Homing peptide-directed decorin conjugates for the treatment of epidermolysis bullosa

Abstract

The present invention relates to homing peptide-directed decorin conjugates for the treatment of epidermolysis bullosa, and corresponding methods of treatment. The use of novel homing peptides enables the targeting of the conjugates in vivo to specifically home to the skin and skin wounds by systemic administration.

Description

Technical Field

[0001] This invention generally relates to the field of molecular medicine. More specifically, this invention relates to homing peptide-guided core proteoglycan conjugates for the treatment of epidermolysis bullosa, and corresponding treatment methods. Background Technology

[0002] As the largest organ in the human body, the skin presents unique challenges for effective drug delivery. A major challenge associated with local, i.e., transdermal drug delivery is the poor permeability of large molecules into the skin. Diffusion via intercellular lipids offers an option for transdermal administration, but it is limited to the transdermal delivery of lipophilic small molecules. Therefore, systemically administered skin-specific therapeutic agents would represent a significant advance in the treatment of skin diseases, particularly those affecting the entire skin, such as epidermolysis bullosa, a group of rare genetic disorders that cause fragile, blistering skin.

[0003] Denial dystrophic epidermolysis bullosa (RDEB) is caused by a mutation in the COL7A1 gene that encodes type VII collagen (C7). Clinical manifestations include skin erosion and vesicles, laceration scars, pseudosyndactyly, and a high risk of developing into aggressive and rapidly metastatic cutaneous squamous cell carcinoma (cSCC). Although some gene-, cell-, and protein-based therapies have shown promising results in delivering type VII collagen to the skin, challenges remain, and RDEB remains incurable.

[0004] Transforming growth factor β (TGFβ) signaling has been shown to play a crucial role in the development of fibrosis and progression to malignancy in renal fibrosis (RDEB). Previously, it was demonstrated that TGFβ signaling is activated as early as one week after birth in Col7a1- / - mice (Liao et al., 2018, Stem Cells 36: 1839-1850). Therefore, early intervention against TGFβ signaling activation may be beneficial in reducing the disease burden in RDEB. TGFB signaling is also considered a phenotypic regulator in identical twins with the same COL7A1 mutation (Odorisio et al., 2014, Hum Mol Genet 23: 3907-3922). Furthermore, the expression level of proteoglycan core proteoglycan (DCN), a natural TGFβ inhibitor, is significantly elevated in less affected twins. DCN is a structural component of the extracellular matrix (ECM). Dcn- / - mice exhibit irregular collagen fiber formation and significantly reduced skin tensile strength (Reed and Iozzo, 2002, Glycoconj J 19: 249-255). Furthermore, DCN possesses anti-fibrotic and anti-tumor functions by regulating the activity of various growth factors, including inhibition of TGFβ. and Prince, 2015, Biomed Res Iht 2015: 654765; and Ruoslahti, 2019, Br J Pharmacol 176: 16-25). Recently, it has also been demonstrated that upregulation of DCN expression in col7a1- / - mice is one of the mechanisms of action of cord blood-derived unrestricted somatic stem cells (USSCs) (Liao et al., 2018, ibid.). Recently, Cianfarani et al. (2019, Matrix Biol 81: 3-16) reported that systemic administration of lentiviral-driven human DCN expression in a C7-hypomorphic RDEB mouse model expressing residual levels of type VII collagen (C7-hypomorphic allele mice) attenuated TGFβ-induced fibrosis, supporting the role of DCN as a potential therapeutic disease modifier for RDEB.

[0005] Furthermore, DCN binds to and neutralizes connective tissue growth factor (CTGF / CCN2), a downstream mediator of TGFβ fibrosis signaling, and has been identified as a therapeutic target for preventing scar formation (Vial et al. 2011, J BiolChem 286: 24242-24252; Daniels et al. 2003, Am J Pathol 163: 2043-2052). Since the binding sites for TGFβ and CTGF / CCN2 are located in different parts of DCN, DCN can theoretically block both mediators of fibrosis simultaneously. In fact, besides RDEB, the role of DCN in inhibiting TGFβ-driven scar formation has been well-established in many disease models (e.g., renal, pulmonary, and hepatic fibrosis, and skin wound healing) (Odorisio et al. 2014, ibid.; Liao et al. 2018, ibid.; Cianfarani et al. 2019, ibid.). However, despite numerous positive anticancer and fibrotic results in preclinical studies, DCN has not yet entered clinical use as a systemic therapy. To date, the only reported clinical application of DCN is in 12 patients with penetrating ocular lesions, where a single intravitreal injection of 200 or 400 μg of recombinant human DCN appeared to be well-tolerated with no ocular adverse events (Abdullatifet al., 2018, Graefes Arch Clin Exp Ophthalmol 256:2473-2481).

[0006] A common limitation of systemic drug delivery is that only a small portion of the drug reaches its desired location, and systemic side effects are encountered in other organs. Therefore, a key objective of modern drug development is to create organ-specific drugs with minimal adverse effects on other parts of the body. This goal can be achieved by developing drugs that recognize specific epitopes expressed in the affected organ. Alternatively, drugs can be conjugated to target specific organs by affinity ligands, such as vascular homing peptides that recognize tissue-specific or target-specific molecular signatures in the blood vessels of a given organ.

[0007] In vivo screening of phage peptide libraries has identified tissue- or disease-specific molecular signatures (vascular zip codes) in blood vessels that can be targeted by systemically administered affinity ligands (e.g., vascular homing peptides). These studies essentially identify organ- or disease-specific molecular markers in the vascular system of different tissues that enable zip code systems (vascular zip codes) to target and specifically deliver systemically administered therapeutic agents (Ruoslahti et al., 2010, J Cell Biol 188: 759-768; Ruoslahti, 2017, Adv Drug Deliv Rev 110-111: 3-12; Ruoslahti, 2004, Biochem Soc Trans 32: 397-402; Pasqualini and Ruoslahti, 1996, Nature 380(6572): 364-366). The most effective vascular homing peptides for tumor-specific homing and cell / tissue penetration contain a common motif R / KXXR / K (SEQ ID NO: 3) with arginine (or rarely lysine) residues at the C-terminus, hence referred to as the C-terminal regular (CendR) sequence (Ruoslahti, 2017, J Clin Invest 127: 1622-1624; Teesalue et al., 2009, Proc Natl Acad Sci USA 106: 16157-16162; Sugahara et al., 2009, Cancer Cell 16: 510-520; Sugahara et al., 2010, Science 328: 1031-1035). CendR sequences bind to neurociliin-1 (NRP-1), activating extravasation and tissue penetration pathways to deliver peptides, along with their payloads, into the parenchyma of tumor tissue (Ruoslahti, 2017, Adv Drug Deliv Rev 110-111: 3-12; Ruoslahti, 2017, J Clin Invest 127: 1622-1624; Teesalu et al., 2009, PNAS 106(38): 16157-16162). The target selectivity of peptides containing hidden CendRs lies in their binding to primary receptors with tumor-specific expression patterns and in proteolytic activation within the tumor to expose the CendR sequence in the target organ.Since NRP-1 is expressed by endothelial cells in all tissues (Ruoslahti, 2017, Adv Drug Deliv Rev 110-111:3-12), the extravasation and tissue penetration of NRP-1 cannot be limited to cancerous tissues; it can also occur in other diseased or healthy tissues.

[0008] In vivo phage display screening also identified a group of peptides that home to blood vessels that generate blood vessels in skin wounds. and Ruoslahti, 2007, Am J Pathol 171:702-711). Two of the most promising peptides, circulating peptides called CAR (CARSKNKDC; SEQ ID NO: 5) and CRK (CRKDKC; SEQ ID NO: 3), have been used to deliver different therapeutic molecules in a targeted and selective manner. (et al., 2017, ACS Biomaterials Science & Engineering 3: 1273-1282). Interestingly, although the CRK peptide contains the hidden CendR sequence RKDK (SEQ ID NO: 1), it is the only vascular homing CendR peptide that cannot penetrate cells and tissues. andRuoslahti, 2007, Am J Pathol 171: 702-711; Agemy et al., 2010, Blood 116: 2847-2856).

[0009] WO 2008 / 136869 discloses CRK peptides as specific homing elements for targeted delivery of core proteoglycans to skin wounds. The disclosed CRK-core proteoglycan fusion does not hone in non-traumatic skin.

[0010] Therefore, systemically applied but skin-specific therapeutic agents would be a major advancement in the treatment of skin diseases such as epidermolysis bullosa. Summary of the Invention

[0011] This invention provides a homing peptide-guided core proteoglycan conjugate for the treatment of epidermolysis bullosa. The conjugate comprises a core proteoglycan fragment and a homing peptide, wherein the C-terminus of the homing peptide consists of the amino acid sequence RKDK (SEQ ID NO: 1) or CRKDK (SEQ ID NO: 2).

[0012] A method is also provided for treating epidermolysis bullosa in subjects in need by administering an effective amount of a core proteoglycan conjugate containing a core proteoglycan fragment and a homing peptide, wherein the C-terminus of the homing peptide consists of the amino acid sequence RKDK (SEQ ID NO: 1) or CRKDK (SEQ ID NO: 2).

[0013] Due to the homing peptide, this conjugate selectively homs to and penetrates the skin and skin wounds in the body.

[0014] The embodiments and details of the above aspects are set forth in the following figures, detailed descriptions, examples and dependent claims. Attached Figure Description

[0015] The accompanying drawings illustrate several embodiments of the subject matter disclosed in this invention and, together with the description, serve to explain the principles of the compositions and methods disclosed in this invention.

[0016] Figures 1A to 1C The structure of an exemplary recombinant DCN-tCRK protein and its binding to neurocilia-1 are illustrated. Figure 1A This is a schematic diagram of the structure of DCN-tCRK. The signal peptide and propeptide of native DCN were replaced with a purified 6×His tag (I). Following the His-tag are the amino terminus (II), core protein (III), and carboxyl terminus (IV) of the mature DCN proteoglycan. The tCRK peptide (V) was cloned to the carboxyl terminus of the protein. Figure 1B The in vitro binding of DCN-tCRK to neurocilia protein-1 (NRP-1) is shown. DCN-tCRK (left panel) and peptide controls (right panel, positive peptide: RPARPAR (SEQ ID NO: 25) and negative peptide: RPARPARA (SEQ ID NO: 26)) were immobilized in ELISA plates. Bovine serum albumin (BSA) was included as a non-specific protein control for DCN-tCRK and the peptide. WT and mutant NRP1 were labeled with FAM and added to the immobilized plates. Binding of NRP1 was measured based on fluorescence intensity. Error bars represent SEM. Experiments were repeated with triplicate samples, **p < 0.01, ***p < 0.001, ****p < 0.0001, Student's unpaired t-test. Figure 1C The internalization of DCN-tCRK in NRP-1 positive cells is shown. FAM-labeled DCN-tCRK was incubated with NRP-1 positive and negative PC3 and M21 cells, respectively. DCN-tCRK was detected by anti-FAM immunostaining. Cell nuclei were counterstained with DAPI. Representative images from experiments in three independent studies. Scale bar 20 μm.

[0017] Figures 2A to 2DThe generation of recombinant proteins and the characterization of exemplary DCN-tCRK are described. Figure 2A Shown in An example of a purified chromatogram with a large peak following the HisTrap HP column step at Start, where all peak fractions are used for further processing. Figure 2B In the image, purified DCN-tCRK is shown on a Coomassie-stained reduced SDS-Page gel (top) and a Western blot (bottom) alongside prior art DCN. 2 μg and 1 μg of protein were loaded onto the SDS gel; for Western blot analysis, 1 and 0.5 μg of protein were applied. The monomeric form of the protein, as well as forms including GAG side chains, are visible. Figure 2C Dynamic light scattering (DLS) measurements of the hydrodynamic diameter of DCN-tCRK are shown (n=3). Figure 2D The differential scanning calorimetry (DSC) curves of the melting temperature of DCN-tCRK are shown.

[0018] Figure 3 The pharmacokinetics of DCN-tCRK compared to DCN were demonstrated. Healthy Balb / c mice were intravenously injected with 5 mg / kg DCN-tCRK or DCN. Blood samples were collected at eight time points, and human DCN was analyzed using standard ELISA. n = 4 per group.

[0019] Figures 4A to 4D This demonstrates that DCN-tCRK improves survival and homing to the skin in col7A1- / - mice. Figure 4A Kaplan-Meier survival analysis of col7A1- / - mice administered DCN-tCRK (median lifespan: 11 days; n = 21), DCN (median lifespan: 7 days; n = 17), and PBS (median lifespan: 2 days; n = 24) is shown. Figure 4B The results show the quantitative results of DCN and DCN-tCRK levels in the skin of recipient col7a1- / - mice measured using a human core proteoglycan ELISA kit at one, two, and three weeks after intrahepatic administration (n=3 at each time point). DCN levels were not quantified at the three-week time point because no mice survived to that time point after DCN administration. *p<0.05, **p<0.01 Figure 4C Immunohistochemical staining results were shown on the skin of the paws and back of col7a1- / - mice using antihistamine (anti-his). Cell nuclei were counterstained with DAPI. Scale bar: 20 μm. Figure 4DRepresentative double staining results for anti-histidine tag and anti-NRP-1 are shown, and a merged image of DCN-tCRK, DCN, and untreated RDEB skin (counterstained with DAPI) is presented. Scale bar: 25 μm

[0020] Figure 5 Kaplan-Meier survival analysis of col7a1- / - mice is shown, comparing historical survival after administration of dextran / human serum albumin (D / HSA; median lifetime: 3 days; n = 29; historical data Liao et al. 2018, Stem Cell Transl Med, 7: 530-542) with survival after administration of DCN-tCRK (median lifetime: 11 days; n = 21), DCN (median lifetime: 7 days; n = 17), and PBS (median lifetime: 2 days; n = 24).

[0021] Figure 6 illustrates how DCN-tCRK normalizes fibrosis gene markers in RDEB. Figure 6A The relative gene expression in the cluster map of genes with >1.5-fold increased expression in untreated RDEB skin compared to WT is shown. Figure 6B Volcano plots showing log2 fold changes and -log10 p values ​​of gene expression in col7a1- / - mouse skin treated with vector, DCN, and DCN-tCRK relative to WT are presented.

[0022] Figure 7 illustrates the inhibition of fibrosis development in col7a1- / - mice by DCN-tCRK administration. Figure 7A Representative immunohistochemical staining results of CTGF / CCN2 in one-week and two-week-old WT and col7Aa1- / - mice with and without DCN-tCRK treatment are shown. Scale bar: top 50 μm, bottom 25 μm. Figure 7B Sirius red staining results from paw skin of 1- and 2-week-old WT and col7a1- / - mice with and without DCN-tCRK treatment are shown. Sirius red images were obtained using polarized light. Scale bar 25 μm. Figure 7C Quantitative results of the average intensity of Sirius crimson in each field of view obtained with a 20× objective are shown. Eight or more fields of view were obtained per section, and at least four sections were analyzed for each biopsy. Scale bar 25 μm. *p < 0.05, **p < 0.01. Figure 7DRepresentative images of double immunofluorescence staining for collagen type I (COL1, column 1) expression in the skin of two-week-old RDEB and WT mice with and without DCN-tCRK treatment, and for α-smooth muscle actin (αSMA, column 2) and blood vessels (CD31, column 3) in two-week-old WT and col7a1- / - mice. Cell nuclei were counterstained with DAPI. Combined images are shown in column 4. Scale bar: 25 μm. Figure 7E The quantitative results of mean immunostaining intensity of COLI and αSMA expression on skin sections are shown (N=3 in each treatment group). Here, *, ** and *** represent P≤0.05, 0.01 and 0.001, respectively.

[0023] Figure 8 Results of in vitro collagen mesh shrinkage assays are shown. The top image represents normal human fibroblasts and RDEB patient-derived fibroblasts with or without DCN and DCN-tCRK at a final concentration of 75 nM, 48 hours after inoculation with collagen gel. The bottom image shows the shrinkage results of the collagen gel as a percentage of shrinkage compared to the initial area. Data (n=3) are expressed as mean ± SEM. *p<0.05, **p<0.001. Detailed Implementation

[0024] It should be understood that the present invention is not limited to any particular method, manner, reagent, or formulation described herein, as they can vary. It should also be understood that the terminology used in this invention is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the appended claims.

[0025] As used in this invention, the singular expressions “a,” “an,” and “the” refer to one or more. Therefore, unless otherwise stated, singular nouns also carry the meaning of their corresponding plural nouns.

[0026] This invention relates to the therapeutic use of homing peptide-guided core proteoglycan conjugates. More specifically, the invention provides homing peptide-guided core proteoglycan conjugates for treating epidermolysis bullosa, and a method for treating epidermolysis bullosa in a subject by administering an effective amount of the homing peptide-guided core proteoglycan conjugate to the subject in need.

[0027] Epidermolysis bullosa is a rare group of diseases that cause fragile, blistering skin. These blisters may appear from minor injuries or even from heat, friction, scratching, or tape. In severe cases, blisters may occur inside the body, such as on the lining of the mouth or stomach. Epidermolysis bullosa exists in a variety of forms, including acquired and congenital forms, the latter of which can be latent or dominant. Non-limiting examples of epidermolysis bullosa include acquired epidermolysis bullosa, borderline epidermolysis bullosa, simple epidermolysis bullosa, Kindler syndrome, and dystrophic epidermolysis bullosa, including dominant and latent dystrophic epidermolysis bullosa, such as recessive dystrophic epidermolysis bullosa inversa. Any subtype of the examples is also included.

[0028] As used in this invention, the term "subject" refers to an animal subject, preferably a mammalian subject, and more preferably a human subject. In this invention, the term "patient" refers to a human subject.

[0029] As used in this invention, the term "treatment" means administering a conjugate or a pharmaceutical composition containing a conjugate to a subject for the purpose of improving, alleviating, inhibiting or curing epidermolysis bullosa.

[0030] As used in this invention, the term "effective amount" refers to the amount that at least mitigates the harmful effects of epidermolysis bullosa.

[0031] As used herein, the term "core proteoglycan" (DCN) refers to any isoform of a small molecule chondroitin sulfate proteoglycan rich in leucine. It is a multifunctional proteoglycan, for example, regulating collagen fiber formation, preventing tissue fibrosis, promoting tissue regeneration, and acting as an antagonist of TGF-β. In some embodiments, the core proteoglycan is a human core proteoglycan comprising or composed of the amino acid sequence of core proteoglycan isoforms A, B, C, D, or E, having or lacking an N-terminal signal sequence and / or a propeptide. In some embodiments, the core proteoglycan comprises or is composed of any one of SEQ ID Nos: 6-20. Conserved sequence variants and peptide mimics of the aforementioned core proteoglycan species are also included. As used herein, the term "core proteoglycan fragment" refers to a portion of the conjugate of the invention that comprises or is composed of core proteoglycan.

[0032] In some embodiments, the core proteoglycan fragment comprises or is composed of an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, or 60% or any percentage thereof sequence identity with the amino acid sequence of SEQ ID NOs: 6-20, provided that the biological properties of the core proteoglycan are not significantly altered. Such core proteoglycan variants can be produced by the addition, deletion, and / or substitution of one or more amino acids. Means and methods for determining whether a core proteoglycan retains its biological properties are readily available in the art.

[0033] As used in this invention, the percentage of sequence identity between two sequences is a function of the number of common positions shared by the sequences (i.e., identity % = number of common positions / total number of positions × 100). Taking into account the number of gaps and the length of each gap, this requires the introduction of an optimal permutation of the two sequences. The comparison of sequences and the determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms available in the art.

[0034] As used in this invention, the term "homing peptide" broadly refers to any peptide that selectively homs to, i.e., targets, specific cells or tissues in the body, preferentially over other cells or tissues. Therefore, homing peptides can be used as targeted delivery carriers.

[0035] The homing peptide-guided core proteoglycan conjugates used in this invention differ from the known core proteoglycan fusion proteins disclosed in WO 2008 / 136869, at least in terms of the homing peptide used. Prior art core proteoglycan fusion proteins comprise known CRK peptides (CRKDKC; SEQ ID NO: 3), while the novel homing peptide used in this invention has a C-terminus composed of the amino acid sequence RKDK (SEQ ID NO: 1). In some embodiments, the C-terminus of the novel homing peptide used in this invention is composed of CRKDK (SEQ ID NO: 2).

[0036] It has now been surprisingly discovered that truncating the C-terminal cysteine ​​of a known CRK peptide (CRKDKC; SEQ ID NO: 3) alters the peptide's homing specificity. The truncated CKR of tCRK (RKDK, SEQ ID NO: 1; or CRKDK, SEQ ID NO: 2), hereinafter referred to as tCRK, confers the peptide the ability to hom to and penetrate non-traumatic skin while retaining its ability to hom to skin trauma. In other words, CRK peptides selectively hom to skin trauma only, while tCRK peptides selectively hom to and penetrate both traumatic and non-traumatic skin.

[0037] The truncation of the C-terminal cysteine ​​residue of the CRK peptide exposes the hidden CendR (C-terminal regular) sequence R / KXXR / K (SEQ ID NO: 4), namely RKDK (SEQ ID NO: 1) in the tCRK peptide of this invention. Without being limited by any theory, the tCRK peptide can penetrate skin tissue through internalization by dermal microvascular endothelial cells expressing NRP-1 on their cell surface. Interestingly, CRK peptides containing the hidden CendR motif cannot penetrate cells and tissues (…). and Ruoslahti, 2007, Am J Pathol 171: 702-711; Agemy et al., 2010, Blood 116: 2847-2856).

[0038] Therefore, the homing peptide used in the conjugates of the present invention contains a tCRK element at the C-terminus of the homing peptide.

[0039] As used in this invention, the term "C-terminus" (also known as carboxyl terminus, C-terminus, or COOH terminus) refers to the end of an amino acid chain capped by a free carboxyl group (-COOH). Here, the terms "C-terminus" and "C-terminus" are used interchangeably.

[0040] As used in this invention, the term "N-terminus" (also known as the amino terminus, amine terminus, N-terminus, or NH2 terminus) refers to the start of an amino acid chain. The first amino acid in the chain contains a free amino group (-NH2). Here, the terms "N-terminus" and "N-terminus" are used interchangeably. The peptide sequence is written from the N-terminus to the C-terminus.

[0041] As used in this invention, the term "tCRK element" refers to a peptide having the amino acid sequence RKDK (SEQ ID NO: 1) or CRKDK (SEQ ID NO: 2) that selectively homs to the skin and skin wounds in vivo and can penetrate skin tissue. The terms "tCRK element" and "tCRK peptide" are used interchangeably.

[0042] According to the present invention, the tCRK element is located at the C-terminus of the homing peptide used in the present invention. More specifically, the tCRK element is located at the C-terminus of the homing peptide and includes a terminal carboxyl group. In other words, the C-terminus of the homing peptide consists of the amino acid sequence RKDK (SEQ ID NO: 1) or CRKDK (SEQ ID NO: 2). Therefore, the homing peptide containing the tCRK element ends with the amino acid sequence RKDK (SEQ ID NO: 1) or CRKDK (SEQ ID NO: 2).

[0043] In some embodiments, the homing peptide used in this invention comprises either SEQ ID NO: 1 or SEQ ID NO: 2. In some other embodiments, the homing peptide comprises either SEQ ID NO: 1 or SEQ ID NO: 2. In the latter case, the homing peptide comprises an additional amino acid linked to the N-terminus of the tCRK element. However, the C-terminus of such a longer homing peptide is still composed of the tCRK element. In some embodiments, the homing peptide may comprise up to 100 amino acids. In some embodiments, the homing peptide may comprise up to 50 amino acids. In some embodiments, the homing peptide may comprise up to 20 amino acids. In some embodiments, peptide homing may comprise up to 10 amino acids.

[0044] In some embodiments, the homing peptide may be part of a cyclic structure and may be cyclized, for example by disulfide bond cyclization, and then cleaved by a protease to expose the tCRK sequence at the C-terminus of the homing peptide as a CendR peptide.

[0045] As used in this invention, the expression "tCRK-guided core proteoglycan" refers to any core proteoglycan conjugate whose targeted delivery or homing is accomplished by a tCRK homing peptide according to any of the embodiments disclosed in this invention. Non-limiting examples of such conjugates include those in which the core proteoglycan fragment comprises or is composed of the amino acid sequence shown in any one of SEQ ID NO: 6-20, and is linked from its C-terminus to the N-terminus of the tCRK element of SEQ ID NO: 1 or 2, with or without an intermediate linker sequence, such as the sequence shown in SEQ ID NO: 23 or 24. Further examples include conjugates comprising or composed of the amino acid sequence of SEQ ID NO: 21 or 22. Further examples include sequence variants having at least about 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, or 60% sequence identity with the said sequence, as well as their conserved sequence variants and peptide mimics, provided that the targeting specificity and penetration capability of the tCRK element, and the bioactivity of the core proteoglycan, remain substantially unchanged.

[0046] In some embodiments, the tCRK-guided core proteoglycan conjugate may be provided as a fusion protein, but is not limited thereto. Thus, in some embodiments, the conjugate is a "fusion protein" comprising a core proteoglycan fragment fused or linked to the N-terminus of the homing peptide disclosed herein, preferably fused or linked from the C-terminus of the core proteoglycan fragment, having or not having one or more additional amino fragments, such as peptides, oligopeptides, polypeptides, or protein fragments, which may consist of, or contain, natural or non-natural amino acids or peptide mimics. Such one or more additional amino acid fragments may be fused or linked to the N-terminus of the core proteoglycan fragment and / or fused or linked between the C-terminus of the core proteoglycan fragment and the N-terminus of the homing peptide. The additional amino acid fragments may have therapeutic activity, or may be used for purposes such as diagnostics, imaging, or visualization.

[0047] As used herein, the term "peptide" refers to a series of amino acid residues typically linked together by peptide (amide) bonds between the α-amino and carbonyl groups of adjacent amino acids to form an amino acid sequence. Generally, a peptide is defined as a molecule consisting of 2 to 100 amino acids, for example, 2 to 50 amino acids. However, peptides can be further subdivided into oligopeptides having a small number of amino acids (e.g., 2 to 20) and polypeptides having a large number of amino acids (e.g., 20 to 100 or 20 to 50). Proteins are essentially large peptides typically consisting of more than 50 or more than 100 amino acids. Therefore, for simplicity of expression, the term "peptide" as used herein includes any series of native (L-) and / or non-native (D-) amino acid residues linked by peptide bonds and is interchangeable with "oligopeptide," "polypeptide," "protein," and fragments thereof, unless explicitly stated otherwise. Peptide-domimetic forms of peptides are also included.

[0048] The fusion protein used in this invention can have any suitable length, for example, up to 300, 350, 400, 500, 1000, or 2000 residues, or it can have any number of residues including or between said integers. As used in this invention, the term "residue" refers to an amino acid or an amino acid analogue.

[0049] In some embodiments, the fusion protein used in this invention may comprise a small peptide tag that facilitates, for example, purification, separation, and / or detection. Non-limiting examples of suitable affinity tags for purification purposes include polyhistidine tags (His tags), hemagglutinin tags (HA tags), glutathione S-transferase tags (GST tags), biotin tags, avidin tags, and streptavidin tags. Suitable detection tags include, but are not limited to, fluorescent proteins, such as GFP.

[0050] Depending on its length, the fusion protein used in this invention can be prepared by any suitable means, methods, or techniques available in the art, such as by an automated peptide synthesizer or by genetic engineering techniques. For example, an expression vector containing a polynucleotide encoding a core proteoglycan and a tCRK homing peptide can be prepared by genetic engineering and then transfected into a host cell to express the fusion protein. Non-limiting examples of suitable host cells include prokaryotic hosts, such as bacteria (e.g., *Escherichia coli*, bacilli), yeasts (e.g., *Pichia pastoris*, *Saccharomyces cerevisiae*), and fungi (e.g., filamentous fungi), as well as eukaryotic hosts, such as insect cells (e.g., Sf9) and mammalian cells (e.g., CHO cells, HEK cells). The expression vector can be transfected into host cells using a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, including but not limited to electroporation, nuclear transfection, acoustic pore effect, magnetic transfection, heat shock, calcium phosphate precipitation, DEAE-glucan transfection, etc. A variety of expression vectors are readily available in the art, and those skilled in the art can easily select a suitable expression vector based on different variables, such as the host cell used. The fusion protein used in this invention can also be prepared by in vitro protein expression (also known as in vitro translation, cell-free protein expression, cell-free translation, or cell-free protein synthesis). Several cell-free expression systems based on, for example, bacteria, rabbit reticulocytes, CHO, or human lysates are commercially available in the art. In vitro protein expression can be performed in batch reaction or dialysis mode.

[0051] Fusion partners of the fusion protein used in this invention can be directly linked to each other or linked to each other via a linker sequence. The linker sequence can be a peptide linker sequence or a non-peptide linker sequence. If the linker sequence is a peptide linker sequence, it can consist of one or more amino acids. Non-limiting examples of peptide linker sequences include or consist of the amino acid sequence shown in SEQ ID NO: 23 or 24.

[0052] Furthermore, homing peptides can be conjugated to core proteoglycans or any other therapeutic proteins contained in the conjugates or compositions of the present invention via systems such as SpyTag / SpyCatcher.

[0053] As described above, in some embodiments, the fusion proteins used in this invention can be generated using nucleic acid molecules encoding the fusion proteins. These nucleic acid molecules can not only be used for recombinant production of the fusion proteins they encode, but also for gene therapy using methods and techniques available in the art.

[0054] The present invention also envisions conserved sequence variants of natural (L-) and / or non-natural (D-) amino acid and / or peptide mimics comprising fusion proteins for the treatment of epidermolysis bullosa.

[0055] As used in this invention, the term "conserved sequence variant" refers to an amino acid sequence modification that does not significantly alter the biological properties of the associated protein or peptide. Conserved sequence variants include variants resulting from the substitution of one or more similar amino acids well known in the art (e.g., amino acids of similar size or with similar charge properties).

[0056] As used herein, the term "peptide mimic" refers to a peptide-like molecule designed to mimic a given protein or peptide without altering its activity, such as homing specificity. Non-limiting examples of peptide mimics include chemically modified peptides, D-peptide mimics, peptide-like molecules containing non-naturally occurring amino acids, peptide-like molecules, and β-peptides. The term also includes peptide-like molecules that are not linked by natural peptide bonds. Means and methods for producing peptide mimics are readily available in the art.

[0057] tCRK-guided core proteoglycan conjugates for the treatment of epidermolysis bullosa may, as needed, further include one or more covalently (directly or indirectly via linker sequences) or non-covalently linked additional portions, provided that the therapeutic activity of the conjugate is preserved.

[0058] In some embodiments, the additional portion may have its own therapeutic activity, such as anti-inflammatory activity, anti-angiogenic activity, regenerative activity, pro-angiogenic activity, cytotoxic activity, pro-apoptotic activity, antimicrobial activity (e.g., antibacterial activity, antiviral activity, antifungal activity, or antiprotozoal activity), antifibrotic activity, anti-wrinkle activity, antipruritic activity, anti-or neurotransmitter (e.g., histamine) activity, or cytokine activity, or it may be a cytokine inhibitor (e.g., an antagonist, a soluble receptor, a cytokine-binding molecule, or a cytokine that blocks other cytokines), as some non-limiting examples of the potential biological activity or therapeutic effect of the therapeutic portion.

[0059] Therefore, in some embodiments, the additional portion may be a small molecule, such as selected from antihistamines, antibiotics, retinoids, benzoyl peroxide, podophyllotoxin, cytotoxic drugs, and immunomodulators such as corticosteroid derivatives, calcineurin inhibitors, and imiquimod. Furthermore, the additional portion may be a protein moiety, such as antifibrotic TGF-β3, any regenerative or anti-inflammatory growth factor or cytokine, such as interleukin-10 (IL-10), any angiogenic growth factor, such as vascular endothelial growth factor (VEGF), any anti-apoptotic protein, such as bit1, any inflammatory inhibitory enzyme, such as CD73, or any collagen, such as type VII collagen.

[0060] In some embodiments, additional components may be used to facilitate the detection of tCRK-guided core proteoglycan conjugates. Therefore, the conjugate may contain a detectable reagent. As used herein, the term "detectable reagent" refers to any molecule that can be detected directly or indirectly, preferably by non-invasive and / or in vivo visualization techniques. Non-limiting examples of detectable reagents suitable for the conjugates disclosed herein include optical agents, such as fluorescent agents, phosphorescent agents, luminescent agents, such as chemiluminescent agents, and chromogenic agents comprising a variety of organic and / or inorganic small molecules and a variety of fluorescent proteins and their derivatives; radiolabels, such as radionuclides emitting gamma rays, positrons, beta or alpha particles, or X-rays; non-radioactive isotopes, such as cadmium (Gd); ionic and non-ionic contrast agents, such as iodine-based contrast agents; electromagnetic agents, such as magnetic, ferromagnetic, paramagnetic, and / or superparamagnetic reagents; upconversion nanoparticles (UCNPs), resonant particles, quantum dots, and gold particles. Other suitable detectable reagents available in the art. Those skilled in the art can readily select appropriate imaging techniques based on the type and variety of detectable reagents used in the conjugate. These technologies include, but are not limited to, radiographic techniques and isotope techniques, such as positron emission tomography, ultrasound imaging, and magnetic resonance imaging (MRI).

[0061] The detectable reagent can be directly linked to the core proteoglycan conjugate, for example, via covalent bonding, or indirectly linked to the core proteoglycan conjugate, for example, via a binding agent, linker sequence, or chelating agent, such as diethylenetriaminepentaacetic acid (DTPA), 4,7,10-tetraazacyclododecane-N-,N′,N″,N″′-tetraacetic acid (DOTA), and / or metallothionein. Techniques for conjugating or otherwise associating detectable reagents to peptide or protein conjugates are well known in the art. For example, conjugates containing detectable proteins, such as fluorescent proteins (e.g., GFP), can be generated as fusion proteins using recombinant techniques.

[0062] The homing peptide-guided core proteoglycan conjugates disclosed in this invention, more specifically tCRK-guided core proteoglycan conjugates, do not exist in nature.

[0063] In some embodiments, tCRK-guided core proteoglycan conjugates for treating epidermolysis bullosa are provided as pharmaceutical compositions comprising the conjugates and a pharmaceutically or physiologically acceptable carrier to enable in vivo administration.

[0064] As used herein, the term "pharmaceutical composition" broadly refers to a formulation of one or more active ingredients and physiologically suitable components such as carriers, adjuvants, and / or excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the compound to a subject or organism. As used herein, the term "active ingredient" broadly refers to a substance responsible for biological effects, including but not limited to anti-inflammatory effects, anti-angiogenic effects, regenerative effects, pro-angiogenic effects, cytotoxic effects, pro-apoptotic effects, antimicrobial effects (e.g., antibacterial, antiviral, antifungal, or antiprobiotic effects), antifibrotic effects, antipruritic effects, anti-neurotransmitter effects, pro-neurotransmitter effects (e.g., histamine), cytokine-inducing effects, or cytokine-inhibiting effects. As disclosed herein, the term "active ingredient" specifically refers to tCRK-guided core proteoglycans, although the composition and / or conjugate may contain further active agents as described above.

[0065] Pharmaceutical compositions may be formulated as needed using readily available means and methods in the art, such as by conventional methods of mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, embedding, lyophilizing, or similar methods, into, for example, semi-solid or solid dosage forms, solutions, dispersions, or suspensions.

[0066] As used herein, the terms "pharmaceuticalally acceptable" and "physiologically acceptable" are used interchangeably to refer to a substance suitable for administration to a subject or organism without excessive adverse side effects such as toxicity, significant irritation, and / or allergic reactions. In other words, the benefit / risk ratio must be reasonable.

[0067] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier substance or diluent that is combined with the active ingredient to facilitate administration and is physiologically acceptable to the recipient. Pharmaceutically acceptable carriers are readily available in the art and, depending on the intended route of administration, may be selected from, but not limited to, transdermal carriers, transmucosal carriers, enteric carriers, parenteral carriers, and carriers for extended-release formulations. The selected carrier should not eliminate the biological activity and properties of the active ingredient, but should minimize any degradation thereto and minimize adverse side effects for the recipient.

[0068] As used in this invention, the term "excipient" refers to a preferred inert substance added to a pharmaceutical composition to further facilitate the administration of the active ingredient. Typical examples of different types of excipients include, but are not limited to, stabilizers, preservatives, pH adjusters, fillers, thickeners, viscosity modifiers, lubricants, solubilizers, surfactants, sweeteners, flavor masking agents, etc.

[0069] Useful stabilizing excipients include, but are not limited to, surfactants such as polysorbate 20, polysorbate 80, and poloxamer 407; polymers such as polyethylene glycols and polyvinylpyrrolidones; carbohydrates such as sucrose, mannitol, glucose, and lactose; sugar alcohols such as sorbitol, glycerol, propylene glycol, and ethylene glycol; proteins such as albumin; amino acids such as glycine and glutamic acid; fatty acids such as ethanolamine; antioxidants such as ascorbic acid; chelating agents such as EDTA salts; and metal ions such as Ca, Ni, Mg, and Mn. Useful preservatives include, but are not limited to, benzyl alcohol, chlorobutanol, benzalkonium chloride, and possibly parabens. Useful buffering excipients include, but are not limited to, sodium and potassium phosphates, citrates, acetates, and carbonate or glycine buffers, depending on the target pH range. Sodium chloride is also useful as a tension modifier. Other non-limiting examples of excipient materials include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols. As will be readily understood by those skilled in the art, a given excipient may provide more than one function.

[0070] The pharmaceutical composition can be administered in a variety of ways, depending on whether local or systemic treatment is required, and depending on the site of treatment. The route of administration can be, for example, parenteral, intravenous, or local.

[0071] If a composition is used, parenteral administration of the composition is typically administered by injection, such as intravenous, intraperitoneal, subcutaneous, or intramuscular injection. Formulations for parenteral administration are typically sterile aqueous or non-aqueous solutions, suspensions, or emulsions; however, the formulation may also be provided in concentrated or powder form for reconfiguration as needed. Sustained-release or sustained-release formulations are also considered. The means and methods for formulating parenteral administration formulations are readily available in the art, and those skilled in the art can readily select appropriate physiologically suitable carriers, adjuvants, and / or excipients according to the specific circumstances of the desired formulation.

[0072] Non-limiting examples of aqueous carriers used in parenteral and other pharmaceutical preparations include sterile water, water-alcohol solutions, saline, and buffer solutions with physiological pH. Parenteral carriers include sodium chloride solutions, Ringer's glucose solutions, glucose plus sodium chloride solutions, lactose-containing Ringer's solutions, or non-volatile oils. Intravenous carriers include fluid and nutritional supplements, electrolyte supplements, such as carriers based on Ringer's glucose solutions, etc.

[0073] Non-limiting examples of non-aqueous carriers used in parenteral and other pharmaceutical preparations include solvents such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, fish oil, and injectable organic esters such as ethyl oleate.

[0074] If the parenteral preparation is provided as a concentrated solution, dispersion, or powder, the aforementioned aqueous or non-aqueous carrier can be used for reconstitution. The solution for reconstitution can be provided in the same package as the concentrate or powder. If the powder is prepared by lyophilization, the use of cryoprotectants may be beneficial, including but not limited to polymers (e.g., polyvinylpyrrolidone, polyethylene glycol, dextran), sugars (e.g., sucrose, glucose, lactose), amino acids (e.g., glycine, arginine, glutamic acid), and albumin.

[0075] If a composition is used, it can be applied for enteral administration, for example, by oral administration or via percutaneous endoscopic gastrostomy (PEG). Compositions for oral administration include, but are not limited to, powders, granules, capsules, sachets, tablets, and aqueous or non-aqueous solutions and suspensions. The means and methods for formulating formulations for enteral administration are readily available in the art, and those skilled in the art can readily select appropriate physiologically suitable carriers, adjuvants, and / or excipients according to the specific circumstances of the desired formulation.

[0076] If used, the composition can be administered topically, for example, via transdermal, transmucosal, epidermal, intranasal, rectal, vaginal, and via an inhaler. Depending on the route of administration, formulations for topical application may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, and sustained-release or continuous-release formulations or solids. The means and methods for formulating formulations for topical administration are readily available in the art, and those skilled in the art can readily select appropriate physiologically suitable carriers, adjuvants, and / or excipients according to the specific circumstances of the desired formulation.

[0077] Some compositions can be administered as pharmaceutically acceptable acid or base addition salts, formed by reaction with inorganic and organic acids, such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanate, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic and organic bases, such as sodium hydroxide, ammonium hydroxide, and potassium hydroxide, and organic bases such as mono-, di-, trialkyl, and arylamines and substituted ethanolamines.

[0078] The dosage and administration method of the conjugates or pharmaceutical compositions disclosed in this invention can be readily determined by a person skilled in the art in treating skin diseases and conditions, particularly epidermolysis bullosa. Typically, the dosage will vary depending on considerations such as: the age, sex, and general health of the subject to be treated; the type of concurrent treatment, if any; the frequency of treatment and the nature of the desired effect; the severity and type of epidermolysis bullosa; and other variables adjusted by the individual physician. The desired dosage can be administered in one or more applications to achieve the desired results. For example, the pharmaceutical composition can be administered as a single daily dose, or the total daily dose can be administered in divided doses, for example, twice, three, or four times daily. The pharmaceutical composition can be provided, for example, in a unit dosage form or as an extended-release formulation.

[0079] Experimental Section

[0080] Materials and methods

[0081] Cloned core protein glycan fusion protein

[0082] Human core proteoglycan (DCN) cDNA (Krusius and Ruoslahti, 1986, PNAS 83: 7683-787) without the natural signal and propeptide sequence was cloned into the mammalian expression vector pEFIRES-P (Hobbs et al., 1998, Biochem Biophys Res Commun 252: 368-372). The tCRK wound homing peptide cDNA was cloned to the C-terminus of the core proteoglycan flanked by a stop codon. A 6×His tag was cloned to the N-terminus preceding the core proteoglycan. The construct was assembled using the PIPE method (Klock and Lesley, 2009, Methods Mol Biol 498: 91-103). Transformation was performed using NEB 5-α competent *E. coli* (high-efficiency) cells according to the manufacturer's instructions (C2987H; New England Biolabs Ipswich, MA). Plasmid purification (Mini-Prep), PCR purification, and agarose gel purification were performed using a kit from Qiagen (Hilden, Germany). DCN naturally forms dimers (Scott et al., 2004, PNAS 101: 15633-15638).The protein sequence of the monomeric 6×His tag-DCN-tCRK fusion protein is: GHHHHHH DE ASGIGPEVPDDRDFEPSLGPV CPFRCQCHLRVVQCSD LG LDKVPKDLPPDTTLLDLQNNK IT EI KDGDFKNLKNLH AL ILVNNKISKVSPGAFTPLVKL ERLYLSKNQLKELPE KMet PKTLQELRAHENEITKVRKVT FNGLNQ Met IVIELGT NP LKSSGIENGAFQG Met KKLSYIRIADTNITSIPQGLP P SL TELHLDGNKISRVDAASLKGL NNLAKLGLSFNSISAV DN GS LA NTPHLRELHLDNNKLTRVPGG LAEHKYIQVVYL HN NNISVVGSSDFCPPGHNTKKA SYSGVSLFSNPVQYWE IQ PSTFRCVYVRSAIQLGNYKGS EFCRKDK terminated (SEQ ID NO: 21).

[0083] Figure 1A A schematic diagram of the DCN-tCRK fusion protein used in the experimental section is shown. As clearly illustrated in the detailed description, Figure 1A The DCN-tCRK fusion protein is a non-limiting example of a tCRK-guided core proteoglycan conjugate suitable for use in this invention.

[0084] Recombinant protein preparation

[0085] The construct in the pEFIRES-P expression vector was transfected into HEK293F cells via liposome transfection (FuGene 6, Promega, Madison, WI). Positive clones were selected in the presence of 5–160 μg / ml puromycin (HyClone, Thermo Fisher Scientific) in a medium consisting of DMEM Hi-glucose (4.5 g / L), 2 mM L-alanyl-L-glutamine, 100 IU / ml penicillin (all from Sigma Aldrich, St. Louis, MO), and 10% FBS (Gibco, Grand Island, NY). The established cell lines were maintained in a culture containing 10 μg / ml puromycin.

[0086] The validated cells were then resuspended in serum-free OptiCHO medium (Gibco) supplemented with 2 mM L-alanyl-L-glutamine (Sigma) and cultured in square glass flasks mounted on a rotary shaker at 37°C under a 5% CO2 atmosphere. The cells were cultured until they reached 1–2 × 10⁻⁶ cells / year. 6 After reaching a density of [number] cells / ml, they were further cultured at 33°C for 4 days for recombinant protein expression and secretion into the culture medium. Proteins were purified from culture medium using a two-step HisTrap purification protocol on a Start chromatography system (GE Healthcare, Munich, Germany).

[0087] Recombinant protein purification

[0088] Cell culture supernatant was filtered and degassed on ice using a 0.45 μm filter (Corning #430514, Corning, NY). 6×His-labeled proteins were purified using a two-step Ni-NTA-IMAC purification protocol, first with a HisTrap Excel column, then with a HisTrap HP column. The chromatography was performed on a Start chromatography system (GE Healthcare, Munich, Germany) in a 4°C cold oven according to the manufacturer's instructions. Buffers were prepared using a His buffer kit (GE Healthcare / VWR (11-0034-00), and all buffers were filtered and degassed.

[0089] The HisTrap Excel column eluent was diluted to a final imidazole concentration of 30 mM in 20 mM sodium phosphate buffer (pH 7.4) containing 0.5 M NaCl, and then further purified on a HisTrap HP column by washing with 35 mM imidazole and eluting gradient to 300 mM imidazole.Figure 2A (Including an example of such purified chromatograms). Peak fractions were analyzed on SDS NuPAGE 4-12% gradient gels (Life Technologies / Thermo Fisher Scientific, Waltham, MA) and observed using PageBlue protein staining solution (Thermo Fisher Scientific, Waltham, MA).

[0090] Selected peak fractions were combined, dialyzed against cold TBS buffer (pH 7.6) using 50 kDa MWCO Float-A-Lyzers (Fisher Scientific / Spectrum Labs), and then concentrated using 10 kDa MWCO VivaSpin 6-tube (GE Healthcare). Samples were filtered sterile (Ultrafree-MC GV centrifuge filter 0.22 μm, Millipore, Burlington, MA) and protein concentrations were measured at A280 nm using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA). All steps were performed at 4 °C or on ice. Sterile Tween-20 was added to a final concentration of 0.05% before rapid freezing of aliquots at -80 °C to prevent aggregation.

[0091] Recombinant proteins were validated using SDS-PAGE and Western blotting. A BioRad's wet-pot Mini-PROTEAN Trans-blot cell system was used (according to the manufacturer's instructions). PVDF membranes were probed using a mouse primary antibody against human core proteoglycans (MAB143, R&D Systems, Minneapolis, MN) according to the manufacturer's protocol. A secondary horseradish peroxidase-conjugated anti-mouse antibody from Cell Signaling Technology was used. Chemiluminescent blot images were captured using an ImageQuant LAS 4000mini (GE Healthcare).

[0092] Biophysical protein analysis

[0093] Hydrodynamic diameters were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS instrument (Malvern Instruments Ltd, Worchestershire, UK). DCN-tCRK protein samples were diluted 1:5 in TBS buffer. Three 10×10 measurements were performed at 25°C, and the data were analyzed using Zetasizer software v7.11 (Malvern Instruments Ltd) via protein analysis models (non-negative least squares analysis followed by L-Cuve) and volumetric size distribution.

[0094] The unfolding temperatures of DCN-tCRK were determined using a VP-capillary DSC (differential scanning calorimetry) instrument (GE Healthcare, Microcal Inc. / Malvern Instruments Ltd.) in TBS buffer (50 mM Tris-Cl, 150 mM NaCl, pH 7.5) at a protein concentration of 0.2 mg / ml. All solutions were degassed. Samples were heated from 20 °C to 130 °C at a scan rate of 2 °C / min. The feedback mode was set to “low”, and the filtration cycle was 5 seconds. The melting temperature Tm (transition midpoint) was calculated using the Origin 7.0 DSC software suite (Microcal Inc.) via a non-2-state fitting model.

[0095] Using Eksigent 425 NanoLC with Sciex high-speed TripleTOF TM The recombinant DCN-tCRK protein was identified from monomeric gel bands using a 5600+ mass spectrometer coupled with a 5600+ mass spectrometer. After separating the gel bands and Coomassie staining, protein removal was then performed as described in [the original text is missing here]. The reduction (TCEP, 25 mM), alkylation (iodoacetamide, 0.5 M), and trypsin digestion were described in detail in et al., 2018. After trypsin digestion, the peptide was diluted to 14 μl of sample buffer (2% acetonitrile, 0.1% formic acid), and 1 μl of sample was injected into a triple TOF mass spectrometer.

[0096] In vitro binding analysis

[0097] ELISA analysis was used to analyze the in vitro binding of DCN-tCRK and peptides to NRP-1. 96-well, black FLUOTRAC. TMHigh-binding plates (Greiner Bio-One, Kremsmünster, Austria) were coated overnight at 4°C with 100 μL / well of 100 μg / ml DCN-tCRK in PBS. Parallel coatings of 10 μg / well of RPARPAR (SEQ ID NO: 25) and RPAPRARA (SEQ ID NO: 26) peptides were used as positive and negative controls, respectively. BSA was used as an immobilization control. Plates were washed three times with phosphate-buffered saline (PBS) and blocked for 1 hour at 37°C with 300 μL of blocking solution (1×PBS, 1% BSA, 0.1% Tween-20). His-labeled neurocilia protein-1 b1b2 domain (NRP-1WT) and the triple mutant NS346A-E348A-T349A neurocilia protein-1 b1b2 domain (NRP-1 mutant) were expressed and purified as previously described at the Protein Production and Analysis Facility of the Sanford Burnham Prebys Institute for Medical Discovery (La Jolla, CA) (Teesalu et al., 2009, PNAS 106: 16157-16162). Recombinant proteins NRP1 WT, NRP1 mutant, and DCN-tCRK were labeled with FAM (5-(and-6)-carboxyfluorescein, #90024, Biontium Inc, CA, USA) by mixing an amine-reactive FAM dye (diluted in DMSO to a final concentration of 0.2%) with the protein at a 1:10 ratio. The mixed reaction mixture was incubated at room temperature in the dark for 2 hours, followed by ultrafiltration / dialysis with PBS to separate the free dye from the protein. 100 μl of blocking solution of FAM-labeled NRP1 WT or NRP1 mutant protein was added to each well (20 μg / well), and incubated at room temperature for 4–6 hours or overnight at 4°C, followed by washing three times with the blocking solution. After adding 100 μl of PBS to each well, the plate was immediately read in top-reading mode using a fluorescence reader (Flex State II, Molecular Devices; peak excitation = 485 nm, peak emission = 530 nm, cutoff = 515 nm).

[0098] To enable in vitro binding of FAM-DCN-tCRK with NRP-1 positive prostate cancer-3 (PC-3) cells (gifted by the Ruoslahti laboratory at the Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA) and negative melanoma (M21) cells (gifted by the David Cheresh laboratory at the University of California, San Diego), cells were first cultured in a growth medium consisting of 10% fetal bovine serum (FBS) in DMEM high-glucose medium supplemented with penicillin and streptomycin (Gibco). For the experiment, the medium was aspirated, cells were washed twice with warm medium, and fresh medium was added along with 10 μg of FAM-labeled DCN-tCRK recombinant protein. Labeling was performed using the Lightning-Link fluorophore kit (Expedon Ltd, UK) by directly conjugating the DCN-tCRK recombinant protein to fluorophore, according to the manufacturer's protocol. Cells were incubated at 37°C for one hour; the medium was aspirated, cells were washed, and fixed with methanol at -20°C. Cells were washed with PBS and blocked at room temperature (PBS, 1% BSA, 1% FBS, 1% goat serum, 0.05% Tween-20) for 30 minutes, followed by blocking with primary anti-FITC (Invitrogen, CA, USA. Catalog #A-889) for one hour at room temperature. Cells were washed again and secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, USA) was administered in the dark at room temperature for one hour. Cell nuclei were stained with DAPI. Coverslips were fixed onto slides using Fluorosmount-G (Electron Microscopy Sciences, PA, USA) and imaged using a confocal microscope (Olympus FV1200MPE, Tokyo, Japan), and analyzed using an FV10-ASW4.2 indicator.

[0099] Mouse and research approval

[0100] BALB / cJRj mice (Janvier Labs, Le-Genest-Saint-Isles, France) were used for pharmacokinetic studies. Mice were fed standard laboratory pellets and had free access to water. All animal experiments using Balb / cJRj mice were conducted in accordance with protocols approved by the Finnish National Animal Ethics Committee (ESAVI / 6422 / 04.10.07 / 2017).

[0101] An animal model of recessive dystrophic epidermolysis bullosa (RDEB), namely the col7a1- / -RDEB mouse, was used to study skin homing and therapeutic function of DCN-tCRK. The col7a1- / -RDEB mouse was obtained by breeding C57BL6 / J col7a1+ / - mice with a genotype determined by polymerase chain reaction (PCR). The C57BL6 / J col7a1+ / - mice, provided by Dr. Jouni Uitto of Thomas Jefferson University, were developed through out-of-frame deletion targeting the col7a1 gene. All animal studies using col7a1- / -RDEB were conducted using protocols approved by the International Association for Animal Control and Use (IACUC) of the New York Medical College.

[0102] Pharmacokinetics of recombinant proteins

[0103] Recombinant proteins DCN-tCRK or DCN were diluted in Tris-buffered saline (TBS) containing 0.05% Tween-20. Pharmacokinetics of DCN-tCRK and DCN were investigated in 8-week-old Balb / c male mice. Under isoflurane anesthesia, 5 mg / kg of DCN-tCRK or DCN was injected into the tail vein. Blood samples were collected from different tail veins at 15, 30, 60, 2, 4, and 16 hours post-injection. Mice were sacrificed under metoprimidine-ketamine anesthesia at 8 or 24 hours post-injection, and blood samples were collected from the subclavian vein. Samples were mixed with 1 M EDTA, centrifuged at 2000 g for 10 minutes at room temperature, and plasma was stored for analysis. The concentration of human core proteoglycan in plasma samples was determined using a Human DecorinDuoSet ELISA kit (#DY143, R&D Systems) according to the manufacturer's instructions. Venous blood samples from uninjected mice were used in each culture plate to ensure the specificity of the primary antibody.

[0104] In col7a1 - / - Administration of DCN-tCRK and DCN in mice

[0105] pregnant col7a1 + / - Mice were housed individually and monitored daily before birth. Because intravenous injection in newborn mice is technically challenging and often yields inconsistent results, the inventors opted to inject the first dose of DCN-tCRK and DCN (5 μg in 15 μl PBS, equivalent to ~5 mg / kg) into col7a1 within 24 hours of birth. - / -The liver of mice was used because it is a major site of hematopoiesis in fetuses and newborn mice, and human cells have been shown to rapidly enter circulation after intrahepatic injection (Liao et al., 2015, Stem Cells 33: 1807-1817; Liao et al., 2018, Stem Cells Transl Med 7: 530-542). Following this first dose, intraperitoneal administration of protein was repeated every other day until the mice reached 14 days of age (maximum 7 doses). When the mice reached one week of age, the dose was increased to 10 μg. Mice were monitored daily. All experiments were performed using col7a1. - / - All mice were genotyped during sample collection.

[0106] col7a1 - / - Histological and immunohistochemical staining and hDCN quantification in mice

[0107] Dorsal skin and paws (forepaws and hindaws) were excised from selected mice, embedded in Tissue-Tec OCT compound (Sakura Finetek, Torrance, CA), and stored at -80°C. Each sample was cut into 6 μm serial sections. Sirius red staining and CTGF (#ab6992, Abcam, Cambridge, UK) immunohistochemical staining were performed at the Core Histology Laboratory of New York Medical College. For immunochemical staining of his tags, sections were fixed in 4% paraformaldehyde and blocked with MOM blocking agent containing 0.1% Triton (Sigma, St Louis, MO) (Vector Laboratories, Burlingame, CA) (for antibodies produced in mice) or 10% horse serum (GIBCO, Grand Island, NY). The slides were then incubated with their respective primary antibodies, including anti-Col1A (#R1038, Acris, Rockville, MD), anti-αSMA (#14968, Cell Signaling Technology, Danvers, MA), anti-6x-His tag (#R930-25, Thermofisher Scientific, Carlsbad, CA), and anti-NRP-1 (#AF566-SP, R&D Systems, Minneapolis, MN), followed by the corresponding Alexa Fluor 488 secondary antibody (Invitrogen, Carlsbad, CA). The slides were then fixed in Vectashield fixation medium containing DAPI (Vector Laboratories, Burlingame, CA). Images were acquired using the same settings across the groups in each experiment, using a Nikon 90i Eclipse microscope (Nikon Instrument Inc., NY). Immunostaining intensity was measured for each field of view using NIS-Element AR software, following user instructions. RGB images are used for quantitative Sirius red staining, and the threshold is defined by selecting reference points within the image.

[0108] As recommended by the manufacturer, the Human Decorin DuoSet ELISA kit (#DY143.R&DSystems.Minneapolis.MN) was used to verify DCN-tCRK and DCN at col7a1. - / -Homegrown to skin in mice. Tissue biopsy samples were flash-frozen in liquid nitrogen, homogenized with a pre-chilled pestle, and homogenized with lysis buffer (PBS containing 1% Tween 20, a mixture of protease inhibitors, DNase, and RNase). After centrifugation at 12,000g for 10 minutes at 4°C, the supernatant was collected, and the total protein concentration was quantified using the BioRad DC protein assay (BioRad.Hercule.CA). Before use, col7a1 from both treated and untreated DCN-tCRK or DCN samples was... - / - Mouse serum was diluted 1:20 in sample diluent.

[0109] RT 2 PCR analysis of wound healing pathways

[0110] Using RT 2 PCR array (RT) 2 The Profiler PCR Array (QIAGEN. Hilden. Germany) was used to study the expression of genes involved in the mouse wound healing pathway. RT 2 The spectroscopic array contained primers for 84 wound healing genes and 5 housekeeping genes in a 96-well plate, along with genomic DNA, reverse transcription, and PCR positive controls. On day 7, WT, RDEB, and either DCN or DCN-tCRK were injected into col7a1. - / - Total RNA was isolated from the entire forepaw of mice (3 mice per group), and RNA quality and concentration were determined using a NanoDrop 200C (ThermoScientific.Waltham.MA). RNA was treated with a genomic DNA elimination mixture (QIAGEN). RT was used. 2 The First Strand kit (QIAGEN) uses 500 ng of total RNA per sample for reverse transcription. The cDNA synthesis reaction product is then mixed with 2×RT... 2 The SYBR Green Master Mix was pooled, and 25 μl of this mixture was allocated to each well of a 96-well plate. Q-PCR was performed on a QuantStudio 5 Real-Time PCR instrument (AppliedBiosystems, Foster City, CA). CT values ​​were output to an Excel file. Raw data were obtained by analyzing the data using a PCR array data analysis template at the GeneGlobe Data Analysis Center (https: / / www.qiagen.com / us / GeneGlobe). ΔΔC was used. TMethods were used to calculate gene expression. A fold change gene expression threshold of 1.5 and a p-value threshold of 0.05 were used to analyze data between WT pups and untreated / treated pups.

[0111] Collagen lattice shrinkage assay

[0112] As previously described, normal human fibroblasts and RDEB patient-derived fibroblasts were cultured in DMEM supplemented with 10% FBS (Liao et al., 2018, Stem Cells 36: 1839-1850). Collagen meshes were prepared by mixing the cell suspension with neutralized type I rat tail collagen (Advance BioMatrix, Carlsbad, CA). The final collagen concentration was 2.4 mg / ml, and the cell density was 2.1 × 10⁻⁶ cells / ml. 5 Cells / ml. 500 μl of cell / collagen suspension was dispensed into individual wells of a 24-well plate and allowed to cure at room temperature for 30 min. After collagen polymerization, 0.5 ml of DMEM supplemented with 5% FBS was added to each well, and the plate was incubated at 37°C with 5% CO2. After 12 hours of incubation, the gel was gently released from each well using a fine pipette tip, and DCN or DCN-CRK (n = 3 / condition) was added to the wells at a final concentration of 75 μM. Images were acquired at 12 hours (initial area) and 48 hours (shrinkage area), and the area of ​​the gel was quantified using ImageJ.

[0113] Statistical data

[0114] Kaplan-Meier analysis was applied to determine median lifetime, and the Mantel-Cox log-ordination test was used to compare survival between different experimental groups (GraphPad Prism 6). Student's unpaired t-test was used to investigate the binding of DCN-tCRK to NRP-1. A p-value less than 0.05 was considered significant.

[0115] result

[0116] Generation of multifunctional recombinant DCN-tCRK fusion protein

[0117] The inventors designed a DCN-tCRK fusion protein by placing the tCRK peptide at the C-terminus of DCN. Figure 1A Both DCN-tCRK and native DCN are expressed in mammalian cells and purified by chromatography. Figure 2A Both recombinant proteins migrated as sharp bands of approximately 55 kDa. A smear above the bands was observed in SDS-PAGE, and Western blot analysis identified it as DCN (…). Figure 2BThe sharp bands correspond to the core protein, and the smears are caused by the heterogeneity of glycosaminoglycan sulfate chains (mainly chondroitin) attached to the DCN core. Mass spectrometry confirmed the identity of the DCN and C-terminal tCRK sequences (Table 1). Hydrodynamic dimensions indicate that DCN-tCRK exists as a homogeneous and non-aggregated macromolecule with a diameter consistent with the reported DCN dimer (Scott et al., 2003, J Biol Chem 278: 18353). Figure 2C Differential scanning calorimetry produced a peak with a melting temperature (Tm) of 49 °C, indicating that tCRK-DCN will maintain a stable tertiary structure under physiological conditions. Figure 2D ).

[0118] Table 1. Mass spectrometry analysis of human DCN sequence and C-terminal tCRK sequence. Underlined letters The text indicates peptides found to be specific to human DCN, with italicized letters indicating amino acids specific to the C-terminus of the tCRK sequence (CRKDK / RKDK), which is further indicated in bold.

[0119]

[0120]

[0121] DCN-tCRK interacts with NRP-1 in vitro.

[0122] The inventors then investigated whether the tCRK peptide fused with DCN retained its ability to interact with NRP-1. DCN-tCRK was immobilized on ELISA plates and its binding to wild-type (WT) or mutant NRP-1 was tested, with the CendR binding pocket inactivated by a triple mutation (Teesalu et al., 2009, PNAS 106: 16157-16162). DCN-tCRK bound to WT NRP-1 efficiently at significantly higher levels than control bovine serum albumin (p < 0.01), while showing no significant binding to mutant NRP-1. Furthermore, parallel studies using the synthetic RPARPAR (SEQ ID NO: 25) peptide, the prototype CendR peptide, and RPARPARA (SEQ ID NO: 26), a control peptide with a C-terminal capped CendR sequence that did not interact with NRP-1, were used to demonstrate that this binding was CendR sequence-dependent. Figure 1BThe inventors further determined whether DCN-tCRK binds to NRP-1-expressing cells, namely human PC3 prostate cancer cells. The analysis also included M21 melanoma cells that do not express NRP-1. Internalization of DCN-tCRK was observed only in NRP-1-positive PC3 cells but not in NRP-1-negative M21 cells, supporting NRP-1-dependent cell binding and penetration properties. Figure 1C ).

[0123] DCN-tCRK and DCN exhibit similar pharmacokinetics in vivo.

[0124] To determine whether the addition of tCRK peptide had any effect on the circulating half-life of DCN, DCN-tCRK and DCN were administered intravenously in parallel to healthy Balb / c mice, and the amounts of DCN-tCRK and DCN in peripheral blood were quantified by ELISA at different time points within 24 hours of administration. The half-life of DCN-tCRK in the blood was 30 minutes, which was not significantly different from that of DCN. Figure 3 Pharmacokinetic studies have shown that modifying DCN with small vessel homing peptides does not affect the pharmacokinetics of DCN.

[0125] Administration of DCN-tCRK improves col7a1 - / - mouse survival

[0126] In the RDEB animal model col7a1 - / - The therapeutic function and skin homing properties of DCN and DCN-tCRK were evaluated in mice. These mice were bred by raising hybrid litter-born offspring, and col7a1 - / - Mice can be identified at birth based on the presence of hemorrhagic blistering in their skin. Newborn col7a1 mice... - / - Mice were randomly assigned to receive intrahepatic DCN, DCN-tCRK, or PBS (negative control). Surviving mice in each group were given repeated intraperitoneal administration every other day after the first dose until day 14. Here, col7a1 - / - The median lifespan of mice was 2 days after PBS injection and was significantly prolonged to 7 days after DCN administration (p < 0.0001). Figure 4A However, after DCN administration, col7a1 - / -Mouse survival was not statistically significant compared to previous administrations of dextran / human serum albumin (D / HSA), which served as a mediator for stem cell administration and may occasionally increase some receptor col7a1 by regulating fluid homeostasis. - / - mouse survival ( Figure 5 Furthermore, DCN injection did not prolong recipient survival beyond two weeks of age. Importantly, the median lifespan of mice treated with DCN-tCRK was further extended to 11 days, which was significantly superior to the lifespan following PBS (p < 0.0001) or historical D / HSA administration (p < 0.001). Figure 4A and 5 In addition, 85% of the DCN-tCRK-treated mice survived for 7 days, and 20% of these mice survived for more than three weeks before being sacrificed for skin analysis.

[0127] DCN-tCRK homed to col7a1 - / - mouse skin

[0128] Human DCN and DCN-tCRK in the skin of RDEB-positive mice were measured using ELISA at one, two, and three weeks (n = 3 time points for all time points). Figure 4B There was no statistically significant difference between DCN-tCRK and DCN-treated skin at the one-week time point. However, at the two-week time point, the level of DCN-tCRK was significantly higher than that of DCN (3.6-fold, p < 0.05). Figure 4B Furthermore, since the last intraperitoneal administration of DCN-tCRK was performed on day 14, the identification of DCN-tCRK in the skin at three weeks (19.47 ± 12.80 pg / ml) strongly suggests its stability in vivo for at least 7 days.

[0129] Immunohistochemical staining based on histidine tag expression was also performed to analyze the anatomical distribution of DCN-tCRK or DCN in RDEB skin. DCN-tCRK was detected in the dermis of the paw and back skin of RDEB mice at one, two, and three weeks. Figure 4C Furthermore, gastrointestinal (GI) staining in recipient RDEB mice showed no reactivity to anti-his antibodies (data not shown), suggesting skin-specific targeting of DCN-tCRK. Conversely, although ELISA demonstrated the presence of DCN in skin lysates, anti-his immunostaining on DCN-treated RDEB skin (expressed at one-week time points) appeared to be only nonspecific (diffuse). Figure 4CDCN-tCRK homing is provided by NRP-1-dependent cell and tissue penetration, and double staining with anti-his and -NRP-1 confirms that signals from DCN-tCRK are present in or closely adjacent to NRP-1-positive cells in RDEB skin. Figure 4D This further supports the inventor's non-restrictive hypothesis.

[0130] DCN-tCRK therapy inhibits fibrosis in RDEB mice.

[0131] The inventor's recent research has demonstrated that col7a1 begins as early as one week after birth in the interdigital folding of the paw. - / - Significantly enhanced TGFβ signaling was observed in mice. Therefore, in this study, skin biopsy samples at this time point were selected to compare the expression of 84 genes crucial for wound healing response and fibrosis formation (n=3 per group) between WT and vector (D / HSA), DCN, or DCN-tCRK-treated RDEB skin (Table 2). Figure 6A As demonstrated by the cluster plots, more than half of the genes showed a >1.5-fold increase in expression in the injected RDEB skin compared to WT. The relative fold change in gene expression (log2) and p-value (-log10) were also presented in volcano plots, with genes showing significant (p < 0.05) aberrant regulation marked in white in each plot. Figure 6B Genes significantly upregulated in vector RDEB skin involved TGFβ signaling (i.e., Tgfb1, Tgfb3, Ctgf), WNT signaling (Ctnnb1), MAPK1 / MAPK3 signaling (Mapk3), epidermal growth factor receptor signaling (Egfr), ECM remodeling (Ctsg, Plaur), cell adhesion (Itgb3, Itgb5), and inflammation (Il4, Cxcl3, Tnfα). No genes were significantly downregulated in vector RDEB skin compared to DCN-treated RDEB mouse skin, and the overall gene expression profile was similar to that in vector RDEB skin. Figure 6B Even though Tgfb1 expression was no longer significantly abnormal, Tgfbr3 and Ctgf expression were still significantly upregulated in DCN-treated RDEB skin. Some genes, such as Il4, Cxcl3, and Tnfα, were more significantly upregulated in DCN-treated RDEB skin than in the vector control. Figure 6B (and Table 2).

[0132] Importantly, the expression profile of RDEB skin treated with DCN-tCRK was significantly different from that of RDEB skin treated with the vector and DCN, but similar to that of WT skin. Figure 6AAlthough it shows individual differences in the expression of some genes, no genes in the array were significantly dysregulated when compared with WT in DCN-tCRK treated RDEB skin (Figure 6 and Table 2).

[0133] Table 2. Col7a processed by carrier, DCN, and DCN-tCRK - / - The fold change in gene expression in skin relative to WT and P values. N / A indicates that the mean threshold cycle was not determined or was greater than the defined cutoff point. Genes significantly upregulated compared to WT are shown in bold; col7a was only treated in DCN. - / - Genes that are significantly upregulated in the skin are indicated by underscores.

[0134]

[0135]

[0136] Strong expression of CTGF / CCN2 was observed in RDEB-injected skin, and its expression level was significantly reduced after treatment with DCN-tCRK. Figure 7A This supports the development of TGFβ1-mediated fibrosis in untreated RDEB skin and its inhibition by DCN-tCRK treatment. Furthermore, as demonstrated by Sirius red staining, total collagen deposition in injected carrier-treated RDEB skin increased over time, but was significantly reduced in DCN-tCRK-treated mouse skin. Figure 7B and 7C Immunostaining showed that at the two-week time point, the expression of type I collagen (COL1) was significantly increased in carrier-treated skin, while its expression was decreased in DCN-tCRK-treated skin. Figure 7D and 7E Similar results were obtained using immunostaining of myofibroblasts, specifically α-smooth muscle actin (aSMA). Figure 7D ja 7E). Furthermore, most αSMA+ cells in WT and DCN-tCRK-treated RDEB skin were colocalized with blood vessels (CD31 staining), indicating they function as the bulk of vascular smooth muscle cells and pericytes, while αSMA+ cells in carrier-treated RDEB skin were extravascular, i.e., indicating they are myofibroblasts (ja 7E). Figure 7D ).

[0137] To directly demonstrate the anti-fibrotic function of DCN-tCRK, the ability of DCN and DCN-tCRK to inhibit collagen gel contraction in vitro was compared using normal and RDEB-derived fibroblasts. At a low concentration (75 μM) where DCN had no significant effect on collagen contraction, DCN-tCRK inhibited collagen gel contraction in both normal (p < 0.05) and RDEB-derived (p < 0.01) fibroblasts. Figure 8 ).

[0138] discuss

[0139] This invention demonstrates that the C-terminal exposure of the CendR sequence in wound homing peptides provides novel tissue penetration capabilities for the peptides in both normal and injured skin. The conjugation of the tCRK peptide to DCN promotes skin-selective targeting of the therapeutic fusion protein, which exerts anti-fibrotic effects and improves survival in a mouse model of RDEB.

[0140] Experiments have shown that systemic administration of recombinant DCN-tCRK protein improves col7a1 - / - The modified DCN was more effective in improving mouse survival than the unmodified DCN. The exact molecular mechanism is unknown, but without being bound by any theory, it is hypothesized that several different mechanisms may contribute to improved survival. DCN is an anti-inflammatory and anti-fibrotic molecule. Consistent with the inventors' previous finding that TGFβ signaling is activated as early as one week after birth, more than half of the genes associated with fibrosis formation were upregulated in the skin of untreated RDEB mice at a one-week time point. Without being bound by any theory, the improvement in RDEB mouse survival by administering DCN-tCRK may be related to the anti-fibrotic and anti-inflammatory effects of the therapeutic protein.

[0141] Not only did DCN-tCRK (but not DCN) normalize genes directly involved in TGFβ signaling in RDEB skin treated with DCN-tCRK, but DCN-tCRK also normalized genes associated with other signaling pathways, such as β-catenin and EGFR. Wnt / β-catenin and EGFR signaling have both been shown to contribute to fibrosis in various fibrotic diseases through their independent pro-fibrotic mechanisms or through cross-interactions with TGFβ signaling. For example, EGFR activation is required for the pro-fibrotic functions of TGFB and CCN2-mediated fibroblast proliferation and myofibroblast transdifferentiation. DCN can bind to and downregulate EGFR and HGF receptor Met (to inhibit β-catenin expression). After DCN-tCRK administration, these genes were normalized in col7a1. - / -Normalized expression in mouse skin suggests multiple therapeutic functions of DCN-tCRK in RDEB. Conversely, upregulation of pro-inflammatory genes in DCN-treated RDEB skin may indicate therapeutic effects that cannot be maintained by the application of natural DCN.

[0142] In summary, this invention demonstrates that exposure of the hidden CendR sequence provides novel characteristics in wound-targeting peptides for homing to normal skin in addition to wounded skin, and also provides dermal tissue penetration. It also demonstrates that this peptide (tCRK) can be used as a carrier for delivering core proteoglycans and other therapeutic molecules in the treatment of systemic skin diseases, particularly epidermolysis bullosa. SEQUENCE LISTING <110> Tampere University Registered Foundation <120> Homing peptide-guided core proteoglycan conjugates for the treatment of epidermolysis bullosa <130> P79901PNA <160> 26 <170> PatentIn version 3.5 <210> 1 <211> 4 <212> PRT <213> Homo sapiens <400> 1 Arg Lys Asp Lys 1 <210> 2 <211> 5 <212> PRT <213> Homo sapiens <400> 2 Cys Arg Lys Asp Lys 1 5 <210> 3 <211> 6 <212> PRT <213> Homo sapiens <400> 3 Cys Arg Lys Asp Lys Cys 1 5 <210> 4 <211> 4 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa is either Arg or Lys <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa is any amino acid. <220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa is any amino acid. <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa is either Arg or Lys <400> 4 Xaa Xaa Xaa Xaa 1 <210> 5 <211> 9 <212> PRT <213> Homo sapiens <400> 5 Cys Ala Arg Ser Lys Asn Lys Asp Cys 1 5 <210> 6 <211> 358 <212> PRT <213> Homo sapiens <400> 6 Met Lys Ala Thr Ile Ile Leu Leu Leu Leu Ala Gln Val Ser Trp Ala 1 5 10 15 Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 20 25 30 Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro 35 40 45 Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val 50 55 60 Val Gln Cys Ser Asp Leu Gly Leu Asp Lys Val Pro Lys Asp Leu Pro 65 70 75 80 Pro Asp Thr Thr Leu Leu Asp Leu Gln Asn Asn Lys Ile Thr Glu Ile 85 90 95 Lys Asp Gly Asp Phe Lys Asn Leu Lys Asn Leu His Ala Leu Ile Leu 100 105 110 Val Asn Asn Lys Ile Ser Lys Val Ser Pro Gly Ala Phe Thr Pro Leu 115 120 125 Val Lys Leu Glu Arg Leu Tyr Leu Ser Lys Asn Gln Leu Lys Glu Leu 130 135 140 Pro Glu Lys Met Lys Thr Leu Gln Glu Leu Arg Ala His Glu Asn Glu 145 150 155 160 Ile Thr Lys Val Arg Lys Val Thr Phe Asn Gly Leu Asn Gln Met Ile 165 170 175 Val Ile Glu Leu Gly Thr Asn Pro Leu Lys Ser Ser Gly Ile Glu Asn 180 185 190 Gly Ala Phe Gln Gly Met Lys Lys Leu Ser Tyr Ile Arg Ile Ala Asp 195 200 205 Thr Asn Ile Thr Ser Ile Pro Gln Gly Leu Pro Pro Ser Leu Thr Glu 210 215 220 Leu His Leu Asp Gly Asn Lys Ile Ser Arg Val Asp Ala Ala Ser Leu 225 230 235 240 Lys Gly Leu Asn Asn Leu Ala Lys Leu Gly Leu Ser Phe Asn Ser Ile 245 250 255 Ser Ala Val Asp Asn Gly Ser Leu Ala Asn Thr Pro His Leu Arg Glu 260 265 270 Leu His Leu Asp Asn Asn Lys Leu Thr Arg Val Pro Gly Gly Leu Ala 275 280 285 Glu His Lys Tyr Ile Gln Val Val Tyr Leu His Asn Asn Asn Ile Ser 290 295 300 Val Val Gly Ser Ser Asp Phe Cys Pro Pro Gly His Asn Thr Lys Lys 305 310 315 320 Ala Ser Tyr Ser Gly Val Ser Leu Phe Ser Asn Pro Val Gln Tyr Trp 325 330 335 Glu Ile Gln Pro Ser Thr Phe Arg Cys Val Tyr Val Arg Ser Ala Ile 340 345 350 Gln Leu Gly Asn Tyr Lys 355 <210> 7 <211> 342 <212> PRT <213> Homo sapiens <400> 7 Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 1 5 10 15 Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro 20 25 30 Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val 35 40 45 Val Gln Cys Ser Asp Leu Gly Leu Asp Lys Val Pro Lys Asp Leu Pro 50 55 60 Pro Asp Thr Thr Leu Leu Asp Leu Gln Asn Asn Lys Ile Thr Glu Ile 65 70 75 80 Lys Asp Gly Asp Phe Lys Asn Leu Lys Asn Leu His Ala Leu Ile Leu 85 90 95 Val Asn Asn Lys Ile Ser Lys Val Ser Pro Gly Ala Phe Thr Pro Leu 100 105 110 Val Lys Leu Glu Arg Leu Tyr Leu Ser Lys Asn Gln Leu Lys Glu Leu 115 120 125 Pro Glu Lys Met Lys Thr Leu Gln Glu Leu Arg Ala His Glu Asn Glu 130 135 140 Ile Thr Lys Val Arg Lys Val Thr Phe Asn Gly Leu Asn Gln Met Ile 145 150 155 160 Val Ile Glu Leu Gly Thr Asn Pro Leu Lys Ser Ser Gly Ile Glu Asn 165 170 175 Gly Ala Phe Gln Gly Met Lys Lys Leu Ser Tyr Ile Arg Ile Ala Asp 180 185 190 Thr Asn Ile Thr Ser Ile Pro Gln Gly Leu Pro Pro Ser Leu Thr Glu 195 200 205 Leu His Leu Asp Gly Asn Lys Ile Ser Arg Val Asp Ala Ala Ser Leu 210 215 220 Lys Gly Leu Asn Asn Leu Ala Lys Leu Gly Leu Ser Phe Asn Ser Ile 225 230 235 240 Ser Ala Val Asp Asn Gly Ser Leu Ala Asn Thr Pro His Leu Arg Glu 245 250 255 Leu His Leu Asp Asn Asn Lys Leu Thr Arg Val Pro Gly Gly Leu Ala 260 265 270 Glu His Lys Tyr Ile Gln Val Val Tyr Leu His Asn Asn Asn Ile Ser 275 280 285 Val Val Gly Ser Ser Asp Phe Cys Pro Pro Gly His Asn Thr Lys Lys 290 295 300 Ala Ser Tyr Ser Gly Val Ser Leu Phe Ser Asn Pro Val Gln Tyr Trp 305 310 315 320 Glu Ile Gln Pro Ser Thr Phe Arg Cys Val Tyr Val Arg Ser Ala Ile 325 330 335 Gln Leu Gly Asn Tyr Lys 340 <210> 8 <211> 312 <212> PRT <213> Homo sapiens <400> 8 Glu Pro Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu 1 5 10 15 Arg Val Val Gln Cys Ser Asp Leu Gly Leu Asp Lys Val Pro Lys Asp 20 25 30 Leu Pro Pro Asp Thr Thr Leu Leu Asp Leu Gln Asn Asn Lys Ile Thr 35 40 45 Glu Ile Lys Asp Gly Asp Phe Lys Asn Leu Lys Asn Leu His Ala Leu 50 55 60 Ile Leu Val Asn Asn Lys Ile Ser Lys Val Ser Pro Gly Ala Phe Thr 65 70 75 80 Pro Leu Val Lys Leu Glu Arg Leu Tyr Leu Ser Lys Asn Gln Leu Lys 85 90 95 Glu Leu Pro Glu Lys Met Lys Thr Leu Gln Glu Leu Arg Ala His Glu 100 105 110 Asn Glu Ile Thr Lys Val Arg Lys Val Thr Phe Asn Gly Leu Asn Gln 115 120 125 Met Ile Val Ile Glu Leu Gly Thr Asn Pro Leu Lys Ser Ser Gly Ile 130 135 140 Glu Asn Gly Ala Phe Gln Gly Met Lys Lys Leu Ser Tyr Ile Arg Ile 145 150 155 160 Ala Asp Thr Asn Ile Thr Ser Ile Pro Gln Gly Leu Pro Pro Ser Leu 165 170 175 Thr Glu Leu His Leu Asp Gly Asn Lys Ile Ser Arg Val Asp Ala Ala 180 185 190 Ser Leu Lys Gly Leu Asn Asn Leu Ala Lys Leu Gly Leu Ser Phe Asn 195 200 205 Ser Ile Ser Ala Val Asp Asn Gly Ser Leu Ala Asn Thr Pro His Leu 210 215 220 Arg Glu Leu His Leu Asp Asn Asn Lys Leu Thr Arg Val Pro Gly Gly 225 230 235 240 Leu Ala Glu His Lys Tyr Ile Gln Val Val Tyr Leu His Asn Asn Asn 245 250 255 Ile Ser Val Val Gly Ser Ser Asp Phe Cys Pro Pro Gly His Asn Thr 260 265 270 Lys Lys Ala Ser Tyr Ser Gly Val Ser Leu Phe Ser Asn Pro Val Gln 275 280 285 Tyr Trp Glu Ile Gln Pro Ser Thr Phe Arg Cys Val Tyr Val Arg Ser 290 295 300 Ala Ile Gln Leu Gly Asn Tyr Lys 305 310 <210> 9 <211> 250 <212> PRT <213> Homo sapiens <400> 9 Met Lys Ala Thr Ile Ile Leu Leu Leu Leu Ala Gln Val Ser Trp Ala 1 5 10 15 Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 20 25 30 Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro<Q 35 40 45 Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val 50 55 60 Val Gln Cys Ser Asp Leu Glu Leu Gly Thr Asn Pro Leu Lys Ser Ser 65 70 75 80 Gly Ile Glu Asn Gly Ala Phe Gln Gly Met Lys Lys Leu Ser Tyr Ile 85 90 95 Arg Ile Ala Asp Thr Asn Ile Thr Ser Ile Pro Gln Gly Leu Pro Pro 100 105 110 Ser Leu Thr Glu Leu His Leu Asp Gly Asn Lys Ile Ser Arg Val Asp 115 120 125 Ala Ala Ser Leu Lys Gly Leu Asn Asn Leu Ala Lys Leu Gly Leu Ser 130 135 140 Phe Asn Ser Ile Ser Ala Val Asp Asn Gly Ser Leu Ala Asn Thr Pro 145 150 155 160 His Leu Arg Glu Leu His Leu Asp Asn Asn Lys Leu Thr Arg Val Pro 165 170 175 Gly Gly Leu Ala Glu His Lys Tyr Ile Gln Val Val Tyr Leu His Asn 180 185 190 Asn Asn Ile Ser Val Val Gly Ser Ser Asp Phe Cys Pro Pro Gly His 195 200 205 Asn Thr Lys Lys Ala Ser Tyr Ser Gly Val Ser Leu Phe Ser Asn Pro 210 215 220 Val Gln Tyr Trp Glu Ile Gln Pro Ser Thr Phe Arg Cys Val Tyr Val 225 230 235 240 Arg Ser Ala Ile Gln Leu Gly Asn Tyr Lys 245 250 <210> 10 <211> 234 <212> PRT <213> Homo sapiens <400> 10 Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 1 5 10 15 Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro 20 25 30 Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val 35 40 45 Val Gln Cys Ser Asp Leu Glu Leu Gly Thr Asn Pro Leu Lys Ser Ser 50 55 60 Gly Ile Glu Asn Gly Ala Phe Gln Gly Met Lys Lys Leu Ser Tyr Ile 65 70 75 80 Arg Ile Ala Asp Thr Asn Ile Thr Ser Ile Pro Gln Gly Leu Pro Pro 85 90 95 Ser Leu Thr Glu Leu His Leu Asp Gly Asn Lys Ile Ser Arg Val Asp 100 105 110 Ala Ala Ser Leu Lys Gly Leu Asn Asn Leu Ala Lys Leu Gly Leu Ser 115 120 125 Phe Asn Ser Ile Ser Ala Val Asp Asn Gly Ser Leu Ala Asn Thr Pro 130 135 140 His Leu Arg Glu Leu His Leu Asp Asn Asn Lys Leu Thr Arg Val Pro 145 150 155 160 Gly Gly Leu Ala Glu His Lys Tyr Ile Gln Val Val Tyr Leu His Asn 165 170 175 Asn Asn Ile Ser Val Val Gly Ser Ser Asp Phe Cys Pro Pro Gly His 180 185 190 Asn Thr Lys Lys Ala Ser Tyr Ser Gly Val Ser Leu Phe Ser Asn Pro 195 200 205 Val Gln Tyr Trp Glu Ile Gln Pro Ser Thr Phe Arg Cys Val Tyr Val 210 215 220 Arg Ser Ala Ile Gln Leu Gly Asn Tyr Lys 225 230 <210> 11 <211> 204 <212> PRT <213> Homo sapiens <400> 11 Glu Pro Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu 1 5 10 15 Arg Val Val Gln Cys Ser Asp Leu Glu Leu Gly Thr Asn Pro Leu Lys 20 25 30 Ser Ser Gly Ile Glu Asn Gly Ala Phe Gln Gly Met Lys Lys Leu Ser 35 40 45 Tyr Ile Arg Ile Ala Asp Thr Asn Ile Thr Ser Ile Pro Gln Gly Leu 50 55 60 Pro Pro Ser Leu Thr Glu Leu His Leu Asp Gly Asn Lys Ile Ser Arg 65 70 75 80 Val Asp Ala Ala Ser Leu Lys Gly Leu Asn Asn Leu Ala Lys Leu Gly 85 90 95 Leu Ser Phe Asn Ser Ile Ser Ala Val Asp Asn Gly Ser Leu Ala Asn 100 105 110 Thr Pro His Leu Arg Glu Leu His Leu Asp Asn Asn Lys Leu Thr Arg 115 120 125 Val Pro Gly Gly Leu Ala Glu His Lys Tyr Ile Gln Val Val Tyr Leu 130 135 140 His Asn Asn Asn Ile Ser Val Val Gly Ser Ser Asp Phe Cys Pro Pro 145 150 155 160 Gly His Asn Thr Lys Lys Ala Ser Tyr Ser Gly Val Ser Leu Phe Ser 165 170 175 Asn Pro Val Gln Tyr Trp Glu Ile Gln Pro Ser Thr Phe Arg Cys Val 180 185 190 Tyr Val Arg Ser Ala Ile Gln Leu Gly Asn Tyr Lys 195 200 <210> 12 <211> 212 <212> PRT <213> Homo sapiens <400> 12 Met Lys Ala Thr Ile Ile Leu Leu Leu Leu Ala Gln Val Ser Trp Ala 1 5 10 15 Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 20 25 30 Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro 35 40 45 Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val 50 55 60 Val Gln Cys Ser Asp Leu Gly Leu Pro Pro Ser Leu Thr Glu Leu His 65 70 75 80 Leu Asp Gly Asn Lys Ile Ser Arg Val Asp Ala Ala Ser Leu Lys Gly 85 90 95 Leu Asn Asn Leu Ala Lys Leu Gly Leu Ser Phe Asn Ser Ile Ser Ala 1 Val Asp Asn Gly Ser Leu Ala Asn Thr Pro His Leu Arg Glu Leu His 115 120 125 Leu Asp Asn Asn Lys Leu Thr Arg Val Pro Gly Gly Leu Ala Glu His 130 135 140 Lys Tyr Ile Gln Val Val Tyr Leu His Asn Asn Asn Ile Ser Val Val 145 150 155 160 Gly Ser Ser Asp Phe Cys Pro Pro Gly His Asn Thr Lys Lys Ala Ser 165 170 175 Tyr Ser Gly Val Ser Leu Phe Ser Asn Pro Val Gln Tyr Trp Glu Ile 180 185 190 Gln Pro Ser Thr Phe Arg Cys Val Tyr Val Arg Ser Ala Ile Gln Leu 195 200 205 Gly Asn Tyr Lys 210 <210> 13 <211> 196 <212> PRT <213> Homo sapiens <400> 13 Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 1 5 10 15 Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro 020 25 30 Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val 35 40 45 Val Gln Cys Ser Asp Leu Gly Leu Pro Pro Ser Leu Thr Glu Leu His 50 55 60 Leu Asp Gly Asn Lys Ile Ser Arg Val Asp Ala Ala Ser Leu Lys Gly 65 70 75 80 Leu Asn Asn Leu Ala Lys Leu Gly Leu Ser Phe Asn Ser Ile Ser Ala 85 90 95 Val Asp Asn Gly Ser Leu Ala Asn Thr Pro His Leu Arg Glu Leu His 100 105 110 Leu Asp Asn Asn Lys Leu Thr Arg Val Pro Gly Gly Leu Ala Glu His 115 120 125 Lys Tyr Ile Gln Val Val Tyr Leu His Asn Asn Asn Ile Ser Val Val 130 135 140 Gly Ser Ser Asp Phe Cys Pro Pro Gly His Asn Thr Lys Lys Ala Ser 145 150 155 160 Tyr Ser Gly Val Ser Leu Phe Ser Asn Pro Val Gln Tyr Trp Glu Ile 165 170 175 Gln Pro Ser Thr Phe Arg Cys Val Tyr Val Arg Ser Ala Ile Gln Leu 180 185 190 Gly Asn Tyr Lys 195 <210> 14 <211> 166 <212> PRT <213> Homo sapiens <400> 14 Glu Pro Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu 1 5 10 15 Arg Val Val Gln Cys Ser Asp Leu Gly Leu Pro Pro Ser Leu Thr Glu 20 25 30[[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​Glu Ile Gln Pro Ser Thr Phe Arg Cys Val Tyr Val Arg Ser Ala Ile 145 150 155 160 Gln Leu Gly Asn Tyr Lys 165 <210> 15 <211> 172 <212> PRT <213> Homo sapiens <400> 15 Met Lys Ala Thr Ile Ile Leu Leu Leu Leu Ala Gln Val Ser Trp Ala 1 5 10 15 Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 20 25 30 Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro 35 40 45 Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val 50 55 60 Val Gln Cys Ser Asp Leu Gly Leu Asp Lys Val Pro Lys Asp Leu Pro 65 70 75 80 Pro Asp Thr Thr Leu Leu Asp Leu Gln Asn Asn Lys Ile Thr Glu Ile 85 90 95 Lys Asp Gly Asp Phe Lys Asn Leu Lys Asn Leu His Val Val Tyr Leu 100 105 110 His Asn Asn Asn Ile Ser Val Val Gly Ser Ser Asp Phe Cys Pro Pro 115 120 125 Gly His Asn Thr Lys Lys Ala Ser Tyr Ser Gly Val Ser Leu Phe Ser 130 135 140 Asn Pro Val Gln Tyr Trp Glu Ile Gln Pro Ser Thr Phe Arg Cys Val 145 150 155 160 Tyr Val Arg Ser Ala Ile Gln Leu Gly Asn Tyr Lys 165 170 <210> 16 <211> 156 <212> PRT <213> Homo sapiens <400> 16 Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 1 5 10 15 Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro 20 25 30 Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val 35 40 45 Val Gln Cys Ser Asp Leu Gly Leu Asp Lys Val Pro Lys Asp Leu Pro 50 55 60 Pro Asp Thr Thr Leu Leu Asp Leu Gln Asn Asn Lys Ile Thr Glu Ile 65 70 75 80 Lys Asp Gly Asp Phe Lys Asn Leu Lys Asn Leu His Val Val Tyr Leu 85 90 95 His Asn Asn Asn Ile Ser Val Val Gly Ser Ser Asp Phe Cys Pro Pro 100 105 110 Gly His Asn Thr Lys Lys Ala Ser Tyr Ser Gly Val Ser Leu Phe Ser 115 120 125 Asn Pro Val Gln Tyr Trp Glu Ile Gln Pro Ser Thr Phe Arg Cys Val 130 135 140 Tyr Val Arg Ser Ala Ile Gln Leu Gly Asn Tyr Lys 145 150 155 <210> 17 <211> 126 <212> PRT <213> Homo sapiens <400> 17 Glu Pro Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu 1 5 10 15 Arg Val Val Gln Cys Ser Asp Leu Gly Leu Asp Lys Val Pro Lys Asp 20 25 30 Leu Pro Pro Asp Thr Thr Leu Leu Asp Leu Gln Asn Asn Lys Ile Thr 35 40 45 Glu Ile Lys Asp Gly Asp Phe Lys Asn Leu Lys Asn Leu His Val Val 50 55 60 Tyr Leu His Asn Asn Asn Ile Ser Val Val Gly Ser Ser Asp Phe Cys 65 70 75 80 Pro Pro Gly His Asn Thr Lys Lys Ala Ser Tyr Ser Gly Val Ser Leu 85 90 95 Phe Ser Asn Pro Val Gln Tyr Trp Glu Ile Gln Pro Ser Thr Phe Arg 100 105 110 Cys Val Tyr Val Arg Ser Ala Ile Gln Leu Gly Asn Tyr Lys 115 120 125 <210> 18 <211> 75 <212> PRT <213> Homo sapiens <400> 18[[ID=2(5]] Met Lys Ala Thr Ile Ile Leu Leu Leu Leu Ala Gln Val Ser Trp Ala 1 5 10 15 Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 20 25 30 Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro 35 40 45 Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val 50 55 60 Val Gln Cys Ser Asp Leu Gly Cys Leu Pro Ser 65 70 75 <210> 19 <211> 59 <212> PRT <213> Homo sapiens <400> 19 Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 1 5 10 15 Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro 20 25 30 Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val 35 40 45 Val Gln Cys Ser Asp Leu Gly Cys Leu Pro Ser 50 55 <210> 20 <211> 29 <212> PRT <213> Homo sapiens <400> 20 Glu Pro Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu 1 5 10 15 Arg Val Val Gln Cys Ser Asp Leu Gly Cys Leu Pro Ser 20 25 <210> twenty one <211> 345 <212> PRT <213> Artificial sequence <220> <223> Decorative protein-tCRK fusion protein <400> twenty one Gly His His His His His Asp Glu Ala Ser Gly Ile Gly Pro Glu 1 5 10 15 Val Pro Asp Asp Arg Asp Phe Glu Pro Ser Leu Gly Pro Val Cys Pro 20 25 30 Phe Arg Cys Gln Cys His Leu Arg Val Val Gln Cys Ser Asp Leu Gly 35 40 45 Leu Asp Lys Val Pro Lys Asp Leu Pro Pro Asp Thr Thr Leu Leu Asp 50 55 60 Leu Gln Asn Asn Lys Ile Thr Glu Ile Lys Asp Gly Asp Phe Lys Asn 65 70 75 80 Leu Lys Asn Leu His Ala Leu Ile Leu Val Asn Asn Lys Ile Ser Lys 85 90 95 Val Ser Pro Gly Ala Phe Thr Pro Leu Val Lys Leu Glu Arg Leu Tyr 100 105 110 Leu Ser Lys Asn Gln Leu Lys Glu Leu Pro Glu Lys Met Pro Lys Thr 115 120 125 Leu Gln Glu Leu Arg Ala His Glu Asn Glu Ile Thr Lys Val Arg Lys 130 135 140 Val Thr Phe Asn Gly Leu Asn Gln Met Ile Val Ile Glu Leu Gly Thr 145 150 155 160 Asn Pro Leu Lys Ser Ser Gly Ile Glu Asn Gly Ala Phe Gln Gly Met 165 170 175 Lys Lys Leu Ser Tyr Ile Arg Ile Ala Asp Thr Asn Ile Thr Ser Ile 180 185 190 Pro Gln Gly Leu Pro Pro Ser Leu Thr Glu Leu His Leu Asp Gly Asn 195 200 205 Lys Ile Ser Arg Val Asp Ala Ala Ser Leu Lys Gly Leu Asn Asn Leu 210 215 220 Ala Lys Leu Gly Leu Ser Phe Asn Ser Ile Ser Ala Val Asp Asn Gly 225 230 235 240 Ser Leu Ala Asn Thr Pro His Leu Arg Glu Leu His Leu Asp Asn Asn 245 250 255 Lys Leu Thr Arg Val Pro Gly Gly Leu Ala Glu His Lys Tyr Ile Gln 260 265 270 Val Val Tyr Leu His Asn Asn Asn Ile Ser Val Val Gly Ser Ser Asp 275 280 285 Phe Cys Pro Pro Gly His Asn Thr Lys Lys Ala Ser Tyr Ser Gly Val 290 295 300 Ser Leu Phe Ser Asn Pro Val Gln Tyr Trp Glu Ile Gln Pro Ser Thr 305 310 315 320 Phe Arg Cys Val Tyr Val Arg Ser Ala Ile Gln Leu Gly Asn Tyr Lys 325 330 335 Gly Ser Glu Phe Cys Arg Lys Asp Lys 340 345 <210> 22 <211> 368 <212> PRT <213> artificial sequence <220> <223> decoration protein‑tCRK fusion protein <400> 22 Met Lys Ala Thr Ile Ile Leu Leu Leu Leu Ala Gln Val Ser Trp Ala 1 5 10 15 Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 20 25 30 Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro 35 40 45 Serum Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val 50 55 60 Val Gln Cys Ser Asp Leu Gly Leu Asp Lys Val Pro Lys Asp Leu Pro 65 70 75 80 Pro Asp Thr Thr Leu Leu Asp Leu Gln Asn Asn Lys Ile Thr Glu Ile 85 90 95 Lys Asp Gly Asp Phe Lys Asn Leu Lys Asn Leu His Ala Leu Ile Leu 100 105 110 Val Asn Asn Lys Ile Ser Lys Val Ser Pro Gly Ala Phe Thr Pro Leu 115 120 125 Val Lys Leu Glu Arg Leu Tyr Leu Ser Lys Asn Gln Leu Lys Glu Leu 130 135 140 Pro Glu Lys Met Pro Lys Thr Leu Gln Glu Leu Arg Ala His Glu Asn 145 150 155 160 Glu Ile Thr Lys Val Arg Lys Val Thr Phe Asn Gly Leu Asn Gln Met 165 170 175 Ile Val Ile Glu Leu Gly Thr Asn Pro Leu Lys Ser Ser Gly Ile Glu 180 185 190 Asn Gly Ala Phe Gln Gly Met Lys Lys Leu Ser Tyr Ile Arg Ile Ala 195 200 205 Asp Thr Asn Ile Thr Ser Ile Pro Gln Gly Leu Pro Pro Ser Leu Thr 210 215 220 Glu Leu His Leu Asp Gly Asn Lys Ile Ser Arg Val Asp Ala Ala Ser 225 230 235 240 Leu Lys Gly Leu Asn Asn Leu Ala Lys Leu Gly Leu Ser Phe Asn Ser 245 250 255 Ile Ser Ala Val Asp Asn Gly Ser Leu Ala Asn Thr Pro His Leu Arg 260 265 270 Glu Leu His Leu Asp Asn Asn Lys Leu Thr Arg Val Pro Gly Gly Leu 275 280 285 Ala Glu His Lys Tyr Ile Gln Val Val Tyr Leu His Asn Asn Asn Ile 290 295 300 Ser Val Val Gly Ser Ser Asp Phe Cys Pro Pro Gly His Asn Thr Lys 305 310 315 320 Lys Ala Ser Tyr Ser Gly Val Ser Leu Phe Ser Asn Pro Val Gln Tyr 325 330 335 Trp Glu Ile Gln Pro Ser Thr Phe Arg Cys Val Tyr Val Arg Ser Ala 340 345 350 Ile Gln Leu Gly Asn Tyr Lys Gly Ser Glu Phe Cys Arg Lys Asp Lys 355 360 365 <210> twenty three <211> 4 <212> PRT <213> Artificial sequence <220> <223> peptide linkers <400> twenty three Gly Ser Glu Phe 1 <210> twenty four <211> 5 <212> PRT <213> Artificial sequence <220> <223> peptide linkers <400> twenty four Gly Ser Glu Phe Cys 1 5 <210> 25 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 25 Arg Pro Ala Arg Pro Ala Arg 1 5 <210> 26 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 26 Arg Pro Ala Arg Pro Ala Arg Ala 1 5

Claims

1. A homing peptide-guided core proteoglycan conjugate for the treatment of malnutrition-related epidermolysis bullosa, wherein, The conjugate consists of the amino acid sequence of SEQ ID NO:21, having a core proteoglycan fragment linked to the N-terminus of the homing peptide, wherein the C-terminus of the homing peptide consists of the amino acid sequence CRKDK (SEQ ID NO:2).

2. The conjugate according to claim 1, wherein, The conjugate selectively homs to the skin and skin wounds.

3. The conjugate according to claim 1 or 2, wherein, The conjugate is provided in the form of a pharmaceutical composition comprising the conjugate and a pharmaceutically acceptable carrier.

4. The conjugate according to claim 1, wherein, Nutritional bullous epidermolysis can be classified as overt nutritional bullous epidermolysis, latent nutritional bullous epidermolysis, or inverse latent nutritional bullous epidermolysis.

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