Homing peptide-derived decorin complexes for use in the treatment of epidermolysis bullosa

Abstract

The present invention relates to a homing peptide-derived decorin conjugate for use in the treatment of epidermolysis bullosa, and a corresponding method of treatment. The use of a novel homing peptide enables the conjugate to target-specifically home to skin and skin wounds in vivo upon systemic administration.

Description

[Technical Field]

[0001] The present invention relates generally to the field of molecular medicine. More specifically, the present invention relates to homing peptide-derived decorin conjugates for use in the treatment of epidermolysis bullosa and corresponding methods of treatment. [Background technology]

[0002] As the largest organ of the human body, skin poses unique challenges for efficient drug delivery. A major challenge associated with topical, or transdermal, drug delivery is the poor penetration of macromolecules through the skin. Diffusion through intercellular lipids offers an option for transdermal delivery, but is limited to small, lipophilic molecules only. Therefore, systemically administered, yet skin-specific, therapeutic agents would represent a substantial therapeutic advance for the treatment of skin disorders, particularly those affecting the entire skin, such as epidermolysis bullosa, a group of rare genetic disorders that cause fragile, blistering skin.

[0003] Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in the COL7A1 gene, which encodes type 7 collagen (C7). Clinical symptoms include skin erosions and blistering, amputation scars, pseudosyndactyly, and a high risk of developing rapidly metastatic, aggressive cutaneous squamous cell carcinoma (cSCC). While some gene-, cell-, and protein-based therapies have shown promising results in delivering type 7 collagen to the skin, challenges remain, and there is currently no cure for RDEB.

[0004] Transforming growth factor β (TGFβ) signaling has been shown to play an essential role in the development of fibrosis and progression of RDEB to malignancy. Previously, we demonstrated that TGFβ signaling mediates the expression of col7a1. - / -It has been shown to be activated as early as one week after birth in mice (Liao et al., 2018, Stem Cells 36:1839-1850). Therefore, early intervention in activating TGFβ signaling may be beneficial in reducing the disease burden of RDEB. It has also been suggested that TGFβ signaling is a phenotypic modulator in monozygotic twins with the same COL7A1 mutation (Odorisio et al., 2014, Hum Mol Genet 23:3907-3922). Furthermore, the expression level of the proteoglycan decorin (DCN), a natural TGFβ inhibitor, was significantly higher in the unaffected twin. DCN is a structural component of the extracellular matrix (ECM) and is involved in the regulation of Dcn. - / - Mice with DCN exhibit irregular collagen fibrillation and significantly reduced skin tensile strength (Reed and Iozzo, 2002, Glycoconj J 19:249-255). Furthermore, DCN has anti-fibrotic and anti-tumor functions by regulating the activity of multiple growth factors, particularly TGFβ (Jarvinen and Prince, 2015, Biomed Res Int 2015:654-765; Jarvinen and Ruoslahti, 2019, Br J Pharmacol 176:16-25). Recently, upregulation of DCN expression has been shown to be associated with the expression of col7a1. - / - DCN has been shown to be one of the mechanisms of action for the efficacy of unrestricted somatic stem cells (USSCs) derived from mouse umbilical cord blood (Liao et al., 2018, supra). Supporting the role of DCN as a potential therapeutic disease-modifying molecule in RDEB, Cianfarani et al. (2019, Matrix Biol 81:3-16) recently reported that systemic administration of a lentivirus driving expression of human DCN attenuated TGFβ-induced fibrosis in a C7 hypomorphic RDEB mouse model (C7 hypomorphic mice) that expresses residual levels of type 7 collagen.

[0005] Furthermore, DCN binds to and neutralizes connective tissue growth factor (CTGF / CCN2), which has been proposed to be a downstream mediator of TGFβ fibrotic signaling and a therapeutic target for scar prevention (Vial et al., 2011, J Biol Chem 286:24242-24252; Daniels et al., 2003, Am J Pathol 163:2043-2052). Because the binding sites for TGFβ and CTGF / CCN2 are located in different parts of DCN, theoretically, DCN can simultaneously block both mediators of fibrosis. Indeed, the role of DCN in suppressing TGFβ-promoted scar formation is well documented in many disease models, including kidney, lung, and liver fibrosis, as well as skin wound healing, in addition to RDEB (Odorisio et al., 2014, supra; Liao et al., 2018, supra; Cianfarani et al., 2019, supra). However, despite numerous positive anti-cancer and anti-fibrotic results in preclinical studies, DCN has not yet reached clinical use as a systemic treatment. To date, only 12 patients with perforating ocular injuries have been reported to receive DCN in clinical trials. A single intravitreal injection of 200 μg or 400 μg of human recombinant DCN appeared to be well tolerated without any ocular adverse events (Abdullatif et al., 2018, Graefes Arch Clin Exp Ophthalmol 256:2473-2481).

[0006] A common limitation of systemic drug delivery is that only a small fraction of the drug reaches the desired site, and systemic side effects occur in other organs. Therefore, an important goal of modern drug development is to generate drugs that are specific to the target organ with minimal adverse effects on other parts of the body. This goal could be achieved by developing drugs that recognize specific epitopes expressed in the affected organ. Alternatively, drugs can be made target-specific by conjugating them with affinity ligands, such as vascular homing peptides, that recognize tissue- or target-specific molecular features within the vasculature of a particular organ.

[0007] In vivo screening of phage-peptide libraries has confirmed that these tissue- or disease-specific molecular signatures (vascular zip codes) within the vasculature can be targeted by systemically administered affinity ligands, such as vascular homing peptides. These studies essentially establish that the existence of organ- or disease-specific molecular signatures within the vasculature of different tissues allows for a zip code system (vascular zip code) for the target-specific delivery of systemically administered therapeutics (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 efficient vascular homing peptides for tumor-specific homing and cell / tissue penetration contain the consensus motif R / KXXR / K (SEQ ID NO: 3), which is called the C-end Rule (CendR) sequence because it has an arginine (or rarely a lysine) residue at the C-terminus (Ruoslahti, 2017, J Clin Invest 127:1622-1624; Teesalu 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). The CendR sequence binds to neuropilin-1 (NRP-1) and activates extravasation and tissue penetration pathways that deliver the peptide along with its payload into the tumor tissue parenchyma (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).Potential CendR-containing peptides possess target selectivity due to a combination of binding to a primary receptor with a tumor-specific expression pattern and exposure of the CendR sequence in target organs through proteolytic activation within tumors. Because NRP-1 is expressed by endothelial cells in all tissues (Ruoslahti, 2017, Adv Drug Deliv Rev 110-111:3-12), NRP-1-mediated extravasation and tissue penetration are unlikely to be limited to cancer tissues but may occur in other diseased or healthy tissues.

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

[0009] WO 2008 / 136869 discloses a CRK peptide as a specific homing element for targeted delivery of decorin to skin wounds. The disclosed CRK-decorin fusion does not home to unwounded skin.

[0010] Therefore, a therapeutic agent that is systemically administered but specific to the skin would be a substantial therapeutic advance for the treatment of skin diseases such as epidermolysis bullosa. [Primary Technology Documents] [Chartered documents]

[0011]

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[0012]

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[0013] The present specification provides a homing peptide-derived decorin complex for use in treating epidermolysis bullosa, which complex comprises a decorin fragment and a homing peptide, the C-terminus of which consists of the amino acid sequence RKDK (SEQ ID NO: 1) or CRKDK (SEQ ID NO: 2).

[0014] Also provided is a method for treating epidermolysis bullosa in a subject in need thereof by administering an effective amount of a homing peptide-derived decorin complex comprising a decorin 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).

[0015] The homing peptide enables the complex to selectively home to and penetrate skin and skin wounds in vivo.

[0016] Embodiments and details of the above aspects are shown in the following figures, detailed description, examples and dependent claims. [Brief explanation of the drawings]

[0017] The accompanying drawings illustrate several embodiments of the disclosed subject matter and, together with the description, serve to explain the principles of the disclosed compositions and methods.

[0018] [Figure 1A] Figures 1A-1C show the structure of an exemplary recombinant DCN-tCRK protein and its binding to neuropilin-1. Figure 1A is a schematic diagram of the DCN-tCRK structure. The signal peptide and propeptide of native DCN were replaced with a 6xHis tag (I) for purification. The His tag is followed by the amino terminus (II), core protein (III), and carboxyl terminus (IV) of the mature DCN proteoglycan. The tCRK peptide (V) was cloned into the carboxyl terminus of the protein. [Figure 1B]Figures 1A-1C show the structure of an exemplary recombinant DCN-tCRK protein and its binding to neuropilin-1. Figure 1B shows the in vitro binding of DCN-tCRK to neuropilin-1 (NRP-1). 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 on an ELISA plate. Bovine serum albumin (BSA) was included as a nonspecific protein control for DCN-tCRK and peptides. WT and mutant NRP1 were labeled with FAM and added to the immobilized plate. NRP1 binding 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] Figures 1A-1C show the structure of an exemplary recombinant DCN-tCRK protein and its binding to neuropilin-1. Figure 1C shows the internalization of DCN-tCRK in NRP-1-positive cells. FAM-labeled DCN-tCRK was incubated with PC3 and M21 cells, which are positive and negative for NRP-1 expression, respectively. DCN-tCRK was detected by anti-FAM immunostaining. Nuclei were counterstained with DAPI. Representative images are from three independently tested experiments. The scale bar is 20 µm. [Figure 2A] Figures 2A-2D show the production and characterization of an exemplary DCN-tCRK recombinant protein. Figure 2A shows an example of a purification chromatogram after a HisTrap HP column step on an Aekta Start, showing one major peak, the entire peak fraction of which was used for further processing. [Figure 2B]Figures 2A-2D show the production and characterization of an exemplary recombinant DCN-tCRK protein. Figure 2B shows a Coomassie-stained reduced SDS-Page gel (top panel) and Western blot (bottom panel) of purified DCN-tCRK alongside prior art DCN. 2 μg and 1 μg of protein were loaded on the SDS gel, and 1 μg and 0.5 μg of protein were used for Western blot analysis. Both the monomeric form of the protein and a form containing GAG side chains are observed. [Figure 2C] Figures 2A-2D show the production and characterization of an exemplary recombinant protein of DCN-tCRK. Figure 2C shows dynamic light scattering (DLS) measurements (n=3) of the hydrodynamic diameter of DCN-tCRK. [Figure 2D] Figures 2A-2D show the production and characterization of an exemplary recombinant protein of DCN-tCRK. Figure 2D shows the differential scanning calorimetry (DSC) curve for the melting temperature of DCN-tCRK. [Figure 3] Figure 3 shows the pharmacokinetics of DCN-tCRK compared to DCN. Healthy Balb / c mice were intravenously injected with 5 mg / kg of DCN-tCRK or DCN. Blood samples were collected at eight time points and analyzed by standard ELISA for human DCN. n=4 per group. [Figure 4A] Figures 4A-4D show that DCN-tCRK improves survival and homed to the skin in col7a1- / - mice. Figure 4A shows Kaplan-Meier survival analysis of col7a1- / - mice treated with DCN-tCRK (median survival: 11 days, n = 21), col7a1- / - mice treated with DCN (median survival: 7 days, n = 17), and col7a1- / - mice treated with PBS (median survival: 2 days, n = 24). [Figure 4B]Figures 4A-4D show that DCN-tCRK improves survival and homed to the skin of col7a1- / - mice. Figure 4B shows quantification of DCN and DCN-tCRK levels in the skin of treated col7a1- / - mice 1, 2, and 3 weeks after intrahepatic administration, as determined using a Human Decorin ELISA kit (n=3 per time point). 3 weeks after DCN administration, no mice were surviving, so DCN levels were not quantified at this time point. *p<0.05, **p<0.01. [Figure 4C] Figures 4A-4D show that DCN-tCRK improves survival in col7a1- / - mice and homed to the skin. Figure 4C shows immunohistochemical staining using an anti-histidine antibody (anti-His) on both the paw and dorsal skin of col7a1- / - mice. Nuclei were counterstained with DAPI. Scale bar = 20 µm. [Figure 4D] Figures 4A-4D show that DCN-tCRK improves survival and homed to the skin in col7a1 mice. Figure 4D shows representative double staining with anti-histidine tag and anti-NRP-1, as well as merged images (with DAPI counterstaining) of DCN-tCRK-treated, DCN-treated, and untreated RDEB skin. Scale bar = 25 μm. [Figure 5] Figure 5 shows Kaplan-Meier survival analysis of col7a1- / - mice, comparing historical survival rates after dextran / human serum albumin (D / HSA, median survival: 3 days, n=29; historical data, Liao et al., 2018, Stem Cell Transl Med, 7:530-542) with survival rates after DCN-tCRK (median survival: 11 days, n=21), DCN (median survival: 7 days, n=17), and PBS (median survival: 2 days, n=24). [Figure 6A]Figure 6 shows that DCN-tCRK normalizes the fibrotic gene signature of RDEB. Figure 6A shows the relative gene expression in a gene clustergram that showed a >1.5-fold increase in expression in untreated RDEB skin compared to WT. [Figure 6B] Figure 6 shows that DCN-tCRK normalizes the fibrotic gene signature of RDEB. Figure 6B shows volcano plots of the log2 fold change and -log10 p-values ​​of gene expression in the skin of col7a1- / - mice treated with vehicle, DCN, and DCN-tCRK, respectively, relative to WT. [Figure 7A] Figure 7 shows that administration of DCN-tCRK suppressed the development of fibrosis in col7a1 − / − mice. Figure 7A shows representative immunohistochemical staining of CTGF / CCN2 in 1- and 2-week-old WT and col7a1 − / − mice treated with and without DCN-tCRK. Scale bars are 50 μm in the upper panel and 25 μm in the lower panel. [Figure 7B] Figure 7 shows that administration of DCN-tCRK suppressed the development of fibrosis in col7a1 − / − mice. Figure 7B shows picrosirius red staining of paw skin from 1- and 2-week-old WT and col7a1 − / − mice treated with and without DCN-tCRK. Picrosirius red images were acquired using polarized light. The scale bar is 25 μm. [Figure 7C] Figure 7 shows that administration of DCN-tCRK suppressed the development of fibrosis in col7a1 mice. Figure 7C shows quantification of the mean picrosirius red intensity per field acquired with a 20x objective. Quantification of eight or more fields was acquired for each section, and at least four sections were analyzed per biopsy. Scale bar = 25 μm. *p<0.05, **p<0.01. [Figure 7D]Figure 7 shows that administration of DCN-tCRK suppressed the development of fibrosis in col7a1 − / − mice. Figure 7D shows representative photographs of type 1 collagen (COL1) expression (COL1 in the first column), double immunofluorescence staining of α-smooth muscle actin (αSMA in the second column), and blood vessels (CD31 in the third column) in 2-week-old RDEB and WT skin of 2-week-old WT and col7a1 − / − mice treated with and without DCN-tCRK. Nuclei were counterstained with DAPI. Merged images are shown in the fourth column. Scale bars are 25 μm. [Figure 7E] Figure 7 shows that administration of DCN-tCRK suppressed the development of fibrosis in col7a1 mice. Figure 7E shows quantification of the mean immunostaining intensity of COL1 and αSMA expression in skin sections (n ​​= 3 for each treatment group). *, **, and *** represent p < 0.05, 0.01, and 0.001, respectively. [Figure 8] Figure 8 shows the results of an in vitro collagen lattice contraction assay. The upper panel shows representative images of human normal fibroblasts and fibroblasts from an RDEB patient 48 hours after seeding onto collagen gels with or without DCN and DCN-tCRK at a final concentration of 75 nM, respectively. The lower panel shows the contraction rate of the collagen gels, calculated as the percentage contraction compared to the initial area. Data (n = 3) are presented as mean ± SEM. *p < 0.05, **p < 0.001. DETAILED DESCRIPTION OF THE INVENTION

[0019] It is to be understood that this invention is not limited to the particular methodology, protocols, reagents, and formulations described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0020] As used herein, the singular terms "a," "an," and "the" refer to one or more. Thus, singular nouns also refer to the corresponding plural nouns, unless otherwise specified.

[0021] The present invention relates to therapeutic uses of homing peptide-derived decorin conjugates. More specifically, the present invention provides homing peptide-derived decorin conjugates for use in treating epidermolysis bullosa and methods of treating epidermolysis bullosa in a subject in need thereof by administering to the subject an effective amount of a homing peptide-derived decorin conjugate.

[0022] Epidermolysis bullosa is a group of rare diseases that cause fragile, blistering skin. Blisters may appear in response to minor injuries, such as heat, friction, scratching, or adhesive tape. In severe cases, blisters may occur internally, such as on the mucous membranes of the mouth or stomach. Epidermolysis bullosa exists in various forms, including acquired and congenital forms, which may be recessive or dominant. Non-limiting examples of epidermolysis bullosa include epidermolysis bullosa acquisita, junctional epidermolysis bullosa, epidermolysis bullosa simplex, Kindler's syndrome, and dystrophic epidermolysis bullosa, which includes dominant dystrophic epidermolysis bullosa and recessive dystrophic epidermolysis bullosa, such as recessive contralateral dystrophic epidermolysis bullosa. Also included are any subtypes of the above examples.

[0023] As used herein, the term "subject" refers to an animal subject, preferably a mammalian subject, and more preferably a human subject. As used herein, the term "patient" refers to a human subject.

[0024] As used herein, the term "treatment" or "treating" refers to the administration of the complex or a pharmaceutical composition comprising same to a subject for purposes which may include ameliorating, alleviating, inhibiting or curing epidermolysis bullosa.

[0025] As used herein, the term "effective amount" refers to an amount that at least ameliorates the adverse effects of epidermolysis bullosa.

[0026] As used herein, the term "decorin" (DCN) refers to any isoform of a small leucine-rich chondroitin sulfate proteoglycan. Decorin is a multifunctional proteoglycan that, for example, regulates collagen fibrillogenesis, prevents tissue fibrosis, promotes tissue regeneration, and acts as an antagonist of TGF-β. In some embodiments, decorin is human decorin comprising or consisting of the amino acid sequence of decorin isoform A, B, C, D, or E, with or without an N-terminal signal sequence and / or propeptide. In some embodiments, decorin comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 6-20. Conserved sequence variants and peptidomimetics of the above decorin species are also included. As used herein, the term "decorin fragment" refers to a portion of the present complex comprising or consisting of decorin.

[0027] In some embodiments, a decorin fragment comprises or consists of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, or 60%, or any percentage therebetween, sequence identity with the amino acid sequence of SEQ ID NOs: 6-20, provided that the biological properties of decorin are not significantly altered. Such decorin variants may result from the addition, deletion, and / or substitution of one or more amino acids. Means and methods for determining whether the biological properties of decorin are retained are readily available in the art.

[0028] As used herein, the percentage of sequence identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions × 100), taking into account the number of gaps and the length of each gap to be introduced for optimal alignment of the two sequences. The comparison of the sequences of two sequences and determination of the percentage identity can be accomplished using mathematical algorithms available in the art.

[0029] As used herein, the term "homing peptide" refers broadly to any peptide that selectively homes to a target, specific cell or tissue in vivo in preference to other cells or tissues. Thus, homing peptides can be utilized as targeted delivery vehicles.

[0030] The homing peptide-derived decorin complexes used in the present invention differ from the known decorin fusion proteins disclosed in WO 2008 / 136869 at least in terms of the homing peptide used. While the prior art decorin fusion proteins contain the known CRK peptide (CRKDKC, SEQ ID NO: 3), the C-terminus of the novel homing peptides used in the present invention consists of the amino acid sequence RKDK (SEQ ID NO: 1). In some embodiments, the C-terminus of the novel homing peptides used in the present invention consists of CRKDK (SEQ ID NO: 2).

[0031] Surprisingly, it has now been discovered that truncation of the C-terminal cysteine ​​of a known CRK peptide (CRKDKC, SEQ ID NO: 3) alters the homing specificity of this peptide. While the CRK peptide selectively homes to skin wounds, the truncated CKR (RKDK, SEQ ID NO: 1 or CRKDK, SEQ ID NO: 2), hereafter referred to as tCRK, confers to the peptide the ability to home to and penetrate unwounded skin while retaining the ability to home to skin wounds. In other words, the CRK peptide selectively homes only to skin wounds, whereas the tCRK peptide selectively homes to and penetrates both skin wounds and unwounded skin.

[0032] Cleavage of the C-terminal cysteine ​​of the CRK peptide exposes the potential CendR (C-end Rule) sequence R / KXXR / K (SEQ ID NO: 4), i.e., RKDK (SEQ ID NO: 1), of this tCRK peptide. Without being limited by theory, the tCRK peptide can penetrate skin tissues by internalization by dermal microvascular endothelial cells expressing NRP-1 on their cell surface. Interestingly, CRK peptides containing potential CendR motifs cannot penetrate cells and tissues (Jarvinen and Ruoslahti, 2007, Am J Pathol 171:702-711; Agemy et al., 2010, Blood 116:2847-2856).

[0033] Thus, the homing peptide used in the present conjugate contains a tCRK element at the C-terminus of the homing peptide.

[0034] As used herein, the term "C-terminal end" (also known as the carboxyl end, carboxy terminus, C-terminus or COOH end) refers to the end of an amino acid chain that ends in a free carboxyl group (-COOH). As used herein, the terms "C-terminal end" and "C-terminal" are interchangeable.

[0035] As used herein, the term "N-terminal end" (also known as amino terminus, amine terminus, N-terminus, or NH2-terminus) refers to the beginning of an amino acid chain. The first amino acid in the amino acid chain contains a free amine group (-NH2). As used herein, the terms "N-terminal end" and "N-terminal" are interchangeable. Peptide sequences are written from the N-terminus to the C-terminus.

[0036] As used herein, the term "tCRK element" refers to a peptide having the amino acid sequence of RKDK (SEQ ID NO: 1) or CRKDK (SEQ ID NO: 2) that selectively homes to skin and skin wounds in vivo and can penetrate skin tissue. The terms "tCRK element" and "tCRK peptide" are interchangeable.

[0037] In the present invention, the tCRK element is located at the C-terminus of the homing peptide used herein. More specifically, the tCRK element is located at the C-terminal end of the homing peptide and includes the terminal carboxyl group. In other words, the C-terminus of the homing peptide consists of the amino acid sequence of RKDK (SEQ ID NO: 1) or CRKDK (SEQ ID NO: 2). Therefore, a homing peptide containing a tCRK element ends with the amino acid sequence of RKDK (SEQ ID NO: 1) or CRKDK (SEQ ID NO: 2).

[0038] In some embodiments, the homing peptide used in the present invention consists of SEQ ID NO: 1 or SEQ ID NO: 2. In some other embodiments, the homing peptide comprises SEQ ID NO: 1 or SEQ ID NO: 2. In the latter case, the homing peptide contains additional amino acids attached to the N-terminus of the tCRK element. However, the C-terminus of such longer homing peptides still consists of the tCRK element. In some embodiments, the homing peptide can contain up to 100 amino acids. In some embodiments, the homing peptide can contain up to 50 amino acids. In some embodiments, the homing peptide can contain up to 20 amino acids. In some embodiments, the homing peptide can contain up to 10 amino acids.

[0039] In some embodiments, the homing peptide may be part of a cyclic structure and may be cyclized, such as via a disulfide bond, and then cleaved by a protease to expose the tCRK sequence as a CendR peptide at the C-terminus of the homing peptide.

[0040] As used herein, the phrase "tCRK-derived decorin" refers to any decorin conjugate in which targeted delivery or homing is achieved by a tCRK homing peptide according to any one of the embodiments disclosed herein. Non-limiting examples of such conjugates include conjugates in which a decorin fragment comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOS: 6-20 and is linked from the C-terminus of the decorin fragment to the N-terminus of a tCRK element of SEQ ID NOS: 1 or 2, with or without an intervening linker, such as the intervening linker of SEQ ID NOS: 23 or 24. Further examples include conjugates comprising or consisting of the amino acid sequence of SEQ ID NOS: 21 or 22. Still further examples include sequence variants having at least about 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, or 60% sequence identity to the above sequences, as well as conservative sequence variants and peptidomimetics thereof, provided that the homing specificity, penetration capacity of the tCRK element, and biological activity of the decorin remain essentially unchanged.

[0041] In some embodiments, the tCRK-induced decorin complex may be provided for use in the form of, but is not limited to, a fusion protein. Thus, in some embodiments, the complex is a "fusion protein" comprising a decorin fragment, with or without one or more additional amino acid fragments, such as peptides, oligopeptides, polypeptides, or protein fragments, which may consist of or include natural or unnatural amino acids or peptidomimetics, fused or linked, preferably from the C-terminus of the decorin fragment, to the N-terminus of a homing peptide disclosed herein. Such one or more additional amino acid fragments may be fused or linked to the N-terminus of the decorin fragment and / or between the C-terminus of the decorin 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 diagnosis, imaging, or visualization.

[0042] As used herein, the term "peptide" refers to a series of amino acid residues typically connected to one another by peptide (amide) bonds between the alpha-amino and carbonyl groups of adjacent amino acids to form an amino acid sequence. Conventionally, a peptide is defined as a molecule consisting of 2 to 100, e.g., 2 to 50, amino acids. However, peptides can be subdivided into oligopeptides, which have fewer amino acids (e.g., 2 to 20), and polypeptides, which have more amino acids (e.g., 20 to 100 or 20 to 50). Proteins are essentially large peptides, typically consisting of more than 50 or 100 amino acids. Therefore, for simplicity, the term "peptide" as used herein encompasses any peptide bond system of natural (L-) and / or non-natural (D-) amino acid residues and is interchangeable with "oligopeptide," "polypeptide," "protein," and fragments thereof, unless otherwise specified. Peptidomimetic forms of peptides are also encompassed.

[0043] Fusion proteins used in the present invention can have any suitable length, for example, up to 300, 350, 400, 500, 1000, or 2000 residues, or any number of residues inclusive or between the above integers. As used herein, the term "residue" refers to an amino acid or amino acid analog.

[0044] In some embodiments, the fusion proteins used in the present invention may contain small peptide tags that facilitate purification, isolation, 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.

[0045] Fusion proteins used in the present invention may be produced by any suitable means, method, or technique available in the art, depending on their length, such as an automated peptide synthesizer or by genetic engineering. For example, an expression vector containing polynucleotides encoding decorin and a tCRK homing peptide may be produced 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, Bacillus), yeast (e.g., Pichia postulis, 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, HER cells). The expression vector may be transfected into host cells by various techniques commonly used for introducing foreign DNA into prokaryotic or eukaryotic host cells, including, but not limited to, electroporation, nucleofection, sonoporation, magnetofection, heat shock, calcium phosphate precipitation, DEAE-dextran transfection, etc. Various expression vectors are readily available in the art, and those skilled in the art can easily select an appropriate expression vector depending on different variables, such as the host cell used. The fusion proteins used in the present invention can be produced 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 bacteria, rabbit reticulocytes, CHO, or human lysates are commercially available in the art. In vitro protein expression may be carried out in either a batch reaction or dialysis mode.

[0046] The fusion partners of the fusion protein used in the present invention may be directly linked to each other or may be linked via a linker. The linker may be a peptide linker or a non-peptide linker. If the linker is a peptide linker, it may be composed of one or more amino acids. Non-limiting examples of peptide linkers include or consist of the amino acid sequence set forth in SEQ ID NO: 23 or 24.

[0047] Additionally, the homing peptide may be attached to decorin or any other therapeutic protein included in the complex or composition via a system such as SpyTag / SpyCatcher.

[0048] Thus, the fusion proteins used in the present invention may, in some embodiments, be produced using nucleic acid molecules encoding the fusion proteins, which may be used not only for the recombinant production of the fusion proteins they encode, but also for gene therapy by means and methods available in the art.

[0049] Conservative sequence variants of the above fusion proteins, including natural (L-) and / or unnatural (D-) amino acids and / or peptidomimetics, are also envisioned for use in the treatment of epidermolysis bullosa.

[0050] As used herein, the term "conservative sequence variant" refers to amino acid sequence modifications that do not significantly alter the biological properties of the protein or peptide being discussed. Conservative sequence variants include variants resulting from the substitution of one or more amino acids with similar amino acids known in the art (e.g., amino acids of similar size or amino acids with similar charge characteristics).

[0051] As used herein, the term "peptidomimetic" refers to a peptide-like molecule designed to mimic a given protein or peptide without altering its activity, e.g., homing specificity. Non-limiting examples of peptidomimetics include chemically modified peptides, peptidomimetics of D-peptides, peptide-like molecules containing unnatural amino acids, peptoids, and β-peptides. Also included within the term are molecules that resemble peptides but are not connected via natural peptide linkages. Means and methods for producing peptidomimetics are readily available in the art.

[0052] The tCRK-inducing decorin complex for the treatment of epidermolysis bullosa may further comprise one or more additional moieties, optionally covalently (directly or indirectly via a linker) or non-covalently linked, provided that the therapeutic activity of the complex is maintained.

[0053] In some embodiments, the additional moiety may have its own therapeutic activity, for example, anti-inflammatory activity, anti-angiogenic activity, regenerative activity, pro-angiogenic activity, cytotoxic activity, pro-apoptotic activity, antimicrobial activity (e.g., antibacterial, antiviral, antifungal, or antiprotozoal activity), antifibrotic activity, anti-wrinkle activity, anti-pruritic activity, mediator (e.g., histamine) inhibitory activity, mediator promoting activity, or cytokine activity, or the additional moiety may be a cytokine inhibitor (e.g., an antagonist, soluble receptor, cytokine binding molecule, or cytokine that blocks other cytokines), to name a few non-limiting examples of potential biological activities or therapeutic effects of the therapeutic moiety.

[0054] Thus, in some embodiments, the additional moiety may be a small molecule, e.g., a small molecule selected from antihistamines, antibiotics, retinoids, benzoyl peroxide, podophyllotoxin, cytotoxic drugs, and immune modulators such as corticosteroid derivatives, calcineurin inhibitors, and imiquimod. Furthermore, the additional moiety may be a protein moiety, e.g., anti-fibrotic 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 anti-inflammatory enzyme, such as CD73, or any collagen, such as type 7 collagen.

[0055] In some embodiments, additional moieties may be used to facilitate detection of the tCRK-induced decorin complex. Accordingly, the complex may include a detectable agent. As used herein, the term "detectable agent" 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 agents suitable for use in the disclosed complexes include various organic and / or inorganic small molecules, optical agents such as fluorescent, phosphorescent, and luminescent agents (e.g., chemiluminescent, chromogenic) including various fluorescent proteins and their derivatives, radioactive labels such as radionuclides that emit gamma rays, positrons, beta, or alpha particles, or X-rays, non-radioactive isotopes such as gadolinium (Gd), ionic and non-ionic contrast agents such as iodine-based contrast agents, electromagnetic agents such as magnetic, ferromagnetic, paramagnetic, and / or superparamagnetic agents, upconversion nanoparticles (UCNPs), resonant particles, quantum dots, and gold particles. Further suitable detectable agents are available in the art. Those skilled in the art can easily select an appropriate imaging technique depending on the type and species of detectable agent used in the conjugate. Such techniques include, but are not limited to, radiological techniques, isotopic techniques such as positron emission tomography, ultrasound imaging, and magnetic resonance imaging (MRI).

[0056] The detectable agent may be directly bound to the decorin conjugate, such as by a covalent bond, or may be indirectly associated, such as through a binder, linker, or chelator, such as diethylenetriaminepentaacetic acid (DTPA), 4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA), and / or metallothionein. Techniques for conjugating or otherwise linking detectable agents to peptide or protein conjugates are well known in the art. For example, conjugates including a detectable protein, such as a fluorescent protein (e.g., GFP), can be produced as a fusion protein by recombinant techniques.

[0057] None of the disclosed homing peptide-induced decorin complexes, and more specifically, the tCRK-induced decorin complexes, occur in nature.

[0058] In some embodiments, the tCRK-inducing decorin complex for use in treating epidermolysis bullosa is provided in the form of a pharmaceutical composition comprising the complex and a pharmaceutically or physiologically acceptable carrier permitting administration in vivo.

[0059] As used herein, the term "pharmaceutical composition" broadly refers to a formulation of one or more active ingredients with physiologically suitable ingredients, such as carriers, adjuvants, and / or excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to a subject or organism. As used herein, the term "active ingredient" broadly refers to a substance that exerts a biological effect, including, but not limited to, an anti-inflammatory effect, an anti-angiogenic effect, a regenerative effect, a pro-angiogenic effect, a cytotoxic effect, a pro-apoptotic effect, an antimicrobial effect (e.g., an antibacterial effect, an antiviral effect, an antifungal effect, or an antiprotozoal effect), an antifibrotic effect, an antipruritic effect, a mediator-inhibiting effect, a mediator (e.g., histamine)-promoting effect, a cytokine-inducing effect, or a cytokine-inhibiting effect. In the context of the present disclosure, the term "active ingredient" specifically refers to tCRK-induced decorin, although the composition and / or complex may also include additional active agents.

[0060] The pharmaceutical compositions may be formulated, for example, as semi-solid or solid preparations, solutions, dispersions or suspensions, as appropriate, using means and methods readily available in the art, for example, conventional mixing, dissolving, granulating, dragee-making, micronizing, emulsifying, encapsulating, entrapping, lyophilizing or similar processes.

[0061] As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" are used interchangeably and refer to a substance that is suitable for administration to a subject or organism without undue adverse side effects, such as toxicity, significant irritation, and / or allergic response. In other words, the benefit / risk ratio must be reasonable.

[0062] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier substance or diluent with which an active ingredient is combined to facilitate administration and which is physiologically acceptable to the recipient. Pharmaceutically acceptable carriers are readily available in the art and may be selected, depending on the intended route of administration, from the group consisting of, but not limited to, transdermal carriers, transmucosal carriers, enteral carriers, parenteral carriers, and sustained-release carriers. The selected carrier should not abolish the biological activity and properties of the active ingredient, minimize degradation of the active ingredient, and minimize adverse side effects to the recipient.

[0063] As used herein, the term " excipient " preferably refers to the inert substance that is added to pharmaceutical compositions to facilitate the administration of active ingredients.The typical examples of various types of excipients include but are not limited to stabilizers, preservatives, pH adjusters, fillers, thickeners, viscosity adjusters, lubricants, solubilizers, surfactants, sweeteners, flavor enhancers, etc.

[0064] Useful stabilizing excipients include, but are not limited to, surfactants such as polysorbate 20, polysorbate 80, and poloxamer 407; polymers such as polyethylene glycol and povidone; 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, chlorbutanol, benzalkonium chloride, and, in some cases, parabens. Useful buffer excipients include, but are not limited to, sodium and potassium phosphate, citrate, acetate, and carbonate or glycine buffers, depending on the target pH range. Sodium chloride is also useful as an isotonicity adjuster. Additional excipient materials include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. As will be readily understood by one of skill in the art, a given excipient may serve more than one function.

[0065] The pharmaceutical compositions can be administered in a number of ways depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be, for example, parenteral, enteral or topical.

[0066] Parenteral administration of the composition, if used, is generally by injection, for example, intravenous, intraperitoneal, subcutaneous, or intramuscular injection. Formulations for parenteral administration are typically sterile aqueous or non-aqueous solutions, suspensions, or emulsions, although formulations may also be provided in concentrated form or in the form of a powder to be reconstituted as needed. Sustained-release or sustained-release formulations are also contemplated. Means and methods for formulating formulations for parenteral administration are readily available in the art, and those skilled in the art can easily select appropriate physiologically suitable carriers, adjuvants, and / or excipients depending on the desired characteristics of the formulation.

[0067] Non-limiting examples of aqueous carriers for parenteral and other pharmaceutical formulations include sterile water, water-alcohol solutions, physiological saline, and buffered solutions at physiological pH. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride solution, Ringer's lactose, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose solution, and the like.

[0068] Non-limiting examples of non-aqueous carriers for parenteral and other pharmaceutical formulations include solvents such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, fish oil, and injectable organic esters such as ethyl oleate.

[0069] When the parenteral formulation is provided as a concentrated solution or dispersion or as a powder, the above-mentioned aqueous or non-aqueous carrier may be used for reconstitution. The solution for reconstitution may be provided in the same package as the concentrate or powder. When lyophilization is used to prepare the powder, it may be beneficial to use a cryoprotectant, including but not limited to polymers (e.g., povidone, polyethylene glycol, dextran), sugars (e.g., sucrose, glucose, lactose), amino acids (e.g., glycine, arginine, glutamic acid), and albumin.

[0070] Enteral administration of the composition, if used, may be by oral administration or administration via percutaneous endoscopic gastrostomy (PEG). Compositions for oral administration include, but are not limited to, powders, granules, capsules, sachets, tablets, aqueous or non-aqueous solutions and suspensions. Means and methods for formulating preparations for enteral administration are readily available in the art, and one skilled in the art can easily select appropriate physiologically suitable carriers, adjuvants and / or excipients depending on the desired characteristics of the preparation.

[0071] If used, topical administration of the composition may be by transdermal, transmucosal, epicutaneous, intranasal, rectal, vaginal, inhalant, or other route. Depending on the route of administration, formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, sustained- or sustained-release formulations, or solids. Means and methods for formulating formulations for topical administration are readily available in the art, and one skilled in the art can easily select appropriate physiologically suitable carriers, adjuvants, and / or excipients depending on the desired characteristics of the formulation.

[0072] Some of the compositions can be administered as pharmaceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and with 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 bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and with organic bases such as mono-, di-, tri-alkyl, aryl amines, and substituted ethanol amines.

[0073] Dosages and administration schedules for the conjugates or pharmaceutical compositions disclosed herein can be readily determined by those skilled in the clinical art of treating skin diseases and conditions, particularly epidermolysis bullosa. Generally, dosages will depend on considerations of the age, sex, and general health of the subject, the type of concurrent therapy, if any, the frequency of treatment and the nature of the desired effect, the severity and type of epidermolysis bullosa in question, and other variables tailored by the individual physician. To achieve the desired results, the desired dosage can be administered in one or more applications. For example, the pharmaceutical composition may be administered in a single daily dose, or the total daily dosage may be administered in divided doses, e.g., two, three, or four times daily. The pharmaceutical composition may be provided, for example, in a unit dosage form or sustained-release formulation. [Example]

[0074] Experimental Department Materials and Methods Cloning of decorin fusion proteins Human decorin (DCN) cDNA (Krusius and Ruoslahti, 1986, PNAS 83:7683-787), lacking the native signal and propeptide sequences, 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 into the C-terminus of decorin adjacent to the stop codon. A 6xHis tag was cloned into the N-terminus preceding decorin. This construct was assembled using the PIPE method (Klock and Lesley, 2009, Methods Mol Biol 498:91-103). For transformation, NEB 5-alpha competent E. coli (high efficiency) cells (C2987H, New England Biolabs, Ipswich, MA) were used according to the manufacturer's instructions. Kits from Qiagen (Hilden, Germany) were used for plasmid purification (Mini-Prep), PCR purification, and agarose gel purification. DCN naturally forms dimers (Scott et al., 2004, PNAS 101:15633-15638). The protein sequence of the monomeric 6xHis tag-DCN-tCRK fusion protein is GHHHHHHDEASGIGPEVPDDRDFEPSLGPVCPFRCQCHLRVVQCSDLGLDKVPKDLPPDTTLLDLQNNKITEIKDGDFKNLKNLHALILVNNKISKVSPGAFTPLVKLERLYLSKNQLKELPEKMetPKTLQELRAHENEITKVRKVTFNGLNQMetIVIELGTNPLKSSGIENGAFQGMetKKLSYIRIADTNITSIPQGLPPSLTELHLDGNKISRVDAASLKGLNNLAKLGLSFNSISAVDNGSLANTPHLRELHLDNNKLTRVPGGLAEHKYIQVVYLHNNNISVVGSSDFCPPGHNTKKASYSGVSLFSNPVQYWEIQPSTFRCVYVRSAIQLGNYKGSEFCRKDKstop (SEQ ID NO: 21).

[0075] A schematic diagram of the DCN-tCRK fusion protein used in the experimental section is shown in Figure 1A. As clearly indicated in the detailed description, the DCN-tCRK fusion protein of Figure 1A is a non-limiting example of a tCRK-derived decorin complex suitable for use in the present invention.

[0076] Recombinant protein production The pEFIRES-P expression vector construct was transfected into HEK293F cells by lipofection (FuGene 6, Promega, Madison, WY). Positive clones were selected in medium consisting of DMEM high 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) in the presence of 5–160 μg / ml puromycin (HyClone, Thermo Fisher Scientific). Established cell lines were maintained in medium containing 10 μg / ml puromycin.

[0077] The confirmed cells were then resuspended in serum-free OptiCHO medium (Gibco) supplemented with 2 mM L-alanyl-L-glutamine (Sigma) and cultured in a square glass bottle mounted on a rotary shaker at 37°C in a 5% CO atmosphere. When the cells reached a density of 1–2 × 10 6 After reaching a density of 10 cells / ml, cells were cultured for an additional 4 days at 33°C for recombinant protein expression and secretion into the medium. The protein was purified from the medium using a two-step HisTrap purification protocol on an Aekta Start chromatography system (GE Healthcare, Munich, Germany).

[0078] Recombinant protein purification Cell culture supernatants were filtered and degassed on ice through a 0.45 μm filter unit (Corning #430514, Corning, NY). The 6xHis-tagged proteins were purified by Ni-NTA-IMAC using a two-step purification protocol, first using a HisTrap Excel column and then a HisTrap HP column, on an Aekta Start chromatography system (GE Healthcare, Munich, Germany) in a 4°C cold cabinet according to the manufacturer's instructions. Buffers were prepared using the His Buffer Kit (GE Healthcare / VWR (11-0034-00)). All buffers were filtered and degassed.

[0079] The HisTrap Excel column eluate was diluted with 20 mM sodium phosphate buffer (pH 7.4) containing 0.5 M NaCl to a final imidazole concentration of 30 mM, and then further purified on a HisTrap HP column with a 35 mM imidazole wash and a gradient elution up to 300 mM imidazole (Figure 2A includes an example of such a purification chromatogram). Peak fractions were analyzed on an SDS NuPAGE 4-12% gradient gel (Life Technologies / Thermo Fisher Scientific, Waltham, MA) and visualized with PageBlue Protein Staining Solution (Thermo Fisher Scientific, Waltham, MA).

[0080] Selected peak fractions were pooled and dialyzed against cold TBS buffer (pH 7.6) using a 50 kDa MWCO Float-A-Lyzer (Fisher Scientific / Spectrum Labs) and then concentrated in a 10 kDa MWCO VivaSpin 6 tube (GE Healthcare). Samples were filter-sterilized (Ultrafree-MC GV Centrifugal Filter 0.22 μm, Millipore, Burlington, MA), and protein concentrations were measured at A280 nm using a Nanodrop (Thermo Fisher Scientific, Waltham, MA). All steps were performed at 4°C or on ice. To prevent aggregation, sterile Tween-20 was added to a final concentration of 0.05%, and aliquots were quickly frozen at -80°C.

[0081] Recombinant proteins were confirmed by SDS-PAGE and Western blotting using the BioRad wet-tank Mini-PROTEAN Trans-Blot Cell system (according to the manufacturer's instructions). PVDF membranes were probed with a primary mouse antibody against human decorin (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 4000 mini (GE Healthcare).

[0082] Biophysical analysis of proteins Hydrodynamic diameter was measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS instrument (Malvern Instruments Ltd, Worcestershire, UK). DCN-tCRK protein samples were diluted 1:5 with TBS buffer. Triplicate 10 × 10 s measurements were performed at 25°C. Data were analyzed using Zetasizer software v7.11 (Malvern Instruments Ltd.) using a protein analysis model (non-negative least-squares analysis followed by L-cube) and volumetric size distribution.

[0083] The unfolding temperature of DCN-tCRK was 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 to 130 °C at a scan rate of 2 °C / min. The feedback mode was set to "low" and the filter period was 5 seconds. The melting temperature (Tm) (transition midpoint) was calculated using the Origin 7.0 DSC software suite (Microcal Inc.) using a non-2-state fitting model.

[0084] The expressed recombinant DCN-tCRK protein was identified from the monomeric gel band using an Exigent 425 NanoLC coupled to a Sciex high-speed TripleTOF™ 5600+ mass spectrometer. After isolation of the gel band and Coomassie stain removal, the protein was subjected to reduction (TCEP, 25 mM), alkylation (iodoacetamide, 0.5 M), and trypsin digestion, as described in detail in Vaehaetupa et al., 2018. After trypsin digestion, peptides were diluted in 14 μl of sample buffer (2% acetonitrile, 0.1% formic acid), and 1 μl of sample was injected into the triple TOF mass spectrometer.

[0085] In vitro binding assay ELISA assays were used to analyze the in vitro binding of DCN-tCRK and peptides to NRP-1. 96-well black FLUOTRAC™ 600 high-binding plates (Greiner Bio-One, Kremsmuenster, Austria) were coated with 100 μg / ml DCN-tCRK in PBS at 100 μL / well and incubated overnight at 4°C. 10 μg / well of RPARPAR (SEQ ID NO: 25) and RPAPRARA (SEQ ID NO: 26) peptides were coated in parallel 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 with 300 μl of blocking solution (1x PBS, 1% BSA, 0.1% Tween-20) at 37°C for 1 hour. His-tagged neuropilin-1 b1b2 domain (NRP-1 WT) and triple mutant NS346A-E348A-T349A neuropilin-1 b1b2 domain (NRP-1 mutant) were expressed and purified at the Protein Production and Analysis Facility at the Sanford Burnham Prebys Medical Discovery Institute (La Jolla, CA) as previously described (Teesalu et al., 2009, PNAS 106:16157-16162). Recombinant proteins NRP1 WT, NRP1 mutants, and DCN-tCRK were labeled with FAM (5-(and-6)-carboxyfluorescein, #90024, Biotium, CA, USA) by mixing the proteins with the amine-reactive FAM dye (diluted in DMSO to a final concentration of 0.2%) at a ratio of 1:10. The reaction mixture was incubated at room temperature in the dark for 2 hours, followed by ultrafiltration / dialysis against PBS to separate the free dye from the proteins.One hundred microliters of FAM-labeled NRP1 WT or NRP1 mutant protein in blocking solution was added to each well (20 μg / well), incubated for 4–6 hours at room temperature or overnight at 4°C, and washed three times with blocking solution. After adding 100 μl of PBS to each well, the plate was immediately read in top-read mode using a fluorescence reader (Flex Station II, Molecular Devices, peak excitation = 485 nm, peak emission = 530 nm, cutoff = 515 nm).

[0086] To test in vitro binding of FAM-DCN-tCRK to NRP-1-positive prostate cancer 3 (PC-3) cells (gift from the Ruoslahti laboratory at Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA) and NRP-1-negative melanoma (M21) cells (gift from the David Cheresh laboratory at University of California, San Diego, La Jolla, CA), the cells were first cultured in 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, the 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 by directly conjugating the DCN-tCRK recombinant protein to fluorescein using the Lightning-Link Fluorescein Kit (Expedon Ltd, UK) according to the manufacturer's protocol. Cells were incubated at 37°C for 1 hour, the medium was aspirated, the cells were washed, and the cells were fixed with methanol at -20°C. The cells were washed with PBS and blocked for 30 minutes at room temperature (PBS, 1% BSA, 1% FBS, 1% goat serum, 0.05% Tween-20), followed by blocking with primary anti-FITC (Invitrogen, CA, USA, Cat. No. A-889) for 1 hour at room temperature. The cells were washed and then incubated with secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, USA) for 1 hour in the dark at room temperature. Nuclei were stained with DAPI. Coverslips were mounted onto glass slides with Fluoromount-G (Electron Microscopy Sciences, PA, USA), imaged with a confocal microscope (Olympus FV1200MPE, Tokyo, Japan), and analyzed with the FV10-ASW4.2 viewer.

[0087] Mice and study approval BALB / cJRj mice (Janvier Labs, Le-Genest-Saint-Isle, France) were used for the pharmacokinetic studies. Mice were fed standard laboratory pellets and water ad libitum. All animal experiments using BALB / cJRj mice were performed according to a protocol approved by the National Animal Ethics Committee of Finland (ESAVI / 6422 / 04.10.07 / 2017).

[0088] To study the skin-homing and therapeutic functions of DCN-tCRK, we used an animal model of recessive dystrophic epidermolysis bullosa (RDEB), namely col7a1 - / - RDEB mice were used. C57BL6 / J col7a1 with genotype determined by polymerase chain reaction (PCR). + / - By breeding mice, col7a1 - / - RDEB mice were generated using C57BL6 / J col7a1 mice, kindly provided by Dr. Jouni Uitto at Thomas Jefferson University. + / - Mice were developed by targeted ablation of the col7a1 gene by an out-of-frame deletion. col7a1 - / - All animal studies using RDEB were performed using protocols approved by the Institutional Animal Care & Use Committee (IACUC) of New York Medical College.

[0089] Pharmacokinetics of recombinant proteins Recombinant DCN-tCRK or DCN proteins were diluted in Tris-buffered saline (TBS) containing 0.05% Tween-20. The pharmacokinetics of DCN-tCRK and DCN were studied in 8-week-old Balb / c male mice. 5 mg / kg of either DCN-tCRK or DCN was injected into the tail vein under isoflurane anesthesia. Separate tail vein blood samples were collected at 15, 30, 60, 2, 4, and 16 hours after injection. Eight or 24 hours after injection, mice were sacrificed under medetomidine-ketamine anesthesia, and blood samples were collected from the subclavian vein. The samples were mixed with 1 M ethylenediaminetetraacetic acid (EDTA) and centrifuged at 2000 g for 10 minutes at room temperature. Plasma was saved for analysis. The concentration of human-derived decorin in plasma samples was measured using the Human Decorin DuoSet ELISA Kit (#DY143, R&D Systems) according to the manufacturer's instructions. Venous blood samples from uninjected mice were used on each plate to ensure the specificity of the primary antibody.

[0090] col7a1 - / - Administration of DCN-tCRK and DCN in mice col7a1 during pregnancy + / - Mice were housed individually and monitored daily until birth. Because intravenous injection of newborn mice is technically challenging and often yields inconsistent results, we administered the first dose of DCN-tCRK and DCN (5 μg in 15 μl PBS, corresponding to approximately 5 mg / kg) to col7a1 mice within 24 hours after birth. - / -We chose to inject mice into the liver because the liver is the primary site of hematopoiesis in fetal and newborn mice, and human cells have been shown to rapidly enter the 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 initial dose, the protein was administered intraperitoneally (ip) every other day (for a maximum of seven doses) until the mice reached 14 days of age, at which point the dose was increased to 10 μg. Mice were monitored daily. All experimental col7a1 - / - Mice were genotyped at the time of sample collection.

[0091] col7a1 - / - Histological and immunohistochemical staining and hDCN quantification in mice The dorsal skin and paws (front and rear) of selected mice were excised, embedded in Tissue-Tec OCT Compound (Sakura Finetek, Torrance, CA), and stored in a -80°C freezer. Serial 6-μm sections were cut for each specimen. Picrosirius red staining and CTGF (#ab6992, Abcam, Cambridge, UK) immunohistochemical staining were performed at the Core Histology Lab at New York Medical College. For immunohistochemistry staining of the His tag, sections were fixed in 4% paraformaldehyde and blocked with MOM blocking reagent (Vector Laboratories, Burlingame, CA) (for antibodies raised in mouse) or 10% horse serum (GIBCO, Grand Island, NY) containing 0.1% Triton (Sigma, St. Louis, MO). Next, slides were incubated with 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 antibodies (Invitrogen, Carlsbad, CA). Slides were then mounted with Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA). Images were acquired using a Nikon 90i Eclipse microscope (Nikon Instrument Inc., NY) with the same settings across different groups in each set of experiments.The intensity of immunostaining per field was measured using NIS-Elements AR software according to the user guide. RGB images were used to quantify picrosirius red staining, and a threshold was defined by selecting reference points within the image.

[0092] col7a1 was detected using the human decorin DuoSet ELISA kit (#DY143, R&D Systems, Minneapolis, MN) according to the manufacturer's recommendations. - / - Homing of DCN-tCRK and DCN to mouse skin was measured. Tissue biopsies were snap-frozen in liquid nitrogen, pulverized with a pre-cooled pestle, and homogenized in lysis buffer (1% Tween 20, protease inhibitor cocktail, DNase, and RNase in PBS). After centrifugation at 12,000 g for 10 minutes at 4°C, the supernatant was collected and total protein concentration was quantified using a BioRad DC protein assay (BioRad, Hercules, CA). The col7a1 activity was measured with and without DCN-tCRK or DCN administration. - / - Serum from mice was diluted 1:20 in sample diluent before application to the assay.

[0093] RT 2 Analysis of wound healing pathways by Profiler PCR RT 2 The expression of genes involved in wound healing pathways in mice was studied using the Profiler PCR Array (QIAGEN, Hilden, Germany). 2 The Profiler Array contains 84 wound healing genes, 5 housekeeping genes, genomic DNA, reverse transcription controls, and PCR positive control primers in a 96-well plate. On day 7, WT mice, RDEB mice, and col7a1 mice injected with DCN or DCN-tCRK were used. - / -Total RNA was isolated from the whole forepaws of mice (3 per group). RNA quality and concentration were measured using a NanoDrop 200C (ThermoScientific, Waltham, MA). RNA was treated with genomic DNA removal mix (QIAGEN). RT 2 500 ng of total RNA from each sample was subjected to reverse transcription using the First Strand kit (QIAGEN). The cDNA synthesis reaction was reverse transcribed using 2x RT. 2 The mixture was mixed with SYBR Green Master Mix, and 25 μl of the cocktail was dispensed into each well of a 96-well plate. Q-PCR was performed on a QuantStudio5 Real-Time PCR instrument (Applied Biosystems, Foster City, CA). C values ​​were exported to an Excel file. The resulting raw data were analyzed using the PCR Array Data Analysis Template available at the GeneGlobe Data Analysis Center (https: / / www.qiagen.com / us / geneglobe). ΔΔC T Gene expression was calculated using the method. A threshold of 1.5 for fold-change gene expression values ​​and a p-value threshold of 0.05 were used to analyze data between WT and untreated / treated pups.

[0094] Collagen lattice contraction assay Human normal fibroblasts and fibroblasts derived from RDEB patients were cultured in DMEM supplemented with 10% FBS as previously described (Liao et al., 2018, Stem Cells 36:1839-1850). Collagen lattices were made by mixing the cell suspension with neutralized rat tail type I collagen (Advance BioMatrix, Carlsbad, CA). The final collagen concentration was 2.4 mg / ml, and the cell density was 2.1 × 10 5The cell / collagen suspension was aliquoted into a single well of a 24-well plate and allowed to solidify for 30 minutes at room temperature. 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 from each well was gently released with a fine pipette tip, and DCN or DCN-CRK was added to a final concentration of 75 μM (n = 3 per condition). Images were taken at 12 hours (initial area) and 48 hours (contracted area), and the gel area was quantified using Image J.

[0095] statistics Kaplan-Meier analysis was used to measure median survival time, and the log-rank (Mantel-Cox) test was used to compare survival rates between different experimental groups (GraphPad Prism 6). Student's unpaired t-test was used to study the binding of DCN-tCRK to NRP-1. A p value of less than 0.05 was considered significant.

[0096] result Generation of multifunctional recombinant DCN-tCRK fusion protein We engineered a DCN-tCRK fusion protein by placing a tCRK peptide at the C-terminus of DCN (Fig. 1A). Both DCN-tCRK and native DCN were expressed in mammalian cells and purified using chromatography (Fig. 2A). Both recombinant proteins migrated as a sharp band of approximately 55 kDa on SDS gel electrophoresis, with a smear above the band that was detected as DCN by Western blot analysis (Fig. 2B). This sharp band corresponds to the core protein, and the smear is caused by 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 size analysis indicates that DCN-tCRK exists as a uniform, 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 produces a sharp peak at a melting temperature (Tm) of 49°C, suggesting that tCRK-DCN maintains a stable tertiary structure under physiological conditions (Figure 2D).

[0097] [Table 1]

[0098] DCN-tCRK interacts with NRP-1 in vitro Next, we investigated whether the tCRK peptide fused to DCN retained its ability to interact with NRP-1. DCN-tCRK was immobilized on an ELISA plate and tested for binding to wild-type (WT) or mutant NRP-1 in which the CendR-binding pocket was abolished by a triple mutation (Teesalu et al., 2009, PNAS 106:16157-16162). DCN-tCRK bound effectively to WT NRP-1 at significantly higher levels than to the control bovine serum albumin (BSA) (p<0.01), whereas no significant binding to mutant NRP-1 was observed (p>0.05) (Figure 1B). Parallel studies using the synthetic RPARPAR (SEQ ID NO: 25) peptide, the original CendR peptide, RPARPARA (SEQ ID NO: 26), and a control peptide with a C-terminally capped CendR sequence that cannot interact with NRP-1 further reinforced the dependency of binding on the CendR sequence (Figure 1B). We further determined whether DCN-tCRK binds to cells that express NRP-1, namely, human PC3 prostate cancer cells. M21 melanoma cells, which do not express NRP-1, were also included in this assay. Internalization of DCN-tCRK was observed only in NRP-1-positive PC3 cells, but not in NRP-1-negative M21 cells (Figure 1C), confirming its NRP-1-dependent cell binding and penetration properties.

[0099] DCN-tCRK and DCN exhibited similar in vivo pharmacokinetics. To determine whether the addition of the tCRK peptide affected the circulating half-life of DCN, DCN-tCRK and DCN were injected intravenously in parallel into healthy Balb / c mice, and the amounts of DCN-tCRK and DCN in the peripheral blood at different time points within 24 hours after administration were quantified by ELISA. The blood half-life of DCN-tCRK was 30 minutes, which was not significantly different from that of DCN (Figure 3). This pharmacokinetic study suggests that modification of DCN with a small vascular-homing peptide does not affect the pharmacokinetics of DCN.

[0100] DCN-tCRK administration was associated with col7a1 - / - Improves mouse survival The therapeutic function and skin-homing properties of DCN and DCN-tCRK were investigated in the col7a1 animal model of RDEB. - / - These mice were generated by breeding heterozygous littermates and were characterized by the expression of col7a1 - / - Mice can be identified at birth based on the appearance of hemorrhagic blisters on the skin. - / - Mice were randomly assigned to receive intrahepatic administration of DCN, DCN-tCRK, or PBS (negative control). After the initial administration, surviving mice in each group were repeatedly administered intraperitoneally every other day until day 14. - / - The median survival time of mice was 2 days after PBS injection and was significantly prolonged to 7 days after DCN administration (p<0.0001) (Figure 4A). However, the median survival time of mice after DCN administration was significantly longer than that of col7a1. - / - The survival rate of mice was not statistically significant compared with previous administration of dextran / human serum albumin (D / HSA), which was used as a vehicle for stem cell administration and may have a role in reducing the number of col7a1 cells in some patients, possibly by regulating fluid balance. - / - DCN-tCRK treatment sporadically improved the survival rate of treated mice (Figure 5). Furthermore, DCN injection did not extend the survival of treated mice beyond 2 weeks of age. Importantly, the median survival time of mice after DCN-tCRK treatment was further extended to 11 days, significantly better than the median survival time after PBS administration (p<0.0001) or previous D / HSA administration (p<0.001) (Figures 4A and 5). Furthermore, 85% of DCN-tCRK-treated mice survived for 7 days, and 20% of these mice survived beyond 3 weeks of age before being sacrificed for skin analysis.

[0101] DCN-tCRK is col7a1 - / - Homing to mouse skin Using ELISA assays, human DCN and DCN-tCRK were quantified in the skin of treated RDEB mice at 1 week (wk), 2 weeks, and 3 weeks (n = 3, all time points) (Fig. 4B). At 1 week, there was no statistically significant difference between DCN-tCRK-treated and DCN-treated skin. However, at 2 weeks, the level of DCN-tCRK was significantly higher than that of DCN (3.6-fold, p < 0.05) (Fig. 4B). Furthermore, when the final ip administration of DCN-tCRK was administered on day 14, the detection of DCN-tCRK in the skin at 3 weeks (19.47 ± 12.80 pg / ml) strongly suggests that it is stable for at least 7 days in vivo.

[0102] We also performed immunohistochemical staining based on histidine tag expression to analyze the anatomical distribution of DCN-tCRK or DCN in RDEB skin. DCN-tCRK was detected in the dermis of both the paw and dorsal skin of RDEB mice at 1, 2, and 3 weeks (Figure 4C). Furthermore, staining of the gastrointestinal (GI) tract of treated RDEB mice showed no reactivity with anti-His antibodies (data not shown), suggesting that DCN-tCRK specifically targets the skin. In contrast, although ELISA demonstrated the presence of DCN in skin lysates, anti-His immunostaining in DCN-treated RDEB skin at the 1-week time point appeared to be nonspecific (diffuse) (Figure 4C). Anti-His and anti-NRP-1 double staining showed that the signal from DCN-tCRK was located within or near NRP-1-positive cells in RDEB skin (Fig. 4D), further supporting our non-limiting hypothesis that DCN-tCRK homing is due to NRP-1-dependent cell and tissue penetration.

[0103] DCN-tCRK treatment suppresses the fibrotic response in RDEB mice According to recent research by the present inventors, col7a1 - / -Mice have demonstrated a significant increase in TGFβ signaling beginning in the interdigital folds of the paw as early as 1 week after birth. Therefore, in this study, skin biopsies from this time point were selected to compare the expression of 84 genes central to the wound healing response and fibrosis formation between WT skin and RDEB skin treated with vehicle (D / HSA), DCN, or DCN-tCRK (n = 3 per group) (Table 2). As shown in the clustergram in Figure 6A, more than half of the genes in vehicle-injected RDEB skin showed a >1.5-fold increase in expression compared to WT. The relative fold change (log2) and p-value (-log10) of gene expression are also shown as volcano plots. Significantly (p < 0.05) dysregulated genes are marked in white in each plot (Figure 6B). Genes significantly upregulated in vehicle RDEB skin were involved in TGFβ signaling (i.e., Tgfb1, Tgfbr3, 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 vehicle RDEB skin compared with WT. In skin from DCN-treated RDEB mice, the overall gene expression profile was similar to that in vehicle RDEB skin (Figure 6B). In DCN-treated RDEB skin, Tgfb1 expression was no longer significantly abnormal, whereas Tgfbr3 and Ctgf expression remained significantly upregulated. Several genes, such as Il4, Cxcl3, and Tnfα, were significantly upregulated in DCN-treated RDEB skin compared with vehicle controls (Fig. 6B and Table 2).

[0104] Importantly, the expression profile in DCN-tCRK-treated RDEB skin was significantly different from that in vehicle- and DCN-treated RDEB skin and resembled that in WT skin (Figure 6A). Although the expression of some genes showed interindividual differences, not all genes in the array were significantly upregulated in DCN-tCRK-treated RDEB skin compared to WT (Figure 6 and Table 2).

[0105] [Table 2(1)] [Table 2(2)] [Table 2(3)]

[0106] Strong expression of CTGF / CCN2 was observed in vehicle-injected RDEB skin, and its expression level significantly decreased after treatment with DCN-tCRK (Figure 7A), confirming the onset of TGFβ1-mediated fibrosis in untreated RDEB skin and its suppression by DCN-tCRK treatment. Furthermore, as shown by picrosirius red staining, overall collagen deposition increased over time in vehicle-injected RDEB skin but significantly decreased in the skin of DCN-tCRK-treated mice (Figures 7B and 7C). Immunostaining showed that at 2 weeks (w), type I collagen (COL1) expression was significantly increased in vehicle-treated skin and decreased in DCN-tCRK-treated skin (Figures 7D and 7E). Similar results were obtained with immunostaining for myofibroblasts, i.e., α-smooth muscle actin (αSMA, Figures 7D and 7E). Furthermore, most of the αSMA + cells in WT- and DCN-tCRK-treated RDEB skin colocalized with blood vessels (CD31 staining), indicating their identity as vascular smooth muscle cells and pericytes, whereas the αSMA + cells in vehicle-treated RDEB skin were located outside of blood vessels, i.e., were myofibroblasts ( Figure 7D ).

[0107] To directly demonstrate the antifibrotic function of DCN-tCRK, we compared the inhibitory ability of DCN and DCN-tCRK on collagen gel contraction in vitro using both normal and RDEB-derived fibroblasts. At a low concentration (75 μM) at which DCN had no significant effect on collagen contraction, DCN-tCRK inhibited collagen gel contraction in both normal (p < 0.05) and RDEB-derived fibroblasts (p < 0.01) (Fig. 8).

[0108] Consideration Herein, we demonstrate that C-terminal exposure of the CendR sequence in a wound-homing peptide reveals a novel tissue-penetrating function for the peptide in normal and wounded skin. Conjugation of the tCRK peptide to DCN facilitates selective targeting of a therapeutic fusion protein to the skin, which exerts antifibrotic effects and improves survival in a mouse model of RDEB.

[0109] The above experiments demonstrated that systemic administration of DCN-tCRK recombinant protein significantly inhibited col7a1 - / - The study showed that DCN-tCRK was more effective than unmodified DCN in improving the survival rate of RDEB mice. The exact molecular mechanism is unknown, but without being limited by theory, it is believed that several different mechanisms may contribute to this improved survival rate. DCN is an anti-inflammatory and anti-fibrotic molecule. Consistent with our previous findings that TGFβ signaling is activated as early as one week after birth, the expression of more than half of the genes associated with fibrosis was upregulated in the skin of untreated RDEB mice at one week. Without being limited by theory, the improved survival rate of RDEB mice with DCN-tCRK administration is likely related to the anti-fibrotic and anti-inflammatory effects of the therapeutic protein.

[0110] Not only were genes directly involved in TGFβ signaling normalized in RDEB skin treated with DCN-tCRK (but not DCN), but genes associated with other signaling pathways, such as β-catenin and EGFR, were also normalized by DCN-tCRK administration. Both Wnt / β-catenin signaling and EGFR signaling have been shown to contribute to fibrogenesis in multiple fibrotic diseases, either through independent profibrotic mechanisms or through crosstalk with TGFβ signaling. For example, EGFR activation is required for the profibrotic function of TGFβ and CCN2-mediated fibroblast proliferation and myofibroblast transdifferentiation. DCN can bind to and downregulate EGFR and the HGF receptor Met (to suppress β-catenin expression). Col7a1 expression after DCN-tCRK administration was also normalized. - / -These normalized gene expression in mouse skin suggest multiple therapeutic functions of DCN-tCRK in RDEB. Therefore, the upregulation of proinflammatory genes in DCN-treated RDEB skin may represent a therapeutic effect that was not sustained by administration of native DCN.

[0111] In summary, we demonstrate here that the exposure of cryptic CendR sequences represents a novel feature of a wound-targeting peptide that homes to normal skin in addition to wounded skin and provides dermal tissue penetration. We also demonstrate that this peptide (tCRK) can function as a vehicle for delivering decorin and other therapeutic molecules in the treatment of systemic dermal diseases, particularly epidermolysis bullosa.

Claims

1. A homing peptide-induced decorin complex, the complex comprising a decorin fragment and a homing peptide, the C-terminus of the homing peptide consisting of the amino acid sequence CRKDK (SEQ ID NO: 2); the decorin fragment is linked to the N-terminus of the homing peptide; The complex, wherein the decorin fragment comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence represented by any one of SEQ ID NOs: 6 to 20, provided that the biological properties of decorin are maintained.

2. The complex of claim 1, which selectively homes to skin and skin wounds.

3. 3. The conjugate of claim 1 or 2, wherein the conjugate is a peptidomimetic, the peptidomimetic having a protein sequence that includes at least one component selected from the group consisting of chemically modified peptides, D-peptides, unnatural amino acids, peptoids, and β-peptides.

4. 3. The complex of claim 1 or 2, wherein the complex is a fusion protein comprising or consisting of SEQ ID NO: 21 or 22.

5. The complex of any one of claims 1 to 4, further comprising one or more additional moieties attached to the complex.

6. The conjugate of claim 5 , wherein the one or more additional moieties comprises a therapeutic agent.

7. 7. The conjugate of claim 6, wherein the therapeutic agent is an anti-inflammatory agent, an angiogenesis inhibitor, a regenerative agent, a pro-angiogenesis agent, a cytotoxic agent, a pro-apoptotic agent, an antibacterial agent, an anti-fibrotic agent, an anti-wrinkle agent, an anti-pruritic agent, a mediator inhibitor, a mediator promoter, a cytokine, or a cytokine inhibitor.

8. The conjugate of claim 7 , wherein the therapeutic agent is a peptide, polypeptide, protein, or small molecule.

9. The complex of claim 5 , wherein the one or more additional moieties comprises a detectable agent.

10. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

11. A pharmaceutical composition for the treatment of a systemic skin disease or disorder, comprising the complex of any one of claims 1 to 9.

12. The pharmaceutical composition of claim 11, wherein the systemic skin disease or disorder is epidermolysis bullosa.

13. The pharmaceutical composition according to claim 12, wherein the epidermolysis bullosa is epidermolysis bullosa acquisita, junctional epidermolysis bullosa, epidermolysis bullosa simplex, dystrophic epidermolysis bullosa, dominant dystrophic epidermolysis bullosa, recessive dystrophic epidermolysis bullosa, recessive contra-dystrophic epidermolysis bullosa, Kindler syndrome, or a subtype thereof.

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