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• W3027722708 abstract "Systemic skin-selective therapeutics would be a major advancement in the treatment of diseases affecting the entire skin, such as recessive dystrophic epidermolysis bullosa (RDEB), which is caused by mutations in the COL7A1 gene and manifests in transforming growth factor-β (TGF-β)-driven fibrosis and malignant transformation. Homing peptides containing a C-terminal R/KXXR/K motif (C-end rule [CendR] sequence) activate an extravasation and tissue penetration pathway for tumor-specific drug delivery. We have previously described a homing peptide CRKDKC (CRK) that contains a cryptic CendR motif and homes to angiogenic blood vessels in wounds and tumors, but it cannot penetrate cells or tissues. In this study, we demonstrate that removal of the cysteine from CRK to expose the CendR sequence confers the peptide novel ability to home to normal skin. Fusion of the truncated CRK (tCRK) peptide to the C terminus of an extracellular matrix protein decorin (DCN), a natural TGF-β inhibitor, resulted in a skin-homing therapeutic molecule (DCN-tCRK). Systemic DCN-tCRK administration in RDEB mice led to inhibition of TGF-β signaling in the skin and significant improvement in the survival of RDEB mice. These results suggest that DCN-tCRK has the potential to be utilized as a novel therapeutic compound for the treatment of dermatological diseases such as RDEB. Systemic skin-selective therapeutics would be a major advancement in the treatment of diseases affecting the entire skin, such as recessive dystrophic epidermolysis bullosa (RDEB), which is caused by mutations in the COL7A1 gene and manifests in transforming growth factor-β (TGF-β)-driven fibrosis and malignant transformation. Homing peptides containing a C-terminal R/KXXR/K motif (C-end rule [CendR] sequence) activate an extravasation and tissue penetration pathway for tumor-specific drug delivery. We have previously described a homing peptide CRKDKC (CRK) that contains a cryptic CendR motif and homes to angiogenic blood vessels in wounds and tumors, but it cannot penetrate cells or tissues. In this study, we demonstrate that removal of the cysteine from CRK to expose the CendR sequence confers the peptide novel ability to home to normal skin. Fusion of the truncated CRK (tCRK) peptide to the C terminus of an extracellular matrix protein decorin (DCN), a natural TGF-β inhibitor, resulted in a skin-homing therapeutic molecule (DCN-tCRK). Systemic DCN-tCRK administration in RDEB mice led to inhibition of TGF-β signaling in the skin and significant improvement in the survival of RDEB mice. These results suggest that DCN-tCRK has the potential to be utilized as a novel therapeutic compound for the treatment of dermatological diseases such as RDEB. A general limitation in systemic drug delivery is that only a small fraction of drug reaches its desired location and systemic side effects are encountered in other organs. Thus, a critical goal of modern drug development is to generate drugs to be target organ-specific, with minimal adverse effects in the other parts of the body. This goal could be achieved by developing drugs that recognize a specific epitope expressed in the affected organ. Alternatively, drugs can be converted to be target-specific by conjugation with an affinity ligand such as a homing peptide.1Järvinen T.A.H. Rashid J. Valmari T. May U. Ahsan F. Systemically administered, target-specific therapeutic recombinant proteins and nanoparticles for regenerative medicine.ACS Biomater. Sci. Eng. 2017; 3: 1273-1282Crossref PubMed Scopus (12) Google Scholar, 2Ruoslahti E. Bhatia S.N. Sailor M.J. Targeting of drugs and nanoparticles to tumors.J. Cell Biol. 2010; 188: 759-768Crossref PubMed Scopus (720) Google Scholar, 3Ruoslahti E. Tumor penetrating peptides for improved drug delivery.Adv. Drug Deliv. Rev. 2017; 110-111: 3-12Crossref PubMed Scopus (225) Google Scholar In vivo screening of phage peptide libraries has identified organ- or disease-specific molecular signatures in the vascular tissues, enabling a postal code system (vascular zip codes) for target-specific delivery of systemically administered therapeutics.2Ruoslahti E. Bhatia S.N. Sailor M.J. Targeting of drugs and nanoparticles to tumors.J. Cell Biol. 2010; 188: 759-768Crossref PubMed Scopus (720) Google Scholar, 3Ruoslahti E. Tumor penetrating peptides for improved drug delivery.Adv. Drug Deliv. Rev. 2017; 110-111: 3-12Crossref PubMed Scopus (225) Google Scholar, 4Ruoslahti E. Vascular zip codes in angiogenesis and metastasis.Biochem. Soc. Trans. 2004; 32: 397-402Crossref PubMed Scopus (125) Google Scholar The most efficient vascular homing peptides for tumor-specific cell and tissue penetration contain a consensus motif R/KXXR/K, with an arginine (or rarely lysine) residue at the C terminus, thus called a C-end rule (CendR) sequence.5Ruoslahti E. Access granted: iRGD helps silicasome-encased drugs breach the tumor barrier.J. Clin. Invest. 2017; 127: 1622-1624Crossref PubMed Scopus (9) Google Scholar, 6Teesalu T. Sugahara K.N. Kotamraju V.R. Ruoslahti E. C-end rule peptides mediate neuropilin-1-dependent cell, vascular, and tissue penetration.Proc. Natl. Acad. Sci. USA. 2009; 106: 16157-16162Crossref PubMed Scopus (529) Google Scholar, 7Sugahara K.N. Teesalu T. Karmali P.P. Kotamraju V.R. Agemy L. Girard O.M. Hanahan D. Mattrey R.F. Ruoslahti E. Tissue-penetrating delivery of compounds and nanoparticles into tumors.Cancer Cell. 2009; 16: 510-520Abstract Full Text Full Text PDF PubMed Scopus (805) Google Scholar, 8Sugahara K.N. Teesalu T. Karmali P.P. Kotamraju V.R. Agemy L. Greenwald D.R. Ruoslahti E. Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs.Science. 2010; 328: 1031-1035Crossref PubMed Scopus (800) Google Scholar The CendR sequence binds to neuropilin-1 (NRP-1), activating an extravasation and tissue penetration pathway that delivers the peptide along with its payload into the parenchyma of the tumor tissue.3Ruoslahti E. Tumor penetrating peptides for improved drug delivery.Adv. Drug Deliv. Rev. 2017; 110-111: 3-12Crossref PubMed Scopus (225) Google Scholar,5Ruoslahti E. Access granted: iRGD helps silicasome-encased drugs breach the tumor barrier.J. Clin. Invest. 2017; 127: 1622-1624Crossref PubMed Scopus (9) Google Scholar,8Sugahara K.N. Teesalu T. Karmali P.P. Kotamraju V.R. Agemy L. Greenwald D.R. Ruoslahti E. Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs.Science. 2010; 328: 1031-1035Crossref PubMed Scopus (800) Google Scholar As NRP-1 is expressed by the endothelial cells in all tissues,3Ruoslahti E. Tumor penetrating peptides for improved drug delivery.Adv. Drug Deliv. Rev. 2017; 110-111: 3-12Crossref PubMed Scopus (225) Google Scholar peptides containing cryptic CendR owe their target selectivity to a combination of binding to primary receptor with a tumor-specific expression pattern, and to a proteolytic activation to expose the CendR sequence in the target organ.5Ruoslahti E. Access granted: iRGD helps silicasome-encased drugs breach the tumor barrier.J. Clin. Invest. 2017; 127: 1622-1624Crossref PubMed Scopus (9) Google Scholar, 6Teesalu T. Sugahara K.N. Kotamraju V.R. Ruoslahti E. C-end rule peptides mediate neuropilin-1-dependent cell, vascular, and tissue penetration.Proc. Natl. Acad. Sci. USA. 2009; 106: 16157-16162Crossref PubMed Scopus (529) Google Scholar, 7Sugahara K.N. Teesalu T. Karmali P.P. Kotamraju V.R. Agemy L. Girard O.M. Hanahan D. Mattrey R.F. Ruoslahti E. Tissue-penetrating delivery of compounds and nanoparticles into tumors.Cancer Cell. 2009; 16: 510-520Abstract Full Text Full Text PDF PubMed Scopus (805) Google Scholar, 8Sugahara K.N. Teesalu T. Karmali P.P. Kotamraju V.R. Agemy L. Greenwald D.R. Ruoslahti E. Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs.Science. 2010; 328: 1031-1035Crossref PubMed Scopus (800) Google Scholar Being the largest organ of the human body, skin presents unique challenges for efficient drug delivery. The primary challenge related to local, i.e., transdermal, drug delivery is the poor penetration of macromolecules into the skin. Diffusion through intercellular lipids provides a transdermal delivery option, but it is limited only for the delivery of small lipophilic molecules. Therefore, systemically administered, yet skin-specific therapeutics would be a substantial therapeutic advance for the treatment of skin diseases, particularly those that affect the entire skin, such as recessive dystrophic epidermolysis bullosa (RDEB). RDEB is caused by mutations in the COL7A1 gene that encodes type VII collagen (C7).9Hilal L. Rochat A. Duquesnoy P. Blanchet-Bardon C. Wechsler J. Martin N. Christiano A.M. Barrandon Y. Uitto J. Goossens M. et al.A homozygous insertion-deletion in the type VII collagen gene (COL7A1) in Hallopeau-Siemens dystrophic epidermolysis bullosa.Nat. Genet. 1993; 5: 287-293Crossref PubMed Scopus (109) Google Scholar, 10Bruckner-Tuderman L. McGrath J.A. Robinson E.C. Uitto J. Progress in epidermolysis bullosa research: summary of DEBRA International Research Conference 2012.J. Invest. Dermatol. 2013; 133: 2121-2126Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 11Kiuru M. Itoh M. Cairo M.S. Christiano A.M. Bone marrow stem cell therapy for recessive dystrophic epidermolysis bullosa.Dermatol. Clin. 2010; 28 (xii–xiii): 371-382Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 12Nyström A. Bernasconi R. Bornert O. Therapies for genetic extracellular matrix diseases of the skin.Matrix Biol. 2018; 71-72: 330-347Crossref PubMed Scopus (16) Google Scholar Clinical manifestations include skin erosions and blistering, mutilating scarring, pseudosyndactyly, and a high risk of developing aggressive and rapidly metastasizing cutaneous squamous cell carcinomas (cSCCs).10Bruckner-Tuderman L. McGrath J.A. Robinson E.C. Uitto J. Progress in epidermolysis bullosa research: summary of DEBRA International Research Conference 2012.J. Invest. Dermatol. 2013; 133: 2121-2126Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar,11Kiuru M. Itoh M. Cairo M.S. Christiano A.M. Bone marrow stem cell therapy for recessive dystrophic epidermolysis bullosa.Dermatol. Clin. 2010; 28 (xii–xiii): 371-382Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar,13Uitto J. Toward treatment and cure of epidermolysis bullosa.Proc. Natl. Acad. Sci. USA. 2019; 116: 26147-26149Crossref Scopus (9) Google Scholar, 14Rashidghamat E. McGrath J.A. Novel and emerging therapies in the treatment of recessive dystrophic epidermolysis bullosa.Intractable Rare Dis. Res. 2017; 6: 6-20Crossref PubMed Scopus (50) Google Scholar, 15Nyström A. Bruckner-Tuderman L. Injury- and inflammation-driven skin fibrosis: the paradigm of epidermolysis bullosa.Matrix Biol. 2018; 68-69: 547-560Crossref PubMed Scopus (39) Google Scholar, 16Cianfarani F. Zambruno G. Castiglia D. Odorisio T. Pathomechanisms of altered wound healing in recessive dystrophic epidermolysis bullosa.Am. J. Pathol. 2017; 187: 1445-1453Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Although novel gene-, cell-, and protein-based therapies have demonstrated promising results in delivering C7 to the skin, challenges remain and there is still no cure for RDEB.13Uitto J. Toward treatment and cure of epidermolysis bullosa.Proc. Natl. Acad. Sci. USA. 2019; 116: 26147-26149Crossref Scopus (9) Google Scholar, 14Rashidghamat E. McGrath J.A. Novel and emerging therapies in the treatment of recessive dystrophic epidermolysis bullosa.Intractable Rare Dis. Res. 2017; 6: 6-20Crossref PubMed Scopus (50) Google Scholar, 15Nyström A. Bruckner-Tuderman L. Injury- and inflammation-driven skin fibrosis: the paradigm of epidermolysis bullosa.Matrix Biol. 2018; 68-69: 547-560Crossref PubMed Scopus (39) Google Scholar, 16Cianfarani F. Zambruno G. Castiglia D. Odorisio T. Pathomechanisms of altered wound healing in recessive dystrophic epidermolysis bullosa.Am. J. Pathol. 2017; 187: 1445-1453Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar TGF-β signaling has been demonstrated to play an essential role in the development of fibrosis and in the progression to malignancy in RDEB.17Knaup J. Gruber C. Krammer B. Ziegler V. Bauer J. Verwanger T. TGFβ-signaling in squamous cell carcinoma occurring in recessive dystrophic epidermolysis bullosa.Anal. Cell. Pathol. (Amst.). 2011; 34: 339-353Crossref PubMed Google Scholar, 18Odorisio T. Di Salvio M. Orecchia A. Di Zenzo G. Piccinni E. Cianfarani F. Travaglione A. Uva P. Bellei B. Conti A. et al.Monozygotic twins discordant for recessive dystrophic epidermolysis bullosa phenotype highlight the role of TGF-β signalling in modifying disease severity.Hum. Mol. Genet. 2014; 23: 3907-3922Crossref PubMed Scopus (62) Google Scholar, 19Nyström A. Thriene K. Mittapalli V. Kern J.S. Kiritsi D. Dengjel J. Bruckner-Tuderman L. Losartan ameliorates dystrophic epidermolysis bullosa and uncovers new disease mechanisms.EMBO Mol. Med. 2015; 7: 1211-1228Crossref PubMed Scopus (103) Google Scholar Our previous study demonstrated that TGF-β signaling is activated as early as a week after birth in col7a1−/− mice.20Liao Y. Ivanova L. Zhu H. Plumer T. Hamby C. Mehta B. Gevertz A. Christiano A.M. McGrath J.A. Cairo M.S. Cord blood-derived stem cells suppress fibrosis and may prevent malignant progression in recessive dystrophic epidermolysis bullosa.Stem Cells. 2018; 36: 1839-1850Crossref PubMed Scopus (11) Google Scholar Thus, an early intervention on the activation of TGF-β signaling may be beneficial in reducing disease burden in RDEB. TGF-β signaling has also been suggested to be a phenotypic modulator in monozygotic twins with identical COL7A1 mutations.18Odorisio T. Di Salvio M. Orecchia A. Di Zenzo G. Piccinni E. Cianfarani F. Travaglione A. Uva P. Bellei B. Conti A. et al.Monozygotic twins discordant for recessive dystrophic epidermolysis bullosa phenotype highlight the role of TGF-β signalling in modifying disease severity.Hum. Mol. Genet. 2014; 23: 3907-3922Crossref PubMed Scopus (62) Google Scholar Moreover, the expression level of a proteoglycan decorin (DCN), a natural TGF-β inhibitor, was significantly higher in the less affected twin. DCN is a structural constituent of extracellular matrix (ECM), and Dcn−/− mice exhibit irregular collagen fibril formation and significantly reduced tensile strength in skin.21Reed C.C. Iozzo R.V. The role of decorin in collagen fibrillogenesis and skin homeostasis.Glycoconj. J. 2002; 19: 249-255Crossref PubMed Scopus (292) Google Scholar Furthermore, DCN has anti-fibrotic and anti-tumor functions by regulating activities of multiple growth factors, among them inhibitory action on TGF-β.22Järvinen T.A. Prince S. Decorin: a growth factor antagonist for tumor growth inhibition.BioMed Res. Int. 2015; 2015: 654765Crossref PubMed Scopus (65) Google Scholar,23Järvinen T.A.H. Ruoslahti E. Generation of a multi-functional, target organ-specific, anti-fibrotic molecule by molecular engineering of the extracellular matrix protein, decorin.Br. J. Pharmacol. 2019; 176: 16-25Crossref PubMed Scopus (19) Google Scholar We recently also demonstrated an upregulation of DCN expression as one of the mechanisms of action for the effects of cord blood-derived unrestricted somatic stem cells (USSCs) in col7a1−/− mice.20Liao Y. Ivanova L. Zhu H. Plumer T. Hamby C. Mehta B. Gevertz A. Christiano A.M. McGrath J.A. Cairo M.S. Cord blood-derived stem cells suppress fibrosis and may prevent malignant progression in recessive dystrophic epidermolysis bullosa.Stem Cells. 2018; 36: 1839-1850Crossref PubMed Scopus (11) Google Scholar Supporting the role of DCN as a potential therapeutic disease-modifying molecule for RDEB, Cianfarani et al.24Cianfarani F. De Domenico E. Nyström A. Mastroeni S. Abeni D. Baldini E. Ulisse S. Uva P. Bruckner-Tuderman L. Zambruno G. et al.Decorin counteracts disease progression in mice with recessive dystrophic epidermolysis bullosa.Matrix Biol. 2019; 81: 3-16Crossref PubMed Scopus (27) Google Scholar recently reported that systemic administration of lentivirus driving the expression of human DCN (hDCN) attenuated TGF-β-induced fibrosis in a C7-hypomorphic RDEB mouse model that expresses a residual level of C7 (C7-hypomorphic mice). Our past in vivo phage display screens identified a panel of peptides that home to angiogenic blood vessels in skin wounds.25Järvinen T.A. Ruoslahti E. Molecular changes in the vasculature of injured tissues.Am. J. Pathol. 2007; 171: 702-711Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Two of the most promising peptides, cyclic peptides dubbed CAR (CARSKNKDC) and CRK (CRKDKC), have been utilized to deliver different therapeutic molecules in a target-selective fashion.1Järvinen T.A.H. Rashid J. Valmari T. May U. Ahsan F. Systemically administered, target-specific therapeutic recombinant proteins and nanoparticles for regenerative medicine.ACS Biomater. Sci. Eng. 2017; 3: 1273-1282Crossref PubMed Scopus (12) Google Scholar Interestingly, whereas CRK peptide contains a cryptic CendR sequence, RKDK, it is the only peptide among the vascular-homing CendR peptides that is not capable of penetrating cells and tissues.25Järvinen T.A. Ruoslahti E. Molecular changes in the vasculature of injured tissues.Am. J. Pathol. 2007; 171: 702-711Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar,26Agemy L. Sugahara K.N. Kotamraju V.R. Gujraty K. Girard O.M. Kono Y. Mattrey R.F. Park J.H. Sailor M.J. Jimenez A.I. et al.Nanoparticle-induced vascular blockade in human prostate cancer.Blood. 2010; 116: 2847-2856Crossref PubMed Scopus (128) Google Scholar In this study, we demonstrate that C-terminal exposure of the cryptic CendR-sequence of CRK, i.e., truncated CRK (tCRK; CRKDK), confers the peptide the ability to home to and penetrate normal skin while retaining its ability to home to skin wounds. This novel targeting specificity can be used for therapeutic benefit; that is, recombinant DCN-tCRK fusion protein had a superior therapeutic effect compared to native DCN in a col7a1−/− RDEB mouse model that completely lacks expression of C7. We previously characterized the homing of CAR and CRK peptides to skin wounds at different phases of wound healing.25Järvinen T.A. Ruoslahti E. Molecular changes in the vasculature of injured tissues.Am. J. Pathol. 2007; 171: 702-711Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar As CRK contains a cryptic CendR motif, but is not capable of penetrating cells and tissues,25Järvinen T.A. Ruoslahti E. Molecular changes in the vasculature of injured tissues.Am. J. Pathol. 2007; 171: 702-711Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar,26Agemy L. Sugahara K.N. Kotamraju V.R. Gujraty K. Girard O.M. Kono Y. Mattrey R.F. Park J.H. Sailor M.J. Jimenez A.I. et al.Nanoparticle-induced vascular blockade in human prostate cancer.Blood. 2010; 116: 2847-2856Crossref PubMed Scopus (128) Google Scholar we set out to investigate whether truncation of CRK by removing the last cysteine residue to expose the CendR motif at the C terminus would change its homing and/or tissue-penetration properties. We first determined the homing of tCRK to wounded skin at day 7 at the peak of angiogenesis. Similar to our previous report on CAR and CRK,25Järvinen T.A. Ruoslahti E. Molecular changes in the vasculature of injured tissues.Am. J. Pathol. 2007; 171: 702-711Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar the phage carrying tCRK peptide homed to the wound 112 ± 71.2-fold higher than the nonrecombinant control phage (p < 0.001, n = 8) (Figure 1A). The wound homing of tCRK was not statistically different from phage displaying CAR peptide (101 ± 89.3-fold higher than negative control, n = 5), but it was significantly better than the CRK phage (15.5 ± 8.19-fold, p < 0.05, n = 8) (Figure 1A). Our previous studies also showed that CAR, but not CRK, homes to early angiogenic blood vessel sprouts in day 5 wounds.25Järvinen T.A. Ruoslahti E. Molecular changes in the vasculature of injured tissues.Am. J. Pathol. 2007; 171: 702-711Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar In this study, we observed that tCRK also homes to the day 5 wound at a significantly higher level than the nonrecombinant phage (6.14 ± 1.41-fold, p < 0.05, n = 9) (Figure S1). As wound healing progresses, the granulation tissue rich in angiogenic blood vessels gradually converts to scar tissue with a limited number of blood vessels. The homing of CRK and CAR were diminished to 13.6 ± 2.66-fold (p < 0.001, n = 7) and 20.7 ± 4.72-fold (p < 0.001, n = 7) when compared to the control phage at the excisional wound on day 14 (Figure 1B). In contrast, there was an ~35-fold enrichment of tCRK peptide at this time point (34.3 ± 9.78-fold, p < 0.001, n = 7). These results show that tCRK homes to skin wounds at different phases of wound healing and is able to home to wounds that have matured to scar tissue. There was no overrepresentation of the phages displaying tCRK, CAR, or CRK peptides at other organs, including lung, heart, spleen, kidney, and liver (Figure 1C). Surprisingly, in addition to homing to the wound, tCRK was also detected in normal skin, 3 cm or farther from the edge of the wound (Figure 1C), and the enrichment (11.2 ± 9.16-fold) was remarkably higher than any other studied phage clones. To exclude possible influence of a nearby wound on the tCRK homing to normal skin, we investigated the skin homing property of tCRK in mice without wounds. Indeed, about 20-fold (20.5 ± 5.67-fold, p < 0.001, n = 7) enrichment of tCRK compared to the control phage was seen in the normal skin (Figure 1D). This was significantly higher (p < 0.05) than that of CRK (5.04 ± 2.88-fold). To confirm that tCRK peptide indeed homes to both normal skin and skin wounds, we generated nanoparticles, iron oxide nanoworms (IONWs), and coupled them with/without tCRK-homing peptides. IONWs were synthesized and characterized as described previously.27Park J.H. von Maltzahn G. Ruoslahti E. Bhatia S.N. Sailor M.J. Micellar hybrid nanoparticles for simultaneous magnetofluorescent imaging and drug delivery.Angew. Chem. Int. Ed. Engl. 2008; 47: 7284-7288Crossref PubMed Scopus (274) Google Scholar Fluorescent tCRK, a scrambled CendR peptide (negative control dubbed PRP), or FAM label alone was coated on the IONWs through a thioether bond between the cysteine thiol from the peptide and the maleimide on the IONWs.28Park J.H. von Maltzahn G. Zhang L. Schwartz M.P. Ruoslahti E. Bhatia S.N. Sailor M.J. Magnetic iron oxide nanoworms for tumor targeting and imaging.Adv. Mater. 2008; 20: 1630-1635Crossref PubMed Scopus (475) Google Scholar The IONWs were injected into mice with either excision wounds with splints or without splints. Only tCRK-coated IONWs, but not control peptide or FAM-coated IONWs, were detected throughout the blood vessels of the normal dermis (taken farther than 5 cm from the wounds) (Figure 2). In both excision wound models, strong accumulation of tCRK-coated IONWs was detected in hypervascular regions of the wounds, whereas these hypervascular regions were almost devoid of control peptide or FAM-coated IONWs (Figure S2). These results further confirmed that exposure of cryptic CendR sequence facilitates tCRK to be not only a potent wound-homing peptide but also a peptide homing to normal skin. We next engineered DCN-tCRK fusion protein by placing tCRK peptide at the C terminus of DCN (Figure 3A). Both DCN-tCRK and native DCN were expressed in mammalian cells and purified using chromatography (Figure S3A). Both recombinant proteins migrated as sharp bands at about 55 kDa with a smear above the band in SDS gel electrophoresis and detected as DCN by western blot analysis (Figure S3B). The sharp band corresponds to the core protein, and the smear is caused by heterogeneity in the glycosaminoglycan sulfate chain (mostly chondroitin) attached to the DCN core. Mass spectrometry validated the identity of DCN and the C-terminal tCRK sequence (Table S1). The hydrodynamic size indicates that DCN-tCRK exists as homogeneous and non-aggregated macromolecules with a diameter consistent with the reported dimer of DCN29Scott P.G. Grossmann J.G. Dodd C.M. Sheehan J.K. Bishop P.N. Light and X-ray scattering show decorin to be a dimer in solution.J. Biol. Chem. 2003; 278: 18353-18359Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar (Figure S3C). Differential scanning calorimetry produced a sharp peak with a melting temperature (Tm) of 49°C, suggesting that tCRK-DCN will maintain a stable tertiary structure at a physiological condition (Figure S3D). We next investigated whether the tCRK peptide fused to DCN retains its ability to interact with NRP-1. We immobilized DCN-tCRK on ELISA plates and tested its binding to wild-type (WT) or mutant NRP-1, where the CendR-binding pocket was disabled by a triple mutation.6Teesalu T. Sugahara K.N. Kotamraju V.R. Ruoslahti E. C-end rule peptides mediate neuropilin-1-dependent cell, vascular, and tissue penetration.Proc. Natl. Acad. Sci. USA. 2009; 106: 16157-16162Crossref PubMed Scopus (529) Google Scholar DCN-tCRK effectively binds to WT NRP-1 at a significantly higher level than the control bovine serum albumin (BSA) (p < 0.01), whereas it showed no significant binding to the mutant NRP-1 (p > 0.05) (Figure 3B). Furthermore, parallel studies with a synthetic RPARPAR peptide, a prototypic CendR peptide, and RPARPARA, a control peptide with a C-terminally capped CendR sequence and unable to interact with NRP-1, were used to fortify that the binding is dependent on CendR sequence (Figure 3B). We further determined whether DCN-tCRK binds to the cells that express NRP-1, i.e., human PC3 prostate carcinoma cells. M21 melanoma cells that do not express NRP-1 were also included in the assay. Supporting the NRP-1-dependent cell binding and penetration properties, internalization of DCN-tCRK was observed only in the NRP-1-positive PC3 cells, but not in the NRP-1-negative M21 cells (Figure 3C). To determine whether the addition of tCRK peptide had any effect on the circulation half-life of DCN, DCN-tCRK and DCN were injected intravenously in parallel in healthy BALB/c mice, and their amount in peripheral blood at different time points within 24 h of administration was quantitated by ELISA. The half-life of DCN-tCRK in blood was 30 min and was not significantly different from that of DCN (Figure S4). The pharmacokinetic studies suggest that modification of DCN with small vascular-homing peptide does not influence the pharmacokinetics of DCN. We next evaluated the therapeutic function and skin-homing properties of DCN and DCN-tCRK in col7a1−/− mice, an animal model of RDEB. These mice are generated by breeding of the heterozygous littermates, and col7a1−/− mice can be identified at birth based on the manifestation of hemorrhagic blistering in the skin.30Liao Y. Ivanova L. Zhu H. Yahr A. Ayello J. van de Ven C. Rashad A. Uitto J. Christiano A.M. Cairo M.S. Rescue of the mucocutaneous manifestations by human cord blood derived nonhematopoietic stem cells in a mouse model of recessive dystrophic epidermolysis bullosa.Stem Cells. 2015; 33: 1807-1817Crossref PubMed Scopus (13) Google Scholar The newborn col7a1−/− mice were randomly divided to receive DCN, DCN-tCRK, or PBS (negative control) via intrahepatic administration. Repeated intraperitoneal (i.p.) administration was performed to the surviving mice within each group every other day after the first dose until day 14. In this study, the median lifespan of col7a1−/− mice was 2 days after PBS injection and it was significantly prolonged to 7 days after administration of DCN (p < 0.0001) (Figure 4A). However, the survival of col7a1−/− mice after DCN administration was not statistically significant as compared to a historical administration of dextran/human serum albumin (D/HSA), which was used as the vehicle for stem cell administration and sporadically increased the survival of some col7a1−/− recipient mice likely by adjusting fluid balance (Figure S5).31Liao Y. Ivanova L. Sivalenka R. Plumer T. Zhu H. Zhang X. Christiano A.M. McGrath J.A. Gurney J.P. Cairo M.S. Efficacy of human placental-derived stem cells in collagen vii knockout (recessive dystrophic epidermolysis bullosa) animal model.Stem Cells Transl. Med. 2018; 7: 530-542Crossref PubMed Scopus (5) Google Scholar Moreover, DCN injections did not extend the survival of the recipients beyond 2 weeks of age. Importantly, the median lifespan of the mice after DCN-tCRK treatment was further extended to 11 days, which was significantly better than that after either PBS (p < 0.0001) or historical D/HSA administration (p < 0.001) (Figures 4A and S5). In addition, 85% of DCN-tCRK-treated mice reached 7 days of survival, and 20% of these mice survived past 3 weeks of age and were subsequently sacrificed for skin analyses. We next utilized an ELISA assay to quantitate hDCN and DCN-tCRK in the skin of recipient RDEB mice at 1, 2, and 3 weeks (n = 3 for all time points) (Figure 4B). There was no statistically significant difference between DCN-tCRK- and DCN-treated skin at the 1-week time point. However, the level of DCN-tCRK at the 2-week time point was significantly higher than that of DCN (3.6-fold, p < 0.05) (Figure 4B). In addition, as the last i.p. administration of DCN-tCRK was conducted on day 14, identification of DCN-tCRK in the 3-week skin (19.47 ± 12.80 pg/mL) is highly suggestive of its stability in vivo for at least 7 days. We also performed immunohistochemical staining based on the expression of histidine tag to analyze the anatomical distribution of DCN-tCRK or DCN in the RDEB skin. DCN-tCRK was detected in the dermis of both the paw and dorsal skin of the RDEB mice at 1," @default.
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