WO2017189826A1 - Use of cell-permeable peptides and non-peptide carriers conjugated with nemo binding domain cargo sequence for the treatment of dry eye disease and uveitis - Google Patents

Abstract

Provided herein are compositions and methods for treating dry eye disease and uveitis through administration of cell permeable peptide conjugates linked with NFKB essential modulator (NEMO) binding domain. These peptides facilitate entry into tissues, cells and the nucleus of cells thus allowing the NEMO binding domain to reach its site of action for therapeutic purposes.

Description

USE OF CELL-PERMEABLE PEPTIDES AND NON-PEPTIDE CARRIERS CONJUGATED WITH NEMO BINDING DOMAIN CARGO SEQUENCE FOR THE TREATMENT OF DRY EYE DISEASE AND UVEITIS

PRIORITY CLAIM

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 62/329,540, filed April 29, 2016, the entire contents of which is incorporated herein by references.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 26, 2017, is named 89352- 8005_WO00_SL.TXT and is 52,185 bytes in size.

TECHNICAL FIELD

[0003] The embodiments disclosed herein relate generally to compositions and methods for treating dry eye disease and uveitis through administration of cell permeable peptide conjugates linked with NFKB essential modulator (NEMO) binding domain. These peptides facilitate entry into tissues, cells and the nucleus of cells thus allowing the NEMO binding domain to reach its site of action for therapeutic purposes.

BACKGROUND

[0004] In diseases with manifestations due to aberrant gene function or deficient function, genes may be regulated through the direct delivery of biologically active molecules, such as nucleic acids, peptides and proteins, to their intracellular and intranuclear sites of action to influence gene expression either directly or indirectly through interference with transcription, translation or transcription factor production and action and also missing or defective protein products may be replaced to provide these types of molecules in individuals with germ-line or somatic mutations. The direct replacement of biologically important proteins in genetically deficient individuals is hampered by both (i) the inability of these proteins to reach intracellular sites and tissue sites such as the central nervous system (CNS) where they normally function and (ii) by the immunogenicity of these proteins.

SUMMARY

[0005] The embodiments disclosed herein, together with a range of modifications, provide compositions for conjugates, including fusion proteins, and methods of using them to treat a variety of conditions. The conjugates or fusion proteins incorporate a peptide-based or non-peptide based carrier to translocate NEMO binding domain (NBD) polypeptides, which are not naturally associated with the peptide-based or non-peptide based carriers, across cell membranes to their intended sites of action.

[0006] According to one aspect of the disclosure, the conjugate comprises a first region, having a sequence described in any of SEQ ID Nos. 2 to 65 or variant or portion thereof, conjugated to a second region not naturally associated with the first region, wherein the second region is a NEMO binding domain having a sequence set forth in any one of SEQ ID No. 1 or a variant or variant or portion thereof.

[0007] According to another aspect of the disclosure, the conjugate comprises a first region comprising a non-peptide carrier, conjugated to a second region not naturally associated with the first region, wherein the second region is a NEMO binding domain having a sequence set forth in any one of SEQ ID No. 1 or a variant or variant or portion thereof. In some embodiments, 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. In one preferred embodiment, the non-peptide carrier is an inositol-based carrier.

[0008] According to another aspect of the disclosure, the conjugate is prepared by culturing a host cell transformed with an expression vector comprising a nucleic acid encoding a conjugate of any one of the embodiments described and disclosed herein under conditions which provide for the expression of the conjugate within the host cell; and recovering the conjugate by affinity purification.

[0009] In another aspect the disclosure provides a composition comprising a conjugate of any one of the embodiments described and disclosed herein and a pharmaceutically acceptable carrier. [0010] In yet another aspect, the disclosure provides methods of treating a dry eye in a subject comprising administering a formulation comprising the conjugate of any one of the embodiments described and disclosed herein to the subject, for example, a human.

[0011] In still another aspect, the disclosure provides methods of treating uveitis in a subject comprising administering a formulation comprising the conjugate of any one of the embodiments described and disclosed herein to the subject, for example, a human.

[0012] 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.

[0013] In one embodiment, the conjugate is in the form of a fusion protein.

[0014] In one embodiment, the conjugate further comprises a linker sequence between the first and second regions.

[0015] In another embodiment, the first and second regions combined have a polypeptide sequence selected from the group consisting of SEQ ID Nos: 66-129, or a variant thereof.

[0016] In one embodiment, an initiating methionine residue of the sequence is removed prior to use as an active and/or therapeutic agent.

[0017] In one embodiment, the first region is linked to the second region with a peptidic bond. In another 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."

[0018] In one embodiment, the first region is derived from the human gene. In another embodiment, the second region includes a NEMO binding domain polypeptide sequence derived from a human gene or variant thereof

[0019] 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.

[0020] In another embodiment a conjugate can comprise: (a) a first region comprising a peptide or non-peptide with cell permeabilizing capabilities linked to

(b) a second region comprising a NEMO binding domain polypeptide sequence.

[0021] In another embodiment a composition comprises a conjugate or fusion protein that may take any of the following forms: an eye drop or other ophthalmic composition for local or injectable use; a topical composition; or an injectable composition, including an injectable implant for sustained release.

[0022] Other embodiments comprise methods to prevent immunogenicity of otherwise antigenic proteins delivered as conjugates or fusion proteins to their intended cytosolic or nuclear sites of action.

[0023] These and other embodiments will be disclosed in further detail below.

DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 shows that topical or intra-peritoneal delivery of different cell permeable peptide conjugates linked with the NFKB essential modulator (NEMO) binding domain reduces inflammatory cells in the anterior chamber in an endotoxin-induced uveitis (EIU) in vivo mouse model.

DETAILED DESCRIPTION

[0025] Several of the disclosed embodiments and their modifications relate to novel conjugates, which may take the form of fusion proteins, each comprising a NEMO binding domain peptide linked to a peptide (e.g., cell penetrating peptide (CPP)) or a non- peptide.

[0026] Cargo peptides include both small synthetic peptides and larger proteins such as antibodies or the binding regions of antibodies into cells for therapeutic purposes. (M.J. May et al. "Selective inhibition of NF-κΒ activation by a peptide that blocks the interaction of NEMO with the ΙκΒ kinase complex" Science 289: 1550-54, Sept. 1, 2000)

[0027] The terms "peptide(s)," "protein(s)," and "polypeptide(s)" are used synonymously. [0028] 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.

[0029] 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.

[0030] A person of ordinary skill in the molecular biology/biotechnology art would appreciate that numerous variations of the sequences shown in Table 3 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 m the art (as described in Hames et al, Nucleic Acid Hybridisation. (1985) IRL Press, Oxford, UK). Extent of homology is often measured in terms of percentage of identity between the sequences compared.

[0031] 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.

[0032] 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.

[0033] 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), (He: Leu, Val), (Leu: He, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Trp: Tyr), (Tyr: Trp, Phe), and (Val: He, 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.

[0034] "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.

[0035] First Region: The first region of the conjugate or fusion protein embodiments disclosed may a polypeptide sequence having cell permeabilizing activity, such as SEQ ID Nos. 2 through 65, or variants or portions thereof. Each of the sequences is listed below in Table 3 along with the abbreviation of the name of the gene from which it is derived (SEQ ID Nos. 2 through 65). Cargo sequences may or may not be of human origin depending on their intended function.

[0036] In some embodiments, the first region is a peptide-based carrier, for example, TAT-based peptides, RNA-binding peptides, DNA-binding peptides, cyclic peptides, arginine-rich peptides, cyclodextrins, dynorphin neuropeptides, antibacterial peptide, and antimicrobial peptides (AMP).

[0037] The first region can be protein-derived (e.g., HIV Tat, penetratin, VP- 22, and pVEC), chimeric (e.g., Transportan, Pep-1, MPG, and M-918), de novo designed (e.g., Octa/nona-arginine, MAP, KADY, and KALA). In some embodiments, the first region is a o-amino acid based peptide. In other embodiments, the first region is a β- peptide analogue, for example, of Tat peptides and oligoarginine

[0038] 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 1 below:

[0039] Table 1 : Conservative Amino Acid Substitutions.

ALIPHATIC Non-polar GAP ILV

Polar-uncharged CSTM NQ

Polar - charged DE RK

AROMATIC HFWY OTHER N

[0040] 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 65 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. 2-65 (Table 3) or variant or portion thereof can be used in a fusion protein or conjugate herein.

[0041] In some embodiments, the first region is a cyclic peptide such as the membrane permeable peptide scaffolds described in Hewitt et al, (2015) JACS 137(2):715- 721.

[0042] In some embodiments, the first region is a arginine-rich peptides, for example, as described in SEQ ID No. 16 and described in Futaki et al. (2001) J. Biol Chem 276(8):5836-5840. In some embodiments, the number of arginine residues in between 4 and 16. In a preferred embodiment, the arginine-rich peptide comprises 8 arginine residues.

[0043] In some embodiments, the first region is cyclodextrin-based. The cyclodextrins can be natural (e.g., a-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin), chemically modified derivatives thereof (e.g., hydroxypropyl^-cyclodextrin (ΗΡ-β-CD), randomly methylated^-cyclodextrin (RM- β-CD), and sulfobutylether- β-cyclodextrin (SBE- β-CD), polymerized cyclodextrins (e.g., epichlorohydrin- β-cyclodextrin and carboxy methyl epichlorohydrin β cyclodextrin) and others as described in Gidwani et al. (2015) BioMed Research International 198268.

[0044] In some embodiments, the first region is a dynorphin neuropeptide or derivative thereof, for example, big dynorphin and dynorphin A, as described in Marinova et al. (2005) J Biol Chem 280(28):26360-26370. Dynorphin neuropeptides are short, highly basic peptides that have a number of functions in the brain, both opioid and nonopioid. In some embodiments, the first region is a dynorphin neuropeptide-based polypeptide sequence as described in any one of SEQ ID Nos. 52-55.

[0045] In some embodiments, the first region is a prion protein or derivatives thereof, as described in Lofgren et al. (2008) FASEB 22(7):2177-2184. These peptides have been characterized as cell penetrating peptides (CPPs) and include a signal sequence of about 22, mostly hydrophobic residues) followed by 6 N-terminal, mostly basic, residues. In some embodiments, the first region is a prion-based polypeptide sequence as described in any one of SEQ ID Nos. 56-61. [0046] In some embodiments, the first region is an antibacterial peptide (e.g., LL-37), as described in Zhang et al. (2010) Biochimica et Biophysica Acta 1798(12):2201- 2208 In one embodiment, the first region is an antibacterical-based polypeptide sequence as described in any one of SEQ ID No. 62.

[0047] In some embodiments, the first region is a antimicrobial peptide (AMP), for example, Magainin 2 Buforin 2, as described in Henriques et al. (2006) Biochemical J. 399(1): 1-7. AMPs are short sequences of cationic and hydrophobic peptides wherein the cationic residues enhance electrostatic binding the highly negatively charged membrane of their target and the hydrophobic residues drive the entry of the peptide through the lipid bilayer. In some embodiments, the first region is a prion-based polypeptide sequence as described in any one of SEQ ID Nos. 63-65.

[0048] In some embodiments, the first region comprises a non-peptide carrier, for example, cationic peptoids (e.g., N-substituted glycine oligomer), peptide nucleic acids (PNA), aminoglycoside antiobiotics, heterocyclic guanidinium oligomers, an inositol-based carriers, as described in Chung et al. (2007) Int. J. Pharma 354: 16-22. In one embodiment, the non-peptide carrier consists of multiple units of guandine residues attached to a scaffold structure (monomer or dimer of carbohydrate or cyclitol), as described in Chung et al. In some embodiments, the cyclitol is inositol.

[0049] Enhanced drug delivery systems combination of conjugatesdisclosed herein with other carriers, for example, liposomes, polymers, cationic peptides, nanoparticles, and viral carriers. 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-1- lysine (PLL), a synthetic chiral polymer, or PEG can be added to the conjugates.

[0050] Second Region Peptides or Proteins: The second region of the conjugate or fusion protein embodiments disclosed comprises a NEMO binding domain polypeptide sequence as set forth in SEQ ID No. 1 or a variant thereof. 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.

[0051] 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.

[0052] 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 the treatment. Diseases which may be treated include, but are not limited to: cancer, neurological diseases, inherited diseases, heart disease, stroke, arthritis, viral and bacterial infections, and diseases of the immune system. Suitable therapeutic genes include those coding for tumor suppressor proteins, enzymes, pro-drug activating enzymes, immunomodulatory molecules, antibodies, engineered immunoglobulin-like molecules, conjugates, hormones, membrane proteins, vasoactive proteins or peptides, cytokines, chemokines, anti-viral proteins, antisense RNA and ribozymes.

[0053] The fusion protein or conjugate embodiments disclosed 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.

[0054] 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, Perussis toxin, E. coli toxin toxins, Shigatoxin or Shiga-like toxin.

[0055] The first binding domain may be modified to target cell sites other than the nucleus. [0056] 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.

[0057] The term "conjugate" or "conjugates" herein comprises a category of structures, including fusion proteins, in which the first region is conjugated directly 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 first region 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.

[0058] The first 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, a disulfide bond that will be reduced intracellularly to release the cargo, and 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. 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. [0059] Table 2 - showing partial listing of linkers known in the art, adapted from Chen et al, 2012.

(GGGGS)_n, where n = 1, 2, 3, or 4 (SEQ ID NO: 131)

(Gly)_n, where n = 6 or 8 (SEQ ID NO: 132)

(EAAAK)_n where n = 1 - 3 (SEQ ID NO: 133)

A(EAAAK)₄ALEA(EAAAK)₄ A (SEQ ID NO: 134)

PAPAP (SEQ ID NO: 135)

AEAAAKEAAAKA (SEQ ID NO: 136)

(Ala-Pro)_n where n = 10 - 34 aa (SEQ ID NO: 137)

VSQTSKLTRjAETVFPDV (SEQ ID NO: 138)

PLGjLWAC (SEQ ID NO: 139)

RVLjAEA (SEQ ID NO: 140); EDVVCCjSMSY (SEQ ID NO: 141); GGIEGRjGS (SEQ ID NO: 142)

TRHRQPRjGWE (SEQ ID NO: 143); AGNRVRRjSVG (SEQ ID NO: 144); RRRRRRRjRjR (SEQ ID NO: 145)

GFLGj (SEQ ID NO: 146)

i = cleavable at this location

[0060] 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.

[0061] 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.

[0062] "Expression vectors" or "plasmids" (used interchangeably herein) may be used for producing 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. [0063] 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.

[0064] 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 (T_m) of the hybrid which decreases approximately 1 to 1.5[deg.] 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.

[0065] 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.

[0066] The phrase "moderate stringency" refers to conditions equivalent to hybridization in the above described solution, except that the temperature is at about 60- 62[deg.] C. In that case the final wash is performed at the hybridization temperature in 1*SSC, 0.1% SDS. [0067] Low stringency refers to conditions equivalent to hybridization in the above described solution at about 50-52[deg.] C. In that case, the final wash is performed at the hybridization temperature in 2*SSC, 0.1% SDS.

[0068] It is understood that these conditions may be adapted and duplicated using a variety of buffers, e.g., formamide-based buffers, and temperatures. Denhardt'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.

[0069] In one embodiment, the 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.

[0070] In another embodiment, a host cell is transformed with the expression vector.

[0071] In yet another embodiment a nucleic acid sequence encodes the conjugate or fusion protein for the purposes of synthesis and manufacture by recombinant technology.

[0072] In some embodiments, the 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.

[0073] 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.

[0074] In other embodiments, the 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.

[0075] 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.

[0076] Purification methods known in the art may be used in the process of preparing 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, NJ:Humana Press. The 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 pepentrating peptides (CPPs) and disclosed herein.

[0077] The ability of 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 denaturating agents (urea or guanidinuim) may prevent or inhibit translocation. This conformation-dependent property may be preserved by purifying the peptide conjugate under native conditions.

[0078] In one 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 (NEMO binding domain polypeptide sequence) regions are purified from a bacterial lysate. In other embodiments, the conjugate or fusion protein is purified from a plant cell lysate. In yet another embodiment, the conjugate or fusion protein is purified from an eukaryotic cell lysate,culture medium or fermentation broth.

[0079] One embodiment is a method for preparing a conjugate or fusion protein, comprising:

(a) culturing the host cell under conditions which provide for the expression of the conjugate from the expression vector within the host cell; and

(b) recovering 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 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.

[0080] 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 65 or a variant thereof; and

(b) a second region comprising a NEMO binding domain polypeptide 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.

[0081] 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 conjugate or fusion protein; this allows for both positive and negative purification steps.

[0082] 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 FLAG-tags, myc-tags, His-, HHHHHHGS (SEQ ID NO: 147), 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.

[0083] In one embodiment, the amino acid tail or ligand is fused to the C- terminus of 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 conjugate.

[0084] 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 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.

[0085] In another embodiment, the method of purifying a conjugate comprises fusing an amino acid tail or ligand to the conjugate or fusion protein, which tail is capable of binding to at least one substrate while impurities bind only to a second substrate; the conjugate is contacted with the other substrate such that the conjugate is not bound and remaining impurities are bound to the other substrate.

[0086] According to another embodiment, a method for producing and purifying a conjugate or fusion protein comprising the cell penetrating peptide, non-peptide, or variant or portion thereof and a NEMO binding domain protein comprises culturing the host cell for the expression of the conjugate or fusion protein from the expression vector and subsequently recovering the conjugate or fusion protein using affinity purification techniques known in the art.

[0087] 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.

[0088] Further embodiments include pharmaceutical compositions comprising the conjugates or fusion proteins of the embodiments disclosed herein, and methods of use of the conjugates or fusion proteins in the preparation of a medicament for the treatment of a disease.

[0089] In one embodiment, a conjugate or fusion protein comprises a biologically active peptide or protein that is a functional enzyme, linked to the cell penetrating peptide (CPP), non-peptide, or variant or portion thereof.

[0090] Other embodiments comprise any of a variety of formulations for treating conditions or diseases identified.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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) in the form of a topical lotion, solution, cream, ointment or dusting powder; (2) be formulated as eye drops or for intraocular or intravitreal injection. [0095] In one embodiment, the conjugate comprises:

a first region comprising a structure derived from human genes having a sequence described in any of SEQ ID Nos. 1-65, wherein 1 - 20, 1 - 15, or 1 - 10 amino acid residues are substituted, deleted, added, and/or inserted; and

a second region is a functional or regulatory polypeptide or protein not naturally associated with the first region.

[0096] In one embodiment, the conjugate comprises:

a first region comprising a structure derived from human genes having a sequence described in any of SEQ ID Nos. 1-65, wherein 1 - 20, 1 - 15, or 1 - 10 amino acid residues are substituted, deleted, added, and/or inserted and wherein said amino acid has maintains the cell permeabilizing activity; and

a second region is a functional or regulatory NBD polypeptide or protein not naturally associated with the first region.

[0097] In one embodiment, the active concentration of fusion protein or conjugate in cell culture is less than about 115 μΜ, less than about 100 μΜ, less than about 90 μΜ, less than about 80 μΜ, less than about 70 μΜ, less than about 65 μΜ, less than about 60 μΜ, less than about 55 μΜ, less than about 50 μΜ, less than about 45 μΜ, less than about 40 μΜ, less than about 35 μΜ, less than about 30 μΜ, less than about 25 μΜ, less than about 20 μΜ, less than about 15 μΜ, less than about 10 μΜ, less than about 5 μΜ, or less than about 1 μΜ for example, less than about 1 μΜ to about 3 μΜ, less than about 1 μΜ to about 6 μΜ, less than about 1 μΜ to about 8 μΜ, less than about 1 μΜ to about 15 μΜ, less than about 1 μΜ to about 25 μΜ, less than about 1 μΜ to about 50 μΜ, less than about 1 μΜ to about 70 μΜ, less than about 1 μΜ to about 85 μΜ, less than about 1 μΜ to about 110 μΜ, less than about 10 μΜ to about 110 μΜ, less than about 15 μΜ to about 70 μΜ, less than about 15 μΜ to about 60 μΜ, less than about 20 μΜ to about 55 μΜ, or less than about 25 μΜ to about 45 μΜ.

[0098] In other embodiments, the dosage delivered (daily or as required) in mouse models (per 20 g mouse) through (1) an intraocular, an intravitreal, an intravenous (i.v.) or intraperitoneal (i.p.) injection, or (2) a topical formulation, is at least 500 μg, at least 450 μg, at least 400 μg, at least 350 μg, at least 300 μg, at least 250 μg, at least 200 μg, at least 150 μg, at least 100 μg, at least 80 μg, at least 70 μg, at least 60 μg, at least 50 μg, at least 40 μg, at least 30 μg, at least 20 μg, at least 10 μg, or at least 1 μg, for example, about 1 μg to about 500 μg, about 10 μg to about 450 μg, about 20 μg to about 400 μg about 30 μg to about 350 μg, about 30 μg to about 200 μg, about 30 μg to about 100 μg, about 40 μg to about 300 μg, about 40 μg to about 200 μg, about 50 μg to about 100 μg, about 50 μg to about 90 μg, about 55 μg to about 85 μg, about 60 μg to about 80 μg, about 60 μg to about 100 μg; about 1 μg to about 200 μg; about 1 μg to about 100 μg, about 1 μg to about 90 μg, about 1 μg to about 80 μg, about 1 μg to about 70 μg, about 1 to about 60 μg, about 1 to about 50 μg, about 1 to about 40 μg, about 1 to about 30 μg, about 1 to about 20 μg, about 1 to about 15 μg, about 1 to about 12 μg, about 1 to about 10 μg, about 1 to about 8 μg about 1 to about 6 μg, about 1 to about 4 μg, or about 1 to about 3 μg.

[0099] In other embodiments, the dosage delivered (daily or as required) through an intraocular, intravitreal, intravenous (i.v.) or 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.

[00100] 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.

[00101] In other embodiments the dosage delivered (daily or as required) through a topical formulation in humans (70 kg weight) is less than about 70 μg, less than about 50 μg, less than about 45 μg, less than about 40 μg, less than about 30 μg, less than about 25 μg, less than about 20 μg, less than about 15 μg, less than about 12 μg, less than about 10 μg, less than about 8 μg, less than about 4 μg, less than about 2 μg, or less than about 1 μg, for example, about 1 μg to about 50 μg, about 5 μg to about 40 μg, about 8 μg to about 30 μg, about 10 μg to about 20 μg, or about 12 μg to about 15 μg, about 8 μg to about 12 μg, about 5 μg to about 9, about 3 μg to about 6 μg, about 2 μg to about 5 μg, or less than about 1 μg to about 3 μg.

[00102] 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, 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 μg, at least about 450 μg, at least about 400 μg, at least about 350 μg, at least about 300 μg, at least about 250 μg, at least about 200 μg, at least about 150 μg, at least about 100 μg, at least about 80 μg, at least about 70 μg, at least about 60 μg, at least about 50 μg, at least about 40 μg, at least about 30 μg, at least about 20 μg, at least about 10 μg, or at least about 1 μg, for example, between about 1 μg to about 750 μg; about 1 μg to about 500 μg, about 10 μg to about 450 μg, about 20 μg to about 400 μg about 30 μg to about 350 μg, about 30 μg to about 200 μg, about 30 μg to about 100 μg, about 40 μg to about 300 μg, about 40 μg to about 200 μg, about 50 μg to about 100 μg, about 50 μg to about 90 μg, about 55 μg to about 85 μg, about 60 μg to about 80 μg, about 60 μg to about 100 μg; about 1 μg to about 200 μg; about 1 μg to about 100 μg, about 1 μg to about 90 μg, about 1 μg to about 80 μg, about 1 μg to about 70 μg, about 1 to about 60 μg, about 1 to about 50 μg, about 1 to about 40 μg, about 1 to about 30 μg, about 1 to about 20 μg, about 1 to about 15 μg, about 1 to about 12 μg, about 1 to about 10 μg, about 1 to about 8 μg about 1 to about 6 μg, about 1 to about 4 μg, about 1 μg to about 3 μg, about 1 μg to about 1 mg, about 1 μg to about 2 mg, about 1 μg to about 5 mg; about 1 μg 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.

[00103] In one embodiment, the formulation is administered locally ( e.g. intravitreal injection, intraocular injection, topical) 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.

[00104] In one embodiment, upon treatment of one or more human or animal subjects with any of the fusion protein or conjugate embodiments disclosed, the subject(s) will exhibit one or more of the following outcomes:

(a) restoration of tear secretion towards normal levels;

(b) restoration of visual acuity as determined through standard clinical methodology;

(c) prevention of an increase in graded vitreous haze and/or graded anterior chamber cells;

(d) prevention of deterioration of visual acuity;

(e) reduced activation of NF-DB regulated genes.

[00105] 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.

[00106] In another embodiment comprising a fusion protein or 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) an inhibition of NF-κΒ transcriptional activity;

(b) a reduction in goblet cell infiltration in the epithelial cornea;

(c) an attenuation of the loss of goblet cells in the conjunctiva;

(d) restoration of tear secretion towards normal levels;

(e) restoration of visual acuity as determined through standard clinical methodology;

(f) prevention of an increase in graded vitreous haze and/or graded anterior chamber cells;

(g) prevention of deterioration of visual acuity;

(h) reduced activation of NF-DB regulated genes.

[00107] In another embodiment, the treatment with a formulation comprising a fusion protein and/or 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.

[00108] In another embodiment, upon treatment with a formulation comprising a fusion protein and/or 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: (a) an inhibition in NF-κΒ 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);

(b) a reduction in goblet cell infiltration in the epithelial cornea 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);

(c) an attenuation of the loss of goblet cells in the conjunctiva 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);

(d) restoration of tear secretion towards normal levels as measured by the Schirmer test, with an increase in Schirmer wetting of more than 20 mm, more than 15 mm, more than 10 mm, more than 5 mm, for example between 5 mm and 20 mm, 10 mm and 20 mm, 5 mm and 15 mm, 5mm and 10 mm or 15 mm to 20 mm (actual % change or median % change compared to baseline or control);

(e) the restoration of visual acuity as determined through standard clinical methodology 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);

(f) prevention of a clinically significant increase in graded vitreous haze and/or graded anterior chamber cells based on commonly accepted clinical criteria known to those expert in the field.

(g) prevention of deterioration of a clinically significant change in visual acuity based on commonly used and accepted criteria known to those expert in the field;

(h) reduced activation of NF-DB 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).

[00109] According to the embodiments disclosed and their modifications, the conjugates or 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 dry eye (keratoconjuntivitis sicca) and uveitis.

Table 3: Pe tide Se uence Identities.

Descriptor or

Gene from

Sequence

which the Sequence

ID No.

Sequence was

Derived

2 HIV-1 Tat MEP VDPRLEPWKHPGS QPKT ACTNC YCKKC CFH protein CQVCFITKALGISYGRKKRRQRRRAHQNSQTHQA

SLSKQPTSQPRGDPTGPKE

3 Tat (47-57) YGRKKRRQRRR

4 Tat (49-57) RKKRRQRRR

5 Tat (48-60) GRKKRRQRRRPPQ

6 D-Tat GRKKRRQRRRPPQ

7 Tat (49-60) RKKRRQRRRPPQ

8 R₉-Tat GRRRRRRRRRPPQ

9 Penetratin RQIKIWF QNRRMKWKK

10 VP22 DAATATRGRSAASRPTERPRAPARSASRPRRVD

11 pVEC LLIILRRRIRKQAHAHSK

12 />ISL RVIRVWFQNKRCKDKK

13 hCT (9-32) LGTYTQDFNKFHTFPQTAIGVGAP

derived

peptide

14 Transportan GWTLNSAGYLLGKINLKALAALAKKIL

(TP)

15 TP10 AGYLLGKINLKALAALAKKIL

16 Poly-R R_X (X=4-16)

peptides

17 MAP KLALKLALKALKAALKLA

18 Pep-1 K ETWW ETWWTEW SQPKKKRKV

19 Pep-2 KETWFETWFTEWSQPKKKRKV

20 MPG GALFLGWLGAAGSTMGAPKKKRKV

21 KALA WEAKLAKALAKALAKHLAKALAKALKACEA

22 ppTGl GLFKALLKLLKSLWKLLLKA

23 ppTG20 GLFRALLRLLRSLWRLLLRA

24 PTD4 YARAAARQARA

25 PTD5 RRQRRTS KLMKRGG

26 M-918 MVTVLFRRLRIRRACGPPRVRV

27 KM) KQINNWFINQRKRHWK Descriptor or

Gene from

Sequence

which the Sequence ID No.

Sequence was

Derived

28 Hph-1 YARVRRRGPRR

29 HIV-1 Rev TRQARRNRRRRWRERQR

(34-50)

30 ANP (43-58) RQIKIWF QNRRMKWKK

[Antennapedi

a]

31 POD GGG[ ARKKAAKA] ₄

32 S413-PV ALWKTLLKKVLKAPKKKRKV

33 S41 CVQWSLLRGYQPC

34 KADY Ac-GLWRALWRLLRSLWRLLWKA

35 P28 Azurin LSTAADMQGVVTDGMASGLDKDYLKPDD (50-77)

36 Vectocell® CVKRGLKLRHVRPRVTRMDV

peptide

37 SV40-NLS PKKKRKV

38 Nucleoplasmi KRPAAIKKAGQAKKKK

n-NLS

39 FHV coat (35- RRRRNRTRRNRRRVR

49)

40 BMV Gag (7- KMTRAQRRAAARRNRWTAR

25)

41 HTLV-II Rex TRRQRTRRARRNA

(4-16)

42 CCMV Gag KLTRAQRRAAARKNKRNTR

43 P22 N (14-30) NAKTRRHERRRKLAIER

44 λ N (1-22) MDAQTRRRERRAEKQAQWKAAN

45 φ21 N (12-29) TAKTRYKARRAELIAERR

46 Yeast PRP6 TRRNKRNRIQEQLNRK

(129-144)

47 Human U2AF SQMTRQARRLYV

(142-153)

48 Human cFos KRRIRRERNKMAAAKSRNRRRELTDT (139-164) Descriptor or

Gene from

Sequence

which the Sequence

ID No.

Sequence was

Derived

49 Human cJun RIKAERKRMRNRIAASKSRKRKLERIAR

(252-279)

50 Yeast GCN4 KRARNTEAARRSRARKLQRMKQ

51 Pen-Arg RQIRIWFQNRRMRWRR

52 Dyn A YGGFLRRIRPKLKWDNQ

53 Dyn B YGGFLRRQFKVVT

54 Big Dyn YGGFLRRIRPKLKWDNQKRYGGFLRRQFKVVT

55 Big Dyn 6-26 RRIRPKLKWDNQKRYGGFLRR

56 Mouse PrP (1- MANLGYWLLALFVTMWTDVGLCKKRPKP

28)

57 Mouse PrP (1- MLLADCFPLTPGLNKVLWGTVAKRWYMK

28R

58 Mouse PrP VGLCKKRPKPGG

(19-30)

59 Mouse PrP KKRPKP

(23-28)

60 Mouse PrP KKRPKPGGWNTGGSRYPGQGSPGGNRYP

(23-50)

61 Bovine PrP MVKSKIGSWILVLFVAMWSDVGLCKKRPKP (1-30)

62 LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTE

S

63 Magainin 2 GIGKFLHSAKKFGKAFVGEIMNS

64 Buforin 2 TRS SRAGLQFPVGRVHRLLRK

65 Apidaecins RP(V/I)YIPQPRPPHPR (conserved sequence among class members) and sequences described in US Patent No. 9,556,228 which is incorporated by reference in its entirety

66 HIV-1 Tat- MEP VDPRLEPWKHPGS QPKT ACTNC YCKKC CFH

NBD (Entire CQVCFITKALGISYGRKKRRQRRRAHQNSQTHQA sequence: SLSKQPTSQPRGDPTGPKETALDWSWLQTE combined SEQ

ID Nos. 1 and

2) Descriptor or

Gene from

Sequence

which the Sequence ID No.

Sequence was

Derived

67 Tat (47-57)- YGRKKRRQRRRTALDWSWLQTE NBD (Entire

sequence:

combined SEQ

ID Nos. 1 and

3)

68 Tat (49-57)- RKKRRQRRRTALDWSWLQTE

NBD (Entire

sequence:

combined SEQ

ID Nos. 1 and

4)

69 Tat (48-60)- GRKKRRQRRRPPQTALDWSWLQTE

NBD (Entire

sequence:

combined SEQ

ID Nos. 1 and

5)

70 D-Tat -NBD GRKKRRQRRRPPQTA OWSW QTE

(Entire

sequence:

combined SEQ

ID Nos. 1 and

6)

71 Tat (49-60)- RKKRRQRRRPPQTALDWSWLQTE

NBD (Entire

sequence:

combined SEQ

ID Nos. 1 and

7)

72 Rg-Tat-NBD GRRRRRRRRRPPQTALDWSWLQTE

(Entire

sequence:

combined SEQ

ID Nos. 1 and

8) Descriptor or

Gene from

Sequence

which the Sequence

ID No.

Sequence was

Derived

73 Penetratin- RQIKIWFQNRRMKWKKTALDWSWLQTE

NBD (Entire

sequence:

combined SEQ

ID Nos. 1 and

9)

74 VP22-NBD DAATATRGRSAASRPTERPRAPARSASRPRRVDT

(Entire ALDWSWLQTE

sequence:

combined SEQ

ID Nos. 1 and

10)

75 pVEC-NBD LLIILRRRIRKQAHAHSKTALDWSWLQTE

(Entire

sequence:

combined SEQ

ID Nos. 1 and

11)

76 pISL-NBD RVIRVWFQNKRCKDKKTALDWSWLQTE

(Entire

sequence:

combined SEQ

ID Nos. 1 and

12)

77 hCT (9-32) LGTYTQDFNKFHTFPQTAIGVGAPTALDWSWLQT derived E

peptide -NBD

(Entire

sequence:

combined SEQ

ID Nos. 1 and

13)

78 Transportan GWTLNSAGYLLGKINLKALAALAKKILTALDWS (TP)-NBD WLQTE

(Entire

sequence:

combined SEQ

ID Nos. 1 and

14)

[00110] 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

[00111] Uveitis is a common inflammatory condition of the eye. The many versions of uveitis can be sight-threatening, ocular inflammatory diseases. Endotoxin- induced uveitis (EIU) is thought to be an animal model of human uveitis. Immunosuppressants used to treat human uveitis (e.g., dexamethasone and Pred Forte (prednisolone acetate)) are effective in such an animal model.

[00112] Conjugates comprised of Hox D12 linked to the cargo NBD peptide (PPL-003) (SEQ ID No. 130) and conjugates comprised of Antennapedia (Antp) homeodomain linked to the cargo NBD peptide (PPL-004) (SEQ ID No. 94) were tested in an in vivo murine pharmacology model where animals were challenged with intravenous endotoxin (LPS) injection. Conjugates were delivered either topically or intra-peritoneally. In the topical group, mice were pretreated with a 25 μg dose/eye of either PPL-003 or PPL- 004 30 minutes prior to challenge by LPS (100 μg i.p.). On day 1, at 6- and 12-hours post- LPS challenge, mice received another 25 μg dose/eye of either PPL-003 or PPL-004. On day 3, mice received another 3 doses of 25 μg dose/eye of either PPL-003 or PPL-004. In the intra-pertineal group, mice were pretreated with 1 dose of 2,000 μg of either PPL-003 or PPL-004 30 minutes prior to challenge by LPS (100 μg i.p.). On day 3, mice received another dose of 2,000 μg of either PPL-003 or PPL-004.

[00113] Following treatment, infiltrated leukocyte numbers were compared between treatment groups. As shown in Fig. 1, mice receiving either topical or intraperitoneal injections of PPL-003 or PPL-004 showed a statistically significant reduction in infiltrated leukocytes, as compared to vehicle controls. Surprsingly, treatment with PPL- 003 or PPL-004 were similar or better than dexamethasone (i.p.) or Pred Forte (topical).

[00114] Together this data suggests that NBD conjugates having different cell penetrating peptides (e.g., Hox D12 or Antp) can be used as effective treatments for uveitis. It is further contemplated that alternative embodiments comprising other cell penetrating peptides, for example, any of the other SEQ ID Nos. 2 through 65, or variants or portions thereof, conjugated to the NBD peptide can be useful in the treatment of uveitis.

[00115] For certain comparisons between groups reported in the studies described in the examples above, significance was tested at/? < 0.05. [00116] 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.

Claims

CLAIMS We claim:

1. A conjugate comprising:

a first region comprising a polypeptide sequence as described in any one of SEQ ID

Nos. 2 to 65 or a variant thereof; and

a second region comprising a NEMO binding domain polypeptide sequence as set forth in SEQ ID No. 1 or a variant thereof.

2. A conjugate comprising:

a first region comprising a non-peptide carrier; and

a second region comprising a NEMO binding domain polypeptide sequence as set forth in SEQ ID No. 1 or a variant thereof.

3. The conjugate of claim 1 or claim 2 wherein the second region is conjugated to the C-terminus of the first region.

4. The conjugate of claim 1 or claim 2 wherein the second region is conjugated to the N-terminus of the first region.

5. The conjugate of claim 1 or claim 2 in the form of a fusion protein.

6. The conjugate of claim 1 or claim 2 further comprising a linker sequence between the first and second regions.

7. The conjugate of claim 1 wherein the first and second regions combined have a polypeptide sequence selected from the group consisting of SEQ ID Nos: 66-129, or a variant thereof.

8. The conjugate of claim 7 wherein an initiating methionine residue of the sequence is removed prior to use as an active and/or therapeutic agent.

9. The conjugate of claim 2, 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.

10. The conjugate of claim 1 or claim 2 wherein the first region is linked to the second region with a peptidic bond.

11. The conjugate of claim 1 or claim 2 wherein the second region is conjugated to the first region via a non-peptidic bond.

12. The conjugate of claim 11 wherein the first region and the second region are attached via "click chemistry."

13. The conjugate of claim 1 or claim 2 wherein the first region comprises a polypeptide sequence derived from a human gene.

14. The conjugate of claim 1 or claim 2 wherein the second region includes a NEMO binding domain polypeptide sequence derived from a human gene or variant thereof.

15. The conjugate of claim 1 or claim 2 further comprising at least one molecular cargo selected from the group consisting of liposomes, polymers, cationic peptides, nanoparticles, adeno-associated virus (AAV) and calcium.

16. 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 claim 1 or claim 2 under conditions which provide for the expression of the conjugate within the host cell; and

recovering the conjugate by affinity purification.

17. A composition comprising the conjugate of claim 1 or claim 2 and a pharmaceutically acceptable carrier.

18. The composition of claim 17 wherein the composition is in the form of an eye drop formulation other ophthalmic composition for local or injectable use, a topical composition, or an injectable composition including injectable implants for sustained release.

19. A method of treating a dry eye in a subject comprising administering a formulation comprising the conjugate of claim 1 or claim 2 to the subject.

20. A method of treating uveitis in a subject comprising administering a formulation comprising the conjugate of claim 1 or claim 2 to the subject.

21. The method of claim 19 or claim 20 wherein the subj ect is a human.