P53 PEPTIDE DISRUPTERS OF F0X04:P53 PROTEIN BINDING, VARIANTS AND CONJUGATES THEREOF.

CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of priority to U. S. Provisional Patent Application No. 62/912,480 filed on October 8, 2019, which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

[0002] This application contains a Sequence Listing, which was submitted in ASCII format via USPTO EFS-Web, and is hereby incorporated by reference in its entirety. The ASCII copy, created on September 25, 2020 is named 130268-8002 Sequence Listing.txt and is 136 kilobytes in size.

TECHNICAL FIELD

[0003] The embodiments disclosed herein relate generally to compositions and methods for causing senolysis and/or treating an age-related disease through administration of a p53 peptide disrupter of F0X04:p53 protein binding, a cell permeable variant of the same, a conjugate of the same linked to cell-permeable peptides and/or non-peptide carriers, and compositions and/or formulation of the same. p53 peptide disrupters of F0X04:p53 protein binding, variants thereof, and conjugates thereof facilitate entry into tissues, cells, and the nucleus of cells thus allowing the p53 peptide disrupter of F0X04:p53 protein binding to reach its site of action for therapeutic purposes.

BACKGROUND

[0004] Cellular senescence occurs when normal cellular lifecycles are altered, and aged and/or damaged cells fail to proceed through otherwise normal stages (e.g., checkpoints) of the cell cycle. These aberrant behaviors result in abnormal cells pathologically persisting in a subject rather than undergoing either programmed cell death (e.g., apoptosis) or non- programmed cell death (e.g., necrosis). A key regulator of the cell cycle at certain checkpoints is tumor protein 53 (“p53”). When localized in the nucleus rather than translocating to the mitochondria, p53 enables cells to avoid cell death-associated checkpoints resulting in senescence. Forkhead box protein 04 transcription factor (“F0X04”) anchors p53 in the nucleus via direct binding, and its expression is upregulated during senescence. Disruption of F0X04:p53 binding in mouse models resulted in significant inhibition of P16 expression, a validated marker of cellular senescence (MP Baar et al, Cell, 169, 132-147, 2017; Bourgeois and Madl, FEBS Letters, 592, 2083-2097, 2018).

SUMMARY

[0005] The embodiments disclosed herein, together with a range of modifications, provide peptides, conjugates, fusion proteins, compositions comprising the peptides, conjugates, and fusion proteins, formulations comprising peptides, conjugates, and fusion proteins, and methods of using the foregoing to cause senolysis and/or treat a variety of conditions. In various embodiments, the peptides, conjugates, or fusion proteins incorporate a peptide-based or non peptide-based carrier to translocate p53 polypeptides that bind FOX04 at endogenous p53 binding sites, which are not naturally associated with the peptide-based or non-peptide-based carriers, across cell membranes to their intended sites of action. The embodiments disclosed herein further include variants of the peptides, conjugates, fusion proteins, compositions, and formulations.

[0006] In one embodiment, the p53 peptide comprises a sequence described in any of SEQ ID Nos. 2 to 188 or a variant thereof. In another embodiment, the p53 peptide comprises any one of SEQ ID Nos. 2 to 76 or a variant thereof. In still another embodiment, the p53 peptide comprises any one of SEQ ID Nos. 168 to 178 or a variant thereof. In yet another embodiment, the p53 peptide comprises any one of SEQ ID Nos. 77 to 167, SEQ ID Nos. 179 to 188, or a variant thereof. In one embodiment, the p53 peptide comprises SEQ ID No. 171. In one embodiment, the p53 peptide comprises SEQ ID No. 182.

[0007] In some embodiments, the variant is a cell-permeable peptide variant of a sequence described in any of SEQ ID Nos 2 to 188.

[0008] In one embodiment, the p53 peptide includes at least one D analogue and/or D-retro inverso (DRI) analogue. In one embodiment, the p53 peptide includes at least one stapled and/or cyclic modification. In one embodiment, the p53 peptide includes a least one post-translational modification selected from the group consisting of methylation, glycosylation, sialylation, acetylation, and phosphorylation. [0009] In one embodiment, the conjugate comprises a sequence as described in any one of SEQ ID Nos. 77 to 151 or a variant thereof. In one embodiment, the conjugate comprises a first region conjugated to a second region not naturally associated with the first region and having a sequence described in any of SEQ ID Nos. 2 to 76 and 168 to 178 or variant or portion thereof, wherein the second region has a sequence set forth in SEQ ID No. 1 or a variant or a portion thereof. In one embodiment, the second region is a HOX D12 peptide or a variant or a portion thereof.

[0010] In one embodiment, the second region is conjugated to the C-terminus of the first region. In another embodiment, the second region is conjugated to the N-terminus of the first region.

[0011] In another embodiment, the conjugate further comprises a non-peptide carrier. In some embodiments, the non-peptide carrier is selected from a cationic peptoid, a peptide nucleic acid, aminoglycoside antibiotic, a heterocyclic guanidinium oligomer, and an inositol-based carrier.

[0012] In another embodiment, the peptide, conjugate, and/or fusion protein is prepared by culturing a host cell transformed with an expression vector comprising a nucleic acid encoding a p53 peptide, conjugate, and/or fusion protein of any one of the embodiments described and disclosed herein under conditions which provide for the expression of the p53 peptide, conjugate, and/or fusion protein within the host cell; and recovering the p53 peptide, conjugate, and/or fusion protein by affinity purification.

[0013] In another embodiment, a composition is provided comprising a p53 peptide, conjugate, and/or fusion protein of any one of the embodiments described and disclosed herein and a pharmaceutically acceptable carrier.

[0014] In another embodiment, methods of treating an age-related disease in a subject are provided, the method comprising administering the p53 peptide, conjugate, and/or fusion protein of any one of the embodiments described and disclosed herein, a formulation containing the p53 peptide, conjugate, and/or fusion protein of any one of the embodiments described and disclosed herein, or a composition containing the p53 peptide, conjugate, and/or fusion protein of any one of the embodiments described and disclosed herein to the subject, for example, a human subject.

[0015] In another embodiment, methods of causing senolysis in a subject are provided, the method comprising administering the p53 peptide, conjugate, and/or fusion protein of any one of the embodiments described and disclosed herein, a formulation containing the p53 peptide, conjugate, and/or fusion protein of any one of the embodiments described and disclosed herein, or a composition containing the p53 peptide, conjugate, and/or fusion protein of any one of the embodiments described and disclosed herein to the subject, for example, a human subject.

[0016] In another embodiment, compositions comprising a p53 peptide, a conjugate, a fusion protein, and/or a combination thereof according to any one of the embodiments of the present technology for use in treating an age-related disease in a subject, for example, a human subject, are provided.

[0017] In another embodiment, compositions comprising a p53 peptide, a conjugate, a fusion protein, and/or a combination thereof according to any one of the embodiments of the present technology for use in causing senolysis in a subject, for example, a human subject, are provided.

[0018] In one embodiment, the conjugate is in the form of a fusion protein.

[0019] In another embodiment, the conjugate further comprises a linker sequence between the first and second regions.

[0020] In another embodiment, the first and second regions combined have a polypeptide sequence selected from the group consisting of SEQ ID Nos: 77-151, or a variant thereof.

[0021] In one embodiment, an initiating methionine residue of the sequence is removed prior to use as an active and/or therapeutic agent.

[0022] In one embodiment, the first region is linked to the second region with a peptidic bond. In yet another embodiment, the first region and the second region are attached via “click chemistry.”

[0023] In one embodiment, the first region is derived from the human gene. In another embodiment, the second region includes a p53 peptide sequence derived from a human gene or variant thereof.

[0024] In another embodiment, the conjugate further comprises at least one molecular cargo selected from the group consisting of liposomes, polymers, cationic peptides, nanoparticles, adeno-associated virus (AAV) and calcium.

[0025] In another embodiment a composition is provided that comprises a p53 peptide, a conjugate, and/or a fusion protein that may take any of the following forms: a composition for local or injectable use; a topical composition; an oral composition; an inhaled composition, or an injectable composition such as a subcutaneous injection, an intravenous injection, an intramuscular injection, an intraperitoneal injection, or an injectable implant for sustained release. In some embodiments, the composition comprises a pharmaceutically acceptable carrier.

[0026] Other embodiments comprise methods to prevent immunogenicity of otherwise antigenic proteins delivered as p53 peptides, conjugates, or fusion proteins to their intended cytosolic or nuclear sites of action.

[0027] These and other embodiments will be disclosed in further detail below.

DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 illustrates that delivery of a p53 peptide distrupter of FOXO:p53 protein binding (“disrupter”) results in apoptosis of senescent cells (e.g., senolysis).

[0029] FIGS. 2A-2G illustrate a method of using a peptide microarray to develop p53 peptide disrupters of protein-protein interactions, such as those described and disclosed herein.

[0030] FIG. 3 is a schematic diagram illustrating that at least one of the novel p53 peptide disrupters of F0X04:p53 protein binding, such as those described and disclosed herein, disrupts F0X04:p53 binding.

[0031] FIG. 4A shows cellular in vitro results showing that, in FOX04-Flag tagged HEK293 transiently transfected cells, FOX04 co-immunoprecipitates with endogenous p53. FIG. 4B demonstrates biochemical in vitro results showing that co-incubation of full-length human GST-tagged p53 and His-tagged FOX04 co-immunoprecipitated

[0032] FIGS. 5A-5H shows that certain p53 peptide disrupters of F0X04:p53 protein binding, such as those described and disclosed herein, bind to epitope-tagged FOX04 at a p53 binding site (FIGS. 5A and 5E-5H) and that substitution of alanine for other amino acid residues in certain p53 peptide disrupters of F0X04:p53 protein binding results in loss of FOX04 binding at the p53 binding site (FIGS. 5B-5D).

[0033] FIG. 6 shows that substitution of alanine for other amino acid residues in certain p53 peptide disrupters of F0X04:p53 protein binding, such as those described and disclosed herein, results in identification of certain false positive binding interactions shown in FIGS. SA SH. [0034] FIGS. 7A-7C demonstrate an N and C truncation analysis and a dual N-C truncation analysis resulting in a minimum amino acid sequence for binding to p53 at P ¹⁵¹ -R ¹⁷⁵ .

[0035] FIG. 8 illustrates a three-dimensional crystal structure of p53.

[0036] FIGS. 9A and 9B show results of a rational point substitution analysis to assess sequence optimization of F0X04:p53 disrupter peptide G ¹⁵⁴ TRVRAMAIYKQ ¹⁶⁵ .

[0037] FIGS. 10A-10C show results of an N and C truncation analysis resulting in a minimum amino acid sequence for binding to p53 at H ²⁹⁶ -K ³²⁰ .

[0038] FIGS. 11A and 11B show results of a rational point substitution analysis to assess sequence optimization of F0X04:p53 disrupter peptide S ³⁰³ TKRALPNNTS ³¹³ .

[0039] FIGS. 12A-12C show results of an N and C truncation analysis, and a dual N-C truncation analysis resulting in a minimum amino acid sequence for binding to p53 at E ³⁴⁶ -Q ³⁷⁵ .

[0040] FIGS. 13A and 13B illustrate a downstream K370 residue investigation analysis resulting in a minimum amino acid sequence for binding to p53 at E ³⁴⁶ -Q ³⁷⁵ .

[0041] FIGS. 14A and 14B show results of a rational point substitution analysis to assess sequence optimization of F0X04:p53 disrupter peptide R ³⁶³ AHSSHLKS ³⁷¹ .

[0042] FIGS. 15A and 15B show results of a D-amino and D-retro inverso sequence analysis of certain p53 peptide disrupters of F0X04:p53 protein binding.

[0043] FIG. 16 shows direct binding kinetics of epitope-tagged FOX04 and a p53 peptide by fluorescent polarization.

[0044] FIGS. 17A-17H show direct binding kinetics of epitope-tagged FOX04 and p53 peptides corresponding to a p53 DNA binding domain by fluorescent polarization.

[0045] FIGS. 18A-18D show direct binding kinetics of epitope-tagged FOX04 and p53 peptides corresponding to a p53 nuclear localization domain by fluorescent polarization.

[0046] FIGS. 19A-19H show direct binding kinetics of epitope-tagged FOX04 and p53 peptides corresponding to a p53 C-terminal negative regulatory domain by fluorescent polarization.

[0047] FIGS. 20 A and 20B show direct binding kinetics of epitope-tagged FOX04 and a scrambled negative control peptide by fluorescent polarization. DETAILED DESCRIPTION

[0048] The novel p53 peptide disrupters of F0X04:p53 protein binding described herein are p53 peptides which are configured for intracellular delivery and nuclear translocation. The novel p53 peptide disrupters of F0X04:p53 protein binding specifically bind to FOX04 at a site, or a portion thereof, where a p53 protein otherwise naturally binds to FOX04 following intracellular delivery or in vitro. Once bound to FOX04 at the native p53 binding site, the p53 peptide disrupters of F0X04:p53 protein binding prevent binding of FOX04 and p53. p53 peptide disrupters of F0X04:p53 protein binding prevent binding of FOX04 and p53 regardless of whether FOX04 and p53 are endogenously expressed, exogenously expressed, or a combination thereof. Without intending to be bound by any particular theory, it is thought that disruption of FOX04 and p53 binding with p53 peptide disrupters of F0X04:p53 protein binding allows p53 to translocate out of the nucleus and to the mitochondria to induce apoptosis in senescent cells (e.g., senolysis).

[0049] Several of the disclosed embodiments and their modifications relate to novel p53 peptide disrupters of F0X04:p53 protein binding, and conjugates of these novel p53 peptide disrupters of F0X04:p53 protein binding, which may take the form of fusion proteins, each comprising a F0X04:p53 disrupter peptide linked to another peptide (e.g., cell penetrating peptide (CPP)) or a non-peptide. In some embodiments, the p53 peptide disrupters of F0X04:p53 protein binding are cell-permeable, or are modified into a cell-permeable peptide.

[0050] In some embodiments, novel p53 peptide disrupters of F0X04:p53 protein binding comprise a polypeptide sequence shown in Table 1 and described in any of SEQ ID Nos. 2 to 76 or a variant thereof or any one of SEQ ID Nos. 168 to 178 or a variant thereof. In one embodiment, the novel F0X04:p53 disrupter peptide comprises SEQ ID No. 171, or a variant thereof. In another embodiment, the novel F0X04:p53 disrupter peptide comprises SEQ ID No. 182, or a variant thereof.

[0051] The terms “peptide(s),” “protein(s),” and “polypeptide(s)” are used synonymously.

[0052] The term “p53” refers to tumor protein 53 or variants or portions thereof.

[0053] The term “FOX04” refers to forkhead box protein 04 transcription factor or variants or portions thereof.

[0054] The terms “p53 peptide disrupters of F0X04:p53 protein binding”, “F0X04:p53 disrupter peptides”, “disrupter peptides”, “disrupter”, and “p53 peptides” are used synonymously. Non-limiting examples thereof are provided below in Table 1 as SEQ ID NOs. 2 to 76 and SEQ ID Nos. 168 to 177.

Table 1: p53 Peptide Disrupters of F0X04:p53 Protein Binding, HOX D12 Peptide, and Other Peptide Sequence Identities.

[0055] FIG. 1 illustrates that delivery of a disrupter results in senolysis. As shown in the left panel, in response to stress-induced DNA-damage, p53 is localized at certain specific sites on the damaged DNA (e.g., scars). There, FOX04 binds to p53 and prevents cytosolic translocation and subsequent mitochondrial mediated apoptosis and cell cycle arrest resulting in cellular senescence. As shown in the right panel, targeted apoptosis of senescent cells (TASC) occurs following specific binding of a cell penetrating peptide disrupter to FOX04. Once the cell penetrating peptide disrupter is bound to FOX04, the endogenous F0X04:p53 interaction is displaced, allowing p53 to effectively translocate out of the nucleus into the cytoplasm and potentiate apoptotic-mediated signalling pathways. Senescent cells are thereby eliminated and a senescence-associated secretory phenotype in the subject is reduced.

[0056] Disrupter peptides can be cell-penetrating or otherwise conjugated to a cell- penetrating peptide as described in greater detail below. In some embodiments, the disrupter peptides having SEQ ID Nos. 2 to 76 and 168 to 177 listed in Table 1 have cell permeabilizing activity. In other embodiments, one or more variations to amino acid SEQ ID Nos. 2 to 76 and 168 to 177 listed in Table 1 are otherwise modified to have cell-permeabilizing activity.

[0057] A person of ordinary skill in the molecular biology/biotechnology art would appreciate that numerous variations of the sequences shown in Tables 1 to 4 would fall within the embodiments disclosed herein. As used herein, homology refers to identity or near identity of nucleotide or amino acid sequences. As is understood in the art, nucleotide mismatches can occur at the third or wobble base in the codon without causing amino acid substitutions in the translated polypeptide sequence. Also, minor nucleotide modifications (e.g., substitutions, insertions or deletions) in certain regions of the gene sequence can be tolerated whenever such modifications result in changes in amino acid sequence that do not alter functionality of the final gene product. Homologs of specific DNA sequences may be identified by those skilled in the art using the test of cross-hybridization of nucleic acids under conditions of stringency as is well understood in the art (as described in Hames et al., Nucleic Acid Hybridization, (1985) IRL Press, Oxford, UK). Extent of homology is often measured in terms of percentage of identity between the sequences compared.

[0058] The term “variant” refers to a polypeptide or protein that differs from a reference polypeptide or protein but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall (homologous) and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

[0059] Modifications and changes can be made in the structure of the polypeptides and proteins of this disclosure and still result in a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide’s or protein’s biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide or protein sequence and nevertheless obtain a polypeptide or protein with like properties.

[0060] Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take one or more of the foregoing characteristics into consideration are well known to those of skill in the art and include, but are not limited to (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (lie: Leu, Val), (Leu: lie, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Trp: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Embodiments of this disclosure, therefore, consider functional or biological equivalents of a polypeptide or protein as set forth above. In particular, embodiments of the polypeptides and proteins can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide and protein of interest.

[0061] Polypeptides may include amino acid residues including natural and/or non-natural amino acid residues, such as in the D-form “D analogue” or D-retro inverso “DRI analogues” at one or more than one amino acid residue in the polypeptide, and/or at least one stapled and/or cyclic modification. The terms also include post-translational modifications of the polypeptide, for example, methylation, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some embodiments, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0062] The term “acidic residue” refers to amino acid residues in D- or L-form having sidechains comprising acidic groups. Exemplary acidic residues include D and E.

[0063] The term “amide residue” refers to amino acids in D- or L-form having sidechains comprising amide derivatives of acidic groups. Exemplary residues include N and Q.

[0064] The term “aromatic residue” refers to amino acid residues in D- or L-form having sidechains comprising aromatic groups. Exemplary aromatic residues include F, Y, and W.

[0065] The term “basic residue” refers to amino acid residues in D- or L-form having sidechains comprising basic groups. Exemplary basic residues include H, K, and R.

[0066] The term “hydrophilic residue” refers to amino acid residues in D- or L-form having sidechains comprising polar groups. Exemplary hydrophilic residues include C, S, T, N, and Q.

[0067] The term “nonfunctional residue” refers to amino acid residues in D- or L-form having sidechains that lack acidic, basic, or aromatic groups. Exemplary nonfunctional amino acid residues include M, G, A, V, I, L and norleucine (Nle).

[0068] The term “neutral hydrophobic residue” refers to amino acid residues in D- or L- form having sidechains that lack basic, acidic, or polar groups. Exemplary neutral hydrophobic amino acid residues include A, V, L, I, P, W, M, and F. [0069] The term “polar hydrophobic residue” refers to amino acid residues in D- or L-form having sidechains comprising polar groups. Exemplary polar hydrophobic amino acid residues include T, G, S, Y, C, Q, and N.

[0070] The term “hydrophobic residue” refers to amino acid residues in D- or L-form having sidechains that lack basic or acidic groups. Exemplary hydrophobic amino acid residues include A, V, L, I, P, W, M, F, T, G, S, Y, C, Q, and N.

[0071] “Identity,” as known in the art, is a relationship between two or more polypeptide or protein sequences, as determined by comparing the sequences. In the art, “identity” also refers to the degree of sequence relatedness between polypeptides or proteins, as determined by the match between strings of such sequences. “Identity” can be readily calculated by known bioinformational methods.

[0072] Several of the disclosed embodiments and their modifications relate to novel conjugates, which may take the form of conjugates and/or fusion proteins, each comprising a 60- amino acid peptide or variant or portion thereof (SEQ ID No. 1, Table 1) derived from human HOX genes or from other human genes containing a homeodomain with cell permeabilizing properties, linked to a second region that is at least one of the biologically active “cargo” peptides disclosed herein. For example, the second region includes at least SEQ ID Nos. 2 to 76 and 168 to 177 of Table 1. In some embodiments, the 60-amino acid human HOX D12 homeodomain (“first region”; SEQ ID No. 1) is linked to a biologically-active cargo peptide (“second region”; any one of SEQ ID Nos. 2 to 76 and 168 to 177). The SEQ ID Nos. of Table 2 include non- limiting examples of conjugates comprising aHOX D12 first region and a F0X04:p53 disrupter peptide second region. In some embodiments, the N-terminus of the first region is linked to the C-terminus of the second region. The links in both embodiments may be a simple peptide bond or a linker sequence known in the art provided that function of the fusion protein or conjugate is not compromised by its addition.

[0073] The term “human homeodomain” refers to (1) human HOX-derived homeodomains such as the HOX D12 sequence shown in Table 1 as SEQ ID No. 1, or variants or portions thereof; and (2) any other human homeodomain that has cell permeabilizing activity, or variants or portions thereof.

[0074] The human HOX genes and other homeodomain coding genes are important in embryonic development and include small regions homologous to the gene antennapedia (Antp) but where the majority of amino acids are included in uniquely human sequences that facilitate their function. The protein sequence of antennapedia is characterized by the presence of a 60- amino acid motif (homeodomain) that binds to specific DNA target elements. The Antp homeodomain (see US Pat. No. 7,968,512) and much smaller sequences from the Antp gene have been shown to facilitate the entry of biologically active (“cargo”) peptides into tissues and cells to reach their site of action for therapeutic purposes.

[0075] While the homeodomain and smaller regions of Antp can be used to translocate proteins, including functional and regulatory proteins in vitro and in animal models, peptide sequences from human HOX genes and certain other human homeodomain sequences can also facilitate transport of biologically active molecules such as peptides, proteins and nucleotides to their intracellular and intranuclear sites of action (i) without provoking an unwanted immune response that may reduce exposure and/or effectiveness of the conjugate or may produce an adverse event, and (ii) with greater efficiency than Antp conjugates and fusion proteins. The ability to deliver biologically active molecules including the expression product of a gene of interest directly into a cell has wide applicability, particularly in the medical field. HOX and other human homeodomain peptides are also able to translocate nucleic acids. This is especially advantageous for applications utilizing a mechanism of gene regulation.

[0076] The term “conjugate” or “conjugates” herein comprises a category of structures, including fusion proteins, in which the first region, a 60-amino acid human homeodomain sequence or variant or portion thereof, is conjugated directly to the second region via a peptide bond or other type of bond including both covalent and non-covalent bonds. Conjugates may include a linker region that connects the homeodomain sequence to a second region, a functional or regulatory peptide or protein (“cargo” peptide) that is not naturally associated with the first region. Any of a wide variety of linkers (short, connecting sequences) known in the art may be utilized to form the conjugate provided that function of the conjugate is not compromised by its addition. Thus, translocation of the second region is enabled through a cellular or nuclear membrane. For example, see a wide variety of linkers known in the art in Chen et al. “Fusion protein linkers: property, design and functionality.” Advanced Drug Delivery Reviews. http://dx.doi.Org/10.1016/J.addr.2012.09.039. In alternative embodiments the term “fusion protein” is used to refer to a particular subcategory of conjugate that exists when no such linkers are used to form the conjugate and the domains are linked entirely by peptide bonds.

[0077] Cargo peptides include both small synthetic peptides and larger proteins such as antibodies or the binding regions of antibodies delivered into cells for therapeutic purposes. [0078] First Region: The first region of the conjugate or fusion protein embodiments disclosed may comprise a natural or synthetic 60-amino acid peptide or variant or portion thereof derived from the HOX D12 gene or any other human homeodomain that has cell permeabilizing activity, such as SEQ ID No. 1. Cargo sequences may or may not be of human origin depending on their intended function.

[0079] In some embodiments, the conjugates disclosed herein can be combined with other carriers, for example, liposomes, polymers, cationic peptides, nanoparticles, and viral carriers. Cell penetrating peptide (CPP)-modified liposomes can improve efficacy of cell penetration. CPPs can be attached to the liposome by any method known in the art, for example, polyethylene glycol (PEG) spacer or via liposomal NGPE. The CPPs can be conjugated to PEGylated magnetic polymeric liposomes. In some embodiments, poly-l-lysine (PLL), a synthetic chiral polymer, or PEG can be added to the conjugates. In some embodiments, conjugates combined with other carriers are enhanced drug delivery systems. In other embodiments, conjugates combined with other carriers are a component of enhanced drug delivery systems.

[0080] Second Region Peptides or Proteins: The second region of the conjugate or fusion protein embodiments disclosed comprises a p53 peptide sequence as set forth in SEQ ID Nos. 2 through 76 and 168 to 177, or variants or portions thereof. In some embodiments, the second region of the conjugate or fusion protein embodiments disclosed may comprise a polypeptide sequence having cell permeabilizing activity. The second region may further comprise any peptide or protein sequence not naturally associated with the first region. The gene encoding the first region may or may not also encode the second region. The second region also may or may not be from the same species as the first region, but the first and second regions will be present in the conjugate or fusion protein embodiments in a manner different from the natural situation. Each of the foregoing sequences is listed above in Table 1 along with the abbreviation of the name of the gene from which it is derived (SEQ ID Nos. 2 to 76 and 168 to 177).

[0081] The second region of the fusion protein or conjugate embodiments may be a peptide or protein of any length as long as it is biologically active on its target when included in the fusion protein or conjugate.

[0082] The conjugate or fusion protein, or variants thereof, comprise a peptide sequence as set forth in SEQ ID Nos. 77 to 151 and 179 to 188, or variants or portions thereof as shown in Table 2.

Table 2: Conjugate or Fusion Protein Sequence Identities.

[0083] The phrase “not naturally associated with” means that entire sequence of the conjugate or fusion protein is not found in nature, and that the entire sequence is not encoded for by a single gene found in nature.

[0084] The phrase “operably linked” means that the first and second region are linked such that the second region is able to translocate a cell membrane. Such linkage may be produced via application of “click” chemistry methods or other methods known in the art or may be incorporated as a fusion protein with a peptide bond between regions.

[0085] In addition, synthetic variants may be used provided that they retain the ability to translocate the membrane. Synthetic variants will generally differ from the naturally-occurring proteins by substitution, particularly conservative substitution. The phrase “conservative amino acid changes” herein means replacing an amino acid from one of the amino acid groups, namely hydrophobic, polar, acidic or basic, with an amino acid from within the same group. An example of such a change is the replacement of valine by methionine and vice versa. Other examples of conservative substitutions may be seen by reference to Table 3 below.

[0086] In one embodiment, the peptide comprises a structure derived from a human p53 gene having a sequence described in any of SEQ ID Nos. 2 to 76 and 168 to 177, wherein 1-20, 1-15, or 1-10 amino acid residues are substituted, deleted, added, and/or inserted and wherein said amino acid does not have cell permeabilizing activity.

[0087] In one embodiment, the peptide comprises a structure derived from a human p53 gene having a sequence described in any of SEQ ID Nos. 2 to 76 and 168 to 177, wherein 1-20, 1-15, or 1-10 amino acid residues are substituted, deleted, added, and/or inserted and wherein said amino acid has cell permeabilizing activity.

[0088] In one embodiment, the peptide, conjugate, or fusion protein comprises a first region comprising a structure derived from (1) the human HOX genes, or (2) other human genes having a sequence described in any of SEQ ID No. 1, wherein 1-20, 1-15, or 1-10 amino acid residues are substituted, deleted, added, and/or inserted; and a second region is a F0X04:p53 disrupter polypeptide or protein not naturally associated with the first region having a sequence described in any of SEQ ID No. 2 to 76 and 168 to 177, or a variant thereof, wherein the peptide, conjugate, or fusion protein has cell-permeabilizing activity that is maintained following substitution, deletion, addition, and/or insertion of 1-20 amino acid residues.

[0089] In one embodiment, the peptide, conjugate, or fusion protein comprises a first region comprising SEQ ID No. 1, wherein 1-20, 1-15, or 1-10 amino acid residues are substituted, deleted, added, and/or inserted; and a second region comprising a F0X04:p53 peptide having a sequence described in any of SEQ ID No. 2 to 76 and 168 to 177, wherein 1-20, 1-15, or 1- lOamino acid residues are substituted, deleted, added, and/or inserted, wherein the peptide, conjugate, or fusion protein has cell-permeabilizing activity that is maintained following substitution, deletion, addition, and/or insertion of 1-20 amino acid residues.

[0090] In one embodiment, the peptide, conjugate, or fusion protein comprises a structure derived from human genes having a sequence described in any of SEQ ID Nos. 77 to 151 and 179 to 188, wherein 1-20, 1-15, or 1-10 amino acid residues are substituted, deleted, added, and/or inserted and wherein the peptide, conjugate, or fusion protein has cell-permeabilizing activity that is maintained following substitution, deletion, addition, and/or insertion of 1-20 amino acid residues.

Table 3: Conservative Amino Acid Substitutions.

[0091] Such variants may be synthesized directly or prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions corresponding to the naturally-occurring sequence either side of the insertion site. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used. Variants that retain at least 50% sequence identity with SEQ ID Nos. 2 through 188 or variants or portions thereof will likely maintain their cell permeability characteristics and retain their human characteristics resulting in low immunogenicity potential. The ability of a naturally occurring or synthetic cell penetrating peptide sequence to translocate the membrane may be tested by routine methods known in the art. Any polynucleotide which encodes the amino acid of SEQ ID Nos. 1 to 76 and SEQ ID Nos. and 168 to 177 (Table 1), SEQ ID Nos. 77 to 171 and 179 to 188 (Table 3) or a variant or a portion thereof can be used in a peptide, a fusion protein, or a conjugate herein.

[0092] In some embodiments, the peptide, fusion protein, or conjugate may comprise further suitable domains known to those skilled in the art. For example, an endoplasmic reticulum retention signal functions to affect the intracellular routing of the internalized conjugate or protein/nucleic acid complex. A suitable endoplasmic retention signal may be a mammalian endoplasmic reticulum retention signal.

[0093] Also present may be a translocation domain which serves to enhance nucleic acid or protein escape from the cellular vesicle system and thus to augment nucleic acid transfer by this route. This domain may serve to reduce or avoid lysosomal degradation after internalization of the protein/nucleic acid into the target cell. Suitable translocation domains are derivable from toxins, particularly bacterial toxins, such as exotoxin A, Colicin A, d-endotoxin, diphtheria toxin, Bacillus anthrax toxin, Cholera toxin, Pertussis toxin, E. coli toxin toxins, Shiga toxin or Shiga- like toxin.

[0094] The first binding domain may be modified to target cell sites other than the nucleus.

[0095] Additionally, or alternatively, a target cell-specific binding domain recognizing a cell surface structure may be present, such as a receptor protein or surface antigen on the target cell.

[0096] The first (HOX or other human homeodomain) region and second (cargo) regions may be linked by a cleavable linker region this may be any region suitable for this purpose provided the function of the conjugate is not compromised by its addition. The cleavable linker region is a protease cleavable linker, although other linkers, cleavable for example by small molecules, may be used. These include Met-X sites, cleavable by cyanogen bromide, Asn-Gly, cleavable by hydroxylamine, Asp-Pro, cleavable by weak acid and Trp-X cleavable by, inter alia, NBS-skatole. Protease cleavage sites require milder cleavage conditions and are found in, for example, factor Xa, thrombin and collagenase. Any of these may be used. The precise sequences are available in the art and the skilled person will have no difficulty in selecting a suitable cleavage site. By way of example, the protease cleavage region targeted by Factor Xa is I E G R. The protease cleavage region targeted by Enterokinase is D D D D K. The protease cleavage region targeted by Thrombin is L V P R G. The cleavable linker region may be one that is targeted by endocellular proteases. Linkers may not be required for function but linkers may be included between first and second regions to allow targeted release of the second region without compromising function or to enhance biological activity of the second region with linker cleavage.

[0097] In one embodiment, the first region is at the N-terminus of the second region. In another embodiment the first region is at the C-terminus of the second region.

Table 4: Partial listing of linkers known in the art, adapted from Chen et al., 2012. _

[0098] The embodiments disclosed allow for potent therapeutic action, including efficient translocation into intracellular sites of action of any of a number of identified peptides for the specified therapeutic treatments. Regardless of the target tissue, such translocation does not provoke an unwanted immune response that may reduce the exposure and/or effectiveness of the conjugate or produce an adverse or undesirable clinical event. The biologically-active peptide of the “second region” is not naturally associated with the human homeodomain sequence or variant or portion thereof (the “first region”) before they are joined.

[0099] Peptide, conjugate, and fusion protein embodiments herein may be produced in accordance with any of the standard molecular biology techniques described in the literature. See, for example, Ausubel et al. (2002) Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 5th Ed. John Wiley & Sons. Manufacturing methods including purification methods that may be used are also disclosed in U.S. Pat. No. 7,968,512, the entirety of which is incorporated herein by reference. [00100] “Expression vectors” or “plasmids” (used interchangeably herein) may be used for producing peptides, conjugates or components thereof to introduce heterologous DNA into host cells, either for expression or replication. Selection by the artisan of the appropriate vector will depend on its intended use, i.e. (DNA amplification or DNA expression), the size of the DNA to be inserted into the vector, and the host cell to be transformed with the vector. Each vector contains various components depending on its intended use, which comprise one or more of: an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence and a signal sequence.

[00101] Sources of nucleic acid may be ascertained by reference to published literature or databanks provided by organizations such as NCBI or EMBL. Identification of sequences of interest may be accomplished by using BLAST, BLAT, or other homology search algorithms. Further, nucleic acid encoding the desired first or second region may be obtained from academic or commercial sources where such sources are willing to provide the material or by synthesizing or cloning the appropriate sequence where only the sequence data are available. Generally, this may be done by reference to literature sources which describe the cloning of the gene in question. Alternatively, where limited sequence data are available or where it is desired to express a nucleic acid homologous or otherwise related to a known nucleic acid, exemplary nucleic acids can be characterized as those nucleotide sequences which hybridize to the nucleic acid sequences known in the art.

[00102] The phrase “stringency of hybridization” refers to conditions under which polynucleic acids hybrids are stable. Such conditions are evident to those of ordinary skill in the art. Also as understood by persons skilled in the art, the stability of hybrids is reflected in the melting temperature (Tm) of the hybrid which decreases approximately 1 to 1.5°C. with every 1% decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. The hybridization reaction typically is performed under conditions of higher stringency, followed by washes of varying stringency.

[00103] As used herein, the phrase “high stringency” refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 1 M Na+ at 65- 68[deg.] C. High stringency conditions can be provided, for example, by hybridization in an aqueous solution containing 6*SSC, 5* Denhardt’s, 1% SDS (sodium dodecyl sulphate), 0.1 Na+ pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as nonspecific competitor. Following hybridization, high stringency washing may be done in several steps, with a final wash (about 30 minutes) at the hybridization temperature in 0.2-0.1*SSC, 0.1% SDS.

[00104] The phrase “moderate stringency” refers to conditions equivalent to hybridization in the above described solution, except that the temperature is at about 60-62°C. In that case the final wash is performed at the hybridization temperature in 1*SSC, 0.1% SDS.

[00105] Low stringency refers to conditions equivalent to hybridization in the above described solution at about 50-52°C. In that case, the final wash is performed at the hybridization temperature in 2*SSC, 0.1% SDS.

[00106] It is understood that these conditions may be adapted and duplicated using a variety of buffers, e.g., formamide-based buffers, and temperatures. Denhardf s solution and SSC are well known to those of skill in the art as are other suitable hybridization buffers (see, e.g. Sambrook, et al, eds. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York or Ausubel, et al, eds. (1990) Current Protocols in Molecular Biology, John Wiley & Sons, Inc.). Optimal hybridization conditions must be determined empirically, as the length and the GC content of the probe also play a role.

[00107] In one embodiment, the peptide, conjugate, or fusion protein may be produced through use of an expression vector comprising the nucleic acid sequence and a promoter for recombinant synthesis in, for example, plant cells (including algae), in bacteria such as E. coli, or in eukaryotic cells such as Chinese Hamster Ovary (CHO) cells or yeast cells.

[00108] In another embodiment, a host cell is transformed with the expression vector.

[00109] In yet another embodiment a nucleic acid sequence encodes the peptide, conjugate, or fusion protein for the purposes of synthesis and manufacture by recombinant technology.

[00110] In some embodiments, the peptide, conjugate, or fusion protein is non-denatured, meaning it may exist in its native state, the form in which the protein occurs in the intact cell in its three-dimensional structure.

[00111] The term “non-denatured” may also, but need not, imply a specific non-denaturing step. Denaturing alters the three-dimensional shape of the protein molecule without rupture of its peptide bonds; disulfide bonds may be ruptured, or certain groups in the protein may be chemically modified if such processes are also accompanied by changes in its overall three- dimensional structure. [00112] In other embodiments, the peptide, conjugate, or fusion protein is renatured, a process by which the denatured protein is returned to its original conformation prior to denaturation. For peptides, reversible denaturation is generally brought about by disulfide reducing agents and urea, and for nucleic acid, by heat and salts.

[00113] Purification methods known in the art may be used in the process of preparing peptides, conjugates, and fusion proteins according to the embodiments disclosed, for example, as described in Zachariou, M. (2010) Affinity Chromatography: Methods and Protocols, 2nd Ed. Totoua, NTHumana Press. The peptide, conjugate, or fusion protein can be obtained from bacterial or eukaryotic cell lysates, as denaturing reagents, small changes in pH, and differences in osmolarity may have an effect on the translocation properties of the peptides. Conditions for obtaining and purifying the human cell penetrating peptides (CPPs) and disclosed herein.

[00114] The ability of peptides and peptide conjugates to translocate across the cell surface membrane may be dependent on the conformation of the recombinant proteins. For example, translocation of the polypeptide by using either bacteria cell extracts or purified proteins exposed to small amounts of detergent (ionic and non-ionic) or denaturing agents (urea or guanidinuim) may prevent or inhibit translocation. This conformation-dependent property may be preserved by purifying the peptide or peptide conjugate under native conditions.

[00115] In one embodiment, peptide is purified from a bacterial lysate. In another embodiment, both the first (the peptide-based carrier (e.g., TAT-based, RNA-binding peptides, DNA-binding peptides, cyclic peptides, arginine-rich peptides, cyclodextrins, dynorphin neuropeptides, antibacterial peptide, and antimicrobial peptides (AMP), variants or portions thereof) and second (p53 polypeptide sequence) regions are purified from a bacterial lysate. In other embodiments, the peptide, conjugate, or fusion protein is purified from a plant cell lysate. In yet another embodiment, the peptide, conjugate or fusion protein is purified from a eukaryotic cell lysate, culture medium or fermentation broth.

[00116] One embodiment is a method for preparing a peptide, a conjugate, or a fusion protein, comprising: (a) culturing the host cell under conditions which provide for the expression of the peptide or the conjugate from the expression vector within the host cell; and (b) recovering the peptide or the conjugate, which recovery comprises (i) fusing an amino acid tail or other specific ligand to the conjugate, which tail is capable of binding to at least one substrate and not to another substrate, and wherein the conjugate or fusion protein is caused to bind via the tail to at least one substrate, and wherein components of the host cell do not bind to this substrate, and (ii) putting into contact the peptide or the conjugate and remaining components of the host cell with the other substrate such that the conjugate is not bound and the remaining components of the host cell are bound to the other substrate.

[00117] Another embodiment is a method for preparing a conjugate or fusion protein comprising: (i) culturing a host cell, transformed with an expression vector comprising nucleic acid, operably linked to a promoter, encoding a fusion protein where (a) a first region comprising a polypeptide sequence as described in any one of SEQ ID Nos. 2 to 76 and 168 to 178 or a variant thereof; and (b) a second region comprising a P53 peptide sequence as set forth in SEQ ID No. 1 or a variant thereof; and (ii) recovering the fusion protein, which method comprises fusing an amino acid tail or other ligand to the conjugate, which tail is capable of binding to at least one substrate and not to another substrate, and wherein the conjugate is caused to bind via the tail to at least one substrate such that components of the host cell do not bind to this substrate; and the conjugate is contacted with the other substrate such that the conjugate is not bound and remaining components of the host cell are bound to the other substrate.

[00118] In another embodiment as part of the affinity purification process, the embodiments include the use of a tail or ligand that is attached to the peptide, the conjugate, or the fusion protein; this allows for both positive and negative purification steps.

[00119] For all combined first and second region sequences, additional amino acid sequences can be added to either the amino or carboxy termini in order to facilitate purification. Such sequences may include GST-tags, FLAG-tags, myc-tags, His-, HHHHHHGS, the latter utilizing a GS linker, and other similar tags known to those in the art. In addition, ligands such as the biotin-acceptor protein together with the active BirA protein may be used. For sequences that include an N-terminal initiating methionine, if a N-terminal purification domain is added the methionine will be on the N-terminal of the purification domain instead of at the N-terminal of the peptide or non-peptide first region. For any tag, a peptide linker known in the art may be used and removed after purification with a specific protease.

[00120] In one embodiment, the amino acid tail or ligand is fused to the C-terminus of the peptide or the conjugate. For example, as described in WO 2015/025217 in FIG. 4 a biologically-active cargo peptide (2), linked on its C-terminus (6) through either a peptide bond (13) or a linker sequence known in the art (12) to the N-terminus (3) of the human homeodomain “first region” (1). A GS linker (14) is shown attaching a His-tag (“tail”) to the C-terminus (6) of the cargo peptide (2). Any linker may be used provided it does not compromise the function of the peptide or the conjugate.

[00121] In one embodiment, the immobilized substrate is a nickel or cobalt column, avidin column, or an antibody column with affinity for the amino acid tail. In another embodiment, the peptide or the conjugate’s amino acid tail is serially brought into contact with at least two immobilized substrates with which the tail has affinity, in which case the nickel or cobalt column and/or avidin and/or antibody may be used in any order.

[00122] In another embodiment, the method of purifying a peptide, a conjugate, or a fusion protein comprises fusing an amino acid tail or ligand to the peptide, the conjugate, or the fusion protein, which tail is capable of binding to at least one substrate while impurities bind only to a second substrate; the peptide, the conjugate, or the fusion protein is contacted with the other substrate such that the peptide, the conjugate, or the fusion protein is not bound and remaining impurities are bound to the other substrate.

[00123] According to another embodiment, a method for producing and purifying a peptide, a conjugate, or a fusion protein comprising the cell penetrating peptide, non-peptide, or variant or portion thereof and a p53 peptide comprises culturing the host cell for the expression of the peptide, the conjugate, or the fusion protein from the expression vector and subsequently recovering the peptide, the conjugate, or the fusion protein using affinity purification techniques known in the art.

[00124] When the second region is a DNA binding domain, a complex with nucleic acid may be formed by mixing the conjugate formed with the nucleic acid.

[00125] A bacterial expression construct can be generated as described in WO 2015/025217 however, other methods are also contemplated and are known by one of ordinary skill in the molecular biology /biotechnology art.

[00126] Further embodiments include pharmaceutical compositions comprising the peptides, conjugates, or fusion proteins of the embodiments disclosed herein, and methods of use of the peptides, conjugates, or fusion proteins in the preparation of a medicament for the treatment of a disease.

[00127] In one embodiment, a peptide, a conjugate, or a fusion protein comprises a biologically active peptide or protein that is a functional enzyme, linked to the CPP, non-peptide, or variant or portion thereof. [00128] Other embodiments comprise any of a variety of formulations for treating conditions or diseases identified. Peptide, fusion protein, or conjugate embodiments delivering one or more therapeutic molecules such as genes or proteins may be used alone or in combination with other treatments or components of a treatment. Diseases which may be treated include, but are not limited to aging and/or age-related diseases, such as cancer, neurodegeneration, neurological diseases (e.g., Parkinson’s disease, dementia such as but not limited to Alzheimer’s disease), inherited diseases, metabolic disease, cardiovascular disease, cerebrovascular disease (e.g., stroke), high blood pressure (e.g., hypertension), diabetes, arthritis (e.g., osteoarthritis), respiratory disease (e.g., chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis), viral and bacterial infections, and diseases of the immune system. In some embodiments, the age-related diseases include age specific illnesses associated with senescence. Treatment may also include, but is not limited to, affecting a subject’s healthspan such as by increasing a subject’s healthspan. As used herein, “healthspan” refers to a length of time that a subject is healthy. As used herein, “increasing a subject’s healthspan” refers to a healthspan of a subject that is longer in duration compared to another subject’s healthspan.

[00129] In some embodiments, the methods of treating conditions or diseases in a subject, including treating an age-related disease or causing senolysis, comprise administering to the subject a therapeutically effective amount of a p53 peptide, a conjugate, a fusion protein, and/or a combination thereof according to the present technology. A “therapeutically effective amount” of a p53 peptide, a conjugate, a fusion protein, and/or a combination thereof as used herein is an amount of the p53 peptide, conjugate, fusion polypeptide, and/or combination thereof that produces a desired effect in a subject for treating and/or preventing a condition, e.g., a therapeutic effect. In certain embodiments, the therapeutically effective amount is an amount of the p53 peptide, conjugate, fusion polypeptide, and/or combination thereof that yields maximum therapeutic effect. In other embodiments, the therapeutically effective amount yields a therapeutic effect that is less than the maximum therapeutic effect. For example, a therapeutically effective amount may be an amount that produces a therapeutic effect while avoiding one or more side effects associated with a dosage that yields maximum therapeutic effect. A therapeutically effective amount for a particular composition will vary based on a variety of factors, including, but not limited to, the characteristics of the therapeutic composition (e.g., activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (e.g., age, body weight, sex, disease type and stage, medical history, general physical condition, responsiveness to a given dosage, and other present medications), the nature of any pharmaceutically acceptable carriers, excipients, and preservatives in the composition, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely, by monitoring a subject’s response to administration of the p53 peptide, conjugate, fusion polypeptide, and/or combination thereof and adjusting the dosage accordingly. For additional guidance, see, for example, Remington: The Science and Practice of Pharmacy, 22nd ed., Pharmaceutical Press, London, 2012, and Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 12th ed., McGraw-Hill, New York, NY, 2011, the entire disclosures of which are incorporated by reference herein.

[00130] Composition embodiments disclosed herein may comprise a pharmaceutically acceptable carrier, diluent or excipient. The term “pharmaceutically acceptable carrier diluent or excipient” refers to any substance, not itself a therapeutic agent, used as a carrier or vehicle for delivery of a therapeutic agent to a subject or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a unit dose of the composition, and that does not produce unacceptable toxicity or interaction with other components in the composition.

[00131] The choice of pharmaceutically acceptable carrier, excipient or diluent may be selected based on the formulation and the intended route of administration, as well as standard pharmaceutical practice. Such compositions may comprise any agents that may aid, regulate, release or increase entry into the body compartment, tissue, intracellular or intranuclear target site, such as binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilizing agent(s), or other agents. An injectable implant for the sustained release of the protein may also be used to obtain prolonged exposure and action. The term “sustained release” refers to formulations from which the conjugate is released at a slow rate allowing for a longer period of exposure at active concentrations.

[00132] The compositions comprising one or more conjugates or fusion proteins disclosed herein can be administered, depending on condition to be treated or other considerations, in any number of ways, for example without limitation, by any one or more of the following: (1) inhalation; (2) in the form of a suppository or pessary; (3) in the form of a topical lotion, solution, cream, ointment or dusting powder; (4) by use of a skin patch; (5) orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavoring or coloring agents; (6) injected parenterally, for example intracavemosally, intravenously, intramuscularly or subcutaneously; (7) for ophthalmic diseases, they may be formulated as eye drops or for intraocular injection; (8) for parenteral administration, they may be in the form of a sterile aqueous solution or injectable implant which may contain other substances, for example, with adequate salt or monosaccharide content to make the solution isotonic with blood or substances that allow slow release; (9) for buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner; and (10) for inhalation, they may be in the form of an aerosol, a gas, a liquid, or a combination thereof, which may contain other substances, for example, with saline or other carriers, solvents, or the like useful with conventional methods and conventional substances for formulating compositions for inhalation through a nasal passage.

[00133] In one embodiment, the active concentration of a peptide, a fusion protein, or a conjugate in cell culture is less than about 115 mM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 65 pM, less than about 60 pM, less than about 55 pM, less than about 50 pM, less than about 45 pM, less than about 40 pM, less than about 35 pM, less than about 30 pM, less than about 25 pM, less than about 20 pM, less than about 15 pM, less than about 10 pM, less than about 5 pM, or less than about 1 pM for example, less than about 1 pM to about 3 pM, less than about 1 pM to about 6 pM, less than about 1 pM to about 8 pM, less than about 1 pM to about 15 pM, less than about 1 pM to about 25 pM, less than about 1 pM to about 50 pM, less than about 1 pM to about 70 pM, less than about 1 pM to about 85 pM, less than about 1 pM to about 110 pM, less than about 10 pM to about 110 pM, less than about 15 pM to about 70 pM, less than about 15 pM to about 60 pM, less than about 20 pM to about 55 pM, or less than about 25 pM to about 45 pM.

[00134] In other embodiments, the dosage delivered (daily or as required) in mouse models (per 20 g mouse) through (1) a subcutaneous (s.c.), an intravenous (i.v.), intramuscular (i.m), or intraperitoneal (i.p.) injection, (2) oral, or (3) a topical formulation, is at least 500 pg, at least 450 pg, at least 400 pg, at least 350 pg, at least 300 pg, at least 250 pg, at least 200 pg, at least 150 pg, at least 100 pg, at least 80 pg, at least 70 pg, at least 60 pg, at least 50 pg, at least 40 pg, at least 30 pg, at least 20 pg, at least 10 pg, or at least 1 pg, for example, about 1 pg to about 500 pg, about 10 pg to about 450 pg, about 20 pg to about 400 pg about 30 pg to about 350 pg, about 30 pg to about 200 pg, about 30 pg to about 100 pg, about 40 pg to about 300 pg, about 40 pg to about 200 pg, about 50 pg to about 100 pg, about 50 pg to about 90 pg, about 55 pg to about 85 pg, about 60 pg to about 80 pg, about 60 pg to about 100 pg; about 1 pg to about 200 pg; about 1 mg to about 100 pg. about 1 pg to about 90 pg, about 1 pg to about 80 pg, about 1 pg to about 70 pg, about 1 to about 60 pg, about 1 to about 50 pg, about 1 to about 40 pg, about 1 to about 30 pg, about 1 to about 20 pg, about 1 to about 15 pg, about 1 to about 12 pg, about 1 to about 10 pg, about 1 to about 8 pg about 1 to about 6 pg, about 1 to about 4 pg, or about 1 to about 3 pg.

[00135] In other embodiments, the dosage delivered (daily or as required) through a subcutaneous (s.c ). an intravenous (i.v.), an intramuscular (i.m), or an intraperitoneal (i.p.) injection in a mouse model is at least 100 mg/kg, less than about 80 mg/kg, less than about 45 mg/kg, less than about 40 mg/kg, less than about 30 mg/kg, less than about 25 mg/kg, less than about 20 mg/kg, less than about 15 mg/kg, less than about 12 mg/kg, less than about 10 mg/kg, less than about 8 mg/kg, less than about 4 mg/kg, less than about 2 mg/kg, or less than about 1 mg/kg, for example, less than about 1 mg/kg to about 50 mg/kg, about 5 mg/kg to about 40 mg/kg, about 8 mg/kg to about 30 mg/kg, about 10 mg/kg to about 20 mg/kg, or about 12 mg/kg to about 15 mg/kg, about 8 mg/kg to about 12 mg/kg, about 5 mg/kg to about 9 mg/kg, about 3 mg/kg to about 6 mg/kg, about 2 mg/kg to about 5 mg/kg, about 2 mg/kg to about 4 mg/kg, about 1 mg/kg to about 3 mg/kg, about 1 mg/kg to about 2.0 mg/kg.

[00136] In other embodiments, the dosage delivered (daily or as required) through topical formulation in humans and in mouse models is less than about 5% wt/vol, less than about 4.5% wt/vol, less than about 3.5% wt/vol, less than about 2.5% wt/vol, less than about 1.5% wt/vol, less than about 0.5% wt/vol, less than about 0.4% wt/vol, less than about 0.3% wt/vol, less than about 0.2%, less than about 0.1% wt/vol, less than about 0.09% wt/vol, less than about 0.08% wt/vol, less than about 0.07% wt/vol, less than about 0.06% wt/vol, less than about 0.05% wt/vol, less than about 0.04% wt/vol, less than about 0.03% wt/vol, less than about 0.02% wt/vol, less than about 0.01% wt/vol, less than about 0.008% wt/vol, less than about 0.006% wt/vol, less than about 0.004% wt/vol, or less than about 0.002% wt/vol, for example between about 0.002% wt/vol and about 5% wt/vol, about 0.01% wt/vol and about 4% wt/vol, about 0.05% wt/vol and about 3% wt/vol, about 0.02% wt/vol and about 2.5% wt/vol, about 0.03% wt/vol and about 2% wt/vol, about 0.05% wt/vol and about 1% wt/vol, about 0.06% wt/vol and about 0.9% wt/vol, about 0.07% wt/vol and about 0.6% wt/vol, about 0.08% wt/vol and about 0.4% wt/vol, about 0.09% wt/vol and about 0.2% wt/vol or about 0.09 wt/vol and about 0.1% wt/vol.

[00137] In other embodiments the dosage delivered (daily or as required) through a topical formulation in humans (70 kg weight) is less than about 70 pg, less than about 50 pg, less than about 45 mg, less than about 40 pg. less than about 30 pg, less than about 25 pg, less than about 20 pg, less than about 15 pg, less than about 12 pg, less than about 10 pg, less than about 8 pg, less than about 4 pg, less than about 2 pg, or less than about 1 pg, for example, about 1 pg to about 50 pg, about 5 pg to about 40 pg, about 8 pg to about 30 pg, about 10 pg to about 20 pg, or about 12 pg to about 15 pg, about 8 pg to about 12 pg, about 5 pg to about 9, about 3 pg to about 6 pg, about 2 pg to about 5 pg, or less than about 1 pg to about 3 pg.

[00138] In other embodiments, the dosage delivered (daily or as required) to humans (based on 70 kg weight) through an intravenous, subcutaneous, intraperitoneal, or intramuscular injection, or injectable implant for the sustained release, or topical formulation is at least about 600 mg, at least about 500 mg, at least about 450 mg, at least about 400 mg, at least about 350 mg, at least about 300 mg, at least about 250 mg, at least about 200 mg, at least about 150 mg, at least about 125 mg, at least about 100 mg, at least about 75 mg, at least about 50 mg, at least about 25 mg, at least about 20 mg, at least about 15 mg, at least about 10 mg, at least about 5 mg, at least about 1 mg, at least about 500 pg, at least about 450 pg, at least about 400 pg, at least about 350 pg, at least about 300 pg, at least about 250 pg, at least about 200 pg, at least about 150 pg, at least about 100 pg, at least about 80 pg, at least about 70 pg, at least about 60 pg, at least about 50 pg, at least about 40 pg, at least about 30 pg, at least about 20 pg, at least about 10 pg, or at least about 1 pg, for example, between about 1 pg to about 750 pg; about 1 pg to about 500 pg, about 10 pg to about 450 pg, about 20 pg to about 400 pg about 30 pg to about 350 pg, about 30 pg to about 200 pg, about 30 pg to about 100 pg, about 40 pg to about 300 pg, about 40 pg to about 200 pg, about 50 pg to about 100 pg, about 50 pg to about 90 pg, about 55 pg to about 85 pg, about 60 pg to about 80 pg, about 60 pg to about 100 pg; about 1 pg to about 200 pg; about 1 pg to about 100 pg, about 1 pg to about 90 pg, about 1 pg to about 80 pg, about 1 pg to about 70 pg, about 1 to about 60 pg, about 1 to about 50 pg, about 1 to about 40 pg, about 1 to about 30 pg, about 1 to about 20 pg, about 1 to about 15 pg, about 1 to about 12 pg, about 1 to about 10 pg, about 1 to about 8 pg about 1 to about 6 pg, about 1 to about 4 pg, about 1 pg to about 3 pg, about 1 pg to about 1 mg, about 1 pg to about 2 mg, about 1 pg to about 5 mg; about 1 pg to about 10 mg; about 1 mg to about 10 mg, about 1 mg to about 15 mg; about 2 mg to about 20 mg, about 3 mg to about 30 mg, about 4 mg to about 40 mg, about 5 mg to about 50 mg, about 5 mg to about 80 mg, about 5 mg to about 110 mg, about 10 mg to about 150 mg, about 10 mg to about 80 mg, about 20 mg to about 70 mg, about 20 mg to about 60 mg, about 30 mg to about 60 mg, about 120 mg to about 190 mg, about 130 mg to about 180 mg, about 130 mg to about 200 mg, about 140 mg to about 250 mg, about 180 mg to about 300 mg, about 190 mg to about 350 mg, about 220 mg to about 400 mg, about 250 mg to about 425 mg, about 280 mg to about 460 mg, about 300 mg to about 480 mg, about 350 mg to about 490 mg, about 380 mg to about 550 mg, about 400 mg to about 580 mg, about 480 mg to about 590 mg, or about 520 mg to about 600 mg.

[00139] In other embodiments, the dosage delivered (daily or as required) to humans (based on 70 kg weight) through oral formulation, nasal formulation (e.g., inhalation), or topical formulation is at least about 600 mg, at least about 500 mg, at least about 450 mg, at least about 400 mg, at least about 350 mg, at least about 300 mg, at least about 250 mg, at least about 200 mg, at least about 150 mg, at least about 125 mg, at least about 100 mg, at least about 75 mg, at least about 50 mg, at least about 25 mg, at least about 20 mg, at least about 15 mg, at least about 10 mg, at least about 5 mg, at least about 1 mg, at least about 500 pg, at least about 450 pg, at least about 400 pg, at least about 350 pg, at least about 300 pg, at least about 250 pg, at least about 200 pg, at least about 150 pg, at least about 100 pg, at least about 80 pg, at least about 70 pg, at least about 60 pg, at least about 50 pg, at least about 40 pg, at least about 30 pg, at least about 20 pg, at least about 10 pg, or at least about 1 pg, for example, between about 1 pg to about 750 pg; about 1 pg to about 500 pg, about 10 pg to about 450 pg, about 20 pg to about 400 pg about 30 pg to about 350 pg, about 30 pg to about 200 pg, about 30 pg to about 100 pg, about 40 pg to about 300 pg, about 40 pg to about 200 pg, about 50 pg to about 100 pg, about 50 pg to about 90 pg, about 55 pg to about 85 pg, about 60 pg to about 80 pg, about 60 pg to about 100 pg; about 1 pg to about 200 pg; about 1 pg to about 100 pg, about 1 pg to about 90 pg, about 1 pg to about 80 pg, about 1 pg to about 70 pg, about 1 to about 60 pg, about 1 to about 50 pg, about 1 to about 40 pg, about 1 to about 30 pg, about 1 to about 20 pg, about 1 to about 15 pg, about 1 to about 12 pg, about 1 to about 10 pg, about 1 to about 8 pg about 1 to about 6 pg, about 1 to about 4 pg, about 1 pg to about 3 pg, about 1 pg to about 1 mg, about 1 pg to about 2 mg, about 1 pg to about 5 mg; about 1 pg to about 10 mg; about 1 mg to about 10 mg, about 1 mg to about 15 mg; about 2 mg to about 20 mg, about 3 mg to about 30 mg, about 4 mg to about 40 mg, about 5 mg to about 50 mg, about 5 mg to about 80 mg, about 5 mg to about 110 mg, about 10 mg to about 150 mg, about 10 mg to about 80 mg, about 20 mg to about 70 mg, about 20 mg to about 60 mg, about 30 mg to about 60 mg, about 120 mg to about 190 mg, about 130 mg to about 180 mg, about 130 mg to about 200 mg, about 140 mg to about 250 mg, about 180 mg to about 300 mg, about 190 mg to about 350 mg, about 220 mg to about 400 mg, about 250 mg to about 425 mg, about 280 mg to about 460 mg, about 300 mg to about 480 mg, about 350 mg to about 490 mg, about 380 mg to about 550 mg, about 400 mg to about 580 mg, about 480 mg to about 590 mg, or about 520 mg to about 600 mg.

[00140] In other embodiments, the dosage delivered (daily or as required) to humans (based on 70 kg weight) through any formulation other than an intravenous, subcutaneous, or intramuscular injection or injectable implant for the sustained release, oral formulation, nasal formulation (e.g., inhalation), or topical formulation is at least about 600 mg, at least about 500 mg, at least about 450 mg, at least about 400 mg, at least about 350 mg, at least about 300 mg, at least about 250 mg, at least about 200 mg, at least about 150 mg, at least about 125 mg, at least about 100 mg, at least about 75 mg, at least about 50 mg, at least about 25 mg, at least about 20 mg, at least about 15 mg, at least about 10 mg, at least about 5 mg, at least about 1 mg, at least about 500 pg, at least about 450 pg, at least about 400 pg, at least about 350 pg, at least about 300 pg, at least about 250 pg, at least about 200 pg, at least about 150 pg, at least about 100 pg, at least about 80 pg, at least about 70 pg, at least about 60 pg, at least about 50 pg, at least about 40 pg, at least about 30 pg, at least about 20 pg, at least about 10 pg, or at least about 1 pg, for example, between about 1 pg to about 750 pg; about 1 pg to about 500 pg, about 10 pg to about 450 pg, about 20 pg to about 400 pg about 30 pg to about 350 pg, about 30 pg to about 200 pg, about 30 pg to about 100 pg, about 40 pg to about 300 pg, about 40 pg to about 200 pg, about 50 pg to about 100 pg, about 50 pg to about 90 pg, about 55 pg to about 85 pg, about 60 pg to about 80 pg, about 60 pg to about 100 pg; about 1 pg to about 200 pg; about 1 pg to about 100 pg, about 1 pg to about 90 pg, about 1 pg to about 80 pg, about 1 pg to about 70 pg, about 1 to about 60 pg, about 1 to about 50 pg, about 1 to about 40 pg, about 1 to about 30 pg, about 1 to about 20 pg, about 1 to about 15 pg, about 1 to about 12 pg, about 1 to about 10 pg, about 1 to about 8 pg about 1 to about 6 pg, about 1 to about 4 pg, about 1 pg to about 3 pg, about 1 pg to about 1 mg, about 1 pg to about 2 mg, about 1 pg to about 5 mg; about 1 pg to about 10 mg; about 1 mg to about 10 mg, about 1 mg to about 15 mg; about 2 mg to about 20 mg, about 3 mg to about 30 mg, about 4 mg to about 40 mg, about 5 mg to about 50 mg, about 5 mg to about 80 mg, about 5 mg to about 110 mg, about 10 mg to about 150 mg, about 10 mg to about 80 mg, about 20 mg to about 70 mg, about 20 mg to about 60 mg, about 30 mg to about 60 mg, about 120 mg to about 190 mg, about 130 mg to about 180 mg, about 130 mg to about 200 mg, about 140 mg to about 250 mg, about 180 mg to about 300 mg, about 190 mg to about 350 mg, about 220 mg to about 400 mg, about 250 mg to about 425 mg, about 280 mg to about 460 mg, about 300 mg to about 480 mg, about 350 mg to about 490 mg, about 380 mg to about 550 mg, about 400 mg to about 580 mg, about 480 mg to about 590 mg, or about 520 mg to about 600 mg.

[00141] In one embodiment, the formulation is administered at intervals of 6 hours, 12 hours, daily or every other day or on a weekly or monthly basis to elicit the desired benefit or otherwise provide a therapeutic effect. In another embodiment, the formulation is administered as required to elicit the desired benefit or otherwise provide a therapeutic effect.

[00142] In one embodiment, upon treatment of one or more human or animal subjects with any of the peptide, fusion protein, or conjugate embodiments disclosed, the subject(s) will exhibit one or more of the following outcomes:

(a) prevention of and/or reduction in localized and/or systemic inflammation;

(b) prevention of and/or reduction in deterioration of cognitive ability;

(c) prevention of and/or reduction in cancer;

(d) prevention of and/or reduction in a neurological disease;

(e) prevention of and/or reduction in an inherited disease;

(f) prevention of and/or reduction in cardiovascular disease;

(g) prevention of and/or reduction in cerebrovascular disease;

(h) prevention of and/or reduction in high blood pressure;

(i) prevention of and/or reduction in diabetes;

(j) prevention of and/or reduction in arthritis;

(k) prevention of and/or reduction in chronic obstructive pulmonary disease;

(l) prevention of and/or reduction in viral infections;

(m) prevention of and/or reduction in bacterial infections;

(n) prevention of and/or reduction in disease of the immune system;

(o) prevention of and/or reduction in idiopathic pulmonary fibrosis;

(p) reduced activation of FOX04 regulated genes;

(q) reduced activation of p53 regulated genes;

(r) an inhibition of FOX04 transcriptional activity; (s) an inhibition of p53 activity;

(t) an increase in senolysis; and

(u) reduced cellular senescence.

[00143] In another embodiment, the patient will be treated over a period, for example, of about 1 day through the lifetime of the patient, over a period of about 1 day to about 200 weeks, about 1 day to about 100 weeks, about 1 day to about 80 weeks, about 1 day to about 50 weeks, about 1 day to about 40 weeks, about 1 day to about 20 weeks, about 1 day to about 15 weeks, about 1 day to about 12 weeks, about 1 day to about 10 weeks, about 1 day to about 5 weeks, about 1 week to about 4 weeks, about 2 weeks to about 3 weeks, about 1 day to about 2 weeks, about 1 week, about 1 to 5 days, about 1 to 3 days, or about 1 to 2 days.

[00144] In another embodiment comprising a peptide, a fusion protein, or a conjugate formulation utilized in any of the proposed studies in the examples provided, in other research and treatment, including animal research for human and animal applications, and veterinary treatment, the treatment group members, or the treatment group(s) will exhibit one or more of the following outcomes, each compared to baseline or control, unless otherwise indicated:

(a) prevention of and/or reduction in localized and/or systemic inflammation;

(b) prevention of and/or reduction in deterioration of cognitive ability;

(c) prevention of and/or reduction in cancer;

(d) prevention of and/or reduction in a neurological disease;

(e) prevention of and/or reduction in an inherited disease;

(f) prevention of and/or reduction in cardiovascular disease;

(g) prevention of and/or reduction in cerebrovascular disease;

(h) prevention of and/or reduction in high blood pressure;

(i) prevention of and/or reduction in diabetes;

(j) prevention of and/or reduction in arthritis;

(k) prevention of and/or reduction in chronic obstructive pulmonary disease;

(l) prevention of and/or reduction in viral infections;

(m) prevention of and/or reduction in bacterial infections; (n) prevention of and/or reduction in disease of the immune system;

(o) prevention of and/or reduction in idiopathic pulmonary fibrosis;

(p) reduced activation of F0X04 regulated genes;

(q) reduced activation of p53 regulated genes;

(r) an inhibition of F0X04 transcriptional activity;

(s) an inhibition of p53 activity;

(t) an increase in senolysis; and

(u) reduced cellular senescence.

[00145] In another embodiment, the treatment with a formulation comprising a peptide, a fusion protein and/or a conjugate embodiment disclosed in clinical studies will extend over a period, for example, of about 1 day to about 52 weeks, about 1 day to about 26 weeks, about 1 day to about 16 weeks, about 1 day to about 12 weeks, about 1 day to about 10 weeks, about 1 day to about 5 weeks, about 1 week to about 4 weeks, about 2 weeks to about 3 weeks, about 1 day to about 2 weeks, about 1 week, about 1 to 6 days, about 1 to 4 days, or about 1 to 2 days.

[00146] In another embodiment, upon treatment with a formulation comprising a peptide, a fusion protein and/or a conjugate embodiments disclosed, the (1) patient(s) or (2) treatment group(s) as disclosed in the studies in the examples, including experimental animals such as mice in animal models, exhibit one or more of the following outcomes compared to controls:

[00147] (a) prevention of and/or reduction in localized and/or systemic inflammation of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00148] (b) prevention of and/or reduction in deterioration of cognitive ability of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00149] (c) prevention of and/or reduction in cancer of at least about 99%, at least about

95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00150] (d) prevention of and/or reduction in a neurological disease of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00151] (e) prevention of and/or reduction in an inherited disease of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00152] (1) prevention of and/or reduction in cardiovascular disease of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00153] (g) prevention of and/or reduction in cerebrovascular disease of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00154] (h) prevention of and/or reduction in high blood pressure of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00155] (i) prevention of and/or reduction in diabetes of at least about 99%, at least about

95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00156] (j) prevention of and/or reduction in arthritis of at least about 99%, at least about

95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00157] (k) prevention of and/or reduction in chronic obstructive pulmonary disease of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00158] (1) prevention of and/or reduction in viral infections of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00159] (m) prevention of and/or reduction in bacterial infections of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00160] (n) prevention of and/or reduction in disease of the immune system of at least about

99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00161] (o) prevention of and/or reduction in idiopathic pulmonary fibrosis of at least about

99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00162] (p) reduced activation of FOX04 regulated genes of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00163] (q) reduced activation of p53 regulated genes of at least about 99%, at least about

95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00164] (r) an inhibition of FOX04 transcriptional activity of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00165] (s) an inhibition of p53 activity of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);

[00166] (t) an increase in senolysis of at least about 99%, at least about 95%, at least about

90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control); and

[00167] (u) reduced cellular senescence of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control).

[00168] According to the embodiments disclosed and their modifications, the peptides, the conjugates, or the fusion proteins may be used alone or in combination with other treatments or components of other treatments. Diseases and disorders or conditions that may be treated include, but are not limited, to age-related diseases. In some embodiments, the age-related diseases include age specific illnesses associated with senescence.

[00169] Various features and embodiments and select modifications will now be described by way of non-limiting examples. In all examples where an initiating methionine is included in a complete sequence to allow recombinant synthesis, the initiating methionine may be removed during purification to derive the active product with the indicated peptide sequence. References to sequence numbers for complete conjugates in paragraphs describing their use are referring to the conjugate after removal of the initiating methionine.

EXAMPLE 1

[00170] Certain age-related diseases involve pathologic cellular senescence and impaired senolysis. The direct interaction between FOX04 and p53 proteins is a novel molecular marker of cellular senescence. Therefore, disrupting the direct interaction between FOX04 and p53 serves as a therapeutic target for treating senescence-associated and age-related diseases. In this example, the FOX04 binding regions on p53 were identified and novel peptides which disrupt F0X04:p53 binding were rationally designed based on the identified binding regions.

[00171] FIGS. 2A-2G illustrates at least seven steps in a method of using peptide microarray technology to develop novel peptide disrupters of protein-protein interactions (PPI) (FIG. 2A). The methods are as follows:

1) Initial peptide screening. Full-length human p53 protein was synthesized in 25mer amino acid segments, spanning the entire length of the protein. Peptides were shifted by 5 amino acids (FIG. 2B).

2) Purified recombinant human full-length FOX04 (His-Tag) protein was overlaid onto p53 full-length peptide microarray and initial binding regions were identified following a standard far-westem experimental procedure (FIGS. 2C and 2D).

3) FOX04 binding was identified in three p53 regions (1) DNA-binding domain (DBD), (2) nuclear localization signal (oligomerization) region (NLS), and (3) C- terminal regulatory domain (CRD) (FIG. 2E).

4) Each region was subjected to alanine substitution analyses and truncation analyses to identify essential binding amino acids (e.g., hot spots) and the minimum binding sequences required for FOX04 binding (FIG. 2F). 5) Minimum binding sequences identified in step (4) were used to design three p53 peptides in which F0X04 binds: GTRVRAMAIYKQ (DBD peptide; SEQ ID No. 168), STKRALPNNTS (NLS peptide; SEQ ID No. 170) AND RAHSSHLKS (CRD domain peptide; SEQ ID No. 171) (FIG. 2F).

6) Each peptide from step (5) was subjected to rational point substitution analyses, where each amino acid was substituted for every other natural L-amino acid and changes in F0X04 binding were assessed (FIG. 2F).

7) p53 peptides were also assessed as their respective D-amino (D) and D-Retro Inverso (DRI) amino acid sequences. Binding to F0X04 following D-amino and D-Retro Inverso amino acid sequence changes was monitored (FIG. 2F).

8) Results from rational point substitution analyses and D/DRI-amino acid analyses led to the generation of a peptide library containing ten novel p53-based peptides (see Table 5) (FIG. 2G).

9) The binding kinetics of all p53 peptides with F0X04 was assessed using a Fluorescent Polarization assay in order to refme/narrowing down potential leading compounds within library (see Table 5).

Table 5: p53 Peptide Library.

[00172] Using this method, a rapid analysis (approximately 16 weeks) of more than 2000 peptides led to the generation of a highly selective p53-based peptide library having peptides of 12 amino acids or less. A 9 amino acid p53 peptide (RAHSSHLKS, SEQ ID No. 171) demonstrated the ability to bind F0X04 with less than lOnM affinity.

[00173] FIG. 3 is a schematic diagram illustrating which protein-protein interactions between p53 and F0X04 that at least one of the novel peptide disrupters of the present technology disrupts.

[00174] FIGS. 4A and 4B demonstrate interaction between an epitope-tagged F0X04 peptide and an epitope-tagged p53 peptide using ex vivo co-immunoprecipitation. Cell lysates from HEK293 cells expressing His epitope tagged p53 (p53-His) and FLAG epitope tagged FOX04 (FOX04-Flag) were used to demonstrate the interaction in FIG. 4A where p53-His was used to immunoprecipitated and FOX04-FLAG was used to co-immunoprecipitate. As shown in FIG. 4B, an in vitro biochemical protein pull down assay was used to show that p53-GST (GST tagged p53) pulled down FOX04-His following co-incubation and co-precipitation.

[00175] An initial mapping of full-length p53 binding regions is illustrated in FIGS. 5A-5H and shows that certain p53 peptides bind to epitope-tagged FOX04. FOX04 binds two peptides within the p53 DBD, NLS, and CRD (FIG. 5A). Substitution of alanine for other amino acid residues in certain p53 peptides, at R ¹⁵⁶ , R ¹⁵⁸ , K ¹⁶⁴ , R ³⁶³ and K ³⁷⁰ results in loss of FOX04 binding (FIGS. 5B-5D). The full results of the initial mapping are shown in FIGs. 5E-5H. Some of the peptides shown in FIGs. 5A-5H were FIG. 6 shows that substitution of alanine for other amino acid residues in certain p53 peptides results in identification of certain false positive peptides listed in FIGS. 5A-5H.

[00176] As shown in FIGS. 7A-7C, an N and C truncation analysis, and a dual N-C truncation analysis was performed to determine a minimum amino acid sequence for binding to p53 at P ¹⁵¹ - R ¹⁷⁵ . The three-dimensional crystal structure of p53 is shown in FIG. 8, for reference.

[00177] Further data analyses of truncation and alanine substitution results led to the rational design of peptide GTRVRAMAIYQK (SEQ ID No. 179) as the minimal sequence in the DBD of p53. This peptide (SEQ ID No. 179) was analysed using rational point substitution.

[00178] FIGS. 9A and 9B demonstrate results of a rational point substitution analysis to assess sequence optimization of p53 peptide G ¹⁵⁴ TRVRAMAIYKQ ¹⁶⁵ (SEQ ID No. 168). As shown, multiple single amino acid substitutions demonstrate an increase in FOX04 binding to SEQ ID No. 168 vs. control peptides. Both R ¹⁵⁸ and M ¹⁶⁰ are superficial amino acids and GTRVRALAIYKQ (M ¹⁶⁰ L)(SEQ ID No. 169) was included in the finalised p53 peptide library, along with native GTRVRAMAIYKQ (SEQ ID No. 168) sequence Notable substitutions and the associated fold increase in F0X04 binding include, but are not limited to: [1] R ¹⁵⁸ F (1.88- fold increase), [2] R ¹⁵⁸ Y (1.87-fold), [3] M ¹⁶⁰ F (1.6-fold), [4] M ¹⁶⁰ I (1.82-fold), [5] M ¹⁶⁰ L (1.82- fold), [6] A ¹⁶¹ E (1.85-fold).

[00179] As shown in FIGS. 10A-10C, an N and C truncation analysis and assessment of GSTKR substitution resulted in a minimum amino acid sequence for binding to p53 at H ²⁹⁶ - K ³²⁰ . The combined substitution of GSTKR demonstrated greater than a 50% loss in FOX04 binding. Additional data analyses of truncation and alanine substitution results led to the rational design of peptide STKRALPNNTS (SEQ ID No. 170) as the short sequence nuclear localization signal containing a p53 binding peptide.

[00180] A rational point substitution analysis was performed on SEQ ID No. 170 to assess sequence optimization. The results are shown in FIGS. 11 A and 1 IB. Substitution of positively charged K ³⁰⁵ or R ³⁰⁶ with negatively charged D or E acidic amino acids ablates FOX04 binding. These residues are likely important to binding of FOX04 to the p53 peptide. With only small to modest increases in FOX04 binding observed following single amino acid substitutions, the greatest being a 1.28-fold increase at K ³⁰⁵ R, only the native sequence SEQ ID No. 170 was included in a p53 peptide library.

[00181] FIGS. 12A-12C demonstrate that an N and C truncation analysis and a dual N-C truncation analysis results in a minimum amino acid sequence for binding to p53 at E ³⁴⁶ - Q ³⁷⁵ . As shown in FIGS. 12A-12C, binding of FOX04 to the p53 peptide was lost immediately after K ³⁷⁰ truncation. The minimum reliable binding sequence on the p53 peptide was estimated to be between HSSHLK (SEQ ID No. 179) and RAHSSHLK (SEQ ID No. 180) and includes amino acids downstream of K ³⁷⁰ .

[00182] As shown in FIGS. 13A and 13B, an investigation analysis of residues downstream of K ³⁷⁰ resulted in a minimum amino acid sequence for binding to p53 at E ³⁴⁶ - Q ³⁷⁵ following addition of downstream residues S ³⁷¹ , K ³⁷² , and K ³⁷³ to the original proposed minimum binding sequences. The strongest FOX04 binding was identified with peptide RAHSSHLKS (SEQ ID NO. 171).

[00183] FIGS. 14A and 14B demonstrate results of a rational point substitution analysis to assess sequence optimization of p53 peptide R ³⁶³ AHSSHLKS ³⁷¹ (SEQ ID NO. 171). FOX04 binding assessed following single amino acid substitution with every other existing L-amino acid. The experiment was duplicated and F0X04 binding to each of the single residue substituted SEQ ID NO. 171 was compared to control peptides. Substitutions that increase binding significantly were considered for inclusion in the p53 peptide library. The spot average was n=9, the mean was 69.23 A.U., the standard deviation (STDEV) was 8.3 A.U., and the standard error of the mean (SEM) was 2.77 A.U.

[00184] FIGS. 15A and 15B demonstrate results of a D-amino and D-retro inverso sequence analysis of certain p53 peptides for L vs. D vs. DRI amino acids (FIG. 15 A) and a peptide microarray (FIG. 15B). The optimal minimum binding sequences from the three identified p53 regions in which FOX04 binds were compared with their D-amino and D-RI sequence analogues. As shown, FOX04 binding was maintained or reduced. Since binding to FOX04 still occurred, even if minimally, all L-amino p53-peptides DRI analogues were in the p53 peptide library.

[00185] As shown in FIG. 16, direct binding kinetics of epitope-tagged FOX04 and a p53 peptide by fluorescent polarization occurred with SEQ ID NO. 171 (RAHSSHLKS) p53 C- Terminal NRD peptide versus a negative scrambled peptide control. The FOX04 binding affinity of PGL-131 was 8nM (n=3). The direct binding kinetics were obtained by co-incubating purified FOX04-His full-length human protein was with N-terminal FAM Tagged p53 peptides. Fluorescent polarization of p53 peptides was measured following incubation with increasing concentrations of FOX04-His protein from 5nM to 1.25mM. This range of FOX04-His protein concentration was used to identify high affinity binding peptides. A scrambled control peptide demonstrates no direct binding, representative of an appropriate negative control.

[00186] Additional direct binding kinetics as determined by fluorescent polarization are shown in FIGS. 17A-17H (His-tagged FOX04 and p53 peptides corresponding to a p53 DBD), FIGS. 18A-18D (His-tagged FOX04 and p53 peptides corresponding to a p53 NLS), FIGS. 19A-19H (epitope-His FOX04 and p53 peptides corresponding to a p53 C-terminal NRD), and FIGS. 20A and 20B (scrambled negative control peptide). More specifically, FIGs. 17B and 19B are binding curves indicative of peptide-protein binding. As shown in FIG. 17B, peptide GTRVRAMAIYKQ (SEQ ID No. 168) binds to His-tagged FOX04 and, as shown in FIG. 19B, peptide RAHSSHLKS (SEQ ID No. 171) binds to His-tagged FOX04.

[00187] From the foregoing, it will be appreciated that specific embodiments have been described herein for purposes of illustration, but that various modifications may be made without deviating from the spirit and scope of the disclosure. Accordingly, the disclosure is not limited except as by the appended claims.

[00188] A peptide comprising a sequence as described in any one of SEQ ID Nos. 2 to 188 or a variant thereof.

[00189] The peptide of paragraph 188 wherein the peptide comprises any one of SEQ ID Nos. 2 to 76 or a variant thereof.

[00190] The peptide of paragraph 188 wherein the peptide comprises any one of SEQ ID Nos. 168 to 178 or a variant thereof.

[00191] The peptide of paragraph 188 wherein the peptide comprises any one of SEQ ID Nos. 77 to 167, SEQ ID Nos. 179 to 188, or a variant thereof.

[00192] The peptide of paragraph 188 or paragraph 190, wherein the peptide comprises SEQ

ID No. 171, or a variant thereof.

[00193] The peptide of paragraph 188 or paragraph 191, wherein the peptide comprises SEQ

ID No. 182, or a variant thereof.

[00194] The peptide of any one of paragraph 188 to paragraph 193 wherein the variant is a cell-permeable peptide.

[00195] The peptide of any one of paragraph 188 to paragraph 194 wherein the peptide includes at least one D analogue and/or D-retro inverso (DRI) analogue.

[00196] The peptide of any one of paragraph 188 to paragraph 195 wherein the peptide includes at least one stapled and/or cyclic modification.

[00197] The peptide of any one of paragraph 188 to paragraph 196 wherein the peptide includes a least one post-translational modifications selected from the group consisting of methylation, glycosylation, sialylation, acetylation, and phosphorylation.

[00198] A conjugate comprising a sequence as described in any one of SEQ ID Nos. 77 to 151, SEQ ID Nos. 179 to 188 or a variant thereof.

[00199] A conjugate comprising: a first region comprising a peptide sequence as set forth in SEQ ID No. 1 or a variant thereof; and a second region comprising a polypeptide sequence as described in any one of SEQ ID Nos. 2 to 76 and 168 to 178 or a variant thereof. [00200] A conjugate comprising: a first region comprising a peptide sequence as set forth in SEQ ID No. 1 or a variant thereof; and a second region comprising a peptide set forth in any one of claim 1 to claim 10.

[00201] The conjugate of any one of paragraph 198 to paragraph 200 wherein the second region is conjugated to the C-terminus of the first region.

[00202] The conjugate of any one of paragraph 198 to paragraph 201 wherein the second region is conjugated to the N-terminus of the first region.

[00203] The conjugate of any one of paragraph 198 to paragraph 202 in the form of a fusion protein.

[00204] The conjugate of paragraph 198 to paragraph 203 further comprising a linker sequence between the first and second regions.

[00205] The conjugate of any one of paragraph 198 to paragraph 204 wherein an initiating methionine residue of the sequence is removed prior to use as an active and/or therapeutic agent.

[00206] The conjugate of any one of paragraph 198 to paragraph 205, further comprising a non-peptide carrier.

[00207] The conjugate of paragraph 206 wherein the non-peptide carrier is selected from the group consisting of a cationic peptoid, a peptide nucleic acid, aminoglycoside antibiotic, a heterocyclic guanidinium oligomer, and an inositol-based carrier.

[00208] The conjugate of any one of paragraph 198 to paragraph 207 wherein the first region is linked to the second region with a peptidic bond.

[00209] The conjugate of any one of paragraph 198 to paragraph 208 wherein the second region is conjugated to the first region via a non-peptidic bond.

[00210] The conjugate of paragraph 209 wherein the first region and the second region are attached via “click chemistry.”

[00211] The conjugate of any one of paragraph 198 to paragraph 210 wherein the first region comprises a polypeptide sequence derived from a human gene.

[00212] The conjugate of any one of paragraph 198 to paragraph 211 wherein the second region includes a P53 peptide sequence derived from a human gene or variant thereof. [00213] The conjugate of any one of paragraph 198 to paragraph 212 further comprising at least one molecular cargo selected from the group consisting of liposomes, polymers, cationic peptides, nanoparticles, adeno-associated virus (AAV) and calcium.

[00214] A peptide prepared by a method comprising the steps: culturing a host cell transformed with an expression vector comprising a nucleic acid encoding a peptide according to any one of claim 1 to claim 10 under conditions which provide for the expression of the peptide within the host cell; and recovering the peptide by affinity purification.

[00215] A conjugate prepared by a method comprising the steps: culturing a host cell transformed with an expression vector comprising a nucleic acid encoding a conjugate according to any one of claim 11 to claim 26 under conditions which provide for the expression of the conjugate within the host cell; and recovering the conjugate by affinity purification.

[00216] A composition comprising the peptide of any one of paragraph 188 to paragraph 197 and a pharmaceutically acceptable carrier.

[00217] A composition comprising the conjugate of any one of paragraph 198 to paragraph 213 and a pharmaceutically acceptable carrier.

[00218] The composition of paragraph 216 or paragraph 217 wherein the composition is in the form of an injectable composition.

[00219] The composition of paragraph 216 or paragraph 217 wherein the composition is in the form of an oral composition or an inhaled composition.

[00220] The composition of paragraph 216 or paragraph 217 wherein the composition is in the form of a topical composition.

[00221] A method of treating an age-related disease in a subject comprising administering a formulation comprising the composition of any one of paragraph 216 to paragraph 220 to the subject in need thereof.

[00222] A method of causing senolysis in a subject comprising administering a formulation comprising the composition of any one of paragraph 216 to paragraph 220 to the subject in need thereof.

[00223] A method of treating an age-related disease in a subject comprising administering a formulation comprising the peptide of any one of paragraph 188 to paragraph 197 to the subject in need thereof. [00224] A method of causing senolysis in a subject comprising administering a formulation comprising the peptide of any one of paragraph 188 to paragraph 197 to the subject in need thereof.

[00225] A method of treating an age-related disease in a subject comprising administering a formulation comprising the conjugate of any one of paragraph 198 to paragraph 213 to the subject in need thereof.

[00226] A method of causing senolysis in a subject comprising administering a formulation comprising the conjugate of any one of paragraph 198 to paragraph 213 to the subject in need thereof.

[00227] The method of any one of paragraph 221 to paragraph 226 wherein the subject is a human.

[00228] The composition of any one of paragraph 216 to paragraph 220 for use in treating an age-related disease in a subject in need thereof.

[00229] The composition of any one of paragraph 216 to paragraph 220 for use in causing senolysis in a subject in need thereof.

[00230] A composition comprising the peptide of any one of paragraph 188 to paragraph 197 for use in treating an age-related disease in a subject in need thereof.

[00231] A composition comprising the peptide of any one of paragraph 188 to paragraph 197 for use in causing senolysis in a subject in need thereof.

[00232] A composition comprising the conjugate of any one of paragraph 198 to paragraph 213 for use in treating an age-related disease in a subject in need thereof.

[00233] A composition comprising the conjugate of any one of paragraph 198 to paragraph 213 for use in causing senolysis in a subject in need thereof.

[00234] The composition of any one of paragraph 228 to paragraph 233 wherein the subject is a human.

[00235] A composition for use in treating an age-related disease in a subject in need thereof, the composition comprising a peptide comprising a sequence as described in any one of SEQ ID Nos. 2 to 188 or a variant thereof; a conjugate comprising a sequence as described in any one of SEQ ID Nos. 77 to 151 and 179 to 188 or a variant thereof; a conjugate comprising a first region comprising a peptide sequence as set forth in SEQ ID No. 1 or a variant thereof and a second region comprising a polypeptide sequence as described in any one of SEQ ID Nos. 2 to 76 and 168 to 178 or a variant thereof, or a combination thereof.

[00236] A composition for use in for use in causing senolysis in a subject in need thereof, the composition comprising a peptide comprising a sequence as described in any one of SEQ ID Nos. 2 to 188 or a variant thereof; a conjugate comprising a sequence as described in any one of SEQ ID Nos. 77 to 151 and 179 to 188 or a variant thereof; a conjugate comprising a first region comprising a peptide sequence as set forth in SEQ ID No. 1 or a variant thereof and a second region comprising a polypeptide sequence as described in any one of SEQ ID Nos. 2 to 76 and 168 to 178 or a variant thereof, or a combination thereof.

[00237] The composition of paragraph 235 or paragraph 236 wherein the subj ect is a human.

[00238] The composition of paragraph 235 or paragraph 236 further comprising a pharmaceutically acceptable carrier.

[00239] The composition of paragraph 235 or paragraph 236 wherein the composition is in the form of an injectable composition.

[00240] The composition of paragraph 235 or paragraph 236 wherein the composition is in the form of an oral composition or an inhaled composition.

[00241] The composition of paragraph 235 or paragraph 236 wherein the composition is in the form of a topical composition.