, -SRg, -SORg. -SChRg, -OSO2Rg, -SChORg, =NSO2Rg, and -SOzNRgRh. “Substituted also means any of the above groups in which one or more hydrogen atoms are replaced with -C(=O)Rg, -C(=O)ORg, -C(=O)NRgRh, -CH2SO2Rg, -CFbSChNRgRh. In the foregoing, Rgand Rh are the same or different and independently hydrogen, alkyl, alkenyl, alkynyl. alkoxy, alkylamino, thioalkyd, aryl, aralkyl, cycloalkyl. cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalky nyl. heterocyclyl, V-heterocyclyl, heterocyclylalkyl, heteroaryl, A-heleroaryl and / or heteroarylalkyl. “Substituted” further means any of the above groups in which one or more hydrogen atoms are replaced by a bond to an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkydalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, A-heterocyclyl, heterocyclylalkyl, heteroaryl, .A-hctcroary l and / or heteroarylalkyl group. In addition, each of the foregoing substituents can also be optionally substituted with one or more of the above substituents. Further, those skilled in the art will recognize that “substituted” also encompasses instances in which one or more atoms on any of the above groups are replaced by a substituent listed in this paragraph, and the substituent forms a covalent bond with the CPP, P, or L. For example, in certain embodiments, any of the above groups can be substituted at a first position with a carboxylic acid (i.e. , -C(=O)OH) which forms an amide bond with a lysine in the CPP, or a group can be substituted at a second position with a thiol group which forms a disulfide bond with a cysteine (or amino acid analog having a thiol group).

[0068] As used herein, the term "subject" refers to the target of administration, e.g. a subject. Thus, the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, fish, bird, rodent, or fruit fly. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In some examples, the subject is a mammal. A patient refers to a subject afflicted with a disease or disorder. The term "patient" includes human and veterinary subjects. In some examples of the disclosed methods, the subject has been diagnosed with a need for treatment of cancer, autoimmune disease, and / or inflammation prior to the administering step. In some examples of the disclosed method, the subject has been diagnosed with cancer prior to the administering step. The term subject also includes a cell, such as an animal, for example human, cell.

[0069] As used herein, the term ‘"treat;’ ‘"treating,” “treatment” and variants thereof, refers to the medical management of a patient with the intent to cure, ameliorate, or stabilize a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In some examples, the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease, i.e., arresting its development; or (iii) relieving the disease, i.e., causing regression of the disease.

[0070] As used herein, the term "prevent" or "preventing" refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action.

[0071] As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In some examples, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In some examples, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

[0072] The term "contacting" as used herein refers to bringing a disclosed compound and a target (e.g., a cell, target receptor, transcription factor, or other biological entity) together in such a manner that the compound can affect the activity of the target either directly, i.e., by interacting with the target itself, or indirectly, i.e., by interacting with another molecule, cofactor, factor, or protein on which the activity of the target is dependent.

[0073] As used herein, the terms "effective amount" and "amount effective" refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a "therapeutically effective amount" refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age. body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In some examples, a preparation can be administered in a "prophylactically effective amount"; that is. an amount effective for prevention of a disease or condition.

[0074] The term "pharmaceutically acceptable" describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.

[0075] As used herein, the term "pharmaceutically acceptable carrier" refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose.

[0076] A residue of a chemical species, as used in the specification and concluding claims, refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained from the chemical species. Thus, an amino acid residue in a peptide or protein refers to one or more -OC(O)CH(R)NH- units in the peptide or protein.

[0077] As used herein, the symbol “ ” (hereinafter can be referred to as ”a point of attachment bond”) denotes a bond that is a point of attachment between two chemical entities, one of which is depicted as being attached to the point of attachment bond and the other of which is not depicted as being attached to the point of attachment bond. For example, “ XY-|-

[0078] ” indicates that the chemical entity “XY’‘ is bonded to another chemical entity via the point of attachment bond. Furthermore, the specific point of attachment to the non-depicted chemical entity can be specified by inference. For example, the compound CH3-R3, wherein

[0079] R is H or “XY+ ” infers that when R’ is “XY”, the point of attachment bond is the same bond as the bond by which R3is depicted as being bonded to CH3.

[0080] Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g. each enantiomer and diastereomer, and a mixture of isomers, such as a racemic or scalemic mixture. Compounds described herein can contain one or more asymmetric centers and, thus, potentially give rise to diastereomers and optical isomers. Unless stated to the contrary’, the compounds and compositions disclosed herein include all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof Mixtures of stereoisomers, as well as isolated specific stereoisomers, are also included. During the course of the synthetic procedures used to prepare such compounds, or in using racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers.

[0081] Many organic compounds exist in optically active forms having the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or meaning that the compound is levorotatory'. A compound prefixed with (+) or d is dextrorotatory’. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are non-superimposable mirror images of one another. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Many of the compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms. If desired, a chiral carbon can be designated with an asterisk (*). When bonds to the chiral carbon are depicted as straight lines in the disclosed formulas, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the formula. As is used in the art, when it is desired to specify the absolute configuration about a chiral carbon, one of the bonds to the chiral carbon can be depicted as a wedge (bonds to atoms above the plane) and the other can be depicted as a series or wedge of short parallel lines is (bonds to atoms below the plane). The Cahn-Inglod-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.

[0082] Compounds described herein comprise atoms in both their natural isotopic abundance and in non-natural abundance. The disclosed compounds can be isotopically-labeled or isotopically -substituted compounds identical to those described, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. Examples of isotopes that can be incorporated into compounds disclosed herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as2H,3H,13C,14C,15N,18O,17O,33S,18F and36C1 respectively. Compounds further comprise prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labeled compounds, for example those into which radioactive isotopes such as3H and14C are incorporated, are useful in drug and / or substrate tissue distribution assays. Tritiated, i.e.,3H, and carbon-14, i.e.,14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e..2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labeled compounds and prodrugs thereof can generally be prepared by carrying out the procedures below; by substituting a readily available isotopically labeled reagent for a non- isotopically labeled reagent.

[0083] Disclosed are the components to be used to prepare the compositions disclosed herein as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B. and C are disclosed as well as a class of molecules D. E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B- F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions disclosed herein. Thus, if there are a variety' of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods disclosed herein.

[0084] Polypeptide or polynucleotide molecules of the present disclosure may share a certain degree of sequence similarity' or identity' with the reference molecules (e.g., reference polypeptides or reference polynucleotides), for example, with art-described molecules (e.g., engineered or designed molecules or wild-type molecules). The term ‘"identity;" as known in the art, refers to a relationship between the sequences of two or more polypeptides or polynucleotides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between two sequences as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms’’). Identity' of related peptides can be readily calculated by know n methods. “% identity” as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. Identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. Generally, variants of a particular polynucleotide or polypeptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity' to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al. (1997).” Gapped BLAST and PSI-BLAST: anew generation of protein database search programs,” Nucleic Acids Res. 25:3389-3402). Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981) “Identification of common molecular subsequences.” J Mol. Biol. 147 : 195-197). A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S. B. & Wunsch, C. D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443-453). More recently, a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) was developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm. Other tools are described herein, specifically in the definition of “identity” below.

[0085] As used herein, the term "homology" refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Polymeric molecules (e.g. nucleic acid molecules (e.g. DNA molecules and / or RNA molecules) and / or polypeptide molecules) that share a threshold level of similarity or identity determined by alignment of matching residues are termed homologous. Homology' is a qualitative term that describes a relationship between molecules and can be based upon the quantitative similarity or identity. Similarity or identity is a quantitative term that defines the degree of sequence match between two compared sequences. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered homologous if the polypeptides they7encode are at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology7is determined by the ability' to encode a stretch of at least 4-5 uniquely specified amino acids. Two protein sequences are considered homologous if the proteins are at least 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least 20 amino acids. Homology implies that the compared sequences diverged in evolution from a common origin. The term "homolog" refers to a first amino acid sequence or nucleic acid sequence (e.g., gene (DNA or RNA) or protein sequence) that is related to a second amino acid sequence or nucleic acid sequence by descent from a common ancestral sequence. The term “homolog” may apply to the relationship between genes and / or proteins separated by the event of speciation or to the relationship between genes and / or proteins separated by the event of genetic duplication. “Orthologs” are genes (or proteins) in different species that evolved from a common ancestral gene (or protein) by speciation. Typically, orthologs retain the same function in the course of evolution. “Paralogs” are genes (or proteins) related by duplication within a genome. Orthologs retain the same function in the course of evolution, whereas paralogs evolve new functions, even if these are related to the original one.

[0086] The term “identity7” refers to the overall relatedness between polymeric molecules, for example, between polynucleotide molecules (e.g. DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of the percent identity of two polynucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity7between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleic acid sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology , von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin. A. M., and Griffin. H. G., eds.. Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds.. M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty7of 12 and a gap penalty7of 4. The percent identity between two nucleic acid sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary- computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al.. Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

[0087] Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures.

[0088] Compositions

[0089] Disclosed are peptides and compositions comprising them, which can provide a general vehicle for cytosolic delivery of peptides including: a gene editing polypeptide having at least 90% sequence similarity to SEQ ID Nos: 141, 148-151, or 236-251 linked to a membrane translocation domain having one or more cell penetrating peptide motifs, where at least one cell penetrating peptide motif is from 3 to 10 amino acid residues in length and has at least three arginine and / or lysine residues; or where at least one cell penetrating peptide motif is from 3 to 10 amino acid residues in length and has at least two arginine and / or lysine residues and at least one other cell penetrating peptide motif is from 2 to 8 amino acid residues in length and has at least two hydrophobic residues.

[0090] Nuclear localization sequence (NLS)

[0091] In some embodiments, the peptide can further include a nuclear localization sequence (NLS). The nuclear localization sequence can be linked to a carboxylate group (e.g., C- terminus) in the membrane translocation domain and an amino group (e.g., N-terminus) of a gene editing polypeptide, a carboxylate group (e.g., C-terminus) of a gene editing polypeptide, or any combination thereof. In some embodiments, the nuclear localization sequence can be linked to a carboxylate group (e.g., C-terminus) of a gene editing polypeptide. In some embodiments, the nuclear localization sequence can be linked to a carboxylate group (e.g., C-terminus) in the membrane translocation domain and an amino group (e.g., N-terminus) of a gene editing polypeptide. In some embodiments, the nuclear localization sequence can be linked to a carboxylate group (e.g., C-terminus) in the membrane translocation domain and an amino group (e.g., N-terminus) and a carboxylate group (e.g., C-terminus) of a gene editing polypeptide.

[0092] In some embodiments, the nuclear localization sequence comprises KK, KR, RR, HH, HK, HR, RH, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, RHR, RBR, KKH, KHK, HKK, HRR, HRH, HHR, HBH, HHH, HHHH (SEQ ID NO: 163), KHKK (SEQ ID NO: 164), KKHK (SEQ ID NO: 165), KKKH (SEQ ID NO: 166), KHKH (SEQ ID NO: 167), HKHK (SEQ ID NO: 168), KKKK (SEQ ID NO: 169). KKRK (SEQ ID NO: 170), KRKK (SEQ ID NO: 171), KRRK (SEQ ID NO: 172), RKKR (SEQ ID NO: 173), RRRR (SEQ ID NO: 174), KGKK (SEQ ID NO: 175), KKGK (SEQ ID NO: 176), HBHBH (SEQ ID NO: 177), HBKBH (SEQ ID NO: 178), HBRBH (SEQ ID NO: 179), RRRRR (SEQ ID NO: 180), RBRBR (SEQ ID NO: 181), RBHBR (SEQ ID NO: 182), KKKKK (SEQ ID NO: 183), KKKRK (SEQ ID NO: 184), RKKKK (SEQ ID NO: 185), KRKKK (SEQ ID NO: 186), KKRKK (SEQ ID NO: 187), KKKKR (SEQ ID NO: 188), KBKBK (SEQ ID NO: 189), RKKKKG (SEQ ID NO: 190), KRKKKG (SEQ ID NO: 191), KKRKKG (SEQ ID NO: 192), KKKKRG (SEQ ID NO: 193), RKKKKB (SEQ ID NO: 194), KRKKKB (SEQ ID NO: 195), KKRKKB (SEQ ID NO: 196), KKKKRB (SEQ ID NO: 197), KKKRKV (SEQ ID NO: 198), RRRRRR (SEQ ID NO: 199), HHHHHH (SEQ ID NO: 200), RHRHRH (SEQ ID NO: 201), HRHRHR (SEQ ID NO: 202) KRKRKR (SEQ ID NO: 203), RKRKRK (SEQ ID NO: 204), RBRBRB (SEQ ID NO: 205), KBKBKB (SEQ ID NO: 206), PKKKRKV (SEQ ID NO: 207), PGKKRKV (SEQ ID NO: 208), PKGKRKV (SEQ ID NO: 209), PKKGRKV (SEQ ID NO: 210), PKKKGKV (SEQ ID NO: 211), PKKKRGV (SEQ ID NO: 212) or PKKKRKG (SEQ ID NO: 213), wherein B is beta-alanine. In some embodiments, the nuclear localization sequence comprises PKKKRKVP KKKRKV (SEQ ID NO: 152)

[0093] Membrane Translocation Domain

[0094] The membrane translocation domain portion of the disclosed peptides can be any membrane translocation domain, a peptide sequence that may traverse a lipid bilayer, that has been modified to contain at least one cell penetrating motifs as described herein. Suitable membrane translocation domains include, but are not limited to, membrane translocation domains described in PCT Application No. PCT / US2023 / 064659 which is incorporated herein by reference. Suitable membrane translocation domains may be prepared by the methods described in PCT Application No. PCT / US2023 / 064659 which is incorporated herein by reference. In a preferred example, there are two or three cell penetrating motifs in the membrane translocation domains. For example, at least one cell penetrating peptide motif can be from 3 to 10 amino acid residues in length and have at least three arginine and / or lysine residues, e.g., 4, 5, or 6 arginines and / or lysine residues. Alternatively, at least one cell penetrating peptide motif can be from 3 to 10 amino acid residues in length and have at least two arginine and / or lysine residues and at least one other cell penetrating peptide motif can be from 2 to 8 amino acid residues in length and have at least two hydrophobic residues. When there are two or more cell penetrating peptide motifs, there can be two or more arginine residues and / or lysine residues in a 3 to 10 amino acid span and another cell penetrating peptide motif where there are two or more hydrophobic residues within a 2 to 8 amino acid span. The cell penetrating peptide motifs can be anywhere in the membrane translocation domain.

[0095] In some examples, the membrane translocation domain can be a human membrane translocation domain, such as fibronectin t pe III. In a specific example, the membrane translocation domain has at least 90%, at least 95%, or at least 97% sequence similarity with SEQ. ID. NO.: 118. In other examples, the membrane translocation domain is human fibronectin ty pe III having BC, DE, CD, and FG loops and the cell penetrating peptide motif is in one or more of the BC, DE, CD, or FG loops, e.g., the cell penetrating peptide motif is in two of the BC, DE, CD, or FG loops, in particular the BC and FG loops. These loops can be defined as having the following sequences BC = AVTVR (SEQ ID NO.:31); CD = GGNSPVQ (SEQ ID NO : 32); DE = PGSK (SEQ ID NO : 33); FG = GRGDSPAS (SEQ ID NO.:34).

[0096] In other examples, the membrane translocation domain can be any stably folded protein, which can preferably be efficiently expressed in bacteria. Some additional examples of membrane translocation domains are the nanobody scaffold, DARPin scaffold, and CTPR protein (the consensus tetratricopeptide repeat; Acc. Chem. Res. 2021, 54, 4166-4177).

[0097] Cell Penetrating Peptide Motif

[0098] The cell penetrating peptide (CPP) motif can comprises at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, or at least 6 amino acids, more specifically from 3 to 8, from 3 to 6, from 4 to 8, from 4 to 6, or from 6 to 8 amino acids. In most examples, the CPP motif is substituted into the membrane translocation domain such that the resulting peptide has the same number of amino acids as in native membrane translocation domain. In some examples, at least two, three, four, five, six, or seven amino acids of the CPP motif are adjacent arginine residues. In a preferred, example there are three, four, or five adjacent arginine residues in a CPP motif. In other examples, the arginie residues are not adjacent in the CPP motif. Each amino acid in the CPP motif can independently be a natural or non-natural amino acid. When such adjacent arginine or lysine residues are the CPP motif, then there need not be any additional CPP motifs, e g., those with hydrophobic residues, though such a hydrophobic CPP motif can still be used. When the CPP motif contains two argine residues, then it is preferred that there be another CPP motif with at least two hydrophobic residues within 2 to 8 amino acids.

[0099] In other examples, at least one, at least, two, at least three, or more amino acids of the CPP motif are hydrophobic amino acids, i.e., have hydrophobic side chains. In some examples, the amino acids having hydrophobic side chains are independently selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, naphthylalanine, phenylglycine, homophenylalanine, tyrosine, cy clohexylalanine, piperidine-

[0100] 2-carboxylic acid, cyclohexylalanine, norleucine. 3-(3-benzothienyl)-alanine, 3-(2-quinolyl)- alanine, O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine, S-(4-methylbenzyl)cysteine, N- (naphthalen-2-yl)glutamine, 3-(l,l'-biphenyl-4-yl)-alanine, tert-leucine, or nicotinoyl lysine, each of which is optionally substituted with one or more substituents. In particular examples, each amino acid having a hydrophobic side chain is independently an amino acid having an aromatic side chain. In some embodiments, the amino acid having an aromatic side chain is

[0101] 3-benzothienyl-L-alanine, naphthylalanine, phenylglycine, homophenylalanine, phenylalanine, try ptophan, or tyrosine, each of which is optionally substituted with one or more substituents. Thus, in some examples, the amino acids having hydrophobic side chains are phenylalanine, naphthylalanine, tryptophan, or an analog or derivative thereof naphthylalanine or tryptophan, or analogues or derivatives thereof. In other examples, the CPP motif further comprises at least one phenylalanine, phenylglycine, or histidine, or analogues or derivatives thereof.

[0102] 3-(2-quinolyl)-alanine O-benzylserine 3-(4-(benzyloxy)phenyl)-alanine

[0103] S-(4-methylbenzyl)cysteine A / 5-(naphthalen-2-yl)glutamine 3-(1 ,1'-biphenyl-4-yl)-alanine

[0104] 3-(3-benzothienyl)-alanine

[0105] In some examples, the CPP motif can include any combination of at least three adjacent arginines and either at least two amino acids have a hydrophobic side chain selected from an aryl or heteroaryl, wherein the aryl and heteroaryl are optionally substituted, with a total number of amino acids in the CPP motif in the range of from 5 to about 8 amino acids.

[0106] In some examples, the membrane translocation domain is human fibronectin type III having BC, DE, CD, and FG loops and the CPP is in one or more of the BC, DE, CD, or FG loops. For example, the CPP motif is in two of the BC, DE, CD, or FG loops. In a specific example, the CPP motif is in the BC and either the DE, CD and FG loops, preferably in the BC and FG loops.

[0107] Where there are two or more CPP motifs, one CPP motif can be the 3 to 10 amino acid segment with at least two arginine and / or lysine residues and the other can be a 2 to 8 amino acid segment with at least two hydrophobic residues. For example, the membrane translocation domain can have two or more CPPs and at least one of the motifs is from 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues.

[0108] In an example of this, the membrane translocation domain can be human fibronectin type III having BC, DE, CD, and FG loops, and the CPP motifs can be in the BC loop and have from 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues and a CPP motif can be in the FG loop and have from 3 to 10 amino acid residues and has at least three adjacent arginine and / or lysine residues. Alternatively, the CPP motifs can be in the FG loop and have from 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues and a CPP motif can be in the BC loop and have from 3 to 10 amino acid residues and has at least three adjacent arginine and / or lysine residues.

[0109] When the CPP motif contains from 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues, it can be WW, FF, WF, FW. WWW, FFF, WFW, FWF, WWF, WFF, FWW, FFW, WYW, WWH, YWW, or WYH. It is preferable that this CPP motif be in the BC loop. It is further preferable that this CPP motif be WW, FW, WF, WYW, WWW, WWH, YWW, WYH or YWH.

[0110] The CPP motif with 3 to 10 amino acid residues and has at least three adj acent arginine and / or lysine residues can contain RRR, RRRR (SEQ ID NO: 132), RRRRR (SEQ ID NO: 133). It can also be any combination of arginine and lysine residues. When this CPP motif is in the FG loop it can be 3-10 residues in length and of any combinations of Arg and Lys (and occasionally other non-acidic residues). The CPP motif (e.g., WWWRRRR) (SEQ ID NO: 134) may be alternatively split, so that some of the Arg / Lys residues are moved from the FG loop into the BC loop (e.g., WWWR. . . RRR (SEQ ID NO: 272), WWWRR. .. RR (SEQ ID NO: 273), WWWRRRR. .. (SEQ ID NO: 134), etc ); The CPP motif (e g., WWWRRRR) (SEQ ID NO: 134) may be alternatively split, so that some of the hydrophobic residues are moved from the BC loop to the FG loop (e.g.. WW... WRRR. W... WWRRRR, ... WWWRRRR, etc.) (SEQ ID NOs: 274-278, 134, respectively). The CPP motif (e.g., WWWRRRR) (SEQ ID NO: 134) can be alternatively split, so that either BC or FG loop contains a combination of hydrophobic and positively charged residues (e.g., WWR... WRRR, WWRR... WRR, WWRR... RRW. RRW... WWRR, etc.) (SEQ ID NOs: 279-286, respectively).

[0111] In specific examples, the CPP motif comprises SEQ. ID. NOS.: 104, 105, 11, 112, 113, 114, 115, 116, or 117.

[0112] In some examples, the CPP motif can be or comprise any of the sequences listed in Table 2. In some examples, the cell penetrating peptide can be or comprise the reverse of any of the sequences listed in Table 2.

[0113] Table 2. CPP motif sequences

[0114] <t> = L-naphthylalanine; 4> = D-naphthylalanine; = L -norleucine; r = D-arginine; F = L- phenylalanine; f = D-phenylalanine; q = D-glutamine; X = L-4-fluorophenylalanine; Dap = L-2,3-diaminopropionic acid; Sar, sarcosine; F2Pmp, L-difluorophosphonomethyl phenylalanine; Dod, dodecanoyl; Pra, L-propargylglycine: AzK, Cit, L-citrulline, L-6- Azido-2-amino-hexanoic: Agp, L-2-amino-3-guanidinylpropionic acid; ^Cyclization between Pim and Nlys; “Cyclization between Lys and Glu; ‘'Macrocyclization by multicomponent reaction with aziridine aldehyde and isocyanide; “Cyclization between the main-chain of Gin residue; 'N-terminal amine and side chains of two Dap residues bicyclized with Tm; 'Three Cys side chains bicyclized with tris(bromomethyl)benzene; '’Cyclization by the click reaction between Pra and Azk.

[0115] The chirality of the amino acids can be selected to improve cytosolic uptake efficiency. In some embodiments, at least two of the amino acids have the opposite chirality. In some embodiments, the at least two amino acids having the opposite chirality can be adjacent to each other. In some embodiments, at least three amino acids have alternating stereochemistry relative to each other. In some embodiments, the at least three amino acids having the alternating chirality relative to each other can be adjacent to each other. In some embodiments, at least two of the amino acids have the same chirality. In some embodiments, the at least two amino acids having the same chirality can be adjacent to each other. In some embodiments, at least two amino acids have the same chirality and at least two amino acids have the opposite chirality. In some embodiments, the at least two amino acids having the opposite chirality can be adjacent to the at least two amino acids having the same chirality'. Accordingly, in some embodiments, adjacent amino acids in the cCPP can have any of the following sequences: D-L; L-D; D-L-L-D (SEQ ID NO: 126); L-D-D-L(SEQ ID NO: 127); L-D-L-L-D(SEQ ID NO: 128); D-L-D-D-L(SEQ ID NO: 129); D-L-L-D-L(SEQ ID NO: 130); or L-D-D-L-D (SEQ ID NO: 131). Gene-Editing polypeptide

[0116] Gene editing polypeptide can include, but is not limited to. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) systems including, but not limited to, those described in U.S. Patent Publication No. 2016 / 0298096, 2016 / 0281072, and 2019 / 0153476, and U.S. Patent Nos. 8,697,359, 8,771,945, and 8,945,839, such as Cas9 or variants thereof (e.g., SEQ ID NOs: 141), a dCas9 or variants thereof (e.g., SEQ ID NO: 148), Casl2 or variant thereof (e.g., SEQ ID NO: 150), Casl3 or variants thereof (e.g., SEQ ID NO: 151); a cpfl or variants thereof (e g., SEQ ID NO: 149), adenine base editor (ABE8e) (e.g., SEQ ID NO: 236) (see Gaudelli, N. M. et al. Nat Biotechnol 38, 892-900 (2020)), Cytosine base editor (BE4-Gam) (SEQ ID NO: 237) (see Komor, A. C. et al. Sci Adv 3. eaao4774 (2017)), base editor (both cytosine and adenine base editors) (e.g., SEQ ID NO: 238) (see Zhang, X. et al., Nat Biotechnol 38, 856-860 (2020)), prime editor (PEmax) (SEQ ID NO: 239) (see Chen, P. J. et al. Cell 184, 5635-5652.e5629 (2021)), all protein cytosine base editor (DdCBE, TALEs + CBE) (See Mok, B. Y. et al., Nat Biotechnol 40, 1378-1387 (2022)) (e g., ATP8-DdCBE right TALE repeat (SEQ ID NO: 240) or ATP8- DdCBE left TALE repeat (SEQ ID NO: 241)), Zinc finger nuclease (e.g., zinc finger nuclease left (SEQ ID NO: 242) or zing finger nuclease right (SEQ ID NO: 243)) (see Liu, J., et al., Mol Ther Nucleic Acids 4, e232 (2015)), transcription activator-like nucleases (TALENs) (e.g., SEQ ID NOs: 244-248), TALEN sequence for NTF3 gene (Neurotrophin 3) (e.g., SEQ ID NO: 249) (see Miller, J. C. et al. Nat Biotechnol 29, 143-148 (2011)), meganucleases (e g., M1PCSK9 (SEQ ID NO: 250) and M2PCSK9 (SEQ ID NO: 251)).

[0117] SEQ ID NO: 141 Amino acid sequence of Cas9 DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHER HPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRLIYL AL AHMIKFRGHFLIEGD LNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPG EKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSR FAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGF ANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVD ELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV

[0118] ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPT VAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLII KLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDN EQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENII HLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0119] SEQ ID NO: 148 Amino acid sequence of dCas9

[0120] MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHE RHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQY ADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLP EKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNS RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYE YFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKI ECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKV VDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEH

[0121] PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSE LDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFR KDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDS PTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKD

[0122] LIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE

[0123] DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAE

[0124] NIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG GD

[0125] SEQ ID NO: 149 Amino acid sequence of cpfl

[0126] AASKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLD

[0127] RYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGAA

[0128] GYKSLFKKDIIETILPEAADDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSI

[0129] AFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVL

[0130] TQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNAKTKQALPKFKPLYKQVLSDRES

[0131] LSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAIS

[0132] TISKDIFGEWNLIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQE

[0133] YADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKD

[0134] LLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKP

[0135] YSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKI

[0136] DKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKG

[0137] DMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKV

[0138] SFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQI

[0139] RLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSED

[0140] QYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNI

[0141] VEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVV

[0142] HKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSN

[0143] PCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSI

[0144] ADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFAAA

[0145] KKNNVFAWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSL

[0146] MLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIAR

[0147] KVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVK

[0148] SEQ ID NO: 150 Amino acid sequence of Casl3

[0149] MWISIKTLIHHLGVLFFCDYMYNRREKKIIEVKTMRITKVEVDRKKVLISRDKNGG

[0150] KLVYENEMQDNTEQIMHHKKSSFYKSVVNKTICRPEQKQMKKLVHGLLQENSQEK

[0151] IKVSDVTKLNISNFLNHRFKKSLYYFPENSPDKSEEYRIEINLSQLLEDSLKKQQGTFI CWESFSKDMELYINWAENYISSKTKLIKKSIRNNRIQSTESRSGQLMDRYMKDILNK NKPFDIQSVSEKYQLEKLTSALKATFKEAKKNDKEINYKLKSTLQNHERQIIEELKE NSELNQFNIEIRKHLETYFPIKKTNRKVGDIRNLEIGEIQKIVNHRLKNKIVQRILQEG KLASYEIESTVNSNSLQKIKIEEAFALKFINACLFASNNLRNMVYPVCKKDILMIGEF KNSFKEIKHKKFIRQWSQFFSQEITVDDIELASWGLRGAIAPIRNEIIHLKKHSWKKF FNNPTFKVKKSKIINGKTKDVTSEFLYKETLFKDYFYSELDSVPELIINKMESSKILD YYSSDQLNQVFTIPNFELSLLTSAVPFAPSFKRVYLKGFDYQNQDEAQPDYNLKLNI YNEKAFNSEAFQAQYSLFKMVYYQVFLPQFTTNNDLFKSSVDFILTLNKERKGYAK

[0152] AFQDIRKMNKDEKPSEYMSYIQSQLMLYQKKQEEKEKINHFEKFINQVFIKGFNSFI EKNRLTYICHPTKNTVPENDNIEIPFHTDMDDSNIAFWLMCKLLDAKQLSELRNEMI KFSCSLQSTEEISTFTKAREVIGLALLNGEKGCNDWKELFDDKEAWKKNMSLYVSE ELLQSLPYTQEDGQTPVINRSIDLVKKYGTETILEKLFSSSDDYKVSAKDIAKLHEYD VTEKIAQQESLHKQWIEKPGLARDSAWTKKYQNVINDISNYQWAKTKVELTQVRH LHQLTIDLLSRLAGYMSIADRDFQFSSNYILERENSEYRVTSWILLSENKNKNKYND YELYNLKNASIKVSSKNDPQLKVDLKQLRLTLEYLELFDNRLKEKRNNISHFNYLN GQLGNSILELFDDARDVLSYDRKLKNAVSKSLKEILSSHGMEVTFKPLYQTNHHLKI

[0153] DKLQPKKIHHLGEKSTVSSNQVSNEYCQLVRTLLTMK

[0154] SEQ ID NO: 151 Amino acid sequence of Casl2

[0155] MLPPLKPIAMKERISMLFIEYGEIDVLDGAFVVIDKTGVRTQIPVGSIACLMLEPGTR VSHRAAALAARVGTLLVWVGEAGVRLYASGQPGGARSDRLLYQAKLALDEKARL KVVRKMYAMRFDEEPPAKRSVDQLRGIEGARVRKTYKLLAQRHGVAWKGRSYDP SQWDKSDLPNLCLSAATACLYGITEAAVLAAGYAPAVGFIHTGKPLSFVYDIADIFK FDSVVPIAFQIAARAPAQPERQVRLACRDHFRQSKLLGKIIPAIEEILTAGELEMPKP APDTQPVAIPEEKGLGDAGHRH

[0156] SEQ ID NO: 236 Adenine base editor (ABE8e)

[0157] MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDP TAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDA KTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQS STDSGGS S GGS SGSETPGTSES ATPES SGGS S GGS SEVEFSHEYWMRHALTL AKRAR DEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYD ATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEI

[0158] TEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATP ESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKS RRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEEL LVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKV TVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILE DIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGS PAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREV KVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFV YGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR

[0159] KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKV LSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI HQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV

[0160] SEQ ID NO: 237 Cytosine base editor (BE4-Gam)

[0161] MAKPAKRIKSAAAAYVPQNRDAVITDIKRIGDLQREASRLETEMNDAIAEITEKFAA RIAPIKTDIETLSKGVQGWCEANRDELTNGGKVKTANLVTGDVSWRVRPPSVSIRG MDAVMETLERLGLQRFIRTKQEINKEAILLEPKAVAGVAGITVKSGIEDFSIIPFEQE AGISGSETPGTSESATPESSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEI

[0162] NWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSR AITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNF VNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCH YQRLPPHILWATGLKSGGSSGGSSGSETPGTSESATPESSGGSSGGSGKRNYILGLAI GITS VGYGIIDYETRD VID AGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRV KKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNE VEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKE AKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLM GHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQ KKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELL DQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDEL WHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIK KYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKI KLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENS KKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQK DFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKER NKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQE YKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLN GLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEET GNYLTKYSKKDNGPVIKKJKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDV YLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLI KINGEL YRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKY STDILGNLYEVKSKKHPQIIKKGGSPKKKRKVSSDYKDHDGDYKDHDIDYKDDDD KSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYD ESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKET GKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWA LVIQDSNGENKIKMLSGGSPKKKRKV

[0163] SEQ ID NO: 238 Base editor (both cytosine and adenine base editors)

[0164] MKRTADGSEFESPKKKRKVDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRR DSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCA RHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYC WNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPEAAAKEAAAKEAAAKSEVEF SHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAA GSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDSGG SSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVP VGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTF EPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILAD ECAALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSS GGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDS GETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDK KHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHF LIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLI AQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALV RQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLK EDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT

[0165] LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVRETNNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKV YDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIV WDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE TRIDLSQLGGDSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPES DILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGST

[0166] NLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTS DAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFEPKKKRKVGSGATNFSLLK QAGDVEENPGPSGGSPKKKRKV

[0167] SEO ID NO: 239 (Prime editor (PEmax) MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGN TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVD DSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA KAILSARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMI KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPI LEKMDGTEELLVKLKREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIER MTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRL SRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDS LHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQK NSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN RLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQL LNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTK YDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALI KKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKG NELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS

[0168] TKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVS GGSSGGSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTN DYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPT SQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILL QYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKE GQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPG TLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLG PWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAV EALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNC LDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAK ALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTS EGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITE TPDTSTLLIENSSPSGGSKRTADGSEFESPKKKRKVGSGPAAKRVKLD

[0169] All-protein cytosine base editor (DclCBE, TALEs + CBE)

[0170] SEQ ID NO: 240 ATP 8-DdCBE right TALE repeat

[0171] DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTV AVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLL KIAKRGGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNIGGKQALETVQRLLPV LCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGG KQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLT PQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQ RLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIAS NGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQA HGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQAL ETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHL

[0172] VALACLGGRPALDAVKKGLG

[0173] SEQ ID NO: 241 ATP8-DdCBE left TALE repeat

[0174] DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTV AVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLL KIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQALLPV LCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGG KQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTP EQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQAL LPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNI GGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG LTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETV QALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIA SHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQ

[0175] AHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGRPAL ESIVAQLSRPDPALAALTNDHLVALACLG GRP ALDA VKKGLG Zinc finger nuclease

[0176] SEQ ID NO: 242 Zinc finger nuclease Left

[0177] MGSSHHHHHHSSGLVPRGSHMPKKKRKVLEAAMAERPFQCRICMRNFSDRSNLSR HIRTHTGEKPFACDICGRKFAISSNLNSHTKIHTGSQKPFQCRICMRNFSRSDNLARH IRTHTGEKPFACDICGRKFATSGNLTRHTKIHLRGSQLVKSELEEKKSELRHKLKYV PHEYIELIEIARNPTQDRILEMKVMEFFMKVYGYRGEHLGGSRKPDGAIYTVGSPID YGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFK FLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKF NNGEINF

[0178] SEQ ID NO: 243 Zinc finger nuclease Right

[0179] MGSSHHHHHHSSGLVPRGSHMPKKKRKVLEAAMAERPFQCRICMRNFSRSDNLSV HIRTHTGEKPFACDICGRKFAQKINLQVHTKIHTGEKPFQCRICMRNFSRSDVLSEHI RTHTGEKPFACDICGRKFAQRNHRTTHTKIHLRGSQLVKSELEEKKSELRHKLKYV PHEYIELIEIARNPTQDRILEMKVMEFFMKVYGYRGEHLGGSRKPDGAIYTVGSPID YGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFK FLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKF NNGEINF

[0180] Transcription activator-like nucleases (TALENs)

[0181] SEQ ID NO: 249 TALEN sequence for NTF3 gene (Neurotrophin 3)

[0182] MAPKKKRKVGIHGVPAAVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFT

[0183] HAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTV

[0184] AGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIAS

[0185] NNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQA

[0186] HGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALE TVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVV AIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVL CQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGK QALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP EQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNKGGKQALETVQR LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAH

[0187] GLTPQQVVAIASNKGGRPALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALET VQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVA LACLGGGSGGSGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMK VMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQA DEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLN HITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS

[0188] Meganucleases

[0189] Amino acid sequences of M1PCSK9 (SEQ ID NO: 250) and M2PCSK9 (SEQ ID

[0190] NO: 250), meganucleases that target PCSK9 in macaque liver and leads to stable reduction in serum cholesterol (Wang, L., et al., Nature biotechnology, 36(P>). 717-725 (2018)).

[0191] SEQ ID NO : 250 (M1PCSK9)

[0192] MAPKKKRKVHMNTKYNKEFLLYLAGFVDGDGSIFARILPSQRSKFKHKLRLVFAV YQKTQRRWFLDKLVDEIGVGYVLDSGSVSFYSLSEIKPLHNFLTQLQPFLKLKQKQ ANLVLKIIEQLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLPGS VGGLSPSQASSAASSASSSPGSGISEALRAGAGSGTGYNKEFLLYLAGFVDGDGSIY ARIKPVQRAKFKHELALGFDVTQKTQRRWFLDKLVDEIGVGYVYDKGSVSAYRLS QIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSAKESPDKFLEVCTWVDQIAALN DSKTRKTTSETVRAVLDSLSEKKKSSP

[0193] SEQ ID NO : 251 (M2PCSK9)

[0194] MAPKKKRKVHMNTKYNKEFLLYLAGFVDGDGSIFARIKPSQRSKFKHKLHLVFAV YQKTQRRWFLDKLVDEIGVGYVLDSGSVSFYSLSEIKPLHNFLTQLQPFLKLKQKQ ANLVLKIIEQLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLPGS VGGLSPSQASSAASSASSSPGSGISEALRAGAGSGTGYNKEFLLYLAGFVDGDGSIY ARIKPVQRAKFKHELVLGFDVTQKTQRRWFLDKLVDEIGVGYVYDKGSVSAYRLS QIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSAKESPDKFLEVCTWVDQIAALN DSKTRKTTSETVRAVLDSLSEKKKSSP In some embodiments, gene editing polypeptide can have at least 90% sequence similarity to SEQ ID NOs: 141, 148-151, or 236-251, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence similarity to SEQ ID NOs: 141, 148-151, or 236-251.

[0195] In some examples, the cargo moiety can be linked to the gene editing polypeptide having SEQ ID NOs: 141, 148-151, or 236-251. In some embodiments, the detectable moiety can be linked to a gene editing polypeptide having SEQ ID NOs: 141, 148-151, or 236-251. In some embodiments, the gene editing polypeptide having SEQ ID NOs: 141, 148-151, or 236-251 can be linked to a membrane translocation domain having one or more cell penetrating peptide motifs described herein.

[0196] Cargo moiety

[0197] Also described are peptides as disclosed herein, but that further comprise a cargo moiety linked to the membrane translocation domain. The cargo moiety can be linked to an amino group (e.g., N-terminus). a carboxylate group (e.g., C-terminus), or a side chain of one or more amino acids in the membrane translocation domain.

[0198] When the cargo moiety is attached to the side chain of an amino acid in the membrane translocation domain, the membrane translocation domain includes an amino acid having a side chain with a suitable functional group to form a covalent bond (conjugation) with the cargo, or a side chain which may be modified to provide a suitable functional group (e.g., via conjugation of a linker) that forms a covalent bond with the cargo. In some embodiments, the amino acid on membrane translocation domain which has a side chain suitable conjugation of the cargo is a cysteine residue, glutamic acid residue, an aspartic acid residue, a lysine residue, or a 2,3-diaminopropionic acid residue. In such embodiments, the cargo may be directly conjugated to the side chain of the amino acid (e.g., by forming a disulfide bond with a cysteine residue or an amide bond with a glutamic acid residue or a 2,3- diaminopropionic acid residue) or the cargo may be conjugated to the amino acid side chain through a linker (e.g., PEG).

[0199] The cargo moiety can comprise any cargo of interest, for example a linker moiety, a detectable moiety, a therapeutic moiety, targeting moiety, and the like, or any combination thereof. In some examples, the cargo moiety can comprise one or more additional amino acids (e.g., K, UK, TRV); a linker (e.g., bifunctional linker LC-SMCC); coenzyme A; phosphocoumaryl amino propionic acid (pCAP); 8-amino-3,6-dioxaoctanoic acid (miniPEG); L-2,3-diaminopropionic acid (Dap or J); L-P-naphthylalanine; L-pipecolic acid (Pip); sarcosine; trimesic acid; 7-amino-4-methylcourmarin (Amc); fluorescein isothiocyanate (FITC); L-2-naphthylalanine; norleucine; 2-aminobutyric acid: Rhodamine B (Rho); Dexamethasone (DEX); or combinations thereof.

[0200] In some examples the cargo moiety can comprise any of those listed in Table 3, or derivatives or combinations thereof.

[0201] Table 3. Example cargo moi eties

[0202] *pCAP, phosphocoumaryl amino propionic acid; Q. norleucine; U. 2-aminobutyric acid; D-pThr is D-phosphothreonine, Pip is L-piperidine-2-carboxylate. Detectable moiety

[0203] The detectable moiety can comprise any detectable label. Examples of suitable detectable labels include, but are not limited to, a UV-Vis label, a near-infrared label, a luminescent group, a phosphorescent group, a magnetic spin resonance label, a photosensitizer, a photocleavable moiety, a chelating center, a heavy atom, a radioactive isotope, an isotope detectable spin resonance label, a paramagnetic moiety, a chromophore, or any combination thereof. In some embodiments, the label is detectable without the addition of further reagents.

[0204] In some embodiments, the detectable moiety is a biocompatible detectable moiety', such that the compounds can be suitable for use in a variety of biological applications. “Biocompatible” and “biologically compatible”, as used herein, generally refer to compounds that are. along with any metabolites or degradation products thereof, generally non-toxic to cells and tissues, and which do not cause any significant adverse effects to cells and tissues when cells and tissues are incubated (e.g, cultured) in their presence. The detectable moiety can contain a luminophore such as a fluorescent label or nearinfrared label. Examples of suitable luminophores include, but are not limited to, metal porphyrins; benzoporphyrins; azabenzoporphyrine; napthoporphyrin; phthalocyanine; polycyclic aromatic hydrocarbons such as perylene, perylene diimine, pyrenes; azo dyes; xanthene dyes; boron dipyoromethene, aza-boron dipyoromethene, cyanine dyes, metalligand complex such as bipyridine, bipyridyls, phenanthroline, coumarin, and acetyl acet onates of ruthenium and iridium; acridine, oxazine derivatives such as benzophenoxazine; aza-annulene, squaraine; 8-hydroxyquinoline, polymethines, luminescent producing nanoparticle, such as quantum dots, nanocrystals: carbostynk terbium complex; inorganic phosphor; ionophore such as crown ethers affiliated or derivatized dyes; or combinations thereof. Specific examples of suitable luminophores include, but are not limited to, Pd (II) octaethylporphyrin; Pt (Il)-octaethylporphyrin; Pd (II) tetraphenylporphyrin; Pt (II) tetraphenylporphyrin; Pd (II) meso-tetraphenylporphyrin tetrabenzoporphine; Pt (II) meso- tetrapheny metrylbenzoporphyrin; Pd (II) octaethylporphyrin ketone; Pt (II) octaethylporphyrin ketone; Pd (II) meso-tetra(pentafluorophenyl)porphyrin; Pt (II) mesotetra (pentafluorophenyl) porphyrin; Ru (II) tris(4,7-diphenyl-l,10-phenanthroline) (Ru (dpp)s); Ru (II) tris(l,10-phenanthroline) (Ru(phen)s). tris(2,2’-bipyridine)ruthenium (II) chloride hexahydrate (Ru(bpy)s); erythrosine B; fluorescein; fluorescein isothiocyanate (FITC); eosin; iridium (III) ((N-methyl-benzimidazol-2-yl)-7-(diethylamino)-coumarin)); indium (III) ((benzothiazol-2-yl)-7- (diethylamino)-coumarin))-2-(acetylacetonate); Lumogen dyes; Macroflex fluorescent red; Macrolex fluorescent yellow; Texas Red; rhodamine B; rhodamine 6G; sulfur rhodamine; m-cresol; thymol blue; xylenol blue; cresol red; chlorophenol blue; bromocresol green; bromcresol red; bromothymol blue; Cy2; a Cy3; a Cy5; a Cy5.5; Cy7; 4-nitirophenol; alizarin; phenolphthalein; o-cresolphthalein; chlorophenol red; calmagite; bromo-xylenol; phenol red; neutral red; nitrazine; 3, 4,5,6- tetrabromphenolphtalein; congo red; fluorescein; eosin; 2',7'-dichlorofluorescein; 5(6)- carboxy-fluorecsein; carboxynaphthofluorescein; 8-hydroxypyrene-l,3,6-trisulfonic acid; semi-naphthorhodafluor; semi-naphthofluorescein; tris (4,7-diphenyl-l,10-phenanthroline) ruthenium (II) dichloride; (4,7-diphenyl-l,10-phenanthroline) ruthenium (II) tetraphenylboron; platinum (II) octaethylporphyin; dialkylcarbocyanine; dioctadecylcycloxacarbocyanine; fluorenylmethyloxycarbonyl chloride; 7-amino-4- methylcourmarin (Amc); green fluorescent protein (GFP); and derivatives or combinations thereof. In some examples, the detectable moiety can comprise Rhodamine B (Rho), fluorescein isothiocyanate (FITC), 7-amino-4-methylcourmarin (Amc), green fluorescent protein (GFP), naphthofluorescein (NF), or derivatives or combinations thereof.

[0205] Also disclosed herein are compositions comprising the compounds described herein.

[0206] Also disclosed herein are pharmaceutically-acceptable salts and prodrugs of the disclosed compounds. Pharmaceutically-acceptable salts include salts of the disclosed compounds that are prepared with acids or bases, depending on the particular substituents found on the compounds. Under conditions where the compounds disclosed herein are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts can be appropriate. Examples of pharmaceutically-acceptable base addition salts include sodium, potassium, calcium, ammonium, or magnesium salt. Examples of physiologically-acceptable acid addition salts include hydrochloric, hydrobromic, nitric, phosphoric, carbonic, sulfuric, and organic acids like acetic, propionic, benzoic, succinic, fumaric, mandelic, oxalic, citric, tartaric, malonic, ascorbic, alpha-ketoglutaric, alphaglycophosphoric, maleic, tosyl acid, methanesulfonic, and the like. Thus, disclosed herein are the hydrochloride, nitrate, phosphate, carbonate, bicarbonate, sulfate, acetate, propionate, benzoate, succinate, fumarate, mandelate, oxalate, citrate, tartarate, malonate, ascorbate, alpha-ketoglutarate, alpha-glycophosphate, maleate, tosylate, and mesylate salts. Pharmaceutically acceptable salts of a compound can be obtained using standard procedures well known in the art, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

[0207] Linker

[0208] In various embodiments, the linker is covalently bound to an amino acid on the membrane translocation domain. The linker may be any moiety which conjugates two or more of the membrane translocation domain to the cargo moiety. In some embodiments, the linker can be an amino acid. In other embodiments, the precursor to the linker can be any appropriate molecule which is capable of forming two or more bonds with amino acids in the membrane translocation domain and cargo moiety. Thus, in various embodiments, the precursor of the linker has two or more functional groups, each of which are capable of forming a covalent bond to the membrane translocation domain and cargo moiety. For example, the linker can be covalently bound to the N-terminus, C-terminus. or side chain, or combinations thereof, of any amino acid in the membrane translocation domain. In particular embodiments, the linker forms a covalent bond between the membrane translocation domain and cargo moiety.

[0209] In some embodiments, the linker is selected from the group consisting of at least one amino acid, alkylene, alkenylene, alkynylene, ary l . cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, hctcroar l. ether, each of which can be optionally substituted as defined above. For example, each of these likers can be from 1 to 500 atoms in length, e.g., from 1 to 100, from 1 to 250, from 10 to 200. from 25 to 300 atoms in length. Non-limiting examples of linkers include polyethylene glycol, optionally conjugated to a lysine residue. In other examples, the linker can be a bivalent or trivalent Ci-Cso saturated or unsaturated, straight or branched alkyl, wherein 1-25 methylene groups are optionally and independently replaced by -N(H)-, -N(CI-C4alkyl)-, -N(cycloalkyl)-, -O-, -C(O)-, -C(O)O-. -S-, -S(O)-, -S(O)2-, - S(O)2N(CI-C4alkyl)-, -S(O)2N(cycloalkyl)-, -N(H)C(O)-, -N(CI-C4alkyl)C(O)-, - N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(CI-C4alkyl), -C(O)N(cycloalkyl), aryl, heteroaryl, cycloalkyl, or cycloalkenyl.

[0210] In some embodiments, the linker is covalently bound to the N or C-terminus of an ammo acid on membrane translocation domain, or to a side chain of glutamine, asparagine, or lysine, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group). In particular embodiments, the linker forms a bond with the side chain of glutamine on the CPP motif. In other particular embodiments, the linker described herein has a structure of L-l or L-2:

[0211] L-1 wherein

[0212] AASis a side chain or terminus of an amino acid on the peptide or staple;

[0213] AAc is a side chain or terminus of an amino acid of the cCPP; p is an integer from 0 to 10; and q is an integer from 1 to 50. In other embodiments the linker can be aproteolytically stable peptide sequence, such as (GGS)n, (GGGS)n, (GSS)n, (EAAAK)n, and (PA)n or (PAS)n. where n is 0-100.

[0214] In some embodiments, the linker is capable of releasing the cargo moiety from the membrane translocation domain after the polypeptide conjugate enters the cytosol of the cell. In some embodiments, the linker contains a group, or forms a group after binding to membrane translocation domain and cargo moiety that is cleaved after cytosolic uptake of the polypeptide conjugate to thereby release the cargo moiety. For example, a dithiocyclopeptide containing (LEAGCKNFFPR|SFTSCGSLE (SEQ ID NO: 235) linker sequence having a disulfide bond and a thrombin-sensitive sequence between the two Cys (X. Chen, et al., Biotech 49 (2010) 513-518). Non-limiting examples of physiologically cleavable linking group include carbonate, thiocarbonate, thioether, thioester, disulfide, sulfoxide, hydrazine, protease-cleavable dipeptide linker, and the like.

[0215] For example, in embodiments, the linker is covalently bound to membrane translocation domain through a disulfide bond e.g., with the side chain of cysteine or cysteine analog located in the membrane translocation domain or cargo moiety. In some embodiments, the disulfide bond is formed between a thiol group on a precursor of the linker, and the side chain of cysteine or an amino acid analog having a thiol group on the peptide, wherein the bond to hydrogen on each of the thiol groups is replaced by a bond to a sulfur atom. Non-limiting examples of amino acid analogs having a thiol group which can be used with the polypeptide conjugates disclosed herein are discussed above.

[0216] Therapeutic moiety

[0217] The disclosed compounds can also comprise a therapeutic moiety. In some examples, the cargo moiety comprises a therapeutic moiety. The detectable moiety can be linked to a therapeutic moiety or the detectable moiety can also serve as the therapeutic moiety. Therapeutic moiety refers to a group that when administered to a subject will reduce one or more symptoms of a disease or disorder.

[0218] The therapeutic moiety can comprise a wide variety of drugs, including antagonists, for example enzyme inhibitors, and agonists, for example a transcription factor which results in an increase in the expression of a desirable gene product (although as will be appreciated by those in the art, antagonistic transcription factors can also be used), are all included. In addition, therapeutic moiety includes those agents capable of direct toxicity and / or capable of inducing toxicity towards healthy and / or unhealthy cells in the body. Also, the therapeutic moiety can be capable of inducing and / or priming the immune system against potential pathogens.

[0219] The therapeutic moiety can, for example, comprise an anticancer agent, antiviral agent, antimicrobial agent, ar i -inflammatory agent, immunosuppressive agent, anesthetics, or any combination thereof.

[0220] The therapeutic moiety can comprise an anticancer agent. Example anticancer agents include 13-cis-Retinoic Acid. 2-Amino-6-Mercaptopurine. 2-CdA. 2-Chlorodeoxy adenosine, 5-fluorouracil, 6-Thioguanine, 6-Mercaptopurine, Accutane, Actinomycin-D, Adriamycin, Adrucil, Agrylin, Ala-Cort, Aldesleukin, Alemtuzumab, Alitretinoin, Alkaban-AQ, Alkeran, All-transretinoic acid, Alpha interferon, Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide. Anandron, Anastrozole, Arabinosylcytosine. Aranesp, Aredia, Arimidex, Aromasin, Arsenic trioxide, Asparaginase, ATRA, Avastin, BCG, BCNU, Bevacizumab, Bexarotene, Bicalutamide, BiCNU, Blenoxane, Bleomycin, Bortezomib, Busulfan, Busulfex, C225, Calcium Leucovorin, Campath, Camptosar, Camptothecin-11, Capecitabine, Carac, Carboplatin, Carmustine. Carmustine wafer. Casodex, CCNU, CDDP, CeeNU, Cerubidine, cetuximab. Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen, CPT-11, Cyclophosphamide, Cytadren, Cytarabine, Cytarabine liposomal, Cytosar-U, Cytoxan, Dacarbazine, Dactinomycin, Darbepoetin alfa, Daunomycin, Daunorubicin, Daunorubicin hydrochloride, Daunorubicin liposomal, DaunoXome, Decadron, Delta-Cortef. Deltasone, Denileukin diftitox, DepoCyt. Dexamethasone, Dexamethasone acetate, Dexamethasone sodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil, Doxorubicin, Doxorubicin liposomal, Droxia, DTIC, DTIC-Dome, Duralone, Efudex, Eligard. Ellence, Eloxatin, Elspar, Emcyt. Epirubicin, Epoetin alfa, Erbitux. Erwinia L-asparaginase, Estramustine, Ethyol, Etopophos, Etoposide, Etoposide phosphate, Eulexin, Evista, Exemestane, Fareston, Faslodex, Femara, Filgrastim, Floxuridine, Fludara, Fludarabine, Fluoroplex, Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic Acid, FUDR, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gleevec, Lupron, Lupron Depot. Matulane, Maxidex, Mechlorethamine, -Mechlorethamine Hydrochlorine, Medralone, MedroL Megace, Megestrol, Megestrol Acetate, Melphalan, Mercaptopurine, Mesna, Mesnex, Methotrexate, Methotrexate Sodium, Methylprednisolone, Mylocel, Letrozole, Neosar, Neulasta, Neumega, Neupogen, Nilandron, Nilutamide, Nitrogen Mustard, Novaldex, Novantrone, Octreotide, Octreotide acetate. Oncospar. Oncovin, Ontak, Onxal. Oprevelkin. Orapred, Orasone, Oxaliplatin, Paclitaxel, Pamidronate, Panretin, Paraplatin, Pediapred, PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON, PEG-L-asparaginase, Phenylalanine Mustard, Platinol, Platinol-AQ, Prednisolone. Prednisone, Prelone, Procarbazine, PROCRIT, Proleukin, Prolifeprospan 20 with Carmustine implant, PurinethoL Raloxifene, Rheumatrex, Rituxan, Rituximab, Roveron-A (interferon alfa-2a), Rubex, Rubidomycin hydrochloride, Sandostatin, Sandostatin LAR, Sargramostim, Solu-Cortef, Solu-Medrol, STI-571, Streptozocin, Tamoxifen, Targretin, Taxol. Taxotere, Temodar, Temozolomide, Teniposide, TESPA. Thalidomide, Thalomid. TheraCys, Thioguanine. Thioguanine Tabloid, Thiophosphoamide, Thioplex, Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab, Tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, VePesid, Vesanoid, Viadur, Vinblastine, Vinblastine Sulfate, Vincasar Pfs, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VP-16. Vumon, Xeloda, Zanosar, Zevalin. Zinecard, Zoladex, Zoledronic acid, Zometa, Gliadel wafer, Glivec, GM-CSF, Goserelin, granulocyte colony stimulating factor, Halotestin, Herceptin, Hexadrol, Hexalen, Hexamethylmelamine, HMM, Hycamtin, Hydrea, Hydrocort Acetate. Hydrocortisone, Hydrocortisone sodium phosphate, Hydrocortisone sodium succinate. Hydrocortone phosphate. Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin, Idarubicin, Ifex, IFN-alpha, Ifosfamide, IL 2, IL-11, Imatinib mesylate, Imidazole Carboxamide, Interferon alfa, Interferon Alfa-2b (PEG conjugate), Interleukin 2, Interleukin-11, Intron A (interferon alfa-2b), Leucovorin, Leukeran, Leukine, Leuprolide, Leurocristine, Leustatin, Liposomal Ara-C, Liquid Pred, Lomustine. L- PAM. L-Sarcolysin, Meticorten, Mitomycin. Mitomycin-C. Mitoxantrone. M-Prednisol, MTC, MTX, Mustargen, Mustine, Mutamycin, Myleran, Iressa, Irinotecan, Isotretinoin, Kidrolase, Lanacort, L-asparaginase, and LCR. The therapeutic moiety can also comprise a biopharmaceutical such as, for example, an antibody.

[0221] In some examples, the therapeutic moiety can comprise an antiviral agent, such as ganciclovir, azidothymidine (AZT), lamivudine (3TC), etc.

[0222] In some examples, the therapeutic moiety can comprise an antibacterial agent, such as acedapsone; acetosulfone sodium; alamecin; alexidine; amdinocillin; amdinocillin pivoxil; amicycline; amifloxacin; amifloxacin mesylate; amikacin; amikacin sulfate; aminosalicylic acid; aminosalicylate sodium; amoxicillin; amphomycin; ampicillin; ampicillin sodium; apalcillin sodium; apramycin; aspartocin; astromicin sulfate; avilamycin; avoparcin; azithromycin; azlocillin; azlocillin sodium; bacampicillin hydrochloride; bacitracin; bacitracin methylene disalicylate; bacitracin zinc; bambermycins; benzoylpas calcium; berythromycin; betamicin sulfate; biapenem; biniramycin; biphenamine hydrochloride; bispyrithione magsulfex; butikacin; butirosin sulfate; capreomycin sulfate; carbadox; carbenicillin disodium; carbenicillin indanyl sodium; carbenicillin phenyl sodium; carbenicillin potassium; carumonam sodium; cefaclor; cefadroxil; cefamandole; cefamandole nafate; cefamandole sodium; cefaparole; cefatrizine; cefazaflur sodium; cefazolin; cefazolin sodium; cefbuperazone; cefdinir; cefepime; cefepime hydrochloride; cefetecol; cefixime; cefmenoxime hydrochloride; cefmetazole; cefmetazole sodium; cefonicid monosodium; cefonicid sodium; cefoperazone sodium; ceforanide; cefotaxime sodium; cefotetan; cefotetan disodium; cefotiam hydrochloride; cefoxitin; cefoxitin sodium; cefpimizole; cefpimizole sodium; cefpiramide; cefpiramide sodium; cefpirome sulfate; cefpodoxime proxetil; cefprozil; cefroxadine; cefsulodin sodium; ceftazidime; ceftibuten; ceftizoxime sodium; ceftriaxone sodium; cefuroxime; cefuroxime axetil; cefuroxime pivoxetil; cefuroxime sodium; cephacetrile sodium; cephalexin; cephalexin hydrochloride; cephaloglycin; cephaloridine; cephalothin sodium; cephapirin sodium; cephradine; cetocycline hydrochloride; cetophenicol; chloramphenicol; chloramphenicol palmitate; chloramphenicol pantothenate complex; chloramphenicol sodium succinate; chlorhexidine phosphanilate; chloroxylenol; chlortetracycline bisulfate; chlortetracycline hydrochloride; cinoxacin; ciprofloxacin; ciprofloxacin hydrochloride; cirolemycin; clarithromycin; clinafloxacin hydrochloride; clindamycin; clindamycin hydrochloride; clindamycin palmitate hydrochloride; clindamycin phosphate; clofazimine; cloxacillin benzathine; cloxacillin sodium; cloxyquin; colistimethate sodium; colistin sulfate; coumermycin; coumermycin sodium; cyclacillin; cycloserine; dalfopristin; dapsone; daptomycin; demeclocycline; demeclocycline hydrochloride; demecycline; denofungin; diaveridine; di cl oxacillin; dicloxacillin sodium; dihydrostreptomycin sulfate; dipyrithione; dirithromycin; doxycycline; doxycycline calcium; doxycycline fosfatex; doxycycline hy elate; droxacin sodium; enoxacin; epicillin; epitetracy cline hydrochloride; erythromycin; erythromycin acistrate; ery thromycin estolate; erythromycin ethylsuccinate; erythromycin gluceptate; erythromycin lactobionate; erythromycin propionate; erythromycin stearate; ethambutol hydrochloride; ethionamide; fleroxacin; floxacillin; fludalanine; flumequine; fosfomycin; fosfomycin tromethamine; fumoxicillin; furazolium chloride; furazolium tartrate; fusidate sodium; fusidic acid; gentamicin sulfate; gloximonam; gramicidin; haloprogin; hetacillin; hetacillin potassium; hexedine; ibafloxacin; imipenem; isoconazole; isepamicin; isoniazid; josamycin; kanamycin sulfate; kitasamycin; levofuraltadone; levopropylcillin potassium; lexithromycin; lincomycin; lincomycin hydrochloride; lomefloxacin; Lomefloxacin hydrochloride; lomefloxacin mesylate; loracarbef; mafenide; meclocycline; meclocycline sulfosalicylate; megalomicin potassium phosphate; mequidox; meropenem; methacycline; methacycline hydrochloride; methenamine: methenamine hippurate; methenamine mandelate; methicillin sodium; metioprim; metronidazole hydrochloride; metronidazole phosphate; mezlocillin; mezlocillin sodium; minocycline; minocycline hydrochloride; mirincamycin hydrochloride; monensin; monensin sodiumr; nafcillin sodium; nalidixate sodium; nalidixic acid; natainycin; nebramycin; neomycin palmitate; neomycin sulfate; neomycin undecylenate; netilmicin sulfate; neutramycin; nifuiradene: nifuraldezone; nifuratel; nifuratrone; nifurdazil; nifurimide; nifiupirinol; nifurquinazol; nifurthiazole: nitrocy cline; nitrofurantoin; nitromide; norfloxacin; novobiocin sodium; ofloxacin; onnetoprim; oxacillin; oxacillin sodium; oximonam; oximonam sodium; oxolinic acid; oxytetracycline; oxytetracycline calcium; oxytetracycline hydrochloride; paldimycin; parachlorophenol; paulomycin; pefloxacin; pefloxacin mesylate; penamecillin; penicillin G benzathine; penicillin G potassium; penicillin G procaine; penicillin G sodium; penicillin V; penicillin V benzathine; penicillin V hydrabamine; penicillin V potassium; pentizidone sodium; phenyl aminosalicylate; piperacillin sodium; pirbenicillin sodium; piridicillin sodium; pirlimycin hydrochloride; pivampicillin hydrochloride; pivampicillin pamoate: pivampicillin probenate; polymyxin B sulfate; porfiromycin; propikacin; pyrazinamide; pynthione zinc; quindecamme acetate; quinupristin; racephenicol; ramoplanin; ranimycin; relomycin; repromicin; rifabutin; rifametane; rifamexil; rifamide; rifampin; rifapentine; rifaximin; rolitetracycline; rolitetracycline nitrate; rosaramicin; rosaramicin butyrate; rosaramicin propionate; rosaramicin sodium phosphate; rosaramicin stearate; rosoxacin; roxarsone; roxithromycin; sancycline; sanfetrinem sodium; sarmoxicillin; sarpicillin; scopafungin; sisomicin; sisomicin sulfate; sparfloxacin; spectinomycin hydrochloride; spiramycin; stallimycin hydrochloride; steffimycin; streptomycin sulfate; streptonicozid; sulfabenz; sulfabenzamide; sulfacetamide; sulfacetamide sodium; sulfacytine; sulfadiazine; sulfadiazine sodium; sulfadoxine; sulfalene; sulfamerazine; sulfameter; sulfamethazine; sulfamethizole; sulfamethoxazole; sulfamonomethoxine; sulfamoxole; sulfanilate zinc; sulfanitran; sulfasalazine; sulfasomizole; sulfathiazole; sulfazamet; sulfisoxazole; sulfisoxazole acetyl; sulfisboxazole diolamine; sulfomyxin: sulopenem; sultamricillin: suncillin sodium; talampicillin hydrochloride; teicoplanin; temafloxacin hydrochloride; temocillin; tetracycline; tetracycline hydrochloride; tetracycline phosphate complex; tetroxoprim; thiamphenicol; thiphencillin potassium; ticarcillin cresyl sodium; ticarcillin disodium; ticarcillin monosodium; ticlatone; tiodonium chloride; tobramycin; tobramycin sulfate; tosufloxacin; trimethoprim; trimethoprim sulfate; trisulfapyrimidines; troleandomycin; trospectomycin sulfate: tyrothricin; vancomycin; vancomycin hydrochloride; virginiamycin; or zorbamycin. In some examples, the therapeutic moiety can comprise an anti-inflammatory agent.

[0223] In some examples, the therapeutic moiety can comprise dexamethasone (Dex).

[0224] In other examples, the therapeutic moiety comprises a therapeutic protein. For example, some people have defects in certain enzymes (e.g., lysosomal storage disease). It is disclosed herein to deliver such enzymes / proteins to human cells by linking to the enzyme / protein to one of the disclosed cell penetrating peptides. The disclosed cell penetrating peptides have been tested with proteins (e.g.. GFP, PTP1B, actin, calmodulin, troponin C) and shown to work.

[0225] In some examples, the therapeutic moiety7comprises a targeting moiety7. The targeting moiety7can comprise, for example, a sequence of amino acids that can target one or more enzyme domains. In some examples, the targeting moiety can comprise an inhibitor against an enzyme that can play a role in a disease, such as cancer, cystic fibrosis, diabetes, obesity7, or combinations thereof. For example, the targeting moiety7can comprise any of the sequences listed in Table 4. Table 4. Example targeting moieties

[0226]

[0227] *Fpa, S: L-4-fluorophenylalanine; Pip. 0: L-homoproline; Nle, : L-norleucine;

[0228] Phg, *P L-phenylglycine; F2Pmp, A: L-4-(phosphonodifluoromethyl)phenylalanine; Dap, L- 2,3 -diaminopropionic acid; Nal, O’: L-P-naphthylalanine; Pp, 9: L-pipecolic acid; Sar, S: sarcosine; Tm, trimesic acid.

[0229] The targeting moiety and cell penetrating peptide moiety can overlap. That is, the residues that form the cell penetrating peptide moiety7can also be part of the sequence that forms the targeting moiety', and vice a versa.

[0230] The therapeutic moiety can be attached to the cell penetrating peptide moiety at the ammo group, the carboxylate group, or the side chain of any of the amino acids of the cell penetrating peptide moiety (e.g., at the amino group, the carboxylate group, or the side chain or any of amino acid of the CPP). In some examples, the therapeutic moiety' can be attached to the detectable moiety.

[0231] In some examples, the therapeutic moiety can comprise a targeting moiety that can act as an inhibitor against Ras (e.g., K-Ras), PTP1B, Pinl, Grb2 SH2, CAL PDZ, and the like, or combinations thereof.

[0232] Ras is a protein that in humans is encoded by the RAS gene. The normal Ras protein performs an essential function in normal tissue signaling, and the mutation of a Ras gene is implicated in the development of many cancers. Ras can act as a molecular on / off switch, once it is turned on Ras recruits and activates proteins necessary for the propagation of growth factor and other receptors’ signal. Mutated forms of Ras have been implicated in various cancers, including lung cancer, colon cancer, pancreatic cancer, and various leukemias.

[0233] Protein-tyrosine phosphatase IB (PTP1B) is a prototypical member of the PTP superfamily and plays numerous roles during eukaryotic cell signaling. PTP IB is a negative regulator of the insulin signaling pathway, and is considered a promising potential therapeutic target, in particular for the treatment of type II diabetes. PIP IB has also been implicated in the development of breast cancer.

[0234] Pinl is an enzyme that binds to a subset of proteins and plays a role as a post phosphorylation control in regulating protein function. Pinl activity can regulate the outcome of proline-directed kinase signaling and consequently can regulate cell proliferation and cell survival. Deregulation of Pinl can play a role in various diseases. The up-regulation of Pint may be implicated in certain cancers, and the down-regulation of Pinl may be implicated in Alzheimer’s disease. Inhibitors of Pinl can have therapeutic implications for cancer and immune disorders.

[0235] Grb2 is an adaptor protein involved in signal transduction and cell communication. The Grb2 protein contains one SH2 domain, which can bind tyrosine phosphorylated sequences. Grb2 is widely expressed and is essential for multiple cellular functions. Inhibition of Grb2 function can impair developmental processes and can block transformation and proliferation of various cell types.

[0236] It was recently reported that the activity of cystic fibrosis membrane conductance regulator (CFTR), a chloride ion channel protein mutated in cystic fibrosis (CF) patients, is negatively regulated by CFTR-associated ligand (CAL) through its PDZ domain (CAL-PDZ) (Wolde, M et al. J. Biol. Chem. 2007, 282, 8099). Inhibition of the CFTR / CAL-PDZ interaction was shown to improve the activity of APhe508-CFTR, the most common form of CFTR mutation (Cheng, SH et al. Cell 1990, 63, 827; Kerem, BS et al. Science 1989, 245, 1073), by reducing its proteasome-mediated degradation (Cushing, PR et al. Angew. Chem. Int. Ed. 2010, 49, 9907). Thus, disclosed herein is a method for treating a subject having cystic fibrosis by administering an effective amount of a compound or composition disclosed herein. The compound or composition administered to the subject can comprise a therapeutic moiety that can comprise a targeting moiety that can act as an inhibitor against CAL PDZ. Also, the dcompositions or compositions disclosed herein can be administered with a molecule that corrects the CFTR function.

[0237] Also disclosed herein are compositions comprising the compounds described herein.

[0238] Also disclosed herein are pharmaceutically-acceptable salts and prodrugs of the disclosed compounds. Pharmaceutically-acceptable salts include salts of the disclosed compounds that are prepared with acids or bases, depending on the particular substituents found on the compounds. Under conditions where the compounds disclosed herein are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts can be appropriate. Examples of pharmaceutically-acceptable base addition salts include sodium, potassium, calcium, ammonium, or magnesium salt. Examples of physiologically-acceptable acid addition salts include hydrochloric, hydrobromic, nitric, phosphoric, carbonic, sulfuric, and organic acids like acetic, propionic, benzoic, succinic, fumaric, mandelic, oxalic, citric, tartaric, malonic, ascorbic, alpha-ketoglutaric, alpha- glycophosphoric, maleic, tosyl acid, methanesulfonic, and the like. Thus, disclosed herein are the hydrochloride, nitrate, phosphate, carbonate, bicarbonate, sulfate, acetate, propionate, benzoate, succinate, fumarate, mandelate, oxalate, citrate, tartarate, malonate, ascorbate, alpha-ketoglutarate, alpha-glycophosphate, maleate, tosylate, and mesylate salts. Pharmaceutically acceptable salts of a compound can be obtained using standard procedures well known in the art, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

[0239] Pharmaceutical Compositions

[0240] Described herein are pharmaceutical compositions including an effective amount of a peptide described herein and an acceptable earner.

[0241] The term "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for vetennary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.

[0242] “Excipients'’ include any and all solvents, diluents or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. General considerations in formulation and / or manufacture can be found, for example, in Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton. Pa., 1980), and Remington: The Science and Practice of Pharmacy, 21st Edition (Lippincott Williams & Wilkins, 2005).

[0243] Exemplary excipients include, but are not limited to, any non-toxic, inert solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as excipients include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; com oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. As would be appreciated by one of skill in this art, the excipients may be chosen based on what the composition is useful for. For example, with a pharmaceutical composition, the choice of the excipient will depend on the route of administration, the agent being delivered, time course of delivery of the agent, etc., and can be administered to humans and / or to animals, orally, rectally, parenterally, intracistemally, intravaginally, intranasally, intraperitoneally, topically (as by powders, creams, ointments, or drops), buccally, or as an oral or nasal spray. In some embodiments, the active compounds disclosed herein are administered topically.

[0244] Exemplan ■ diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and combinations thereof.

[0245] Exemplar)' granulating and / or dispersing agents include potato starch, com starch, tapioca starch, sodium starch glycolate. clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, crosslinked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, etc., and combinations thereof.

[0246] Exemplar)' surface active agents and / or emulsifiers include natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat. cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxy vinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate |Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and / or combinations thereof. Exemplary7binding agents include starch (e.g. cornstarch and starch paste), gelatin, sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalactan). alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, etc., and / or combinations thereof.

[0247] Exemplary7preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.

[0248] Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite. Exemplar^' chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g.. sodium edetate. disodium edetate, trisodium edetate. calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0249] Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0250] Exemplar}' alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxy benzoate, and phenylethyl alcohol.

[0251] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, betacarotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid. Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS). sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, NeoIone, Kathon, and Euxyl. In certain embodiments, the preservative is an anti-oxidant. In other embodiments, the preservative is a chelating agent.

[0252] Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen- free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and combinations thereof.

[0253] Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.

[0254] Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, com, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut. mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglycende, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and combinations thereof.

[0255] Additionally, the composition may further comprise a polymer. Exemplary polymers contemplated herein include, but are not limited to, cellulosic polymers and copolymers, for example, cellulose ethers such as methylcellulose (MC), hydroxyethylcellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), methylhydroxyethylcellulose (MHEC). methylhydroxypropylcellulose (MHPC), carboxymethyl cellulose (CMC) and its various salts, including, e.g.. the sodium salt, hydroxyethylcarboxymethylcellulose (HECMC) and its various salts, carboxymethylhydroxyethylcellulose (CMHEC) and its various salts, other polysaccharides and polysaccharide derivatives such as starch, dextran, dextran derivatives, chitosan, and alginic acid and its various salts, carageenan, various gums, including xanthan gum, guar gum, gum arabic. gum karaya, gum ghatti. konjac and gum tragacanth, glycosaminoglycans and proteoglycans such as hyaluronic acid and its salts, proteins such as gelatin, collagen, albumin, and fibrin, other polymers, for example, polyhydroxyacids such as polylactide, poly glycolide, polyl(lactide-co-glycolide) and poly(.epsilon.-caprolactone-co-glycolide)-, carboxyvinyl polymers and their salts (e.g., carbomer), polyvinylpyrrolidone (PVP), polyacrylic acid and its salts, polyacrylamide, polyacrylic acid / acrylamide copolymer, polyalkylene oxides such as polyethylene oxide, polypropylene oxide, poly(ethylene oxidepropylene oxide), and a Pluronic polymer, polyoxy ethylene (polyethylene glycol), polyanhydrides, polyvinylalchol, polyethyleneamine and polypyrridine, polyethylene glycol (PEG) polymers, such as PEGylated lipids (e.g., PEG-stearate, l,2-Distearoyl-sn-glycero-3- Phosphoethanolamine-N-[Methoxy(Poly ethylene glycol)- 1000], 1,2-Distearoyl-sn-glycero- 3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000], and 1.2-Distearoyl-sn- glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-5000]). copolymers and salts thereof.

[0256] Additionally, the composition may further comprise an emulsifying agent. Exemplary emulsifying agents include, but are not limited to, a polyethylene glycol (PEG), a polypropylene glycol, a polyvinyl alcohol, a poly-N-vinyl pyrrolidone and copolymers thereof, poloxamer nonionic surfactants, neutral water-soluble polysaccharides (e.g., dextran, Ficoll, celluloses), non-cationic poly(meth)acrylates, non-cationic polyacrylates, such as poly (meth) acrylic acid, and esters amide and hydroxy alkyl amides thereof, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxy vinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol). sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and / or combinations thereof. In certain embodiments, the emulsify ing agent is cholesterol.

[0257] Liquid compositions include emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the peptide, the liquid composition may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol. 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0258] Injectable compositions, for example, injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be an injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents for pharmaceutical or cosmetic compositions that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. Any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In certain embodiments, the particles are suspended in a carrier fluid comprising 1% (w / v) sodium carboxymethyl cellulose and 0.1% (v / v) Tween 80. The injectable composition can be sterilized, for example, by filtration through a bacteria- retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0259] Solid compositions include capsules, tablets, pills, powders, and granules. In such solid compositions, the particles are mixed with at least one excipient and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar- agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also comprise buffering agents. Solid compositions of a similar ty pe may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0260] Tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0261] Compositions for topical or transdermal administration include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The peptide is admixed with an excipient and any needed preservatives or buffers as may be required.

[0262] The ointments, pastes, creams, and gels may contain, in addition to the peptide, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0263] Powders and sprays can contain, in addition to the peptide, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.

[0264] Transdermal patches have the added advantage of providing controlled delivery of a peptide to the body. Such dosage forms can be made by dissolving or dispensing the nanoparticles in a proper medium. Absorption enhancers can also be used to increase the flux of the peptide across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the particles in a polymer matrix or gel.

[0265] The peptides can be incorporated microparticles, nanoparticles, or combinations thereof that provide controlled release of the peptides and / or additional active agents. For example, the peptides can be incorporated into polymeric microparticles, which provide controlled release of the drug(s). Release of the drug(s) is controlled by diffusion of the drug(s) out of the microparticles and / or degradation of the polymeric particles by hydrolysis and / or enzymatic degradation. Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives.

[0266] Polymers, which are slowly soluble and form a gel in an aqueous environment, such as hydroxypropyl methylcellulose or polyethylene oxide, may also be suitable as materials for drug containing microparticles. Other polymers include, but are not limited to, polyanhydrides, poly(ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3-hydroxybutyrate (PHB) and copolymers thereof, poly-4-hydroxybutyrate (P4HB) and copolymers thereof, poly caprolactone and copolymers thereof, and combinations thereof.

[0267] Alternatively, the peptide can be incorporated into microparticles prepared from materials which are insoluble in aqueous solution or slowly soluble in aqueous solution, but are capable of degrading within the GI tract by means including enzymatic degradation, surfactant action of bile acids, and / or mechanical erosion. As used herein, the term ‘"slowly soluble in water” refers to materials that are not dissolved in water within a period of 30 minutes. Preferred examples include fats, fatty substances, waxes, wax-like substances and mixtures thereof. Suitable fats and fatty' substances include fatty' alcohols (such as laury l, myristyl stearyl, cetyl or cetostearyl alcohol), fatty acids and derivatives, including but not limited to fatty’ acid esters, fatty acid glycerides (mono-, di- and tri-glycerides), and hydrogenated fats. Specific examples include, but are not limited to hydrogenated vegetable oil, hydrogenated cottonseed oil, hydrogenated castor oil, hydrogenated oils available under the trade name Sterotex®, stearic acid, cocoa butter, and steary l alcohol. Suitable waxes and wax-like materials include natural or synthetic waxes, hydrocarbons, and normal waxes. Specific examples of waxes include beeswax, glycowax, castor wax, carnauba wax, paraffins and candelilla wax. As used herein, a wax-like material is defined as any' material, which is normally solid at room temperature and has a melting point of from about 30 to 300° C.

[0268] In some cases, it may be desirable to alter the rate of water penetration into the microparticles. To this end. rate-controlling (wicking) agents may be formulated along with the fats or waxes listed above. Examples of rate-controlling materials include certain starch derivatives (e.g., waxy maltodextrin and drum dried com starch), cellulose derivatives (e.g., hydroxypropylmethyl-cellulose, hydroxypropylcellulose, methylcellulose, and carboxymethyl-cellulose), alginic acid, lactose and talc. Additionally, a pharmaceutically acceptable surfactant (for example, lecithin) may be added to facilitate the degradation of such microparticles.

[0269] Proteins, which are water insoluble, such as zein, can also be used as materials for the formation of drug containing microparticles. Additionally, proteins, polysaccharides and combinations thereof, which are water-soluble, can be formulated with drug into microparticles and subsequently cross-linked to form an insoluble network. For example, cyclodextrins can be complexed with individual drug molecules and subsequently crosslinked.

[0270] Encapsulation or incorporation of peptides into carrier materials to produce peptide- containing microparticles can be achieved through known pharmaceutical formulation techniques. In the case of formulation in fats, waxes or wax -like materials, the carrier material is typically heated above its melting temperature and the drug is added to form a mixture comprising drug particles suspended in the carrier material, drug dissolved in the carrier material, or a mixture thereof. Microparticles can be subsequently formulated through several methods including, but not limited to, the processes of congealing, extrusion, spray chilling or aqueous dispersion. In a preferred process, wax is heated above its melting temperature, drug is added, and the molten wax-drug mixture is congealed under constant stirring as the mixture cools. Alternatively, the molten wax-drug mixture can be extruded and spheronized to form pellets or beads. These processes are known in the art.

[0271] For some carrier materials it may be desirable to use a solvent evaporation technique to produce drug-containing microparticles. In this case drug and carrier material are codissolved in a mutual solvent and microparticles can subsequently be produced by several techniques including, but not limited to, forming an emulsion in water or other appropriate media, spray drying or by evaporating off the solvent from the bulk solution and milling the resulting material.

[0272] In some embodiments, peptides in a particulate form is homogeneously dispersed in a w ater-insoluble or slowly water soluble material. To minimize the size of the drug particles within the composition, the drug powder itself may be milled to generate fine particles prior to formulation. The process of jet milling, known in the pharmaceutical art, can be used for this purpose. In some embodiments, drug in a particulate form is homogeneously dispersed in a wax or wax like substance by heating the wax or wax like substance above its melting point and adding the drug particles while stirring the mixture. In this case a pharmaceutically acceptable surfactant may be added to the mixture to facilitate the dispersion of the drug particles. The particles can also be coated with one or more modified release coatings. Solid esters of fatty acids, which are hydrolyzed by lipases, can be spray coated onto microparticles or drug particles. Zein is an example of a naturally water-insoluble protein. It can be coated onto drug containing microparticles or drug particles by spray coating or by wet granulation techniques. In addition to naturally water-insoluble materials, some substrates of digestive enzymes can be treated with cross-linking procedures, resulting in the formation of nonsoluble networks. Many methods of cross-linking proteins, initiated by both chemical and physical means, have been reported. One of the most common methods to obtain cross-linking is the use of chemical cross-linking agents. Examples of chemical cross-linking agents include aldehydes (gluteraldehyde and formaldehyde), epoxy compounds, carbodiimides, and genipin. In addition to these cross-linking agents, oxidized and native sugars have been used to cross-link gelatin. Cross-linking can also be accomplished using enzymatic means; for example, transglutaminase has been approved as a GRAS substance for cross-linking seafood products. Finally, cross-linking can be initiated by physical means such as thermal treatment, UV irradiation and gamma irradiation.

[0273] Polysaccharides can also be cross-linked to form a water-insoluble network. For many polysaccharides, this can be accomplished by reaction with calcium salts or multivalent cations, which cross-link the main polymer chains. Pectin, alginate, dextran, amylose and guar gum are subject to cross-linking in the presence of multivalent cations. Complexes between oppositely charged polysaccharides can also be formed; pectin and chitosan, for example, can be complexed via electrostatic interactions.

[0274] The peptides described herein can be incorporated into injectable / implantable solid or semi-solid implants, such as polymeric implants. In one embodiment, the peptides are incorporated into a polymer that is a liquid or paste at room temperature, but upon contact with aqueous medium, such as physiological fluids, exhibits an increase in viscosity to form a semi-solid or solid material. Exemplary polymers include, but are not limited to, hydroxyalkanoic acid polyesters derived from the copolymerization of at least one unsaturated hydroxy fatty acid copolymerized with hydroxyalkanoic acids. The polymer can be melted, mixed with the active substance and cast or injection molded into a device. Such melt fabrication require polymers having a melting point that is below the temperature at which the substance to be delivered and polymer degrade or become reactive. The device can also be prepared by solvent casting where the polymer is dissolved in a solvent and the drug dissolved or dispersed in the polymer solution and the solvent is then evaporated. Solvent processes require that the polymer be soluble in organic solvents. Another method is compression molding of a mixed powder of the polymer and the drug or polymer particles loaded with the active agent.

[0275] Alternatively, the peptides can be incorporated into a polymer matrix and molded, compressed, or extruded into a device that is a solid at room temperature. For example, the peptides can be incorporated into a biodegradable polymer, such as polyanhydrides, polyhydroalkanoic acids (PHAs), PLA, PGA. PLGA, polycaprolactone, polyesters, polyamides, polyorthoesters, polyphosphazenes, proteins and polysaccharides such as collagen, hyaluronic acid, albumin and gelatin, and combinations thereof and compressed into solid device, such as disks, wafers, or extruded into a device, such as rods.

[0276] The release of the peptides from the implant can be varied by selection of the polymer, the molecular weight of the polymer, and / or modification of the polymer to increase degradation, such as the formation of pores and / or incorporation of hydrolyzable linkages. Methods for modifying the properties of biodegradable polymers to vary the release profile of the peptides from the implant are well known in the art.

[0277] In some embodiments, the pharmaceutical compositions can be administered locally. In some embodiments, the peptides are incorporated in a delivery system such as gels, nanoparticles, microparticles, or implants such as (e.g., rods, discs, wafers, orthopedic implants) for sustained release. In some embodiments, the peptides can be administered using a local delivery implantable system comprising the peptides incorporated within a gel, nanoparticles, microparticles, or an implant. In some embodiments, the pharmaceutical compositions comprise a delivery system such as gels, nanoparticles, microparticles, or implants such as (e.g., rods, discs, wafers, orthopedic implants) for sustained release of the active agent or a pharmaceutically acceptable salt or derivative thereof.

[0278] Methods of Use

[0279] In some embodiments, the peptide described herein is useful in any in vitro or in vivo application in which it is desirable to modify a polynucleotide, such as in a site-specific, i.e. "targeted", way, for example gene knock-out, gene knock-in, gene editing, gene tagging, sequence replacement, etc., as used in, for example, gene therapy, e.g. to treat a disease or as an antiviral, antipathogenic, or anticancer therapeutic, the production of genetically modified organisms in agriculture, the large scale production of proteins by cells for therapeutic, diagnostic, or research purposes, the induction of iPS cells, biological research, the targeting of genes of pathogens for deletion or replacement, etc.

[0280] The methods described herein may be used to add. i.e. insert or replace, nucleic acid material to a target polynucleotide (e.g. to "knock in" a nucleic acid that encodes for a protein, an siRNA, an miRNA, etc ), to add a tag (e.g., 6xHis, a fluorescent protein (e.g., a green fluorescent protein; a yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), to add a regulatory sequence to a gene (e.g. promoter, polyadenylation signal, internal ribosome entry sequence (IRES), 2A peptide, start codon, stop codon, splice signal, localization signal, etc.), to modify a polynucleotide (e.g., introduce a mutation), and the like.

[0281] In some embodiments, the peptides described herein modifies a target polynucleotide, leading to, for example, polynucleotide cleavage, polynucleotide methylation, polynucleotide damage, polynucleotide repair, etc. In other embodiments, the peptides described herein modifies a target polypeptide associated with a target polynucleotide (e.g., ahistone, a DNA- binding protein, etc.), leading to, for example, histone methylation, histone acetylation, histone ubiquitination, and the like. In some embodiments, cleavage of polynucleotide (e.g., DNA) by the peptide may be used to delete nucleic acid material from a target polynucleotide (e.g., to disrupt a gene that makes cells susceptible to infection (e.g. the CCRS or CXCR4 gene, which makes T cells susceptible to HIV infection), to remove disease-causing trinucleotide repeat sequences in neurons, to create gene knockouts and mutations as disease models in research, etc.) by cleaving the target polynucleotide and allowing the cell to repair the sequence in the absence of an exogenously provided donor polynucleotide. Thus, the methods can be used to knock out a gene (resulting in complete lack of transcription or altered transcription) or to knock in genetic material into a locus of choice in the target polynucleotide (e.g., DNA).

[0282] Described herein are methods of altering expression of at least one gene product comprising contacting a peptide described herein with a target polynucleotide encoding the gene product.

[0283] Transcripts and encoded polypeptides may be collectively referred to as ‘"gene product.”

[0284] Examples of target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide. Examples of target polynucleotides include a disease associated gene or polynucleotide. A "‘disease-associated” gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non disease control. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and / or progression of the disease. A disease- associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease. The transcribed or translated products may be known or unknow n, and may be at a normal or abnormal level. In some embodiments, the target polynucleotide is a viral sequence present in a eukaryotic cell. In some embodiments, the target polynucleotide is a proto-oncogene or an oncogene.

[0285] Examples of disease-associated genes and polynucleotides are available from McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), available on the World Wide Web.

[0286] Examples of disease-associated genes and polynucleotides are listed in Tables A and B. Disease specific information is available from McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), available on the World Wide Web. Examples of signaling biochemical pathway -associated genes and polynucleotides are listed in Table C. Mutations in these genes and pathways can result in production of improper proteins or proteins in improper amounts which affect function. Such genes, proteins and pathways may be the target polynucleotide.

[0287] Table A.

[0288] Table B.

[0289]

[0290] Table C.

[0291] Described herein are also methods for treating and / or preventing cancer in a subject, the methods including administering to the subject an effective amount of a peptides described herein, or an effective amount of a pharmaceutical composition described herein.

[0292] In some embodiments, the peptides, compositions and methods described herein are useful in treating or preventing a cancer. In some embodiments, the compositions herein are used to treat both local and metastatic tumors. In some cases, the cancer is a circulating cancer cell (circulating tumor cell). In some embodiments, the compounds, compositions and methods described herein are useful for treating or preventing metastasis or recurrence of a cancer. In some embodiments, the compounds, compositions and methods described herein are useful for the prevention of recurrence of excised solid tumors. In some embodiments, the compounds, compositions and methods described herein are useful for the prevention of metastasis of excised solid tumors.

[0293] In one aspect, the methods described herein are used to treat cancer, for example, melanoma, lung cancer (including lung adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, bronchogenic carcinoma, non-small-cell carcinoma, small cell carcinoma, mesothelioma); breast cancer (including ductal carcinoma, lobular carcinoma, inflammatory breast cancer, clear cell carcinoma, mucinous carcinoma, serosal cavities breast carcinoma); colorectal cancer (colon cancer, rectal cancer, colorectal adenocarcinoma); anal cancer; pancreatic cancer (including pancreatic adenocarcinoma, islet cell carcinoma, neuroendocrine tumors); prostate cancer; prostate adenocarcinoma; ovarian carcinoma (ovarian epithelial carcinoma or surface epithelial-stromal tumor including serous tumor, endometrioid tumor and mucinous cystadenocarcinoma, sex-cord-stromal tumor); liver and bile duct carcinoma (including hepatocellular carcinoma, cholangiocarcinoma, hemangioma); esophageal carcinoma (including esophageal adenocarcinoma and squamous cell carcinoma); oral and oropharyngeal squamous cell carcinoma; salivary gland adenoid cystic carcinoma; bladder cancer; bladder carcinoma; carcinoma of the uterus (including endometrial adenocarcinoma, ocular, uterine papillary serous carcinoma, uterine clear-cell carcinoma, uterine sarcomas, leiomyosarcomas, mixed mullerian tumors); glioma, glioblastoma, medulloblastoma, and other tumors of the brain; kidney cancers (including renal cell carcinoma, clear cell carcinoma, Wilm's tumor); cancer of the head and neck (including squamous cell carcinomas); cancer of the stomach (gastric cancers, stomach adenocarcinoma, gastrointestinal stromal tumor); testicular cancer; germ cell tumor; neuroendocrine tumor; cervical cancer; carcinoids of the gastrointestinal tract, breast, and other organs; signet ring cell carcinoma; mesenchymal tumors including sarcomas, fibrosarcomas, haemangioma, angiomatosis, haemangiopericytoma, pseudoangiomatous stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma, leiomysarcoma, skin, including melanoma, cervical, retinoblastoma, head and neck cancer, pancreatic, brain, thyroid, testicular, renal, bladder, soft tissue, adenal gland, urethra, cancers of the penis, myxosarcoma, chondrosarcoma, osteosarcoma. chordoma, malignant fibrous histiocytoma, lymphangiosarcoma, mesothelioma, squamous cell carcinoma; epidermoid carcinoma, malignant skin adnexal tumors, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, hypernephroma, cholangiocarcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal cell carcinoma, glioma anaplastic; glioblastoma multiforme,, neuroblastoma, medulloblastoma, malignant meningioma, malignant schwannoma, neurofibrosarcoma, parathyroid carcinoma, medullary carcinoma of thyroid, bronchial carcinoid, pheochromocytoma, Islet cell carcinoma, malignant carcinoid, malignant paraganglioma, melanoma, Merkel cell neoplasm, cystosarcoma phylloide, salivary cancers, thymic carcinomas, and cancers of the vagina among others.

[0294] Methods of Administration

[0295] The compositions as used in the methods described herein can be administered by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the active components described herein can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art including, for example, oral and parenteral routes of administering. As used herein, the term "‘parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrastemal administration, such as by injection. The active agent may be administered by any route. In some embodiments, the active ingredient is administered via a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal. rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and / or drops), mucosal, nasal, bucal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the active ingredient (e.g., its stability in the environment of the gastrointestinal tract), the condition of the subject (e.g., whether the subject is able to tolerate oral administration), etc. Administration of the active components of their compositions can be a single administration, or at continuous and distinct intervals as can be readily determined by a person skilled in the art.

[0296] In certain embodiments, it may be desirable to provide continuous delivery of one or more compounds to a patient in need thereof. For intravenous or intraarterial routes, this can be accomplished using drip systems, such as by intravenous administration. For topical applications, repeated application can be done or a patch can be used to provide continuous administration of the compounds over an extended period of time.

[0297] The peptides may be administered in such amounts, time, and route deemed necessary in order to achieve the desired result. The exact amount of the peptide will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity- of the infection, the particular active ingredient, its mode of administration, its mode of activity, and the like. The active ingredient, whether the active compound itself, or the active compound in combination with an agent, is preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the active ingredient will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity- of the disorder; the activity of the active ingredient employed; the specific composition employed; the age, body w eight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts. The exact amount of a peptide required to achieve a therapeutically or prophy lactically effective amount will vary from subject to subject, depending on species, age. and general condition of a subject, severity of the side effects or disorder, identity of the particular compound(s), mode of administration, and the like. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[0298] Useful dosages of the compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.

[0299] The dosage ranges for the administration of the compositions are those targe enough to produce the desired effect in which the symptoms or disorder are affected. The dosage should not be so targe as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.

[0300] In some embodiments, the peptide as used in the methods described herein may be administered in combination or alternation with one or more additional active agents. Representative examples of additional active agents include antimicrobial agents(including antibiotics, antiviral agents, and antifungal agents), anti-inflammatory agents (including steroids and non-steroidal anti-inflammatory agents), anti-coagulant agents, and anti-cancer agents.

[0301] Exemplary cancer drugs or anti-cancer agents can include, but are not limited to, antimetabolite anti- cancer agents and antimitotic anti-cancer agents, and combinations thereof. Various antimetabolite and antimitotic anti-cancer agents, including single such agents or combinations of such agents, may be employed in the methods and compositions described herein.

[0302] Antimetabohc anti-cancer agents typically structurally resemble natural metabolites, which are involved in normal metabolic processes of cancer cells such as the synthesis of nucleic acids and proteins. The antimetabolites, however, differ enough from the natural metabolites such that they interfere with the metabolic processes of cancer cells. In the cell, antimetabolites are mistaken for the metabolites they resemble, and are processed by the cell in a manner analogous to the normal compounds. The presence of the '‘decoy’’ metabolites prevents the cells from carrying out vital functions and the cells are unable to grow and survive. For example, antimetabolites may exert cytotoxic activity by substituting these fraudulent nucleotides into cellular DNA, thereby disrupting cellular division, or by inhibition of critical cellular enzymes, which prevents replication of DNA.

[0303] In one aspect, therefore, the antimetabolite anti-cancer agent is a nucleotide or a nucleotide analog. In certain aspects, for example, the antimetabolite agent may comprise purine (e.g.. guanine or adenosine) or analogs thereof, or pyrimidine (cytidine or thymidine) or analogs thereof, with or without an attached sugar moiety.

[0304] Suitable antimetabolite anti-cancer agents for use in the present disclosure may be generally classified according to the metabolic process they affect, and can include, but are not limited to, analogues and derivatives of folic acid, pyrimidines, purines, and cytidine. Thus, in one aspect, the antimetabolite agent(s) is selected from the group consisting of cytidine analogs, folic acid analogs, purine analogs, pyrimidine analogs, and combinations thereof.

[0305] In one particular aspect, for example, the antimetabolite agent is a cytidine analog. According to this aspect, for example, the cytidine analog may be selected from the group consisting of cytarabine (cytosine arabinodside), azacitidine (5 -azacytidine), and salts, analogs, and derivatives thereof.

[0306] In another particular aspect, for example, the antimetabolite agent is a folic acid analog. Folic acid analogs or antifolates generally function by inhibiting dihydrofolate reductase (DHFR), an enzyme involved in the formation of nucleotides; when this enzyme is blocked, nucleotides are not formed, disrupting DNA replication and cell division. According to certain aspects, for example, the folic acid analog may be selected from the group consisting of denopterin, methotrexate (amethopterin), pemetrexed. pteropterin, raltitrexed, trimetrexate, and salts, analogs, and derivatives thereof.

[0307] In another particular aspect, for example, the antimetabolite agent is a purine analog. Purine-based antimetabolite agents function by inhibiting DNA synthesis, for example, by interfering with the production of purine containing nucleotides, adenine and guanine which halts DNA synthesis and thereby cell division. Purine analogs can also be incorporated into the DNA molecule itself during DNA synthesis, which can interfere with cell division. According to certain aspects, for example, the purine analog may be selected from the group consisting of acyclovir, allopurinol, 2-aminoadenosine, arabinosyl adenine (ara-A), azacitidine, azathiprine, 8-aza-adenosine, 8-fluoro-adenosine, 8-methoxy-adenosine, 8-oxo- adenosine, cladribine, deoxycoformycin, fludarabine, gancylovir, 8-aza-guanosine, 8-fluoro- guanosine, 8- methoxy-guanosine, 8-oxo-guanosine, guanosine diphosphate, guanosine diphosphate-beta- L-2-aminofucose, guanosine diphosphate-D-arabinose. guanosine diphosphate-2- fluorofucose, guanosine diphosphate fucose, mercaptopurine (6-MP), pentostatin, thiamiprine, thioguanine (6-TG), and salts, analogs, and derivatives thereof.

[0308] In yet another particular aspect, for example, the antimetabolite agent is a pyrimidine analog. Similar to the purine analogs discussed above, pyrimidine-based antimetabolite agents block the synthesis of pyrimidine-containing nucleotides (cytosine and thymine in DNA; cytosine and uracil in RNA). By acting as '‘decoys,” the pyrimidine-based compounds can prevent the production of nucleotides, and / or can be incorporated into a growing DNA chain and lead to its termination. According to certain aspects, for example, the pyrimidine analog may be selected from the group consisting of ancitabine. azacitidine, 6-azauridine, bromouracil (e.g., 5-bromouracil), capecitabine, carmofur, chlorouracil (e.g. 5 -chlorouracil), cytarabine (cytosine arabinoside), cytosine, di deoxy uridine, 3 '-azi do-3 '-deoxy thy mi dine, 3'- dideoxycytidin-2'-ene, 3'-deoxy-3'-deoxythymidin-2'-ene, dihydrouracil, doxifluridine, enocitabine. floxuridine, 5-fluorocytosine, 2-fluorodeoxycytidine, 3-fluoro-3'- deoxythymidine, fluorouracil (e.g., 5 -fluorouracil (also known as 5-FU), gemcitabine, 5- methylcytosine, 5- propynylcytosine, 5-propynylthymine, 5-propynyluracil, thymine, uracil, uridine, and salts, analogs, and derivatives thereof. In one aspect, the pyrimidine analog is other than 5- fluorouracil. In another aspect, the pyrimidine analog is gemcitabine or a salt thereof.

[0309] In certain aspects, the antimetabolite agent is selected from the group consisting of 5- fluorouracil, capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed, and salts, analogs, derivatives, and combinations thereof. In other aspects, the antimetabolite agent is selected from the group consisting of capecitabine. 6- mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed, and salts, analogs, derivatives, and combinations thereof. In one particular aspect, the antimetabolite agent is other than 5-fluorouracil. In a particularly preferred aspect, the antimetabolite agent is gemcitabine or a salt or thereof (e.g.. gemcitabine HC1 (Gemzar®)).

[0310] Other antimetabolite anti-cancer agents may be selected from, but are not limited to, the group consisting of acanthifolic acid, aminothiadiazole, brequinar sodium, Ciba-Geigy CGP-30694, cyclopentyl cytosine, cytarabine phosphate stearate, cytarabine conjugates, Lilly DATHF, Merrel Dow DDFC, dezaguanine, dideoxy cytidine, dideoxy guanosine, didox, Yoshitomi DMDC, Wellcome EHNA. Merck & Co. EX-015, fazarabine. fludarabine phosphate, N-(2'-furanidyl)-5-fluorouracil, Daiichi Seiyaku FO-152, 5-FU-fibrinogen, isopropyl pyrrolizine, Lilly LY-188011; Lilly LY-264618, methobenzaprim, Wellcome MZPES, norspermidine, NCI NSC-127716, NCI NSC-264880, NCI NSC-39661, NCI NSC- 612567, Warner-Lambert PALA, pentostatin, piritrexim, plicamycin, Asahi Chemical PL- AC, Takeda TAC-788, tiazofurin, Erbamont TIF, tyrosine kinase inhibitors, Taiho UFT and uricytin, among others.

[0311] In one aspect, the antimitotic anti-cancer agent is a microtubule inhibitor or a microtubule stabilizer. In general, microtubule stabilizers, such as taxanes and epothilones, bind to the interior surface of the beta-microtubule chain and enhance microtubule assembly by promoting the nucleation and elongation phases of the polymerization reaction and by reducing the critical tubulin subunit concentration required for microtubules to assemble. Unlike mictrotubule inhibitors, such as the vinca alkaloids, which prevent microtubule assembly, the microtubule stabilizers, such as taxanes, decrease the lag time and dramatically shift the dynamic equilibrium between tubulin dimers and microtubule polymers towards polymerization. In one aspect, therefore, the microtubule stabilizer is a taxane or an epothilone. In another aspect, the microtubule inhibitor is a vinca alkaloid.

[0312] In some embodiments, the anti-cancer agent may comprise a taxane or derivative or analog thereof. The taxane may be a naturally derived compound or a related form, or may be a chemically synthesized compound or a derivative thereof, with antineoplastic properties. The taxanes are a family of terpenes, including, but not limited to paclitaxel (Taxol™) and docetaxel (Taxotere™), which are derived primarily from the Pacific yew tree, Taxus brevifolia, and which have activity against certain tumors, particularly breast and ovarian tumors. In one aspect, the taxane is docetaxel or paclitaxel. Paclitaxel is a preferred taxane and is considered an antimitotic agent that promotes the assembly of microtubules from tubulin dimers and stabilizes microtubules by preventing depolymerization. This stability results in the inhibition of the normal dynamic reorganization of the microtubule network that is essential for vital interphase and mitotic cellular functions.

[0313] Also included are a variety of known taxane derivatives, including both hydrophilic derivatives, and hydrophobic derivatives. Taxane derivatives include, but are not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99 / 18113; piperazino and other derivatives described in WO 99 / 14209; taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and U.S. Pat. No. 5,869,680; 6-thio derivatives described in WO 98 / 28288; sulfenamide derivatives described in U.S. Pat. No. 5,821,263; deoxygenated paclitaxel compounds such as those described in U.S. Pat. No. 5,440.056; and taxol derivatives described in U.S. Pat. No. 5,415,869. As noted above, it further includes prodrugs of paclitaxel including, but not limited to, those described in WO 98 / 58927; WO 98 / 13059; and U.S. Pat. No. 5,824.701. The taxane may also be a taxane conjugate such as, for example, paclitaxel-PEG, paclitaxel-dextran, paclitaxel-xylose, docetaxel-PEG, docetaxel- dextran, docetaxel-xylose, and the like. Other derivatives are mentioned in “Synthesis and Anticancer Activity of Taxol Derivatives,” D. G. I. Kingston et al., Studies in Organic Chemistry, vol. 26, entitled “New Trends in Natural Products Chemistry” (1986), Atta-ur-Rabman, P. W. le Quesne, Eds. (Elsevier. Amsterdam 1986). among other references. Each of these references is hereby incorporated by reference herein in its entirety.

[0314] Various taxanes may be readily prepared utilizing techniques known to those skilled in the art (see also WO 94 / 07882, WO 94 / 07881, WO 94 / 07880, WO 94 / 07876, WO 93 / 23555. WO 93 / 10076; U.S. Pat. Nos. 5,294,637; 5,283,253; 5,279.949; 5.274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267) (each of which is hereby incorporated by reference herein in its entirety), or obtained from a variety of commercial sources, including for example, Sigma-Aldrich Co., St. Louis, Mo.

[0315] Alternatively, the antimitotic anti-cancer agent can be a microtubule inhibitor; in one preferred aspect, the microtubule inhibitor is a vinca alkaloid. In general, the vinca alkaloids are mitotic spindle poisons. The vinca alkaloid agents act during mitosis when chromosomes are split and begin to migrate along the tubules of the mitosis spindle towards one of its poles, prior to cell separation. Under the action of these spindle poisons, the spindle becomes disorganized by the dispersion of chromosomes during mitosis, affecting cellular reproduction. According to certain aspects, for example, the vinca alkaloid is selected from the group consisting of vinblastine, vincristine, vindesine, vinorelbine, and salts, analogs, and derivatives thereof.

[0316] The antimitotic anti-cancer agent can also be an epothilone. In general, members of the epothilone class of compounds stabilize microtubule function according to mechanisms similar to those of the taxanes. Epothilones can also cause cell cycle arrest at the G2-M transition phase, leading to cytotoxicity and eventually apoptosis. Suitable epithiolones include epothilone A, epothilone B, epothilone C, epothilone D, epothilone E. and epothilone F, and salts, analogs, and derivatives thereof. One particular epothilone analog is an epothilone B analog, ixabepilone (Ixempra™).

[0317] In certain aspects, the antimitotic anti-cancer agent is selected from the group consisting of taxanes, epothilones, vinca alkaloids, and salts and combinations thereof. Thus, for example, in one aspect the antimitotic agent is a taxane. More preferably in this aspect the antimitotic agent is paclitaxel or docetaxel, still more preferably paclitaxel. In another aspect, the antimitotic agent is an epothilone (e.g., an epothilone B analog). In another aspect, the antimitotic agent is a vinca alkaloid.

[0318] Examples of cancer drugs that may be used in the present disclosure include, but are not limited to: thalidomide; platinum coordination complexes such as cisplatin (cis-DDP), oxaliplatin and carboplatin; anthracenediones such as mitoxantrone; substituted ureas such as hydroxyurea; methylhydrazine derivatives such as procarbazine (N- methylhydrazine, MIH); adrenocortical suppressants such as mitotane (o,p'-DDD) and aminoglutethimide; RXR agonists such as bexarotene; and tyrosine kinase inhibitors such as sunitinib and imatinib.

[0319] Examples of additional cancer drugs include alkylating agents, antimetabolites, natural products, hormones and antagonists, and miscellaneous agents. Alternate names are indicated in parentheses. Examples of alkylating agents include nitrogen mustards such as mechlorethamine, cyclophosphainide, ifosfamide, melphalan sarcolysin) and chlorambucil; ethylenimines and methylmelamines such as hexamethylmelamine and thiotepa; alky 1 sulfonates such as busulfan; nitrosoureas such as carmustine (BCNU), semustine (methyl- CCNU), lomustine (CCNU) and streptozocin (streptozotocin); DNA synthesis antagonists such as estramustine phosphate; and triazines such as dacarbazine (DTIC, dimethyl- triazenoimidazolecarboxamide) and temozolomide. Examples of antimetabolites include folic acid analogs such as methotrexate (amethopterin); pyrimidine analogs such as fluorouracin (5 -fluorouracil, 5-FU, SFU), floxuridine (fluorodeoxyuridine, FUdR), cytarabine (cytosine arabinoside) and gemcitabine; purine analogs such as mercaptopurine (6-mercaptopurine, 6-MP), thioguanine (6-thioguanine, TG) and pentostatin (2'- deoxycoformycin, deoxycoformycin), cladribine and fludarabine; and topoisomerase inhibitors such as amsacrine. Examples of natural products include vinca alkaloids such as vinblastine (VLB) and vincristine; taxanes such as paclitaxel, protein bound paclitaxel (Abraxane) and docetaxel (Taxotere); epipodophyllotoxins such as etoposide and teniposide; camptothecins such as topotecan and irinotecan; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin), doxorubicin, histrelin, bleomycin, mitomycin (mitomycin C), idarubicin. epirubicin; enzymes such as L- asparaginase; and biological response modifiers such as interferon alpha and interlelukin 2. Examples of hormones and antagonists include luteinising releasing hormone agonists such as buserelin; adrenocorticosteroids such as prednisone and related preparations; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol and related preparations; estrogen antagonists such as tamoxifen and anastrozole; androgens such as testosterone propionate and fluoxymesterone and related preparations; androgen antagonists such as flutamide and bicalutamide; and gonadotropin-releasing hormone analogs such as leuprolide. Alternate names and trade-names of these and additional examples of cancer drugs, and their methods of use including dosing and administration regimens, will be known to a person versed in the art.

[0320] In some aspects, the anti-cancer agent may comprise a chemotherapeutic agent. Suitable chemotherapeutic agents include, but are not limited to. alkylating agents, antibiotic agents, antimetabolic agents, hormonal agents, plant-derived agents and their synthetic derivatives, anti-angiogenic agents, differentiation inducing agents, cell growth arrest inducing agents, apoptosis inducing agents, cytotoxic agents, agents affecting cell bioenergetics i.e., affecting cellular ATP levels and molecules / activities regulating these levels, biologic agents, e.g., monoclonal antibodies, kinase inhibitors and inhibitors of growth factors and their receptors, gene therapy agents, cell therapy, e.g., stem cells, or any combination thereof.

[0321] According to these aspects, the chemotherapeutic agent is selected from the group consisting of cyclophosphamide, chlorambucil, melphalan, mechlorethamine, ifosfamide, busulfan, lomustine, streptozocin, temozolomide, dacarbazine, cisplatin, carboplatin, oxaliplatin, procarbazine, uramustine, methotrexate, pemetrexed, fludarabine, cytarabine, fluorouracil, floxuridine, gemcitabine, capecitabine, vinblastine, vincristine, vinorelbine, etoposide, paclitaxel, docetaxel, doxorubicin, daunorubicin. epirubicin. idarubicin, mitoxantrone, bleomycin, mitomycin, hydroxyurea, topotecan, irinotecan, amsacrine, teniposide, erlotinib hydrochloride and combinations thereof. Each possibility represents a separate aspect of the invention.

[0322] Anti-neoplastic agent can be selected from the group consisting of Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Adrucil (Fluorouracil), Afatinib Dimaleate, Afmitor (Everolimus), Akynzeo (Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod), Aldesleukin. Alemtuzumab, Alimta (Pemetrexed Disodium), Aloxi (Palonosetron Hydrochloride), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide. Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi, Avastin (Bevacizumab), Axitinib, Azacitidine, BEACOPP, Becenum (Carmustine), Beleodaq (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, Bevacizumab, Bexarotene. Bexxar (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BiCNU (Carmustine), Bleomycin, Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif (Bosutinib), Bosutinib, Brentuximab Vedotin, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabozantinib-S -Malate, CAF, Campath (Alemtuzumab), Camptosar (Irinotecan Hydrochloride). Capecitabine, CAPOX, Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine. Carmustine Implant. Casodex (Bicalutamide), CeeNU (Lomustine), Ceritinib, Cerubidine (Daunorubicin Hydrochloride), Cervarix (Recombinant HPV Bivalent Vaccine), Cetuximab, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar (Clofarabine), CMF, Cometriq (Cabozantinib-S - Malate), COPP, COPP-ABV, Cosmegen (Dactinomycin), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine, Liposomal, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Dasatinib. Daunorubicin Hydrochloride, Decitabine, Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (Liposomal Cytarabine), DepoFoam (Liposomal Cytarabine), Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), DTIC-Dome (Dacarbazine), Efudex (Fluorouracil), Elitek (Rasburicase). Ellence (Epirubicin Hydrochloride), Eloxatin (Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Enzalutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi), Etopophos (Etoposide Phosphate). Etoposide. Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Evista (Raloxifene Hydrochloride), Exemestane, Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil), Fluorouracil, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI. FOLFIRI-BEVACIZUMAB. FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nonaval ent Vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN. Gemtuzumab Ozogamicin. Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel (Carmustine Implant), Gliadel wafer (Carmustine Implant), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate), Herceptin (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonaval ent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, Hycamtin (Topotecan Hydrochloride), Hyper-CVAD, Ibrance (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride), Idamycin (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), Imatimb Mesylate, Imbruvica (Ibrutinib), Imiquimod, Inlyta (Axitinib), Interferon Alfa-2b, Recombinant, Intron A (Recombinant Interferon Alfa- 2b), Iodine 1 131 Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Istodax (Romidepsin), Ixabepilone. Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepi vance (Palifermin), Keytruda (Pembrolizumab), Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Lenalidomide, Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil). Leuprolide Acetate, Levulan (Aminolevulinic Acid). Linfolizin (Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposome), Liposomal Cytarabine, Lomustine, Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), Lupron Depot- Ped (Leuprolide Acetate), Lupron Depot-3 Month (Leuprolide Acetate), Lupron Depot-4 Month (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megace (Megestrol Acetate), Megestrol Acetate, Mekinist (Trametinib), Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate), Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C). MOPP, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin-stabilized Nanoparticle Formulation), Navelbine (Vinorelbine Tartrate), Nelarabine, Neosar (Cyclophosphamide), Netupitant and Palonosetron Hydrochloride, Neupogen (Filgrastim). Nexavar (Sorafenib Tosylate). Nilotinib, Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride, Ontak (Denileukin Diftitox), Opdivo (Nivolumab), OPPA. Oxaliplatin, Paclitaxel. Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin). Paraplatin (Carboplatin), Pazopanib Hydrochloride, Pegaspargase, Peginterferon Alfa- 2b, PEG-Intron (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, Peg eta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Pralatrexate, Prednisone, Procarbazine Hydrochloride, Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine). Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonaval ent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Rituxan (Rituximab), Rituximab, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Ruxolitinib Phosphate, Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, Somatuline Depot (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon Alfa-2b), Sylvant (Siltuximab), Synovir (Thalidomide), Synribo (Omacetaxine Mepesuccinate), TAC, Tafmlar (Dabrafenib), Talc, Tamoxifen Citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Temodar (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thiotepa, Toposar (Etoposide), Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and Iodine I 131 Tositumomab, Totect (Dexrazoxane Hydrochloride). TPF, Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride), Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Unituxin (Dinutuximab), Vandetanib, VAMP, Vectibix (Panitumumab), VelP, Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (Vinblastine Sulfate), Vemurafenib, VePesid (Etoposide), Viadur (Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, Voraxaze (Glucarpidase), Vorinostat, Votrient (Pazopanib Hydrochloride), Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib), Xeloda (Capecitabine), XELIRI, XELOX, Xgeva (Denosumab), Xofigo (Radium 223 Dichloride). Xtandi (Enzalutamide), Yervoy (Ipilimumab), Zaltrap (Ziv- Aflibercept), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept, Zofran (Ondansetron Hydrochloride), Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic Acid), Zydelig (Idelalisib), Zykadia (Ceritinib), and Zytiga (Abiraterone Acetate).

[0323] Growth factors useful as therapeutic agents include, but are not limited to, transforming growth factor-a (“TGF-a”), transforming growth factors (“TGF-0”), platelet- derived growth factors ( PDGF"). fibroblast growth factors (“FGF”), including FGF acidic isoforms 1 and 2. FGF basic form 2 and FGF 4. 8, 9 and 10, nerve growth factors (“NGF”) including NGF 2.5s, NGF 7. Os and beta NGF and neurotrophins, brain derived neurotrophic factor, cartilage derived factor, bone growth factors (BGF), basic fibroblast growth factor, insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), granulocyte colony stimulating factor (G-CSF), insulin like growth factor (IGF) I and II, hepatocyte growth factor, glial neurotrophic growth factor (GDNF), stem cell factor (SCF), keratinocyte growth factor (KGF), transforming growth factors (TGF), including TGFs alpha, beta, betal, beta2, beta3, skeletal growth factor, bone matrix derived growth factors, and bone derived growth factors and mixtures thereof.

[0324] Immunoglobulins useful in the present disclosure include, but are not limited to, IgG, IgA, IgM, IgD, IgE, and mixtures thereof. Some preferred growth factors include VEGF (vascular endothelial growth factor), NGFs (nerve growth factors), PDGF-AA, PDGF-BB, PDGF-AB, FGFb, FGFa, and BGF

[0325] Other molecules useful as anti-cancer agents include but are not limited to growth hormones, leptin, leukemia inhibitory factor (LIF), tumor necrosis factor alpha and beta, endostatin, thrombospondin, osteogenic protein- 1, bone morphogenetic proteins 2 and 7, osteonectin, somatomedin-like peptide, osteocalcin.

[0326] Tumor antigens can be based on specific mutations (neoepitopes) and those expressed by cancer-germline genes (antigens common to tumors found in multiple patients, referred to herein as ‘‘traditional cancer antigens” or “shared cancer antigens”). In some embodiments, a traditional antigen is one that is known to be found in cancers or tumors generally or in a specific type of cancer or tumor. In some embodiments, a traditional cancer antigen is a nonmutated tumor antigen. In some embodiments, a traditional cancer antigen is a mutated tumor antigen.

[0327] Diagnostic agents include gases; metals; commercially available imaging agents used in positron emissions tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray. fluoroscopy, and magnetic resonance imaging (MRI); and contrast agents. Examples of suitable materials for use as contrast agents in MRI include gadolinium chelates, as well as iron, magnesium, manganese, copper, and chromium. Examples of materials useful for CAT and x-ray imaging include iodine-based materials.

[0328] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

[0329] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

[0330] EXAMPLES

[0331] Example 1: Intracellular Delivery of Gene-Editing Enzymes and Uses Thereof

[0332] A family of highly efficient membrane translocation domains (MTDs) was recently- engineered for the intracellular delivery of essentially any soluble protein cargo (PCT Publication No. WO2023178327). These MTDs are based on the 10thfibronectin type 3 (FN3) domain of human fibronectin, an abundant cell surface protein present in most human cells. They were rendered cell permeable by grafting CPP motifs into their BC and FG loops. One of the most active MTDs, MTD4, has been utilized to efficiently deliver a wide variety of protein cargos into both the cytosol and nucleus of mammalian cells in vitro and in vivo. In this study, MTD4 was genetically^ fused to Cas9 and expressed the MTD4-Cas9 fusion protein in Escherichia coli and purified the fusion protein to near homogeneity7.

[0333] Results

[0334] Cloning, expression, and purification of MTD4-Cas9. The Cas9-coding region of DNA was amplified by the polymerase chain reaction (PCR) with the concomitant incorporation of two nuclear localization sequences (NLS) at the 3’ end of the Cas9 gene. The PCR product was inserted to the C-terminus of MTD4 in a pET15b vector. This cloning procedure generated a construct SEQ ID NO: 140 featuring a six-histidine tag and a thrombin cleavage site at the N-terminus, MTD4, a flexible linker SEQ ID NO: 107 (GGSGGSGGS), Cas9, and two NLS at the C-terminus. Structural simulation of MTD4-Cas9 using Phyre2. an online program that employs sequence homology to predict protein structures, revealed a well-folded 3D structure where both the MTD4 and Cas9 domains are fully exposed to the solvent and have no intramolecular interactions between the two domains (Figure 1).

[0335] MTD4-Cas9 was expressed in E. coll and purified to near homogeneity by ammonium sulfate fractionation and metal affinity chromatography on a Ni-NTA column (Figure 2A- 2C). The overall yield was ~3 mg MTD4-Cas9 from each liter of cell culture.

[0336] Intended Uses

[0337] It is expected that any of the gene-editing proteins or ribonucleoproteins may be rendered cell permeable by fusing one or more MTD to their N-terminus, C-terminus. and / or potentially an internal loop region. MTD-protein fusions have been shown to enter efficiently both animal (PCT Publication No. WO2023178327) and plant cells (PCT Patent Application No. PCT / US2023 / 066615; Attorney Docket No. 103361-267WOI). It is therefore expected that the MTDs may be applied to deliver the gene-editing nucleases into human cells for therapeutic purposes as well as plant cells to generate genetically edited plants.

[0338] Relative to the previous delivery platforms, the MTDs have very high cytosolic delivery' efficiencies. Since they are derived from an abundant human protein, the MTDs are expected to be safe and of low immunogenicity. MTDs are also thermodynamically and proteolytically stable and can be inexpensively produced in E. coll in high yields. The MTDs may provide an effective solution to the intracellular delivery problem.

[0339] SEQ ID NO: 140 (Amino acid sequence of MTD4-Cas9. Bold and Italics, MTD4 sequence; Bold, NLS.)

[0340] MGSSI II II II IHHSSGLNPRGSHMVSDVPRDLEWAATPTSLLISWDAPAWYWR YYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRRRRSKPISINY 7?rGGSGGSGGSELDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEE SFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLS KSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDD LDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEE LLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFR IPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLP NEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKV TVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILE DIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGS PAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREV KVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFV YGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNF LYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ

[0341] SITGLYETRIDLSQLGGDPKKKRKVPKKKRKV

[0342] Experimental Details

[0343] Cloning. The plasmid containing the coding sequence of Cas9 was obtained from Addgene (Plasmid #62374). The SacI endonuclease site within the coding region of Cas9 was first removed by QuikChange site-directed mutagenesis using primers A and B. Next, the modified Cas9 gene was amplified by PCR with primers C and D, resulting in the addition of SacI and Xhol restriction sites at the 5’ and 3’ termini of the Cas9 coding sequence, respectively, and two NLS at the C-terminus. The PCR product was digested with restriction enzymes SacI and Xhol and ligated into plasmid pET15b-MTD4 linearized with the same two enzymes. The authenticity of the DNA construct was confirmed by restriction mapping and sequencing of the coding region for MTD4-Cas9.

[0344] SEQ ID NO: 143 (Primer A (Forward))

[0345] 5’-CGAATCTTGGCGCTCCCGCTGCTTTTAAA-3’

[0346] SEQ ID NO: 144 (Primer B (Reverse))

[0347] 5 ’ -TTTAAAAGC AGCGGGAGCGC C AAGATTC -3 ’ SEQ ID NO: 145 (Primer C (Forward))

[0348] 5 ’ -ATATAAGAGCTCGATAAGAAATACTC AATAGGCTTAGATATCGGC ACAA ATAGCGT-3’

[0349] SEQ ID NO: 146 (Primer D (Reverse))

[0350] 5 ’ -ATTTATCTCGAGTTAC ACTTTACGTTTTTTTTTCGGCACTTTACGTTTTTTT TTCGGGTCACCTCCTAGCTGACTCAAATCAAT-3'

[0351] SEQ ID NO: 147 (DNA sequence of MTD4-Cas9)

[0352] ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGC GCGGCAGCCATATGGTCTCAGATGTACCCAGGGACCTAGAAGTTGTGGCCGCG ACTCCGACCAGCTTGCTGATTTCCTGGGATGCACCGGCGTGGTATTGGCGTTAT TACCGCATTACGTACGGCGAAACCGGTGGTAATAGCCCGGTTCAGGAGTTCACC GTGCCGGGTTCCAAAAGCACCGCTACGATCTCCGGCCTGAAACCGGGTGTAGA CTACACCATCACCGTCTATGCGGTGACCGGCCGTCGTCGACGCAGCAAGCCAAT CAGCATTAACTATCGTACCGGCGGTTCTGGTGGCTCTGGCGGTTCGGAGCTCGA TAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGG TGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATA CAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTG GAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACA CGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCG AAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAA GACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCT TATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCT ACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAG TTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGG ACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACC CTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTA AATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAAT GGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAAT CAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACG ATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGT TTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGT AAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGA TGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCC AGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTT

[0353] ATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTT

[0354] TAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGAT

[0355] TTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCAC

[0356] TTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTA

[0357] AAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTAT

[0358] GTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCT

[0359] GAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCA

[0360] GCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAA

[0361] AAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAA

[0362] TTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCA

[0363] GGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGT

[0364] AACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAG

[0365] TGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCA

[0366] TGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGA

[0367] AGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGAT

[0368] GATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAA

[0369] ACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGAT

[0370] TAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATC

[0371] AGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGAC

[0372] ATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTAC

[0373] ATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTAC

[0374] AGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCA

[0375] GAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCA

[0376] GAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAG

[0377] GAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAA

[0378] AGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAAT

[0379] TAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTT

[0380] CCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCG

[0381] TGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACT

[0382] ATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATT

[0383] TAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCA

[0384] AACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGG

[0385] ATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTA AAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATT CTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAA TGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTT TGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGA GCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAA CTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCT AATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATT TTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAA CAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAAT TCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGG TTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAA AGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTA TGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGAT ATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTG AGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAA GGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGT CATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTT TGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATT TTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATAT AACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTT ATTTACGTTGACGAATCTTGGCGCTCCCGCTGCTTTTAAATATTTTGATACAACA ATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATC CATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGA GGTGACCCGAAAAAAAAACGTAAAGTGCCGAAAAAAAAACGTAAAGTGTAA

[0386] Expression and Purification. Escherichia coli Rosetta (DE3) pLysS cells transformed with pET15b-MTD4-Cas9 were cultured in LB medium supplemented with 75 mg / L ampicillin and 35 mg / mL chloramphenicol at 37 °C. The culture was cooled to 18 °C when ODsoo reached 0 3 and induced by the addition of 0.5 mM IPTG for 18 h at 18 °C. The cells were pelleted by centrifugation. The cell pellet was resuspended in 50 niL of lysis buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 25 mM imidazole, 3 mM p-mercaptoethanol, protease inhibitor cocktail tablets, and 10 mg / 'ml lysozyme] per liter of cell culture. The cells were briefly sonicated and centrifuged at 14,000g for 30 min Ammonium sulfate was gradually added to the soluble cell lysate with constant stirring. Fractionation was carried out in a stepwise manner, starting with 20% saturation, and increasing by 20% at each step until 80% saturation was reached. Each addition was followed by gentle stirring, a short incubation period to allow for protein precipitation and centrifugation at 7.000 g for 10 min to collect precipitates. The protein precipitate obtained at 60% saturation was redissolved in wash buffer [20 mM Tris, 150 mM NaCI, 3 mM p-mercaptoethanol, and pH adjusted to 7 4] and loaded onto a 5-mL Ni-NTA column attached to fast protein liquid chromatography (FPLC). The column was thoroughly washed with wash buffer containing 50 mM imidazole. Protein elution was accomplished by running a linear gradient of 50-500 mM imidazole in the wash buffer over 40 min. Fractions were run on an SDS-PAGE, and selected fractions were combined, concentrated, and subjected to buffer exchange into wash buffer containing 35% glycerol. Prior to cryopreservation, the concentrated protein underwent SDS-PAGE analysis to assess its quality.

[0387] Experimental

[0388] Design, Expression, and Purification ofMTDs. Human Fibronectin Type III (FN3) domain was chosen as the scaffold for Membrane Translocation Domains (MTDs) (Koide, A., et al. (1998) The fibronectin type III domain as a scaffold for binding proteins. J. Mol. Biol. 284(4): 1141-1151). FN3 is a small (90-100 aa), highly stable protein and has been widely used to develop monobodies that bind to target proteins with high affinity and specificity (Chandler, P.G., et al. (2020) Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain. Cells 9(3):610). Previous studies have demonstrated that several loop regions of FN3 are tolerant to mutations (Steven, A., et al. (2012) Design of novel FN3 domains with high stability by a consensus sequence approach, Protein Engineering. Design and Selection, 25(3): 107-1 17). Additionally, FN3 is free of any cysteine or disulfide bond and is thus stable in the intracellular environment. FN3 readily folds into its native form without any physical or chemical assistance and can be produced in Escherichia coli in high yields. Finally, FN3 is derived from an abundant human extracellular protein and is less likely to elicit any immune response.

[0389] The BC, DE, and FG loops of FN3 have previously shown to be highly tolerant to sequence mutations. The GDSPAS (SEQ ID NO: 106) sequence of the FG loop was replaced with RRRWWW (SEQ ID NO: 104) to give MTD1 (Table 5). Together with an arginine residue already in the FG loop, this generates a putative CPP motif (R4W3) without altering the loop size. Similarly, the tetrapeptide AVTV of the BC loop was replaced with WWWRRR (SEQ ID NO: 105) to take advantage of the existing arginine in the loop to form a putative CPP, W3R4 (Table 5). The size of the BC loop in the resulting mutant, MTD2, is increased by 2 residues. To explore the possibility of grafting a CPP motif to the other end of FN3, the CPP motif R4W3 was substituted for the tripeptide NSP in the CD loop to give MTD3. To test the feasibility of grafting a CPP sequence into two different loops, a relatively hydrophobic tripeptide in the BC loop (VTV) was replaced with WYW and a hydrophilic motif in the FG loop (GDSPAS; SEQ ID NO: 106) with RRR to produce MTD4. Finally, MTD5 was generated by switching the WYW and RRR motifs of MTD4. Two mutants, MTD4a and MTD4b, which contain only half of the CPP motif in the BC and FG loops, respectively, were also generated to test the relative importance of the RRRR and WYW motifs. The WYW motif is more hydrophilic than WWW and has previously been reported as the “endosomal escape motif’ of cell-permeable antibodies (Kim, J.-S., et al. (2016) Endosomal acidic pH- induced conformational changes of a cytosol-penetrating antibody mediate endosomal escape. J. Control. Rel. 235: 165-175). The loop insertion mutants were analyzed by an online program, Phyre2, to predict their folded structures based on homology of sequences. All mutants maintained a similar overall folding to wild type FN3, with the CPP motifs displayed on their surfaces and constrained into the “cyclic” topology (Figure 1).

[0390] To further improve the properties of MTD4 (e.g., cell entry efficiency, metabolic stability, and expression yield), the BC and FG loops of FN3 were replaced with different combinations of Y, W, A, and R residues to generate MTD6-10 (Table 5). The total cellular entry efficiency of MTD6-10 was assessed by labeling the MTDs at a unique C-terminal cysteine with tetramethylrhodamine-5-maleimide (TMR). HeLa cells were treated with the TMR-labeled proteins (5 pM) for 2 h and analyzed by live cell confocal microscopy. MTD7TMRand MTD9TMRshowed similar uptake as MTD4TXIR. whereas MTD6TMR, MTD8TMRand MTD10TMRshowed much less cellular entry. Additionally, the isolated yields for MTD6-10 varied from 0.6 to 6.2 mg / L of E. colt cell culture (Table 5). Note that MTD4 and MTD6 differ only slightly in the BC loop sequence (“WYW” vs “YWW”) and yet have dramatic differences in the isolated yields (9.4 mg / L vs 0.9 mg / L) as well as the cell entryefficiency. Similarly, swapping the CPP motifs between the BC and FG loops of MTD4 resulted in a poorly expressed and much less active variant (MTD5 in Table 5). These results demonstrate that the proper folding / stability and high cellular entry efficiency of the MTDs require not only the presence of amphipathic CPP motifs but also their proper presentation on the protein surface. The DNA sequence coding for WT FN3 was chemically synthesized and ligated into prokaryotic expression vector pET-15b. To facilitate protein purification and genetic fusion with cargo proteins, a six-histidine tag and a thrombin cleavage site were added to the N- terminus of FN3, while a flexible linker sequence (GGSGGSGGS; SEQ ID NO: 107) followed by a recognition site for restriction endonuclease SacI and a cysteine was added to its C-terminus (Table 6). All loop insertion mutants were generated by one-step polymerase chain reaction (PCR) method (Qi, D., et al. (2008) A one-step PCR-based method for rapid and efficient site-directed fragment deletion, insertion, and substitution mutagenesis. J. Virol. Meth. 149:85-9020) and expressed in E. coli. Among the mutant proteins, MTD1 failed to produce significant amounts of soluble protein, whereas WT FN3 and MTD2-5 produced soluble proteins in good yields (Table 5). Figures 2A-2B show the expression and purification of MTD4 as an example. All proteins were purified to near homogeneity by metal affinity chromatography on aNi-NTA column.

[0391] Table 5. Loop Sequences and Expression Yields of MTDs *Underlined residues were deleted and bold-faced residues were inserted during mutagenesis.

[0392] Cloning, Expression, and Purification of MTDs. All loop insertion mutants were generated by one-step polymerase-chain reaction (PCR) method (Qi, D., id.). The peptide sequence (Table 6) for each construct was confirmed by sequencing the entire coding region of the plasmid DNA. Pilot-scale protein expression was carried out to check the levels of expression for Mutant proteins, all mutants were expressed in 5 mL E. coli BL21 (DE3) bacterial culture. The induction was carried out in presence of 0.25 mM IPTG at 37 °C. The level of expression was checked by comparing total cell lysate before and after induction on SDS gel (Figures 2A-2B).

[0393] Table 6. Amino Acid Sequences of WT FN3, and MTDs

[0394] Protein SEQ Sequence

[0395] ID

[0396] NO:

[0397] FN3 118 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGET

[0398] GGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTGGSGGS

[0399] GGSELC

[0400] MTD1 119 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGET

[0401] GGNSPVOEFTVPGSKSTATISGLKPGVDYTITVYAVTGRRRRWWWSKPISINYRTGGSGG

[0402] SGGSELC

[0403] MTD2 120 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTSLLISWDAPWWWRRRRYYRIT

[0404] YGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTGG

[0405] SGGSGGSELC

[0406] MTD3 121 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGET

[0407] GGRRRRWWWVOEFTVPGSKSTATISGLKPGVDYTHVYAVTGRGDSPASSKPISINYRTG

[0408] GSGGSGGSELC

[0409] MTD4 122 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEWAATPTSLLISWDAPAWYWRYYRITYGE

[0410] TGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRRRRSKPISINYRTGGSGGSGG SELC

[0411] MTD4a 123 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTSLLISWDAPAWYWRYYRITYGE

[0412] TGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTGGSGG

[0413] SGGSELC

[0414] MTD4b 124 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGET

[0415] GGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRRRRSKPISINYRTGGSGGSGG

[0416] SELC

[0417] MTD4s 252 GSSHHHHHHSSGHMVSDVPRDLEVVAATPTSLLISWDAPAWYWRYYRITYGETGGNSPV

[0418] QEFTVPGSKSTATISGLKPGVDYTITVYAVTGRRRRSKPISINYTTGGSGGSGGSELC

[0419] MTD5 125 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTSLL1SWDAPARRRRYYRITYGET

[0420] GGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGWYWRSKPISINYRTGGSGGSGGS ELC MTD6 135 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTSLLISWDAPAYWWRYYRITYGET

[0421] GGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRRRRSKPISINYRTGGSGGSGGSELC

[0422] MTD7 136 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTSLLISWDAPAWWARYYRITYGET

[0423] GGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRRRRSKPISINYRTGGSGGSGGSELC

[0424] MTD8 137 MGSSHHHHHHSSGEVPRGSHMVSDVPRDI.EVVAATPTSEEISWDAPAWWWRRYYRITYGE

[0425] TGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRRASSKPISINYRTGGSGGSGGSELC

[0426] MTD9 138 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTSLLISWDAPAWWARYYRITYGET

[0427] GGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGWRRRRSKPISINYRTGGSGGSGGSELC

[0428] MTD10 139 MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTSLLISWDAPAWWRRYYRITYGET

[0429] GGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRRWWSKPISINYRTGGSGGSGGSELC

[0430] The large-scale expression conditions were the same as used for small scale, E. coli cells were centrifuged and stored at -80 °C. These cells were lysed using lysis buffer (50 mL of wash buffer, 0.2 mg / mL lysozyme, 2 mM 0-mercaptoethanol, 2 mM PMSF, 2 tablets of Roche complete protease inhibitor cocktail). After homogeneously resuspending the cell palate in lysis buffer the cells were sonicated (Amp. 70%) twice. The crude cell lysate was centrifuged (12000g for 20 min) and the soluble cell lysate was collected. Protein purification was carried out by using fast protein liquid chromatography (FPLC) and the soluble cell lysate was loaded onto a Ni-NTA column (with 15 mM imidazole). The column was exhaustively washed with wash buffer (50 mM Tris, pH 7.4, 300 mM NaCl, 5% glycerol and 50 mM imidazole). Protein was eluted with wash buffer containing a linear gradient of 50-500 mM imidazole (pH 7.4) over 30 min.

[0431] PTEN

[0432] Phosphatase and tensin homolog (PTEN; 47 kD) is a lipid and protein phosphatase that negatively regulates the PI3K / Akt / mTOR signaling pathway and a key tumor suppressor (Y. R. Lee, et al., The functions and regulation of the PTEN tumour suppressor: new modes and prospects. Nat. Rev. Mol. Cell Biol. 19, 547-562 (2018)). Germline mutations in the PTEN gene cause PTEN hamartoma tumor syndrome (PHTS), while somatic mutations of PTEN are frequently found in human cancers (D. J. Marsh, et al.. Mutation spectrum and genotypephenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation. Hum. Mol. Genet. 7, 507-515 (1998)). Intracellular delivery of PTEN provides a potential treatment for both PHTS and cancers.

[0433] MTD4 was fused to the N-terminus of PTEN and produced the fusion protein in E. coli as an insoluble protein, which was subsequently refolded in vitro. Plasmid pET15b-MTD4- PTEN was constructed by amplifying the PTEN gene fragment (amino acids 1-403) by PCR and using plasmid pET-30b-PTEN (Addgene# 20741) as template and DNA primers listed in Table 7. The PCR product was digested with restriction enzymes Sacl and BamHland ligated into pET-15b-MTD4-NL-PTPlB.

[0434] MTD4-PTEN was expressed in E. coll BL21(DE3) Rosetta pLysS cells grown in LB media supplemented with 75 pg / mL ampicillin and 35 pg / mL chloramphenicol. Protein expression was induced by the addition of 0.25 mM IPTG at 18 °C for 20 h. PTEN was expressed in E. coll BL21(DE3) cells grown in LB media supplemented with 50 pg / mL kanamycin at 37 °C until OD600 reached 0.6. The cell culture was cooled on ice for 30 min and protein expression was induced by the addition of 0.25 mM IPTG at 18 °C for 20 h. The cells were pelleted by centrifugation and stored at -80 °C. PTEN was purified from the soluble cell lysate as described for FN3, whereas MTD4-PTEN was refolded from inclusion bodies by using the same protocol as described for FN3-HiBit.

[0435] MDA-MB-468 cells were seeded in a 6-well plate (60 xlO4cells / well) and treated with MTD4-PTEN (or control protein) for 24 h in complete growth medium. The cells were washed with PBS and lysed in Pierce RIPA buffer containing phosphatase and protease inhibitors. After transfer of the proteins onto the membrane as described, the membrane was blocked and probed with anti-p-AKT(S473) and anti-Pan-AKT (Cell Signaling Technologies) antibodies at manufacturer's recommended dilution. Equal sample loading was confirmed by probing the membrane with an anti-GAPDH antibody.

[0436] The recombinant MTD4-PTEN dose-dependently reduced the viability’ of PTEN- deficient MDA-MB-468 (triple-negative breast cancer) cells, with an ICso value of 14 ± 2 nM, whereas MTD4 or PTEN had no effect up to 100 nM concentration (Fig. 2C). Western blot analysis showed dose-dependent reduction in the phospho-Akt level (but not the total Akt level), indicating that MTD4-PTEN down-regulated the PI3K / Akt / mTOR signaling (Fig. 3B).

[0437] SEQ ID NO.: 253 (PTEN)

[0438] MENLYFQCEFTAIIKEIVSRNKRRYQEDGFDLDLTYIYPNIIAMGFPAERLEGVYRN NIDDVVRFLDSKHKNHYKIYNLCAERHYDTAKFNCRVAQYPFEDHNPPQLELIKPF CEDLDQWLSEDDNHVAAIHCKAGKGRTGVMICAYLLHRGKFLKAQEALDFYGEV RTRDKKGVTIPSQRRYVYYYSYLLKNHLDYRPVALLFHKMMFETIPMFSGGTCNP QFVVCQLKVKIYSSNSGPTRREDKFMYFEFPQPLPVCGDIKVEFFHKQNKMLKKDK MFHFWVNTFFIPGPEETSEKVENGSLCDQEIDSICSIERADNDKEYLVLTLTKNDLD KANKDKANRYFSPNFKVKLYFTKTVEEPSNPEASSSTSVTPDVSDNEPDHYRYSDT TDSDPENEPFDEDQHTQITKVLEHHHHHH

[0439] SEQ ID NO. 254 (MTD4-PTEN) MGSSHHHHHHSSGLVPRGSHMVSDVPRDLEVVAATPTSLLISWDAPAWYWRYYRI

[0440] TYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRRRRSKPISINYRTE LTAIIKEIVSRNKRRYQEDGFDLDLTYIYPNIIAMGFP AERLEGVYRNNIDDVVRFLD SKHKNHYKIYNLCAERHYDTAKFNCRVAQYPFEDHNPPQLELIKPFCEDLDQWLSE DDNHVAAIHCKAGKGRTGVMICAYLLHRGKFLKAQEALDFYGEVRTRDKKGVTIP SQRRYVYYYSYLLKNHLDYRPVALLFHKMMFETIPMFSGGTCNPQFVVCQLKVKI

[0441] YSSNSGPTRREDKFMYFEFPQPLPVCGDIKVEFFHKQNKMLKKDKMFHFWVNTFFI PGPEETSEKVENGSLCDQEIDSICSIERADNDKEYLVLTLTKNDLDKANKDKANRYF SPNFKVKLYFTKTVEEPSNPEASSSTSVTPDVSDNEPDHYRYSDTTDSDPENEPFDE DQHTQITKV

[0442] Table 7 List of primers used to perform insertional mutagenesis or cloning. Forward primer (F) and Reverse primer (R)

[0443] MTD4 is thermodynamically and proteolytically stable

[0444] Thermal denaturation of MTD4 revealed a melting temperature (TM) of 53 ± 2 °C (Fig. 4). Although the TM value is lower than that of FN3 (80 ± 3 °C). MTD4 remains a thermodynamically stable protein. The initial MTD4 construct, which contains an N-terminal six-histidine tag followed by a thrombin cleavage site and a C-terminal (GGS)3C linker (Table 6), exhibited a half-life (h / 2) of ~2 h in human serum (FIGs. 5A-5D). Mass spectrometric analysis revealed proteolysis at the thrombin site and between a R / T dipeptide immediately N-terminal to the (GGS)sC linker. Removal of both sites by mutagenesis (which does not affect the cell entry7efficiency) resulted in a variant, MTD4s (“s” for ‘‘stable’'), which remained largely intact after 24 h of incubation in human serum (ti / 2 >24 h; FIGs. 5A-5D).

Claims

WHAT IS CLAIMED IS:

1. A peptide, comprising: a gene-editing polypeptide having at least 90% sequence similarity to SEQ ID No: 141, 148-151, or 236-251 linked to a membrane translocation domain having one or more cell penetrating peptide motifs, where at least one cell penetrating peptide motif is from 3 to 10 amino acid residues in length and has at least three arginine and / or lysine residues; or where at least one cell penetrating peptide motif is from 3 to 10 amino acid residues in length and has at least two arginine and / or lysine residues and at least one other cell penetrating peptide motif is from 2 to 8 amino acid residues in length and has at least two hydrophobic residues.

2. The peptide of claim 1, wherein the membrane translocation domain is human fibronectin type III.

3. The peptide of claim 2, wherein the human fibronectin type III has 90% sequence similarity with SEQ ID NO: 118.

4. The peptide of any of claims 1 -3, wherein the cell penetrating motif has from 3 to 10 adjacent arginine residues.

5. The peptide of any of claims 1-4, wherein the membrane translocation domain is human fibronectin type III having BC, DE, CD, and FG loops and one or more of the BC, DE, CD, or FG loops have cell penetrating peptide motifs.

6. The peptide of any of claims 1-5, wherein the membrane translocation domain is human fibronectin type III having BC, DE, CD, and FG loops and two of the BC, DE, CD, or FG loops have cell penetrating peptide motifs.

7. The peptide of any of claims 1-6, wherein the membrane translocation domain is human fibronectin type III having BC, DE, CD, and FG loops and the BC and either the DE, CD, or FG loops have cell penetrating peptide motifs.

8. The peptide of any of claims 1-6, wherein the membrane translocation domain is human fibronectin type III having BC, DE, CD, and FG loops and the BC and FG loops have cell penetrating peptide motifs.

9. The peptide of claim 8, wherein the cell penetrating peptide motif in the BC loop has from 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues and the cell penetrating peptide motif in the FG loop has from 3 to 10 amino acid residues and has at least two adjacent arginine and / or lysine residues.

10. The peptide of any one of claims 1-9, wherein the cell penetrating peptide motif in the FG loop has from 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues and the cell penetrating peptide motif in the BC loop has from 3 to 10 amino acid residues and has at least two adjacent arginine and / or lysine residues.

11. The peptide of any one of claims 1-10, wherein a second cell penetrating peptide motif is present and is WW, FF, WF, FW, WWW, FFF, WFW, FWF, WWF, WFF, FWW, FFW, WYW, WWH, YWW, or WYH.

12. The peptide of any one of claims 1-11, wherein the cell penetrating peptide motif comprises RRRWWW (SEQ ID NO: 104), WWWRRR (SEQ ID NO: 105), TGRRRRWWWSKPI (SEQ ID NO: 111); APWWWRRRRYY (SEQ ID NO: 112); GGRRRRWWWVQE (SEQ ID NO: 113); APAWYWRYY (SEQ ID NO: 114);TGRRRRSKPI (SEQ ID NO: 115); APARRRRYY (SEQ ID NO: 116); TGWYWRSKPI (SEQ ID NO: 117); SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, or SEQ ID NO: 162.

13. The peptide of any of claims 1-12, wherein the membrane translocation domain comprises SEQ ID NOs: 119, 120, 121, 122, 123, 124, 125, 135, 136, 137, 138, or 139.

14. The peptide of any one of claims 1-12, wherein the cell penetrating peptide motif is APAWYWRYY (SEQ ID NO: 114) and TGRRRRSKPI (SEQ ID NO: 115).

15. The peptide of claim 14, wherein the membrane translocation domain is SEQ ID NO: 122.

16. The peptide of any of claims 1-15, wherein the gene-editing polypeptide having at least 90% sequence similarity to SEQ ID NO: 141, 148-151, or 236-251 is linked to the membrane translocation domain at aN-terminus or C-terminus of the membrane translocation domain, or at a side chain within the membrane translocation domain.

17. The peptide of any of claims 1-16, wherein the gene-editing enzyme polypeptide having at least 90% sequence similarity to SEQ ID NO: 141, 148-151, or 236-251 is linked to the membrane translocation domain at the C-terminus of the membrane translocation domain.

18. The peptide of any of claims 1-17, wherein the peptide further comprises a cargo moiety.

19. The peptide of any one of claims 1-18, wherein the peptide has at least 90% sequence similarity to SEQ ID NO: 140.

20. The peptide of any one of claims 1-19, wherein the peptide further comprises a nuclear localization sequence comprising KK, KR, RR, HH, HK, HR, RH, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR. RHR, RBR, KKH. KHK, HKK. HRR, HRH, HHR, HBH, HHH. SEQ ID NO: 152, or SEQ ID NO: 163-213.

21. The peptide of any one of claims 1-20, wherein the peptide further comprises a nuclear localization sequence SEQ ID NO: 152.

22. A pharmaceutical composition comprising an effective amount of the peptide of any one of claims 1-20 and a pharmaceutically acceptable carrier.

23. A method of altering expression of at least one gene product comprising contacting an effective amount of the pharmaceutical composition of claim 21 or an effective amount of a peptide of any one of claims 1-20 with a target polynucleotide encoding the gene product.

24. A method for treating or preventing cancer, the method comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 21 or an effective amount of a peptide of any one of claims 1-20.

25. The method of claim 24, wherein the cancer comprises lung cancer, metastatic lung cancer, melanoma, breast cancer, colorectal cancer, anal cancer, pancreatic cancer, prostate cancer, ovarian cancer, cervical, liver cancer, kidney cancers, head and neck cancer, esophageal cancer, oral cancer, bladder cancer, brain cancer, brain metastases from lung cancer, stomach cancer, gastrointestinal cancer, testicular cancer, sarcomas, neurofibroma, angiosarcoma, liposarcoma, of neuroblastoma.

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