Collagen 7 Protein Replacement Therapy

Abstract

The present disclosure relates to collagen 7 (Col7) protein replacement therapy for the treatment of skin disorders, specifically dystrophic epidermolysis bullosa (DEB). Recombinant collagen 7 drug products, compositions, and methods of administration and monitoring treatment outcomes are provided.

Description

[Technical field]

[0001] The present disclosure relates to recombinant collagen 7 protein therapy for dystrophic epidermolysis bullosa (DEB), in particular recessive dystrophic epidermolysis bullosa (RDEB). [Background technology]

[0002] Epidermolysis bullosa (EB) encompasses a group of molecularly diverse disorders characterized by the development of blisters following mild mechanical trauma to the skin. Nearly all patients experience severe recurrent blistering or skin rupture, but the severity, extracutaneous manifestations, and clinical course depend on the type of EB. The four main types of inherited EB include simple EB, junctional EB, dystrophic EB (DEB), and Kindler syndrome, which are differentiated based on the level of dermal and / or non-dermal tissues where the protein is absent or where the affected protein is located and where the blisters and their clinical symptoms occur.

[0003] DEB, one of the most severe forms of EB, is characterized by recurrent blistering followed by healing and scarring. In patients with DEB, blistering can be triggered by even minor mechanical trauma due to the extremely fragile nature of the skin. This results in a chronic cycle of blistering, healing, and re-blistering that causes patients to suffer from painful wounds and debilitating scarring of the epidermal tissue.

[0004] DEB is caused by mutations in the COL7A1 gene, which encodes the alpha chain of type VII collagen (Col7), a protein essential for the formation of fibrils that anchor the basement membrane to the underlying dermis. The condition is inherited as either a dominant (DDEB) or recessive (RDEB) form. The recessive form typically presents with a more severe phenotype, and in the most severe generalized RDEB, there may be wounds involving the whole body. The correlation between RDEB genotype and phenotype and COL7A1 mutations is highly variable.

[0005] The extent of skin involvement ranges from localized blistering to widespread involvement of the extremities. It is common for patients with all forms of DEB to have nail dystrophy or loss. Scarring of the hands and feet causing webbing between the fingers, known as pseudosyndactyly, with progression to whiplash deformity in which the fingers or toes are covered with scar tissue, is most characteristic of severe generalized RDEB, while pseudosyndactyly is less frequent in DDEB.

[0006] Wound behavior in DEB, especially in the more severe form of the disease RDEB, has several distinguishing features. Wounds are dynamic and arise due to impairment of the skin's mechanical resistance to external shear forces; they heal but can recur, reflecting a continuing risk of the mechanical insult that led to the initial wound and an alteration of the healing process due to Col7 deficiency. Characteristics of DEB include lifelong skin fragility and healing with scar formation that is thought to be due to effects on two interrelated Col7-dependent mechanisms.

[0007] In addition to skin manifestations, severe forms of DEB can cause erosion and scarring of mucous membranes, such as those of the eyes, mouth and esophagus, genitals, and anus; dental abnormalities are also common. Oral involvement can result in blistering of the lips, fusion of the tongue to the floor of the mouth, and progressive reduction in size of the oral cavity. Esophageal erosions can result in webs and structures that can cause severe dysphagia. As a result, nutritional deficiencies, anemia, and other secondary problems are common. Corneal erosions can lead to scarring and blindness. Blistering and subsequent scarring of the hands and feet leads to pseudosyndactyly, a hallmark of the disorder. The lifetime risk of invasive squamous cell carcinoma is greater than 90%. Other forms of DEB have similar but less pronounced clinical findings.

[0008] Pathogenic variants in the COL7A1 gene result in abnormal, reduced or complete absence of anchoring fibrils in the skin, resulting in DEB. This lack of normal functional anchoring fibrils causes poor epidermal-dermal adhesion in the skin and mucous membranes, marked skin fragility and severe blistering in DEB patients.

[0009] There is no definitive treatment for DEB, and its management focuses on supportive care. Bandaging and infection prevention are the main principles for the management of all forms of EB, with analgesia and antipruritus, nutritional support, and surgical management as needed. The current standard of care using allografts is of very limited benefit in RDEB patients, as 0% of RDEB wounds were reported to be healed 16 weeks after transplantation due to the chronic and dynamic nature of the wounds in RDEB patients. However, there is no uniformly accepted standard of care: dressing selection, pattern, and traction techniques vary between families and institutions. Severely affected patients with extensive cutaneous and extracutaneous manifestations also require a multidisciplinary approach to management. Patients with DEB experience reduced health-related quality of life (HRQL) due to pain, pruritus, reduced activities of daily living, fatigue, weight loss, and the social impact of disease symptoms. Summary of the Invention [Problem to be solved by the invention]

[0010] DEB is a serious systemic disease with no effective treatment. The development of a systemic disease-modifying approach is not known. Because the deteriorating effects of DEB result from a lack of functional Col7, DEB is suitable for systemic protein therapy with recombinant human Col7 (rhCol7). The present disclosure provides rCol7 (e.g., rhCol7) protein therapy for systemic treatment of DEB. [Means for solving the problem]

[0011] The present disclosure relates to collagen 7 (alternatively referred to herein as col7, Col7, or C7), specifically recombinant human collagen 7 (alternatively referred to herein as rhCol7 or rhC7) protein replacement therapy for the treatment of dystrophic epidermolysis bullosa (DEB), particularly recessive DEB (RDEB). The treatment involves systemic administration of recombinant collagen 7 (e.g., rCol7, e.g., rhCol7) material to a subject with DEB via intravenous administration, e.g., intravenous infusion. The present disclosure is based, in part, on the basis that rCol7 protein can be deposited in wounded and non-wounded skin sites via systemic administration, specifically concentrating at the dermal-epidermal junction (DEJ), and thus can be used to treat, prevent, delay the onset of, or alleviate complications and / or symptoms of DEB, and / or prevent the progression of clinical complications and / or symptoms associated with DEB.

[0012] In one aspect, the disclosure provides a method of treating dystrophic epidermolysis bullosa (DEB) in a subject in need thereof, comprising intravenously administering to the subject a recombinant human Col7 (rhCol7) material according to a first dosing regimen and a second dosing regimen, where i) the first dosing regimen comprises administering a first therapeutically effective amount of the material at a first frequency; ii) the second dosing regimen comprises administering a second therapeutically effective amount of the material at a second frequency, where the first frequency is greater than the second frequency, and where administration according to the first dosing regimen occurs before administration according to the second dosing regimen.

[0013] In another aspect, the disclosure provides a method of alleviating, alleviating, reducing, ameliorating, delaying the onset of, slowing the progression of, eliminating, and / or curing a symptom or complication of dystrophic epidermolysis bullosa (DEB) in a subject in need thereof, comprising intravenously administering to the subject a recombinant human Col7 (rhCol7) material according to a first dosing regimen and a second dosing regimen, wherein i) the first dosing regimen comprises administering a first therapeutically effective amount of the material at a first frequency; ii) the second dosing regimen comprises administering a second therapeutically effective amount of the material at a second frequency, the first frequency being greater than the second frequency, and wherein administration according to the first dosing regimen occurs prior to administration according to the second dosing regimen.

[0014] In a further aspect, the disclosure provides a Col7 material (e.g., recombinant human Col7, rhCol7) for use in a method of treating dystrophic epidermolysis bullosa (DEB) in a subject in need thereof, the method comprising administering to the subject the recombinant human Col7 (rhCol7) material according to a first dosing regimen and a second dosing regimen, wherein i) the first dosing regimen comprises administering a first therapeutically effective amount of the material at a first frequency; ii) the second dosing regimen comprises administering a second therapeutically effective amount of the material at a second frequency, the first frequency being greater than the second frequency, and wherein administration according to the first dosing regimen occurs before administration according to the second dosing regimen.

[0015] In a related aspect, the disclosure provides a Col7 material (e.g., recombinant human Col7, rhCol7) for use in a method of alleviating, alleviating, reducing, ameliorating, delaying the onset of, slowing the progression of, eliminating, and / or curing a symptom or complication of dystrophic epidermolysis bullosa (DEB) in a subject in need thereof, the method comprising intravenously administering to the subject the recombinant human Col7 (rhCol7) material according to a first dosing regimen and a second dosing regimen, wherein i) the first dosing regimen comprises administering a first therapeutically effective amount of the material at a first frequency; ii) the second dosing regimen comprises administering a second therapeutically effective amount of the material at a second frequency, the first frequency being greater than the second frequency, and wherein administration according to the first dosing regimen occurs prior to administration according to the second dosing regimen.

[0016] In a related aspect, the disclosure provides for the use of a Col7 material (e.g., recombinant human Col7, rhCol7) for the manufacture of a medicament for the treatment of dystrophic epidermolysis bullosa (DEB) or a symptom or complication thereof, the treatment comprising administering to a subject in need thereof a recombinant human Col7 (rhCol7) material according to a first dosing regimen and a second dosing regimen, where i) the first dosing regimen comprises administering a first therapeutically effective amount of the material at a first frequency; ii) the second dosing regimen comprises administering a second therapeutically effective amount of the material at a second frequency, the first frequency being greater than the second frequency, and where administration according to the first dosing regimen occurs prior to administration according to the second dosing regimen.

[0017] In a related aspect, the disclosure provides use of a Col7 material (e.g., recombinant human Col7, rhCol7) for the manufacture of a medicament for alleviating, alleviating, reducing, ameliorating, delaying the onset of, slowing the progression of, eliminating, and / or curing a symptom or complication of dystrophic epidermolysis bullosa (DEB), wherein the alleviating, alleviating, reducing, ameliorating, delaying the onset of, slowing the progression of, eliminating, and / or curing comprises administering the recombinant human Col7 (rhCol7) material to a subject in need thereof according to a first dosing regimen and a second dosing regimen, wherein i) the first dosing regimen comprises administering a first therapeutically effective amount of the material at a first frequency; ii) the second dosing regimen comprises administering a second therapeutically effective amount of the material at a second frequency, the first frequency being greater than the second frequency, and wherein administration according to the first dosing regimen occurs prior to administration according to the second dosing regimen.

[0018] In some embodiments of the above aspects, symptoms or complications of DEB include skin wounds, including blister formation, itching, pain, skin erosions, scar formation, skin fragility, gangrene, skin aplasia or hypoplasia, skin hypopigmentation, milia, skin infections, cheilitis (e.g., inflammation of the lips), dystrophic fingernails or toenails, dental enamel abnormalities, pseudosyndactyly or camptodactyly of fingers or toes, syndactyly of fingers or toes, flexion contractures of the toes, dental caries, and dysphagia. The conditions may include, but are not limited to, poor oral hygiene, poor oral hygiene, poor oral hygiene (e.g., poor swallowing), grooved or fissured tongue, esophageal stenosis, pharyngeal stenosis, growth disorder, dilated cardiomyopathy, pulmonary interstitial dysmorphology, acute constipation, hearing or vision problems, eczema, glomerulopathy, immunological hypersensitivity, nasolacrimal duct obstruction, anemia, chronic ear infections, corneal erosion, corneal abrasion and scarring, microstomia, osteopenia, ectropion, immunological hypersensitivity, nephrotic syndrome, phimosis, renal failure, urinary retention, ureteral stenosis, stroke, squamous cell carcinoma, or a combination thereof. In some embodiments, a symptom or complication of DEB is skin wounds, including blister formation, skin erosion, scar formation, skin fragility, skin hypoplasia or hypoplasia, cheilitis (e.g., inflammation of the lips), dystrophic fingernails or toenails, pseudosyndactyly or camptodactyly of fingers or toes, syndactyly of fingers or toes, flexion contractures of toes, dysphagia (e.g., inadequate swallowing), esophageal stricture, pharyngeal stricture, or a combination thereof. In some embodiments, a symptom or complication of DEB is skin wounds, including blister formation.

[0019] In some embodiments of the above aspects, the method relieves, alleviates, reduces, improves, or eliminates one or more skin blemishes. In some embodiments of the above aspects, the first dosing regimen continues for at least 1 week. In some embodiments, the first dosing regimen continues for at least 2 weeks. In some embodiments, the first dosing regimen continues for at least 3 weeks. In some embodiments, the first dosing regimen continues for at least 4 weeks. In some embodiments, the first frequency is about every day, about every 2 days, about every 3 days, about every 4 days, about every 5 days, about every 6 days, about every week, about every 10 days, or about every 2 weeks. In some embodiments, the first frequency is every week.

[0020] In some embodiments of the above aspects, the second dosing regimen continues for at least 4 weeks. In some embodiments, the second dosing regimen continues for at least 6 weeks. In some embodiments, the second dosing regimen continues for at least 7 weeks. In some embodiments, the second dosing regimen continues for at least 8 weeks. In some embodiments, the second dosing regimen continues for at least 6 months. In some embodiments, the second dosing regimen continues for at least 1 year. In some embodiments, the second dosing regimen comprises administering the rhCol7 substance to the subject for the subject's lifetime. In some embodiments, the second frequency is about once a week, about once every 10 days, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, or about once a month. In some embodiments, the second frequency is once a week. In some embodiments, the second frequency is once every 2 weeks. In some embodiments, the second dosing regimen is chronic (e.g., continues for the subject's lifetime).

[0021] In some embodiments of the above aspects, the first dosing regimen lasts for 1-4 weeks and the second dosing regimen lasts for at least 6 weeks. In some embodiments, the first dosing regimen lasts for 1 week. In some embodiments, the first dosing regimen lasts for 2 weeks. In some embodiments, the first dosing regimen lasts for 3 weeks. In some embodiments, the first dosing regimen lasts for 4 weeks. In some embodiments, the second dosing regimen lasts for 7 weeks. In some embodiments, the second dosing regimen lasts for at least 6 months. In some embodiments, the second dosing regimen comprises administering the rhCol7 agent to the subject for the life of the subject. In some embodiments, the first dosing regimen lasts for 4 weeks and the second dosing regimen lasts for at least 7 weeks.

[0022] In some embodiments of the above aspect, the first effective amount is about 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2.0 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.6 mg / kg, 2.7 mg / kg, 2.8 mg / kg, 2.9 mg / kg, 3.0 mg / kg, 3.1 mg / kg, 3.2 mg / kg, 3.3 mg / kg, 3.4 mg / kg, 3.5 mg / kg, 3.6 mg / kg, 3.7 mg / kg, 3.8 mg / kg, 3.9 mg / kg, 4.0 mg / kg, 4.1 mg / kg, 4.2 mg / kg, 4.3 mg / kg, 4.4 mg / kg, 4.5 mg / kg, 4.6 mg / kg, 4.7 mg / kg, 4.8 mg / kg, 4.9 mg / kg, 5.0 mg / kg, 5.1 mg / kg, 5.2 mg / kg, 5.3 mg / kg, 5.4 mg / kg, 5.5 mg / kg, 5.6 mg / kg, 5.7 mg / kg, 5.8 mg / kg, 5.9 mg / kg, 6.0 mg / kg In some embodiments, the first effective amount is about 0.1 mg / kg, 3.2 mg / kg, 3.3 mg / kg, 3.4 mg / kg, 3.5 mg / kg, 3.6 mg / kg, 3.7 mg / kg, 3.8 mg / kg, 3.9 mg / kg, 4.0 mg / kg, 4.5 mg / kg, 5.0 mg / kg, 6.0 mg / kg, 7.0 mg / kg, 8.0 mg / kg, 9.0 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg. In some embodiments, the first effective amount is about 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, or 3.0 mg / kg. In some embodiments, the second effective amount is about 0.1 mg / kg. In some embodiments, the first effective amount is about 0.3 mg / kg. In some embodiments, the second effective amount is about 0.5 mg / kg. In some embodiments, the second effective amount is about 1.0 mg / kg. In some embodiments, the second effective amount is about 1.5 mg / kg. In some embodiments, the second effective amount is about 2.0 mg / kg. In some embodiments, the first effective amount is about 3.0 mg / kg.

[0023] In some embodiments of the above aspect, the second effective amount is about 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2.0 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.6 mg / kg, 2.7 mg / kg, 2.8 mg / kg, 2.9 mg / kg, 3.0 mg / kg, 3.1 mg / kg, 3.2 mg / kg, 3.3 mg / kg, 3.4 mg / kg, 3.5 mg / kg, 3.6 mg / kg, 3.7 mg / kg, 3.8 mg / kg, 3.9 mg / kg, 4.0 mg / kg, 4.1 mg / kg, 4.2 mg / kg, 4.3 mg / kg, 4.4 mg / kg, 4.5 mg / kg, 4.6 mg / kg, 4.7 mg / kg, 4.8 mg / kg, 4.9 mg / kg, 5.0 mg / kg, 5.1 mg / kg, 5.2 mg / kg, 5.3 mg / kg, 5.4 mg / kg, 5.5 mg / kg, 5.6 mg / kg, 5.7 mg / kg, 5.8 mg / kg, 5.9 mg / kg, 6.0 mg / kg In some embodiments, the second effective amount is about 0.1 mg / kg, 3.2 mg / kg, 3.3 mg / kg, 3.4 mg / kg, 3.5 mg / kg, 3.6 mg / kg, 3.7 mg / kg, 3.8 mg / kg, 3.9 mg / kg, 4.0 mg / kg, 4.5 mg / kg, 5.0 mg / kg, 6.0 mg / kg, 7.0 mg / kg, 8.0 mg / kg, 9.0 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg. In some embodiments, the second effective amount is about 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, or 3.0 mg / kg. In some embodiments, the second effective amount is about 0.1 mg / kg. In some embodiments, the second effective amount is about 0.3 mg / kg. In some embodiments, the second effective amount is about 0.5 mg / kg. In some embodiments, the second effective amount is about 1.0 mg / kg. In some embodiments, the second effective amount is about 1.5 mg / kg. In some embodiments, the second effective amount is about 2.0 mg / kg. In some embodiments, the second effective amount is about 3.0 mg / kg.

[0024] In some embodiments of the above aspects, the first effective amount is the same as the second effective amount. In some embodiments, the first effective amount and the second effective amount are independently about 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2mg / kg, 1.3mg / kg, 1.4mg / kg, 1.5mg / kg, 1.6mg / kg, 1.7mg / kg, 1.8mg / kg, 1.9mg / kg, 2.0mg / kg, 2.1mg / kg, 2.2mg / kg, 2.3mg / kg, 2.4mg / kg, 2.5mg / kg, 2.6mg / kg, 2.7mg / kg, 2.8mg / kg, 2.9mg / kg, 3.0mg / kg, 3 In some embodiments, the first effective amount and the second effective amount are independently about 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, or 3.0 mg / kg. In some embodiments, the first effective amount and the second effective amount are each about 0.3 mg / kg. In some embodiments, the first effective amount and the second effective amount are each about 3.0 mg / kg.

[0025] In some embodiments of the above aspects, the first dosing regimen continues for at least 1 week, the first frequency is weekly, and the first effective amount is about 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, or 3.0 mg / kg. In some embodiments, the first dosing regimen continues for 4 weeks. In some embodiments, the first effective amount is about 0.3 mg / kg. In some embodiments, the first effective amount is about 3.0 mg / kg. In some embodiments, the second dosing regimen continues for at least 4 weeks, the second frequency is biweekly, and the second effective amount is about 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, or 3.0 mg / kg. In some embodiments, the second dosage regimen continues for at least 7 weeks. In some embodiments, the second dosage regimen is long-term. In some embodiments, the second dosage regimen continues for the patient's lifetime. In some embodiments, the second effective amount is about 0.3 mg / kg. In some embodiments, the second effective amount is about 3.0 mg / kg.

[0026] In some embodiments of the above aspects, intravenous administration is by intravenous infusion or slow push injection. In some embodiments of the above aspects, the subject has recessive dystrophic epidermolysis bullosa (RDEB).

[0027] In some embodiments of the above aspects, the subject has dominant dystrophic epidermolysis bullosa (DDEB). In some embodiments of the above aspects, the subject has a mutation in the COL7A1 gene consistent with a recessive inheritance pattern.

[0028] In some embodiments of the above aspects, the subject is a human. In some embodiments, the subject is under 18 years old. In some embodiments, the subject is at least 6 years old. In some embodiments, the subject is at least 2 years old. In some embodiments, the subject is under 2 years old. In some embodiments, the subject is at least 18 years old.

[0029] In some embodiments of the above aspects, the rhCol7 substance is contained in a pharmaceutical composition comprising 1.2 mg / mL rhCol7 substance, 10 mM sodium phosphate, 5 mM sodium citrate, 100 mM L-arginine, 1.7% sucrose (w / v), 70 mM sodium chloride, and 0.05% (v / v) polysorbate 20 (pH 7.2).

[0030] In some embodiments of the above aspects, the second dosing regimen duration, the second frequency, and / or the second effective amount are determined at least in part based on an evaluation of one or more clinical parameters of the subject performed during or after the first dosing regimen. In some embodiments, the method further comprises evaluating one or more clinical parameters between the first dosing regimen and the second dosing regimen. In some embodiments, the method further comprises evaluating one or more clinical parameters during the first dosing regimen. In some embodiments, the method further comprises evaluating one or more clinical parameters during the second dosing regimen. In some embodiments, the one or more clinical parameters are selected from wound surface area, wound healing, time to chronic wound healing, and time to reblistering. [Brief description of the drawings]

[0031] [Figure 1] FIG. 1 shows representative images of dose-dependent deposition of Col7 in the tongue and skin in Col7a1− / − mice after three intravenous injections. [Figure 2A] FIG. 2A shows representative images (hematoxylin and eosin staining) of tongue tissue in Col7a1− / − mice after a single intravenous administration of rhCol7. [Figure 2B] FIG. 2B is a histogram of a portion of a Col7a1 − / − mouse with closure of the dermal-epidermal separation in the tongue after a single dose of Col7. [Diagram 3] Figure 3 shows PTR-01 exposure-dependent deposition of Col7. All patients received rCol7 supplementation and achieved an increase in Col7 in normal human skin (NHS) of 35% or more. Ph2 patients (black data points) received 4 x 3mg / kg weekly (days 1-29) and 7 x 3mg / kg biweekly (days 42-120), whereas Ph1 patients received only 3 x biweekly PTR-01 doses (days 1-29). *Day 22 and 120 non-wound samples for 202-001 were of poor quality and no data are available, data shown is for wound samples. Patients 02-06, 02-07, 02-08 participated in a Phase 1 / 2 study. NHS: normal human skin. [Figure 4] Figure 4 illustrates the study design for the Phase 2 open-label study of PTR-01. qow: every 2 weeks. [Diagram 5] FIG. 5 shows a comparison of wound assessments at day 120 versus baseline. [Figure 6] Figure 6 shows wound response by percent reduction in wound surface area by Canfield imaging. *N wound images were not available for Day 36 (Wound 6; Patient 201-001) and Day 92 (Wound 1; Patient 201-002); therefore, wound surface areas for these patients at those time points were omitted. Numbers in the shaded bars represent the number of wounds in each percentage category; numbers above each bar are the total number of wounds assessed at that time point. [Figure 7] FIG. 7 shows the wound closure observed in chronic and recurrent wounds. [Figure 8] FIG. 8 shows example wound images demonstrating a clear reduction in wound surface area following PTR-01 treatment. [Figure 9]Figures 9A-9B show individual wound changes from baseline and median wound surface area. AUC: area under the curve (cumulative wound surface area from baseline); N / A: not applicable; *Hodges-Lehman estimate of median difference = -18.01, 95% CI = (-40.35, 7.19); Wilcoxon rank sum p = 0.1776. [Figure 10] 10A-10B show improvements in pain, disease impact, activities of daily living, mood, and essential function as measured by iscorEB-P in patients from a Phase 2 study. IscorEB-P: Patients who provided an assessment of the iscorEB assay. [Figure 11] Figure 11 shows Investigator and Patient Global Impression of Change (GIC) scores by patient and time point. *Data point missing. Patient 203-001 is not shown. The number above each column is the number of patients with that score and the height of the column reflects the IGIC or PGIC score. Note that a decline in perceived global status occurred only in patient 203-002 3 months after the end of treatment. IGI: Investigator Global Impression; PGI: Patient Global Impression. [Figure 12] Figure 12 shows reduction in dermal pro-fibrotic biomarker staining with PTR-01 administration. 1 Scoring was performed on a 3-point scale (1=low; 2=medium; 3=high). N=5 patients (study completers). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The present disclosure provides methods of treating a skin disorder (e.g., dystrophic epidermolysis bullosa (DEB), e.g., recessive DEB) and / or alleviating, ameliorating, reducing, ameliorating, delaying the onset of, delaying the progression of, eliminating, and / or curing a symptom or complication of a skin disorder, e.g., DEB, in a subject (e.g., a human subject) in need thereof. The methods provided herein can include administering to the subject a pharmaceutical composition comprising collagen 7 (e.g., C7, col7, or Col7, e.g., recombinant collagen 7 (rCol7), e.g., recombinant human collagen 7 (rhCol7)), or a functional variant thereof, according to a first dosing regimen and a second dosing regimen. The first dosing regimen can be a "loading" dosing phase in which the subject is provided with a higher dose of collagen 7 and / or is administered more frequently than in the second dosing regimen. The second dosing regimen may be a "maintenance" dosing phase in which the subject is provided with a lower dose of collagen 7 than the first dosing regimen and / or collagen 7 is administered less frequently. The use of distinct dosing regimens may serve to acclimate the subject to therapy, address acute symptoms during the loading phase, and maintain the therapeutic benefits of collagen 7 therapy and prevent the onset or worsening of symptoms during the maintenance phase. The loading phase may last for one or more days, one or more weeks, or one or more months, while the maintenance phase may generally last longer than the loading phase, for weeks, months, or years, and may extend over the life of the subject.

[0033] The details of one or more embodiments of the present disclosure are described in the accompanying detailed description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred materials and methods are currently described. Other features, objects and advantages of the present disclosure will be apparent from the detailed description. In the detailed description, the singular form includes the plural form unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present disclosure belongs. In case of conflict, the present disclosure shall prevail.

[0034] Introduction DEB patients lack functional collagen 7 due to mutations in the COL7A1 gene, which encodes type VII collagen (also called C7 or Col7). Col7 is required to produce anchoring fibrils necessary for epidermal dermal adhesion. Endogenous Col7 forms through a complex supramolecular aggregation process. Specifically, three α-chains associate through their C-terminus to form a homotrimeric molecule that folds into a triple helical conformation within a collagen-like region. Two homotrimers form antiparallel dimers, from which the C-terminal propeptide is proteolytically cleaved by a protease, e.g., bone morphogenetic protein 1. Subsequently, a large number of these antiparallel dimers laterally aggregate to form anchoring fibrils. Col7 acts as the main component of anchoring fibrils that allow attachment of the epidermis to the dermis. Mutations in the COL7A1 gene involved in DEB (eg, RDEB) result in abnormal synthesis of C7 or defective assembly of the protein into anchoring fibrils, resulting in insufficient expression of dermal adhesion.

[0035] Supplementation of abnormal or defective proteins with systemically administered intravenously collagen 7, e.g., recombinant Col7 (rCol7), is predicted to allow the formation of anchoring fibrils and thus result in correction of blistering abnormalities and complications throughout the body in patients with DEB (e.g., RDEB). Intravenous administration of rCol7 in mice has been demonstrated to result in deposition of collagen 7 protein in the skin and improved wound healing (see, e.g., U.S. Patent Publication No. 20140031295, incorporated herein by reference in its entirety).

[0036] The present disclosure relates to disease-modifying Col7 (e.g., rCol7, e.g., rhCol7) replacement therapy for subjects with DEB, e.g., subjects with recessive DEB (RDEB). An rCol7 composition (PTR-01) has been developed by the applicants for use in treating patients with DEB, patients with RDEB. Systemic (e.g., intravenous) delivery of rCol7 is believed to return functional Col7 to the dermal-epidermal basement membrane zone (BMZ), thereby promoting normal anchoring fibril assembly and providing stability to dermal-epidermal adhesion at the stratum densa / upper papillary dermis interface, correcting blister dysplasia and complications in patients with DEB.

[0037] definition The terms used in this disclosure generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used.

[0038] As used herein, the terms "collagen 7", "Col7", "collagen type VII", and "C7" are used interchangeably and refer to collagen 7 protein. Human Col7 (also referred to herein as hCol7) is a large extracellular protein with a mass of approximately 900 kilodaltons (kDa) that is the main component of anchoring fibrils that allow attachment of the epidermis to the dermis. Col7 is a homotrimer composed of three human collagen α1 (type VII) polypeptides (α1 chains). The α1 chain (type VII) is encoded by the COL7A1 gene. Each Col7α1 chain is 2928 amino acids long and contains a central collagen-like domain flanked by two non-collagenous domains: an NC1 domain at the N-terminus and an NC-2 domain at the C-terminus. The three α1 chains associate via their C-terminal NC2 domains to form a homotrimeric molecule that folds into a triple helical conformation within a collagen-like region that contains hydroxylated proline and lysine residues. Two homotrimers result in the formation of an antiparallel dimer with the N-terminus (NC1 domain) present at both ends of the antiparallel dimer. Lateral assembly of the antiparallel dimers results in the formation of anchoring fibrils, which are highly specialized attachment structures in the basement membrane zone (BMZ) and are important for the attachment of the epidermis to the underlying dermis. These anchoring fibrils can also extend from the stratum compactum of the BMZ in the dermal-epidermal junction to the upper papillary layer of the dermis in normal skin. The full-length alpha chain polypeptide of human Col7 comprises the amino acid sequence of SEQ ID NO: 1 (Reference No.: NP_000085), which is naturally encoded by the nucleic acid sequence of SEQ ID NO: 2 (Reference No.: NM_000094). In the context of the present disclosure, Col7 may refer to recombinant collagen 7 protein, or a functional variant thereof. Functional variants of rCol7 may have structural properties (e.g., anchoring fibril formation), binding properties (e.g., binding to collagen IV and / or fibronectin), and / or signaling activity (e.g., transforming growth factor beta (TGF-β) inhibition and / or fibroblast growth factor 2 (FGF2) expression) that are equivalent or substantially similar to full-length Col7.As used herein, the term "recombinant human Col7 (rhCol7)" refers to a recombinant form of human Col7 having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the amino acid sequence of native human Col7 (e.g., SEQ ID NO:1). Recombinant Col7 (e.g., rhCol7) can be produced by recombinant techniques, e.g., by recombinant DNA methods, and produced in a host cell that contains a nucleotide sequence encoding Col7. The host cell can be a mammalian cell that provides the necessary modifications for recombinant Col7.

[0039] As used herein, the terms "recombinant human collagen 7 material", "rhCol7 material" and "rhC7 material" are used interchangeably and refer to a material (e.g., a drug material) that includes a plurality of recombinant Col7α polypeptides and / or functional variants thereof. In some embodiments, the rhCol7 material includes a recombinant Col7α polypeptide having the amino acid sequence of SEQ ID NO:1. The rhCol7 material may be produced by a host cell engineered to express high levels of recombinant Col7 and / or functional variants thereof. The rhCol7 material may be purified from the culture medium of the host cell. Suitable host cells may include primary or transformed cell lines including, but not limited to, fibroblasts, keratinocytes, CHO cells, HEK293 cells, C127 cells, VERO cells, BHK cells, HeLa cells, COS cells, MDCK cells, and the like.

[0040] As used herein, the term "recombinant human Col7 (rhCol7) drug product" refers to a drug formulation that contains rhCol7 material as an active ingredient. The rhCol7 drug product is formulated to maintain Col7 protein stability and is suitable for clinical administration, for example, by intravenous injection and / or infusion. The rhCol7 drug product can be a pharmaceutical composition that contains rhCol7 material.

[0041] As used herein, the term "protein therapy" refers to a medical treatment that supplies or replenishes a protein in a subject in which a particular protein is deficient or absent. The protein may be introduced into the subject via gene therapy, cell therapy, and / or direct protein supplementation. The term "protein supplementation" refers to the introduction of a non-natural purified protein into a subject in which such protein is deficient or absent. The administered protein may be obtained from a natural source, e.g., from a subject having normal protein, e.g., by purification of the protein from isolated tissues or body fluids, e.g., placenta and animal milk, or by recombinant protein expression. Purified recombinant proteins may be produced in vitro, e.g., using genetically engineered cell systems. Protein replacement therapy may also refer to the introduction of purified proteins into subjects who otherwise require or would benefit from administration of purified proteins, e.g., suffer from protein deficiency.

[0042] As used herein, the term "mutant protein" refers to a protein translated from a gene containing one or more genetic mutations that result in an altered protein sequence, a truncated protein fragment, and / or the complete absence of the protein. In contrast, a "wild-type" or "native" protein refers to any protein encoded by a wild-type gene that can have normal functional biological activity when expressed or introduced in vivo.

[0043] As used herein, the terms "wound" and "skin wound" refer to the disruption of the protective function of the skin, e.g., loss of epithelial continuity following injury to the skin or underlying tissue caused by surgery, fissures, lacerations, cuts, lacerations, abrasions, scrapes, scratches, burns, chemical exposure, or as a result of disease, e.g., DEB (e.g., RDEB). Wounds may have uniform or variable depth and morphology. Wounds may be chronic wounds, e.g., chronic open wounds, recurrent wounds, surgical wounds, blisters, ulcers, non-healing wounds, scars, surgical scars, or boils. In the context of this disclosure, wounds associated with DEB (e.g., RDEB) may be chronic open wounds and / or recurrent or acute wounds of different sizes. Chronic open wounds refer to areas that do not heal and remain open for at least 12 weeks. Recurrent wounds refer to areas that partially heal but then easily re-blister. Wounds may occur anywhere on the body, including on the torso, chest, abdomen, arms, hands, fingers, legs, feet, toes, back, buttocks, neck, or head. Wounds may be distinct (e.g., have a definable size or endpoint) and / or continuous (e.g., extend across a swatch of skin and lack a definable size or endpoint).

[0044] As used herein, the terms "disease" and "disorder" refer to a pathological condition of a part, organ, or system of an organism that results from a variety of causes, e.g., autoimmune defects, genetic defects, and / or environmental stresses, and is characterized by an identifiable group of signs or symptoms. As used herein, the term "genetic disease" can be a disease characterized by a protein deficiency. Protein deficiency can be caused by a genetic mutation in a gene encoding such a protein that causes the absence, insufficient amount, or dysfunction of such a protein, or can result from the development of antibodies against the protein. "Skin disease" or "skin disorder" refers to a clinical condition of the skin, e.g., a condition affecting the skin in a subject, e.g., a blistering disorder, an inflammatory skin condition, or a skin cancer. Blistering (blistering) disorders are a heterogeneous group of disorders characterized by the elevation of fluid-filled blistering lesions (blisters) present primarily on the skin and mucous membranes. The blisters can be variable in size, and the specific symptoms and severity of blistering disorders vary between individuals, even between individuals with the same disorder. Exemplary blister formation disorders include, but are not limited to, epidermolysis bullosa acquisita (EBA) and epidermolysis bullosa congenita (EB), e.g., dystrophic EB. EB includes a group of inherited connective tissue disorders that cause blisters on the skin and mucous membranes resulting from genetic defects. DEB is mostly caused by mutations in the COL7A1 gene, which encodes the Col7 protein. To date, about 800 mutations in the COL7A1 gene have been reported. DEB has two patterns of inheritance: autosomal dominant (dominant DEB, DDEB) and autosomal recessive (recessive DEB, RDEB). DDEB is generally associated with reduced Col7 expression caused by a glycine substitution in the collagen-like domain of the collagen alpha 1 (VII) chain. RDEB is usually severe and is mostly caused by the absence or marked reduction of Col7 expression due to a premature stop codon (PTC) in the COL7A1 gene. In some embodiments, DEB (eg, DDEB or RDEB) is diagnosed following a physical examination, laboratory test results (eg, based on a skin biopsy), a medical history review, and / or genetic testing.In some embodiments, a diagnosis of DEB (eg, DDEB or RDEB) is confirmed via genetic testing.

[0045] As used herein, the terms "patient" and "subject" refer to an individual to be treated according to the methods provided herein. The subject may be a human or a non-human mammal, such as a non-human primate, pig, goat, horse, cow, dog, cat, rat, mouse, or rabbit. The subject is preferably a human. The subject may suffer from a skin disorder (e.g., a genodermatosis), such as DEB (e.g., DDEB or RDEB). The subject may have already been diagnosed with a skin disorder (e.g., a genodermatosis), such as DEB (e.g., DDEB or RDEB), and may optionally have undergone treatment therefor. The subject may be known to have a genetic mutation associated with DEB. The subject may be diagnosed with one or more symptoms or complications associated with DEB, such as skin wounds, including blister formation, itching, pain, skin erosions, scar formation, skin fragility, gangrene, skin aplasia or hypoplasia, skin hypopigmentation, milia, skin infections, cheilitis (e.g., inflammation of the lips), dystrophic fingernails or toenails, dental enamel abnormalities, pseudosyndactyly or camptodactyly of fingers or toes, syndactyly of fingers or toes, flexion contractures of the toes, dental caries, dysphagia (e.g., For example, poor swallowing), grooved or fissured tongue, esophageal stenosis, pharyngeal stenosis, growth disorder, dilated cardiomyopathy, pulmonary interstitial dysmorphology, acute constipation, hearing or vision problems, eczema, glomerulopathy, immunological hypersensitivity, nasolacrimal duct obstruction, anemia, chronic ear infections, corneal erosion, corneal abrasion or scarring, oral erosion, microstomia, osteopenia, ectropion, immunological hypersensitivity, nephrotic syndrome, phimosis, renal failure, urinary retention, ureteral stenosis, stroke, or squamous cell carcinoma. In some embodiments, the subject may have one or more symptoms or complications associated with DEB selected from skin wounds including blister formation, scar formation, gangrene, skin aplasia or hypoplasia, skin hypopigmentation, milia, skin infections, cheilitis (e.g., inflammation of the lip), dystrophic fingernails or toenails, dental enamel abnormalities, pseudosyndactyly or camptodactyly of fingers or toes, syndactyly of fingers or toes, flexion contractures of toes, dental caries, dysphagia (e.g., poor swallowing), grooved or fissured tongue, esophageal stenosis, and pharyngeal stenosis.In some embodiments, the subject may have one or more symptoms or complications associated with DEB selected from skin wounds, including blister formation, scar formation, skin hypoplasia or hypoplasia, cheilitis (e.g., inflammation of the lips), dystrophic fingernails or toenails, pseudosyndactyly or camptodactyly of fingers or toes, and syndactyly of fingers or toes. The subject may be of any age or developmental stage. In some embodiments, the subject is an infant or neonate. In some embodiments, the subject is less than about 24 months of age, e.g., less than about 20, 18, 16, 14, 12, 10, 8, 6, 5, 4, 3, 2, or 1 month of age. In some embodiments, the subject is at least about 24 months (e.g., 2 years) old, e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 years of age. In some embodiments, the subject is about 2-5 years old, about 2-6 years old, about 2-8 years old, about 2-10 years old, about 6-10 years old, about 6-12 years old, about 6-18 years old, about 8-12 years old, about 8-18 years old, about 12-18 years old, about 2-13 years old, about 6-13 years old, about 13-18 years old, or any range therein. In some embodiments, the subject is at least 18 years old.

[0046] As used herein, the term "pharmaceutical acceptable" refers to molecular entities and compositions that are physiologically tolerable and typically do not produce adverse reactions when administered to humans. Preferably, as used herein, the term "pharmaceutical acceptable" means approved by a federal or state government regulatory agency, or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, more particularly in humans, or generally recognized as safe for use, for example, in parenteral products.

[0047] As used herein, the terms "therapeutically effective dose" and "effective amount" refer to an amount of a compound sufficient to produce a therapeutic response. In the context of Col7 protein therapy, the terms "therapeutically effective dose" and "effective amount" can refer to an amount of a Col7 substance (e.g., rhCol7 substance), or a drug product or pharmaceutical composition containing the same, sufficient to produce a therapeutic response. A therapeutic response can be any response that a user (e.g., a clinician) recognizes as an effective response to a therapy. Thus, a therapeutic response can generally be an amount that can reduce one or more symptoms and / or complications of a disease or disorder, such as DEB (e.g., DDEB or RDEB), specifically inhibiting to some extent the burden and / or severity of skin wounds; reducing the number of skin wounds; reducing the size of a skin rash; inhibiting, including reducing, slowing, or completely stopping, the formation of blisters; closing chronic open wounds; increasing the healing rate of wounds and blisters; alleviating skin inflammation; and improving Col7 uptake into the epidermal-dermal junction.

[0048] As used herein, "treating," "treat," and "treatment" refer to administering a pharmaceutical composition to a subject having a disease or disorder, or a symptom or complication thereof, such that at least one symptom or complication of the disease or disorder is ameliorated, cured, alleviated, relieved, eliminated, delayed, or reduced. Treating EB (e.g., DEB, e.g., DDEB or RDEB) in a subject refers to administering a pharmaceutical composition (e.g., a pharmaceutical composition comprising a rhCol7 agent described herein) to a subject having EB such that at least one symptom or complication of EB is ameliorated, cured, alleviated, alleviated, eliminated, delayed, or reduced. Symptoms or complications of EB disease that may be targeted for treatment include, but are not limited to, skin wounds, including blister formation; pruritus; pain; lesions (e.g., rectal, anal, urethral lesions and / or mucosal lesions and / or lesions of squamous epithelial tissue); skin erosions; scar formation; skin fragility; gangrene; skin aplasia or hypoplasia; skin hypopigmentation; milia; skin infections; cheilitis (e.g., inflammation of the lips); dystrophic fingernails or toenails; gastrointestinal tract lesions; contractures, e.g., flexion contractures (e.g., of the limbs); pseudosyndactyly or camptodactyly of the hand or foot; carcinoma (e.g., squamous cell carcinoma); blister formation. ;nail and / or dental deformities;esophageal stenosis;pharyngeal stenosis;grooved or fissured tongue;tooth enamel abnormalities;tooth decay;dysphagia (e.g., poor swallowing);eye disorders;hearing or visual impairment;anemia;malnutrition;secondary skin infections;sepsis;hoarseness;urethral stenosis;phimosis;corneal scarring or erosion;corneal abrasion;oral erosions;microstomia;osteopenia;malabsorption;dilated cardiomyopathy;pulmonary interstitial dysmorphology;acute constipation;eczema;glomerulopathy;immunologic hypersensitivity;nasolacrimal duct obstruction;chronic ear infections;ectropion;immunologic hypersensitivity;nephrotic syndrome, phimosis;renal failure;urinary retention;ureteral stenosis;stroke; and growth disorders.

[0049] As used herein, the terms "preventing," "prevent" and "prevention" refer to administering a pharmaceutical composition to a subject, e.g., prior to clinical manifestations of an undesired condition (e.g., a disease or other undesirable condition in a host animal), such that it protects the host from the undesired condition or the onset of the symptom or condition. "Preventing" a disease may also be referred to as "prophylaxis" or "prophylactic treatment." In the context of the present disclosure, one or more symptoms or complications associated with EB, e.g., skin blistering or scarring, may be prevented by administration of a pharmaceutical composition (e.g., a pharmaceutical composition comprising a rhCol7 agent). Scarring in subjects with EB can result in one or more of the following symptoms: contractures, e.g., flexion contractures (e.g., of the limbs); pseudosyndactyly of the hands or feet; carcinomas (e.g., squamous cell carcinoma); rectal lesions; mucosal lesions; blister formation; blister formation after manual trauma; nail or dental deformities; esophageal strictures; eye disorders, anemia, malnutrition; secondary skin infections; sepsis; hoarseness; urethral stricture; phimosis; corneal scarring; malabsorption; and growth disorders. In some embodiments, symptoms or complications of EB disease (e.g., DEB, e.g., DDEB or RDEB) that may be prevented by administration of a pharmaceutical composition (e.g., a pharmaceutical composition comprising an rhCol7 agent) include, for example, skin wounds, including blister formation; pruritus; pain; lesions (e.g., rectal, anal, urethral lesions and / or mucosal lesions and / or lesions of squamous epithelial tissue); skin erosions; scarring; skin fragility; gangrene; skin aplasia or hypoplasia; hypopigmentation of the skin; milia; skin infections; cheilitis (e.g., inflammation of the lips); dystrophic fingernails or toenails; lesions of the gastrointestinal tract; contractures, e.g., flexion contractures (e.g., of the limbs); pseudosyndactyly of the hands or feet. The symptoms may be: camptodactyly; carcinoma (e.g., squamous cell carcinoma); blister formation; nail and / or dental deformities; esophageal stenosis; pharyngeal stenosis; grooved or fissured tongue; dental enamel abnormalities; dental caries; dysphagia (e.g., poor swallowing); eye disorders; hearing or vision problems; anemia; malnutrition; secondary skin infections; sepsis; hoarseness; urethral stenosis; phimosis; corneal scarring or erosion; corneal abrasion; oral erosions; microstomia; osteopenia; malabsorption; dilated cardiomyopathy; pulmonary interstitial dysmorphology; acute constipation; eczema; glomerulopathy; immunological hypersensitivity; nasolacrimal duct obstruction; chronic ear infection; ectropion; immunological hypersensitivity; nephrotic syndrome, phimosis; renal failure; urinary retention; ureteral stenosis; stroke; and growth disorders.

[0050] As used herein, the terms "improve," "increase," "reduce," and "diminish," and their grammatical equivalents, refer to the modulation of a value relative to a baseline measurement, e.g., a measurement in the same individual prior to the initiation of a treatment described herein, or a measurement in a control subject (or control subjects) in the absence of a treatment described herein.

[0051] As used herein, the term "dosing interval" refers to the amount of time that elapses between multiple doses administered to a subject (e.g., a human patient). A dosing interval may alternatively be referred to herein as a dosing frequency (e.g., the frequency at which a dose is administered to a subject). In some embodiments, a single dosing interval is used for the duration of a treatment with a Col7 substance (e.g., rhCol7 substance). Such a dosing interval may be considered to be "fixed" within a given dosing regimen. In some embodiments, multiple dosing intervals are used over the course of a treatment with a Col7 substance (e.g., rhCol7 substance). In some embodiments, the dosing interval is less than about 1 day, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 6 months, or longer. In some embodiments, the dosing interval is an individualized interval determined for a given subject (e.g., a human patient) based on, for example, pharmacokinetic / toxicokinetic (PK / TK) data or other information about the subject, such as age, weight, height, body mass index, disease severity, comorbidities, race, ethnicity, country of origin, genotype, and / or previous response to Col7 replacement therapy. The individualized dosing interval may be the same or different dosing interval as the fixed dosing interval (e.g., under another treatment scheme or for another subject). A treatment regimen comprising administration of a Col7 material (e.g., rhCol7 material) to a subject may include multiple dosing regimens, one or more of which may include a fixed dosing interval described herein, one or more of which may include an individualized dosing interval. In some embodiments, a treatment regimen comprising administration of a Col7 material (e.g., a rhCol7 material) to a subject comprises a first dosing regimen comprising administration of a Col7 material at a first dosing interval (e.g., at a first frequency), and a second dosing regimen comprising administration of the Col7 material at a second dosing interval (e.g., at a second frequency), both of which are fixed dosing intervals having different dosing frequencies, and the first dosing regimen is performed prior to the second dosing regimen.In some embodiments, a treatment regimen comprising administration of a Col7 material (e.g., a rhCol7 material) to a subject comprises a first dosing regimen comprising administration of the Col7 material at a first dosing interval (e.g., at a first frequency) and a second dosing regimen comprising administration of the Col7 material at a second dosing interval (e.g., at a second frequency), wherein the first dosing interval is a fixed dosing interval and the second dosing regimen is an individualized dosing interval, and wherein the first dosing regimen is administered prior to the second dosing regimen. In other embodiments, the treatment regimen comprising administering a Col7 material (e.g., rhCol7 material) to a subject comprises a first dosing regimen comprising administering a Col7 material at a first dosing interval and a second dosing regimen comprising administering a Col7 material at a second dosing interval, wherein the second dosing interval is a fixed dosing interval and the first dosing regimen is an individualized dosing interval, and wherein the first dosing regimen is administered prior to the second dosing regimen. In some embodiments, the dosing regimen comprises administering a dose of a drug material to a subject (e.g., a human patient) more than once a day, about once a day, about every 2 days, about every 3 days, about every 4 days, about every 5 days, about every 6 days, about once a week, about every 2 weeks, about every 3 weeks, about once a month, about every 2 months, about every 3 months, about every 6 months, or less frequently.

[0052] As used herein, the term "clinical parameter" refers to a parameter of clinical measurement. Clinical parameters may relate to complications and / or symptoms associated with DEB, such as the size and number of skin wounds, duration of chronic open wounds, healing rate of wounds and blisters, etc. Clinical parameters may be assessed by visual or physical examination, biopsy, and / or biochemical assays, such as assays of urine, blood, or plasma.

[0053] Drug Substances and Compositions Active substance: rhCol7 The present disclosure relates to intravenous rhCol7 replacement therapy for treating cutaneous and systemic symptoms of dystrophic epidermolysis bullosa (DEB), particularly recessive DEB (RDEB), in a subject. The active drug substance for systemic treatment of the clinical pathology of DEB is recombinant human collagen 7 protein (rhCol7). As described herein, anchoring fibrils formed by the assembly of Col7 dimers facilitate attachment of the epidermis to the underlying dermis. Pathogenic mutations in the gene encoding the collagen alpha 1 (type VII) chain polypeptide (COL7A1) result in abnormal, reduced, or complete absence of anchoring fibrils in the skin, resulting in DEB. This lack of normal functional anchoring fibrils contributes to cutaneous and non-cutaneous symptoms including poor epidermal-dermal adhesion, significant skin fragility, and severe blister formation in the skin and mucosa in DEB patients.

[0054] The direct mechanistic link between genetic defects or absence of Col7 in anchoring fibrils and DEB makes it appropriate to introduce native Col7 or a functional equivalent, such as recombinant Col7 (e.g., rhCol7), to treat DEB patients. Preclinical data demonstrate that intravenous administration of rhCol7 distributes to and is selectively retained in the dermal-epidermal BMZ, forms normal anchoring fibrils, and reverses dermal-epidermal separation by providing stability to dermal-epidermal adhesion at the stratum densa / upper papillary dermis interface, resulting in a statistically significant improvement in survival in a mouse model of DEB.

[0055] Specifically, RDEB is a serious systemic disease with no effective treatment. Current therapies in development are unlikely to address DEB's significant gastrointestinal / genitourinary (GI / GU) morbidity, corneal, and oral manifestations, or the development of squamous cell carcinoma (SCC), the primary cause of death. No other systemic disease-modifying approaches are in development. Recombinant collagen 7 has demonstrated a reasonably favorable toxicological profile, including all observations related to immune-mediated responses to "foreign" proteins. Thus, it is reasonable that intravenous delivery of rhCol7 would return functional Col7 to the dermal-epidermal BMZ. This reversion would promote the assembly of normal anchoring fibrils and provide stability to dermal-epidermal adhesions at the stratum densa / upper dermis papillary interface, which may correct blister dysplasia and complications in patients with DEB.

[0056] According to the present disclosure, the active ingredient of the drug substance is recombinant human α1 chain homotrimer of collagen type VII. In some embodiments, the active drug substance is a recombinant form of Col7 protein produced in engineered cells transfected with an expression plasmid encoding the full-length human collagen VII sequence.

[0057] In some embodiments, the alpha 1 chain (type VII collagen) polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the alpha 1 chain (type VII collagen) polypeptide consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the alpha 1 (type VII collagen) chain polypeptide may comprise an amino acid sequence that is about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 1. In some embodiments, the alpha 1 chain polypeptide may be encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:2. The rhCol7-encoding polynucleotide sequence may be about 75%, 80%, 85%, 86%, 875, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:2. In some embodiments, the rhCol7-encoding polynucleotide sequence may be codon optimized. Codon optimization may function to increase the glycine content of the encoding nucleotide sequence. In some embodiments, the rhCol7-encoding polynucleotide sequence comprises one or more modifications. In some embodiments, the one or more modifications increase the stability of the encoding polynucleotide.

[0058] In some embodiments, the rhCol7 material comprises multiple recombinant Col7 α1 chain polypeptides, and / or functional variants thereof. In some embodiments, the rhCol7 material is produced in a mammalian cell line engineered to express or manufacture rhCol7. In some embodiments, the rhCol7 material is produced using genetically modified mammalian cells. As used herein, the term "genetically modified" refers to a cell that expresses a particular gene product after introduction of a nucleic acid that includes a coding sequence that encodes the gene product. Introduction of the nucleic acid can be accomplished by any method known in the art, including gene targeting and homologous recombination. As used herein, the term also includes cells that have been engineered to express or overexpress a gene product that is not normally expressed by an endogenous gene or cell. In some embodiments, the rhCol7 material is produced in a mammalian cell, for example, a CHO cell. The CHO cell can be engineered to include a polynucleotide encoding the alpha 1 chain (type VII) and at least one polynucleotide encoding a protein that can increase expression of the Col7 alpha 1 chain in the host cell, for example, prolidase, prolyl 4-hydroxylase, and / or heat shock protein 47 (HSP47). The genetically engineered cell line used to produce rhCol7 may be referred to as a Master Cell Bank (MCB). In one preferred embodiment, rhCol7 producing cells may be derived from a transfected Chinese Hamster Ovary CHO-K1 host cell line transfected with a plasmid encoding rhCol7 and the prolyl hydroxylase alpha and beta subunits (P4H) to promote proline hydroxylation, a post-translational modification that stabilizes the rhCol7 structure. In some embodiments, the rhCol7 material is produced by a master cell co-expressing rhCol7 and prolidase as described in U.S. Pat. No. 9,676,837, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the rhCol7 material is produced by a master cell co-expressing rhCol7 and 4-hydroxylase as described in WO2020025129, the contents of which are incorporated herein by reference in their entirety.The rhCol7 material can be produced in cell culture, purified using a series of chromatography and filtration steps, and then processed into a drug product. Each lot of drug material and drug product can be assessed for quality and stability.

[0059] MCB cells expressing rhCol7 can be grown stepwise in vessels of increasing size using batch and / or perfusion culture to reach high cell density in the production bioreactor. Intermediate harvest from the production bioreactor can be harvested daily and clarified. The clarified harvest can be concentrated, frozen, and pooled for later purification to generate rhCol7 material. The clarified and concentrated harvest pool can be purified using multiple chromatography processes. In some instances, three chromatography steps can be used, including mixed mode, cation exchange, and hydrophobic interaction chromatography unit operations. For example, the recombinant product can be purified via a purification process including, but not limited to, ultrafiltration / dialysis (UF / DF) membrane capture, CaptoCore, SP Sepharose, and propolypropylene glycol (PPG) chromatography steps. The purification process may also include one or more viral reduction processes, for example, three orthogonal viral reduction steps including UV-C and detergent (Triton X-100) viral inactivation steps and viral filtration (e.g., Asahi Planova 35N). The processed rhCol7 material is preferably virus-free. The purified product may be concentrated and filtered (e.g., dialyzed) into a formulation buffer. Polysorbate 20 may be added to produce the final drug substance composition.

[0060] In some embodiments, the drug substance comprises one or more salts, surfactants, buffers, amino acids (e.g., non-essential amino acids), cryoprotectants, and / or chelating agents. In some embodiments, the drug substance comprises a non-essential amino acid, which is preferably arginine. In some embodiments, the drug substance comprises a phosphate buffer, which is preferably sodium phosphate. In some embodiments, the drug substance comprises a chelating agent, which is preferably sodium citrate. In some embodiments, the drug substance comprises a cryoprotectant, which is preferably a sugar, e.g., sucrose. In some embodiments, the drug substance comprises sodium chloride. In some embodiments, the drug substance comprises a surfactant, buffers, non-essential amino acids, cryoprotectants, and chelating agents. In some embodiments, the drug substance comprises sodium phosphate, sodium citrate, arginine (e.g., L-arginine), sucrose, sodium chloride, and polysorbate 20. In some embodiments, the drug substance comprises 1.2 mg / mL of recombinant rCol7 in 10 mM sodium phosphate, 5 mM sodium citrate, 100 mM L-arginine, 1.7% sucrose (w / v), 70 mM sodium chloride, and 0.05% (v / v) polysorbate 20, pH 7.2. In some embodiments, the bulk drug substance is 0.2 micrometer (μm) filtered, filled into sterile containers, and stored at ≦−60° C. for drug production.

[0061] The rhCol7 material is preferably free of bacterial and fungal adventitious infectious agents.The structural and functional characteristics of the rhCol7 material can be assessed and compared to the native Col7 protein.

[0062] In some embodiments, the rhCol7 agent is a systemic protein therapy to be delivered via the intravenous route. Formulations and Drug Products The drug product of the present disclosure contains at least one active ingredient, recombinant human Col7 (rhCol7). The rhCol7 material can be produced using MCB cells as discussed herein. The rhCol7 material can be present in the drug product at a concentration of about 0.01 milligrams per milliliter (mg / mL) to 100 mg / mL, about 0.1 mg / mL to 100 mg / mL, about 0.5 mg / mL to 100 mg / mL, about 1 mg / mL to 100 mg / mL, about 10 mg / mL to 100 mg / mL, about 0.01 mg / mL to 50 mg / mL, about 0.1 mg / mL to 50 mg / mL, about 0.5 mg / mL to 50 mg / mL, about 1 mg / mL to 50 mg / mL, about 0.01 mg / mL to 20 mg / mL, about 0. ... The target protein may be present in an amount of about 0.01 mg / mL to 20 mg / mL, about 0.1 mg / mL to 20 mg / mL, about 0.5 mg / mL to 20 mg / mL, about 1 mg / mL to 20 mg / mL, about 10 mg / mL to 50 mg / mL, about 0.01 mg / mL to 20 mg / mL, about 0.1 mg / mL to 20 mg / mL, about 0.5 mg / mL to 20 mg / mL, about 1 mg / mL to 20 mg / mL, about 0.01 mg / mL to 10 mg / mL, about 0.1 mg / mL to 10 mg / mL, about 0.5 mg / mL to 10 mg / mL, or about 1.0 mg / mL to 10 mg / mL.In some examples, the rhCol7 agent is present in the drug product at about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, about 1.1 mg / mL, about 1.2 mg / mL, about 1.3 mg / mL, about 1.4 mg / mL, about 1.5 mg / mL, about 1.6 mg / mL, about 1.7 mg / mL, about 1.8 mg / mL, about 1.9 mg / mL, about 2.0 mg / mL, about 2.1 mg / mL, about 2.2 mg / mL, about 2.3 mg / mL, about 2.4 mg / mL, about 2.5 mg / mL, about 2.6 mg / mL, about 2.7 mg / mL, about 2.8 mg / mL, about 2.9 mg / mL, about 3.0 mg / mL, about 3.1 mg / mL, about 3.2 mg / mL, about 3.4 mg / mL, about 3.5 mg / mL, about 3.6 mg / mL, about 3.7 mg / mL, about 3.8 mg / mL, about 3.9 mg / mL, about 4.0 mg / mL, about 4.1 mg / mL, about 4.2 mg / mL, about 4.3 mg / mL, about 4.4 mg / mL, about 4.5 mg / mL, about 4.6 mg / mL, about 4.7 mg / mL, about 4.8 mg / mL, about 4.9 mg / mL, about 5.0 mg / mL, about 5.1 mg / mL, about 5.2 mg / mL, about 5.3 mg / mL, about 5.4 mg / mL, about 5.5 mg / mL, about 5.6 mg / mL, In some embodiments, the drug product contains recombinant human collagen VII (rhCol7) at a target protein concentration of 1.2 mg / mL, about 2.4 mg / mL, about 2.5 mg / mL, about 2.6 mg / mL, about 2.7 mg / mL, about 2.8 mg / mL, about 2.9 mg / mL, about 3.0 mg / mL, about 3.5 mg / mL, about 4.0 mg / mL, about 4.5 mg / mL, about 5.0 mg / mL, about 5.5 mg / mL, about 6.0 mg / mL, about 6.5 mg / mL, about 7.0 mg / mL, about 7.5 mg / mL, about 8.0 mg / mL, about 8.5 mg / mL, about 9.0 mg / mL, about 9.5 mg / mL, about 10 mg / mL, about 15 mg / mL, or about 20 mg / mL. In some embodiments, the drug product contains recombinant human collagen VII (rhCol7) at a target concentration of 1.2 mg / mL.

[0063] In some embodiments, the rhCol7 drug product comprises at least one pharma- ceutically acceptable excipient, including those commonly used in parenteral biopharmaceutical formulations. In some examples, the excipients added in the rhCol7 drug product include, but are not limited to, dibasic sodium phosphate, sodium citrate dihydrate, sodium chloride, L-arginine HCl, sucrose, and polysorbate 20.

[0064] The buffer may be selected from phosphate buffered saline (PBS) or other suitable buffers. The buffer is suitable for the target pH range, excipients, and surfactants. In some cases, the rhCol7 drug product may have a pH in the range of about pH 6.8 to about pH 7.5. For example, the pH of the rhCol7 drug product may be about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5.

[0065] The rhCol7 drug products provided herein may have significantly improved solubility and reduced viscosity at higher protein concentrations. In some embodiments, agents are added to increase the stability of the drug substance of the product, including, but not limited to, L-arginine (e.g., 100 millimolar (mM) L-arginine) and sucrose.

[0066] In some embodiments, the rhCol7 formulation comprises about 5-200 mM, about 10-150 mM, about 20-150 mM, about 50-150 mM, about 50-100 mM, about 75-100 mM, or about 100 mM L-arginine.

[0067] In some embodiments, the rhCol7 formulation comprises about 10-100 mM, about 75-100 mM, about 50-100 mM, about 15-75 mM, about 35-75 mM, about 50-75 mM, or about 50 mM sucrose.

[0068] In some embodiments, a surfactant is included in the rhCol7 drug product. In some embodiments, the surfactant is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof. In some embodiments, the surfactant is polysorbate 20. In some embodiments, polysorbate 20 is present at a concentration ranging from about 0.01% to 0.2%, e.g., about 0.01% to 0.15%, about 0.01% to 0.1%, about 0.01% to 0.08%, or about 0.01% to 0.06%. In some embodiments, the drug product includes 0.05% polysorbate 20.

[0069] The drug product is formulated to provide acceptable freeze / thaw (F / T) performance to support storage at refrigerated conditions, e.g., either -20°C or ≦-60°C, and formulation conditions that allow for an improved stability profile.

[0070] In some embodiments, the rhCol7 drug product is ready-to-fill. Thus, all drug product excipients are incorporated during the manufacturing stage of the drug substance (i.e., rhCol7 production), e.g., MCB cell culture and culture medium harvesting and purification processes, and there are no additional excipients added during the manufacturing process of the rhCol7 drug product.

[0071] In some embodiments, the rhCol7 drug product of the present disclosure is sterile. In some embodiments, the rhCol7 drug product of the present disclosure is preservative-free. In some embodiments, the rhCol7 drug product is manufactured using aseptic processing techniques. The drug product may be sterile filtered into sterile depyrogenated glass vials, stoppered and sealed in an ISO5 / Class 100 / Grade A area. In-process testing may be performed to ensure sterility throughout the manufacturing process. In-process testing may include integrity testing before and / or after use for bioburden reduction (0.2 micrometer (μm) filtration), bioburden testing before sterile filtration, and / or endotoxin testing. Environmental and microbial monitoring, including particulates, may also be performed throughout the drug product manufacturing process.

[0072] In some embodiments, the rhCol7 drug product is prepared / manufactured directly from rhCol7 material from MCB cell cultures. In some embodiments, frozen rhCol7 material (e.g., culture medium harvested from MCB cultures) is removed from storage, thawed (e.g., in a temperature-controlled water bath), pooled, mixed, and then subjected to bioburden reduction filtration through a 0.5 / 0.2 μm membrane filter unit. Sterile filtration of the pooled bulk drug product can be performed using two consecutive 0.5 / 0.2 μm sterile filter units. The drug product can be aseptically filled into sterile vials (e.g., 10R glass vials with stoppers and flip caps), which are then stoppered and crimped. Filled vials can be 100% visually inspected and defective vials are rejected. All acceptable vials are labeled and packaged at room temperature and then stored at -60°C.

[0073] In some embodiments, the drug formulation containing recombinant human Col7 (rhCol7) material is in the form of a clear to slightly opalescent colorless solution. As a non-limiting example, the rhCol7 drug product is PTR-01, in which the rhCol7 material is formulated at a target concentration of 1.2 mg / mL of recombinant Col7 in 10 mM sodium phosphate, 5 mM sodium citrate, 100 mM L-arginine, 1.7% sucrose (w / v), 70 mM sodium chloride, and 0.05% (v / v) polysorbate 20, pH 7.2. Additional examples of rhCol7 drug products are described in International Publication No. WO2017112757, which is incorporated herein by reference in its entirety.

[0074] In some embodiments, the rhCol7 agent is formulated for systemic administration. In some embodiments, the rhCol7 agent is formulated for intravenous administration, for example, intravenous infusion. In some embodiments, the rhCol7 drug product is an aqueous solution for injection.

[0075] In some embodiments, the rhCol7 composition is supplied as a sterile, preservative-free frozen liquid in 10 mL clear borosilicate glass vials (10R, type I glass). Each vial may have a 20 millimeter (mm) gray butyl rubber stopper and a 20 mm aluminum flip cap. Each vial may be filled with a target volume of 5.5 milliliters (mL) to ensure a draw volume of at least 5.0 mL (i.e., a target weight of a 6.0 mg dose) from each vial. Each vial may be for single use.

[0076] In some embodiments, the rhCol7 drug product is stored at <-60°C and is stable for at least 24 months at <-60°C, or at least 32 months at <-60°C, or at least 36 months at <-60°C, at least 42 months at <-60°C, or at least 48 months at <-60°C. In other embodiments, the rhCol7 drug product may be stored at -20±5°C and is stable for at least 12 months at -20±5°C, or at least 18 months at -20±5°C, or at least 24 months at -20±5°C, or at least 30 months at -20±5°C, or at least 36 months at -20±5°C. In other embodiments, the rhCol7 drug product may be stored at 5±3°C and is stable for up to 1 month.

[0077] The drug product may be clinically prepared as a dosing solution prior to intravenous injection or infusion. During clinical use, the dosing solution may be prepared in a fixed volume in sterile saline (0.9% sodium chloride), with the final dosing solution volume being about 10-1000mL, about 20-1000mL, about 40-1000mL, about 50-1000mL, about 100-1000mL, about 200-1000mL, about 300-1000mL, about 400-1000mL, about 500-1000mL, about 10-500mL, about 20-500mL, about 40-500mL, about 50-500mL, about 100-500mL, about 200-500mL, about 300-500mL, about 400-500mL, about 10-300mL, about 20-300mL, about 40-300mL, about 50-300mL, about 100-300mL, or about 200-300mL, for example, about 10mL, 20mL, 30mL, 40mL, 50mL, 60mL, 70mL, 80mL, 90mL, 100mL, 150mL, 200mL, 250mL, 300mL, 350mL, 400mL, 450mL, 500mL, 600mL, 700mL, 800mL, 900mL, or 1000mL. The dosing solution may be administered intravenously using a dosage formulation in a clinical or hospital facility pharmacy. In some cases, the dosing solution may be prepared in an IV bag, for example, as a PVC IV bag. The dosing solution may be stable for up to 24 hours upon storage at ambient room temperature.

[0078] Diseases and uses The rCol7 substances, drug products, and compositions and methods of the present disclosure may provide preventative, palliative, or therapeutic support for skin wounds, disorders, or diseases in a subject, specifically skin disorders caused by Col7 deficiency, such as dystrophic epidermolysis bullosa (DEB), which includes two major subtypes: recessive dystrophic epidermolysis bullosa (RDEB) and dominant dystrophic epidermolysis bullosa (DDEB). Other skin disorders may include, but are not limited to, skin cancer, psoriasis, lichen planus, lupus, rosacea, eczema, cutaneous candidiasis, cellulitis, impetigo, decubitus ulcers, erysipelas, ichthyosis vulgaris, dermatomyositis, acroderma, stasis dermatitis, burns, and Netherton syndrome.

[0079] In some embodiments, recessive dystrophic epidermolysis bullosa (RDEB) includes all subtypes, such as severe generalized RDEB (formerly known as the Hallopeau-Siemens subtype), other generalized RDEB, inverse RDEB, pretibial RDEB, prurigomatous RDEB, centripetal RDEB, and neonatal transient blistering RDEB (Soro L. et al., J. Anesthel Dermatol. 2015, 8(5):41-46). In other embodiments, dominant dystrophic epidermolysis bullosa (DDEB) includes all subtypes, such as generalized DDEB, acral DDEB, pretibial DDEB, prurigomatous DDEB, onychomycosis, and neonatal transient blistering DDEB (Fine et al., J. Am. Acad. Dermatol. 2008, pp. 58931-950).

[0080] The rhCol7 substances, drug products, and compositions provided herein may provide therapeutic support, including alleviating, preventing, or preventing the progression of one or more complications and symptoms of DEB, particularly RDEB, including skin and systemic symptoms, in a patient.

[0081] According to the present disclosure, the use of the rhCol7 substances, drug products, and compositions provided herein to therapeutically treat a subject having RDEB by systemic administration of an effective amount of the rhCol7 substance may improve, increase, enhance, and / or replenish levels of collagen alpha 1 (type VII) chain polypeptide, in particular levels of Col7 present in the BMZ of the skin.

[0082] In some embodiments, the patient to be treated is diagnosed with RDEB. The patient may be diagnosed with RDEB by genetic testing for a mutation in the COL7A1 gene. In some embodiments, the subject is an adult. For example, the subject may be at least 18 years old. In some embodiments, the subject is an elderly subject. For example, the subject may be at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, or at least 80 years old. In some embodiments, the subject is a pediatric subject under 18 years old. In some embodiments, the subject is less than about 24 months old, for example, less than about 20, 18, 16, 14, 12, 10, 8, 6, 5, 4, 3, 2, or 1 month old. In some embodiments, the subject is at least about 24 months old (e.g., 2 years old), for example, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 years old. In some embodiments, the subject is about 2-5 years old, about 2-6 years old, about 2-8 years old, about 2-10 years old, about 2-12 years old, about 6-10 years old, about 6-12 years old, about 6-18 years old, about 8-12 years old, about 8-18 years old, about 12-18 years old, about 2-13 years old, about 6-13 years old, about 13-18 years old, or any range therein. In some embodiments, the subject is at least 18 years old. In some embodiments, the subject is an infant or neonate (e.g., a newborn). In some embodiments, the subject is less than about 24 months old, e.g., less than about 20, 18, 16, 14, 12, 10, 8, 6, 5, 4, 3, 2, or 1 month old. In some embodiments, the subject is less than about 1 year old. In some embodiments, the pediatric subject is diagnosed with RDEB. In some embodiments, the newborn subject is diagnosed with RDEB.

[0083] In some embodiments, the subject to be treated has chronic wounds, such as chronic open wounds and recurrent wounds. In some embodiments, the subject has chronic open wounds. In some embodiments, the subject has blisters. In other embodiments, the subject has recurrent wounds. In some embodiments, the subject has hypoplasia or hypoplasia of the skin. In some embodiments, the subject has pseudosyndactyly or camptodactyly of the fingers or toes, and syndactyly of the fingers or toes. In some embodiments, the subject has cheilitis. In some embodiments, the subject has dysphagia, fissured or fissured tongue, esophageal stenosis, and / or pharyngeal stenosis. In some embodiments, the subject has chronic or recurrent wounds, blisters, hypoplasia of the skin, hypoplasia of the skin, pseudosyndactyly or camptodactyly of the fingers or toes, syndactyly of the fingers or toes, cheilitis, dysphagia, fissured or fissured tongue, esophageal stenosis, pharyngeal stenosis, or a combination thereof.

[0084] In some embodiments, the subject is suspected of having a mutation in the COL7A1 gene. In some embodiments, the subject has undergone genetic testing to confirm a COL7A1 mutation.

[0085] Methods for Treating Epidermolysis Bullosa The present disclosure provides methods of treating a skin disorder, such as dystrophic epidermolysis bullosa (DEB, e.g., RDEB), or a symptom or complication thereof, using a Col7 material (e.g., rhCol7 material). Treating a subject with the methods provided herein may result in a change, such as an improvement, in the subject's condition as measured by visual inspection, physical examination, laboratory testing, self-report by the subject, or any other useful measure. Treating a subject with the methods provided herein may result in the alleviation, mitigation, reduction, improvement, delay in onset, delay in progression, elimination, and / or cure of one or more symptoms or complications of a skin disorder, such as DEB (e.g., RDEB). The present disclosure also provides methods of alleviating, alleviating, reducing, improving, delay in onset, delay in progression, elimination, and / or cure of one or more symptoms or complications of a skin disorder, such as DEB. Symptoms and complications of DEB (e.g., RDEB) may include, for example, skin and systemic symptoms. Symptoms or complications of DEB include, for example, skin wounds, including blister formation, itching, pain, skin erosions, scar formation, skin fragility, gangrene, skin aplasia or hypoplasia, skin hypopigmentation, milia, skin infections, cheilitis (e.g., inflammation of the lip), dystrophic fingernails or toenails, dental enamel abnormalities, pseudosyndactyly or camptodactyly of fingers or toes, syndactyly of fingers or toes, flexion contractures of the toes, tooth decay, pysphagia, ) (e.g., poor swallowing), grooved or fissured tongue, esophageal stenosis, pharyngeal stenosis, growth disorders, dilated cardiomyopathy, pulmonary interstitial dysmorphology, acute constipation, hearing or vision problems, eczema, glomerulopathy, immunological hypersensitivity, nasolacrimal duct obstruction, anemia, chronic ear infections, corneal erosion, corneal abrasion and scarring, oral erosion, microstomia, osteopenia, ectropion, immunological hypersensitivity, nephrotic syndrome, phimosis, renal failure, urinary retention, ureteral stenosis, stroke, or squamous cell carcinoma.

[0086] In some embodiments, the method of treating DEB (e.g., RDEB), or a symptom or complication thereof, in a subject comprises administering to the subject a Col7 agent (e.g., rhCol7 agent). In some embodiments, the subject has been diagnosed with DEB (e.g., RDEB) or is at risk of developing a severe skin disorder, e.g., DEB.

[0087] In some embodiments, the method of treating DEB (e.g., RDEB) or symptoms or complications thereof in a subject comprises administering to the subject a therapeutically effective amount of a rhCol7 drug product by systemic (e.g., intravenous) administration. In some embodiments, the rhCol7 drug product comprises recombinant human Col7 alpha 1 chain (type VII) as the active drug substance formulated in an aqueous injection solution. In some embodiments, the rhCol7 drug product is PTR-01.

[0088] In some embodiments, prior to receiving rhCol7 protein therapy, subjects with DEB are pre-screened and identified as suitable for treatment with the rhCol7 protein therapy. Pre-screening methods may include analyzing genetic mutations in the COL7A1 gene and / or measuring Col7 deficiency at the dermal-epidermal junction by immunofluorescence (IF) staining. Subjects may further be assessed for the presence of antibodies specific for Col7, e.g., Col7 neutralizing antibodies.

[0089] In some embodiments, the method further comprises analyzing a genetic mutation in the COL7A1 gene in the subject to be treated (e.g., prior to the first administration of rhCol7 protein therapy). In some embodiments, the method comprises selecting a subject for such treatment based on the subject carrying a mutation in the COL7A1 gene consistent with a recessive inheritance pattern (i.e., RDEB). In one aspect, the disclosure provides a method of treating a subject with RDEB comprising: i) identifying a subject carrying a COL7A1 mutation consistent with a recessive inheritance pattern; and ii) intravenously administering to the subject a pharma- ceutical effective amount of a rhCol7 drug product.

[0090] One or more parameters (e.g., complications and clinical symptoms associated with RDEB) may be evaluated following administration of rhCol7 protein therapy. In some embodiments, the one or more parameters are measured at the end of a defined dosing period, e.g., an initial dosing interval. In some embodiments, the one or more parameters are measured at a defined treatment duration, e.g., after 1 month, 3 months, 6 months, 1 year, 2 years, or later. In some embodiments, the one or more parameters are compared to an evaluation of the one or more parameters prior to the initiation of rhCol7 protein therapy. The evaluation of the one or more parameters may be used to determine one or more aspects of the dosing regimen, e.g., dosage, dosing frequency, or duration. In some embodiments, the method further comprises performing or reviewing serial photographic quantitative evaluations of wounds and blisters of the subject being treated. Such evaluations may assess, for example, wound surface area (WSA), percent wound healing, and / or time to re-blistering and may be beneficial in subjects with RDEB given the chronic and dynamic nature of wounds in RDEB patients (Solis D. et al., J Invest Dermatol 2018, Vol. 138, Suppl., p. 97, doi.org / 10.1016 / j.jid.2018.03.580).

[0091] In some embodiments, rhCol7 treatment increases wound healing (e.g., frequency and / or completeness or persistence of wound healing) in subjects with RDEB. In some embodiments, while the subject is on rhCol7, the subject's wound heals faster than it would have otherwise healed or had already healed before the start of treatment. In some embodiments, while the subject is on rhCol7, the subject's wound heals more completely and / or more persistently than it would have otherwise healed or had already healed before the start of treatment. The wound may be a chronic open wound and / or a recurrent wound. In some embodiments, wound healing is sustained. In some embodiments, the majority of visible wounds or lesions are improved. In some embodiments, the improvement in wound lesions is at least a level 2 according to a 7-point Global Impression of Change scale (Table 1). In some embodiments, the improvement in wounds or lesions is assessed via visual inspection by a physician and / or caregiver. In some embodiments, the improvement in wounds or lesions is assessed via patient interview or self-assessment.

[0092] In some embodiments, rhCol7 treatment increases the deposition of rCol7 in the DEJ of skin. The deposition of collagen 7 protein in the DEJ can induce the formation of anchoring fibrils. Thus, the present disclosure further provides a method of inducing the formation of anchoring fibrils in skin, comprising administering rhCol7 to a subject (e.g., as described herein).

[0093] In some embodiments, rCol7 treatment may reduce wound surface area in a subject, including wound surface area of ​​target lesions and / or total body wound surface area. Wound surface area may be measured by imaging the wound area. In some embodiments, skin wounds are reduced in size following rCol7 therapy. Skin wound size may be reduced, for example, by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, skin wounds heal or close completely (e.g., reduced in size by about 100%) in response to rCol7 therapy. In some embodiments, the number and abundance of skin wounds and lesions are reduced in a subject following rCol7 therapy.

[0094] In some embodiments, rCol7 treatment improves skin integrity as assessed, for example, by a decrease in suction blister time and / or localization and / or an increase in time to re-blistering.

[0095] In some embodiments, rCol7 treatment reduces pain associated with skin wounds, for example, pain severity and / or the impact of pain on quality of life, as may be assessed by the modified PROMIS subscales and iscorEB.

[0096] In some embodiments, rCol7 treatment reduces itch or irritation associated with skin wounds, e.g., the severity of the itch or irritation and / or the impact of the itch or irritation on quality of life, as may be assessed by the modified PROMIS subscales and iscorEB.

[0097] In some embodiments, rCol7 treatment reduces or stabilizes systemic features of RDEB including dysphagia (e.g., as measured by the Brief Esophageal Dysphagia Scale and / or oral nutritional intake), corneal symptoms (e.g., as measured by the Eye Symptoms Scale), nutritional status (e.g., as measured by biomarkers Hgb / Hct, total protein / albumin, and / or Fe / TIBC), inflammation (e.g., as measured by markers for inflammation and / or CRP), fibrosis (e.g., as measured by fibrosis biomarkers), DEB-related activity, and / or mental health and social functioning.

[0098] In some embodiments, rCol7 treatment improves the overall quality of life, general health, and disability of subjects with RDEB. In some embodiments, the improvement in quality of life, health, and / or disability is measured by patient interview and / or self-assessment. In some embodiments, the improvement in quality of life, health, and / or disability is measured by caregiver interview and / or assessment.

[0099] In some embodiments, rCoL7 treatment reduces pain associated with RDEB, reduces blisters and / or wound formation, increases the rate of healing of skin blisters and / or skin wounds, increases the rate of wound closure, increases the rate of wound healing, reduces the time to re-blistering and / or re-wounding, increases skin integrity (i.e., reduces the likelihood of tearing or blisters due to mechanical or environmental stress), reduces the number of chronic blisters and / or wounds, reduces wound surface area, including total wound area, reduces the number of concomitant medications required to control RDEB symptoms, reduces the number of infected blisters and / or wounds, reduces pruritus, reduces inflammation associated with RDEB, and / or reduces the incidence of infection. In some embodiments, rCoL7 treatment reduces the risk of anemia, which is typically associated with larger recurrent wound size and longer time to open wound closure in subjects with RDEB.

[0100] Administration For example, the methods provided herein for treating a skin disorder, e.g., DEB (e.g., RDEB or DDEB), and relieving, alleviating, reducing, ameliorating, delaying the onset of, slowing the progression of, eliminating, and / or curing a symptom or complication of a skin disorder, e.g., DEB, can include systemic administration of a Col7 drug substance (e.g., rhCol7 substance). Systemic administration is suitable for delivery of rhCol7 protein to the entire body of a subject's skin, as well as other areas of the body that may be affected by a systemic skin disorder, e.g., DEB (e.g., RDEB or DDEB).

[0101] In some embodiments, systemic administration of a Col7 material (e.g., rhCol7 material) comprises intravenous injection or infusion. As a non-limiting example, the rhCol7 composition can be administered by slow infusion via the intravenous route of administration.

[0102] In some embodiments, the rhCol7 drug product is administered by bolus injection, slow push injection, or intravenous infusion. In some embodiments, administration is by intravenous infusion over a period of time, for example, less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours, less than about 90 minutes, less than about 80 minutes, less than about 70 minutes, less than about 60 minutes, less than about 50 minutes, less than about 45 minutes, less than about 30 minutes, less than about 20 minutes, or less than about 10 minutes. For example, the intravenous infusion can last for about 1-10 minutes, about 1-5 minutes, about 3-10 minutes, about 5-10 minutes, or 10-30 minutes. In some embodiments, the intravenous infusion lasts for about 30 minutes to 4 hours, for example, about 30-60 minutes, about 30-90 minutes, about 1-4 hours, about 1-3 hours, or about 2-4 hours.

[0103] In some embodiments, the rhCol7 protein therapy is administered at a dose of 0.1 mg / kg / min to 5.0 mg / kg / min, for example, about 0.1 mg / kg / min, 0.2 mg / kg / min, 0.3 mg / kg / min, 0.4 mg / kg / min, 0.5 mg / kg / min, 0.6 mg / kg / min, 0.7 mg / kg / min, 0.8 mg / kg / min, 0.9 mg / kg / min, 1.0 mg / kg / min, 1.1 mg / kg / min, 1.2 mg / kg / min, 1.4 mg / kg / min, 1.6 mg / kg / min, 1.8 mg / kg / min, 1.9 mg / kg / min, 1.2 mg / kg / min, 1.4 mg / kg / min, 1.8 mg / kg / min, 1.9 mg / kg / min, 1.2 mg / kg / min, 1.4 mg / kg / min, 1.6 mg / kg / min, 1.8 ... Administered at an infusion rate of 0.2 mg / kg / min, 1.3 mg / kg / min, 1.4 mg / kg / min, 1.5 mg / kg / min, 1.6 mg / kg / min, 1.7 mg / kg / min, 1.8 mg / kg / min, 1.9 mg / kg / min, 2.0 mg / kg / min, 2.5 mg / kg / min, 3.0 mg / kg / min, 3.5 mg / kg / min, 4.0 mg / kg / min, 4.5 mg / kg / min, or 5.0 mg / kg / min.

[0104] Medication Regimen In some embodiments, two or more doses of rhCol7 drug product are administered to the subject. In some embodiments, the dosing interval (e.g., the time between doses during a dosing regimen) is less than about 1 day, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 1 year or more. In some embodiments, the subject is administered a set number of doses of rhCol7 substance in a set period of time. For example, the subject may receive 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses over a set period of time, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 1 year, or other period of time.

[0105] In some embodiments, rhCol7 treatment continues for at least about 1 month, e.g., at least about 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years or more. In some embodiments, rhCol7 treatment continues for the life of the subject.

[0106] In some embodiments, the dosing is based on body weight. In some embodiments, the rhCol7 agent is administered at a dose of about 0.01 milligrams per kilogram of subject (mg / kg) to 50 mg / kg of drug agent, e.g., about 0.05 mg / kg to 50 mg / kg, about 0.1 mg / kg to 50 mg / kg, about 0.5 mg / kg to 50 mg / kg, about 1 mg / kg to 50 mg / kg, about 5 mg / kg to 50 mg / kg, about 0.01 mg / kg to 20 mg / kg, about 0.1 mg / kg to 20 mg / kg, about 0.5 mg / kg to 20 mg / kg, about 1 mg / kg to 20 mg / kg, about 0.01 mg / kg to 10 mg / kg, about 0.1 mg / kg to 10 mg / kg, The rhCol7 agent is administered at a dose of about 0.5 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 3 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.3 mg / kg to about 5 mg / kg, about 0.5 mg / kg to about 5 mg / kg, about 1.0 mg / kg to about 5.0 mg / kg, about 3.0 mg / kg to about 5.0 mg / kg, about 0.01 mg / kg to about 3 mg / kg, about 0.1 mg / kg to about 3 mg / kg, about 0.3 mg / kg to about 3 mg / kg, about 0.5 mg / kg to about 3 mg / kg, about 1 mg / kg to about 3 mg / kg, or any useful range thereof. In some embodiments, the rhCol7 agent is administered at a dose of about 0.3 mg / kg to about 5 mg / kg.In some embodiments, the rhCol7 agent is at about 0.01 mg / kg, about 0.03 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about The compound is administered at a dose of about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.5 mg / kg, about 5.0 mg / kg, about 6.0 mg / kg, about 7.0 mg / kg, about 8.0 mg / kg, about 9.0 mg / kg, about 10 mg / kg, about 15 mg / kg, or about 20 mg / kg.

[0107] In some embodiments, rhCol7 protein treatment involves thawing and diluting frozen or lyophilized rhCol7 material with a pharma- ceutically acceptable carrier suitable for intravenous injection, e.g., sterile saline solution, thereby forming a drug solution; and administering a preselected dose, e.g., about 0.01 mg / kg, about 0.03 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg and withdrawing a volume of the drug solution to provide a dose of about 0.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.5 mg / kg, about 5.0 mg / kg, about 6.0 mg / kg, about 7.0 mg / kg, about 8.0 mg / kg, about 9.0 mg / kg, about 10 mg / kg, about 15 mg / kg, or about 20 mg / kg.

[0108] In some embodiments, a suitable saline solution contains sodium chloride. In some examples, the sodium chloride is present in a concentration ranging from about 0-2% (e.g., about 0-1.5% or 0-1.0%). In some embodiments, the sodium chloride is present in a concentration of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0%. In some embodiments, the saline solution is present in a concentration of greater than about 2.0%. In one example, the rhCol7 drug product is diluted in 0.9% saline to form a drug solution for administration.

[0109] In some embodiments, a single dosing interval is used over the course of therapy with a Col7 substance (e.g., rhCol7 substance). In some embodiments, multiple dosing intervals are used over the course of therapy with a Col7 substance (e.g., rhCol7 substance). In some embodiments, the dosing interval is fixed. In some embodiments, the dosing interval is an individualized interval determined for a given subject, for example, based on pharmacokinetic / toxicokinetic (PK / TK) data or other information about the subject, such as age, weight, height, body mass index, disease severity, comorbidities, race, ethnicity, country of origin, genotype, and / or previous response to Col7 replacement therapy. The combination of individualized dose and dosing interval may be the same or different as for a fixed interval regimen. In some embodiments, a subject initially receives a fixed dosing interval for the first two or more doses, and then switches to an individualized dosing interval depending on follow-up measurements after the first dose. Alternatively, the regimen may be initially in an individualized dosing interval, and then it may switch to a fixed dosing interval.

[0110] In some embodiments, a single dose level is used over the course of therapy with a Col7 substance (e.g., rhCol7 substance). In some embodiments, multiple dose levels are used over the course of therapy with a Col7 substance (e.g., rhCol7 substance). In some embodiments, a dose corresponding to a first dose interval of a first dosing regimen is different from a dose corresponding to a second dose interval of a second dosing regimen. In some embodiments, a first dosing regimen includes dosing a subject at a first dose level and a first dose interval, and a second dosing regimen includes dosing a subject at a second dose level and a second dose interval, and the first dose level and the second dose level are the same. In some embodiments, a first dosing regimen includes dosing a subject at a first dose level and a first dose interval, and a second dosing regimen includes dosing a subject at a second dose level and a second dose interval, and the first dose level and the second dose level are different. In some embodiments, the first dosage regimen precedes the second dosage regimen, and the first dose level is lower than the second dose level. In some embodiments, the first dosage regimen precedes the second dosage regimen, and the first dose level is higher than the second dose level. In some embodiments, the first dosage regimen precedes the second dosage regimen, and the first dosage interval is shorter than the second dosage interval. In some embodiments, the first dosage regimen precedes the second dosage regimen, and the first dosage interval is longer than the second dosage interval.

[0111] In some embodiments, a method of treating a skin disorder (e.g., DEB, e.g., RDEB or DDEB) or alleviating, alleviating, reducing, ameliorating, delaying the onset of, delaying the progression of, eliminating, and / or curing a symptom or complication of a skin disorder, e.g., DEB, comprises administering a Col7 material (e.g., rhCol7 material) according to a first dosage regimen and a second dosage regimen: i) the first dosage regimen comprises administering a first therapeutically effective amount of the material according to a first dosage interval; ii) the second dosage regimen comprises administering a second therapeutically effective amount of the material according to a second dosage interval. The administration according to the first dosage regimen may occur before the administration according to the second dosage regimen. The first dosage regimen may be referred to as a "loading phase" and the second dosage regimen may be referred to as a "maintenance phase". In some embodiments, the first therapeutically effective amount is greater than the second therapeutically effective amount. In some embodiments, the first therapeutically effective amount is less than the second therapeutically effective amount. In some embodiments, the first therapeutically effective amount is the same as the second therapeutically effective amount. In some embodiments, the first dosing interval is the same as the second dosing interval. In some embodiments, the first dosing interval is longer than the second dosing interval (e.g., the first dosing frequency is lower than the second dosing frequency). In some embodiments, the first dosing interval is shorter than the second dosing interval (e.g., the first dosing frequency is higher than the second dosing frequency). In some embodiments, the first dosing interval is a fixed dosing interval and the second dosing interval is an individualized dosing interval. In some embodiments, the first dosing interval is an individualized dosing interval and the second dosing interval is a fixed dosing interval. In some embodiments, the first dosing interval and the second dosing interval are both fixed dosing intervals. In some embodiments, the second dosing regimen lasts longer than the first dosing regimen. In some embodiments, the first dosing regimen lasts longer than the second dosing regimen.

[0112] In some embodiments, the first dosing regimen includes administering at least one dose of the substance to the subject, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more doses. In some embodiments, the first dosing regimen includes administering the Col7 substance to the subject at least once a week, for example, at least 2, 3, 4, 5, 6, or 7 times a week. In some embodiments, the first dosing interval is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days (e.g., 1 week), 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the first dosing regimen includes administering the Col7 substance to the subject once a week. In some embodiments, the first dosing regimen includes administering the Col7 substance to the subject less than once a week, for example, once every 2 weeks, once every 3 weeks, or once every 4 weeks. In some embodiments, the first dosing regimen lasts for 1 to 12 weeks, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.In some embodiments, the first dosing regimen comprises about 0.1 to 50 mg / kg of the Col7 agent, e.g., about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg, about 4.9 mg / kg, about 5.0 mg / kg, about 5.1 mg / kg, about 5.2 mg / kg, about 5.3 mg / kg 4mg / kg, about 2.5mg / kg, about 2.6mg / kg, about 2.7mg / kg, about 2.8mg / kg, about 2.9mg / kg, about 3.0mg / kg, about 3.1mg / kg, about 3.2mg / kg, about 3.3mg / kg, about 3.4mg / kg, about 3.5mg / kg, about 3.6mg / kg, about 3.7mg / kg, about 3.8mg / kg, about 3.9mg / kg, about 4.0mg / kg, about 4.1mg / kg, about 4.2mg / kg, about 4.3mg / kg, about 4.4mg / kg, about 4.5mg / kg, about 4.6mg / kg, about 4.7mg / kg, about 4.8mg / kg, about 4.9mg / kg, about 5.0mg / kg, about 5.2, about 5.4mg / kg 5.0 mg / kg, about 6.0 mg / kg, about 6.2 mg / kg, about 6.4 mg / kg, about 6.6 mg / kg, about 6.8 mg / kg, about 7.0 mg / kg, about 7.5 mg / kg, about 8.0 mg / kg, about 8.5, about 9.0 mg / kg, about 9.5 mg / kg, about 10.0 mg / kg, about 11.0 mg / kg, about 12.0 mg / kg, about 13.0 mg / kg, about 14.0 mg / kg, about 15.0 mg / kg, about 20.0 mg / kg, about 25.0 mg / kg, about 30.0 mg / kg, about 35.0 mg / kg, about 40.0 mg / kg, about 45.0 mg / kg, or about 50.0 mg / kg.

[0113] In some embodiments, the second dosing regimen comprises administering at least one dose of the substance to the subject, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 52, 60, 70, 80, 90, 100 or more doses. In some embodiments, the second dosing regimen continues for at least about one month, for example, at least about 1, 3, 6, 8, 9, 12, 18, 24, 36 or more months. In some embodiments, the second dosing regimen continues from its initiation until the end of the subject's life. In some embodiments, the second dosing regimen comprises administering the Col7 substance to the subject about every day, about every 2 days, about every 3 days, about every 4 days, about every 5 days, about every 6 days, about once a week, about every 2 weeks, about every 3 weeks, about once a month, about every 2 months, about every 3 months, about every 6 months, or less frequently. In some embodiments, the second dosing interval is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days (e.g., 1 week), 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, or more. In some embodiments, the second dosing regimen comprises administering the Col7 substance to the subject about every 2 weeks. In some embodiments, the second dosing regimen comprises administering the Col7 substance to the subject about every 4 weeks.In some embodiments, the second dosing regimen comprises about 0.1 to 50 mg / kg of the Col7 agent, e.g., about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 3.1 mg / kg, about 3.2 ...3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg / kg, about 1.8mg / kg, about 1.9mg / kg, about 2.0mg / kg, about 2.1mg / kg, about 2.2mg / kg, about 2.3mg / kg, about 2.4mg / kg, about 2.5mg / kg, about 2.6mg / kg, about 2.7mg / kg, about 2.8mg / kg, about 2.9mg / kg, about 3.0mg / kg, about 3.1mg / kg, about 3.2mg / kg, about 3.3mg / kg, about 3.4mg / kg, about 3.5mg / kg, about 3.6mg / kg, about 3.7mg / kg, about 3.8mg / kg, About 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg, about 4.9 mg / kg, about 5.0 mg / kg, about 5.2 mg / kg, about 5.4 mg / kg, about 5.6 mg / kg, about 5.8 mg / kg, about 6.0 mg / kg, about 6.2 mg / kg, about 6.4 mg / kg, about 6.6 mg / kg, about 6.8 mg / kg, about 7.0 ... In some embodiments, the present invention relates to a method for administering a dose of about 1 mg / kg, about 7.5 mg / kg, about 8.0 mg / kg, about 8.5 mg / kg, about 9.0 mg / kg, about 9.5 mg / kg, about 10.0 mg / kg, about 11.0 mg / kg, about 12.0 mg / kg, about 13.0 mg / kg, about 14.0 mg / kg, about 15.0 mg / kg, about 20.0 mg / kg, about 25.0 mg / kg, about 30.0 mg / kg, about 35.0 mg / kg, about 40.0 mg / kg, about 45.0 mg / kg, or about 50.0 mg / kg.

[0114] In some embodiments, the second therapeutically effective amount and / or the second dosing interval of the second dosing regimen is determined, at least in part, based on evaluating the subject during or after the first dosing regimen. In some embodiments, the method further comprises evaluating the subject prior to the second dosing regimen, for example, to evaluate the effectiveness of the Col7 material and / or the subject's response to the Col7 material. In some embodiments, the method further comprises evaluating one or more clinical parameters during the first dosing regimen, during the second dosing regimen, and / or between the first and second dosing regimens.

[0115] The methods provided herein may include a first dosing regimen (loading phase) and a second dosing regimen (maintenance phase). The loading phase at the beginning of treatment may include an initial consolidation period, at least in part, due to the severe deficiency of endogenous collagen 7 in subjects with DEB (e.g., RDEB). Without being bound by theory, the consolidation dosing regimen may provide a sufficient amount of rhCol7 to the skin to increase fibril formation. In some embodiments, the loading regimen includes administration of a dose of the rCol7 composition twice a day, every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every week, or every 2 weeks. In some embodiments, the loading regimen includes 2, 3, 4, 5, 6 or more doses, for example, the loading regimen may include 3 or more doses, or 4 or more doses, or 5 or more doses. In some embodiments, the loading dosing regimen continues for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months or more. In some embodiments, the loading dose is about 3 mg / kg of rhCol7.

[0116] The maintenance dosing regimen may include dosing the subject less frequently than the loading dosing regimen. In some embodiments, the maintenance dosing regimen includes administration of a single dose of the rCol7 composition every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every week, every 2 weeks, every 3 weeks, every 4 weeks, every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, or every year. The rCol7 drug product may be administered in the loading and maintenance phases at the same dose or alternatively at different doses. As a non-limiting example, the dose in the loading dosing phase is about 3.0 mg / kg, and the dose administered in the maintenance dosing phase is about 3.0 mg / kg. The maintenance period may last for 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, 2 years, 5 years, 10 years or more, for example, for the life of the patient.

[0117] In some embodiments, the dosing regimen (e.g., the first dosing regimen and / or the second dosing regimen), including the dose frequency, dose, and duration, is personalized according to the subject's response, side effects, and treatment efficacy. In some embodiments, the dosing regimen (e.g., the first dosing regimen and / or the second dosing regimen), including the dose frequency, dose, and duration, is fixed (e.g., not individualized based on subject details).

[0118] In some embodiments, the treatment regimen comprises administering to a patient with RDEB at least one loading dose of about 3.0 mg / kg of the rCol7 composition daily, twice daily, three times daily, or weekly, and at least one maintenance dose of about 3.0 mg / kg weekly. In some embodiments, the dosing interval during the loading phase is about 1 day. In some embodiments, the dosing interval during the loading phase is about 1 week.

[0119] In some embodiments, the treatment regimen comprises administering to a patient with RDEB at least one loading dose of about 3.0 mg / kg of the rCol7 composition daily, twice daily, three times daily, or weekly, and at least one maintenance dose of about 3.0 mg / kg every other week. In some embodiments, the dosing interval during the loading phase is about one day. In some embodiments, the dosing interval during the loading phase is about one week.

[0120] In some embodiments, the treatment regimen comprises administering to a patient with RDEB at least one loading dose of about 3.0 mg / kg of the rCol7 composition daily, twice daily, three times daily, or weekly and at least one maintenance dose of about 3.0 mg / kg monthly. In some embodiments, the dosing interval during the loading phase is about 1 day. In some embodiments, the dosing interval during the loading phase is about 1 week.

[0121] In some embodiments, the treatment regimen comprises administering to a patient with RDEB at least one loading dose of about 3.0 mg / kg of the rCol7 composition daily, twice daily, three times daily, or weekly and at least one maintenance dose of about 3.0 mg / kg every two months. In some embodiments, the dosing interval during the loading phase is about one day. In some embodiments, the dosing interval during the loading phase is about one week.

[0122] In some embodiments, the treatment regimen comprises administering to a patient with RDEB at least one loading dose of about 3.0 mg / kg of the rCol7 composition daily, twice daily, three times daily, or weekly and at least one maintenance dose of about 3.0 mg / kg every three months. In some embodiments, the dosing interval during the loading phase is about one day. In some embodiments, the dosing interval during the loading phase is about one week.

[0123] In some embodiments, the methods of the disclosure include intravenously administering to a subject a Col7 material (e.g., a rhCol7 material) according to a first dosing regimen and a second dosing regimen, the first dosing regimen comprising: (i) administering to a subject about 0.1-5.0 mg / kg, about 0.3-5.0 mg / kg, about 0.5-5.0 mg / kg, about 1.0-5.0 mg / kg, about 0.1-3.0 mg / kg, about 0.3-3.0 mg / kg, about 0.5-3.0 mg / kg, or about 1.0-3.0 mg / kg of Col7 material, e.g., about 0.1 mg / kg, about 0.3 mg / kg, (ii) a first loading dose of about 0.1-5.0 mg / kg, about 0.3-5.0 mg / kg, about 0.5-5.0 mg / kg, about 1.0-5.0 mg / kg, 0.1-3.0 mg / kg, about 0.3-3.0 mg / kg, about 0.5-3.0 mg / kg, or about 1.0-3.0 mg / kg, e.g., about 0.1 mg / kg, about 0.3 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 3.0 mg / kg, or about 4 ... (iii) a first loading dose of about 0.1-5.0 mg / kg, about 0.3-5.0 mg / kg, about 0.5-5.0 mg / kg, about 1.0-5.0 mg / kg, about 0.1-3.0 mg / kg, about 0.3-3.0 mg / kg, about 0.5-3.0 mg / kg, or about 1.0-3.0 mg / kg, e.g., about 0.1 mg / kg, about 0.3 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 3.0 mg / kg, or about 5.0 mg / kg of the Col7 substance. and optionally (iv) a fourth loading dose of about 0.1-5.0 mg / kg, about 0.3-5.0 mg / kg, about 0.5-5.0 mg / kg, about 1.0-5.0 mg / kg, about 0.1-3.0 mg / kg, about 0.3-3.0 mg / kg, about 0.5-3.0 mg / kg, or about 1.0-3.0 mg / kg, e.g., about 0.1 mg / kg, about 0.3 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 3.0 mg / kg, or about 5.0 mg / kg of the Col7 agent, administered in the fourth week of treatment.In some embodiments, at least one of the first loading dose, the second loading dose, the third loading dose, and the optional fourth loading dose is different from the other three. In some embodiments, the first loading dose, the second loading dose, the third loading dose, and the optional fourth loading dose are the same. In some embodiments, the first loading dose is about 3.0 mg / kg. In some embodiments, the second loading dose is about 3.0 mg / kg. In some embodiments, the third loading dose is about 3.0 mg / kg. In some embodiments, the first loading dose, the second loading dose, the third loading dose, and the optional fourth loading dose are each 3.0 mg / kg. In some embodiments, the second dosing regimen (maintenance phase) is implemented after the completion of the first dosing regimen (loading phase). In some embodiments, the second dosing regimen is administered immediately after the completion of the first dosing regimen (e.g., after the administration of the final dose of the first dosing regimen and the corresponding dosing interval). In some embodiments, a drug holiday is used between the first dosing regimen and the second dosing regimen. In some embodiments, the second dosing regimen comprises the administration of a dose of the Col7 substance daily, twice daily, weekly, biweekly, 3 weeks, 4 weeks, monthly, bimonthly, 3 months, 4 months, 5 months, 6 months, or yearly. In some embodiments, the second dosing regimen comprises administration of about 0.1-5.0 mg / kg, about 0.3-5.0 mg / kg, about 0.5-5.0 mg / kg, about 1.0-5.0 mg / kg, about 0.1-3.0 mg / kg, about 0.3-3.0 mg / kg, about 0.5-3.0 mg / kg, or about 1.0-3.0 mg / kg, e.g., about 0.1 mg / kg, about 0.3 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 3.0 mg / kg, or about 5.0 mg / kg of the Col7 material. In some embodiments, the second dosing regimen comprises administration of about 3.0 mg / kg of the Col7 material. In some embodiments, the duration of the second dosing regimen is such that the subject receives the maintenance dose for more than 1 year, or more than 5 years, or more than 10 years, or for the subject's lifetime.

[0124] In some embodiments, a method of treating a skin disorder (e.g., DEB, e.g., RDEB or DDEB) or alleviating, ameliorating, reducing, ameliorating, delaying the onset of, slowing the progression of, eliminating, and / or curing a symptom or complication of a skin disorder, e.g., DEB, in a subject (e.g., a human subject) in need thereof, comprises administering a Col7 material (e.g., rhCol7 material) according to a first dosing regimen and a second dosing regimen: i) a first dosing regimen; The regimen comprises administering a first therapeutically effective amount of the substance according to a first dosing interval; ii) the second dosing regimen comprises administering a second therapeutically effective amount of the substance according to a second dosing interval: a) the first dosing regimen continues for at least one week, the first frequency is weekly, and the first effective amount is about 0.1-10 mg / kg; b) the second dosing regimen continues for at least four weeks, the second frequency is biweekly, and the second effective amount is about 0.1-10 mg / kg. In some embodiments, administration according to the first dosing regimen is performed prior to administration according to the second dosing regimen. In some embodiments, administration according to the first dosing regimen is performed immediately prior to administration according to the second dosing regimen (e.g., as described herein). In some embodiments, there is no gap between the first and second dosing regimens (e.g., the second dosing regimen begins after the final dose of the first dosing regimen is administered and the corresponding dosing interval has elapsed). In some embodiments, there is a gap of at least 1 day, e.g., at least 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks or more, between the first and second dosing regimens. In some embodiments, the first dosing regimen lasts for 4 weeks. In some embodiments, the second dosing regimen lasts for at least 7 weeks. In some embodiments, the second dosing regimen comprises administering the Col7 substance to the subject for the lifetime of the subject. In some embodiments, the first effective amount and the second effective amount are the same.In some embodiments, the first effective amount is about 0.1-5.0 mg / kg, about 0.3-5.0 mg / kg, about 0.5-5.0 mg / kg, about 1.0-5.0 mg / kg, about 0.1-3.0 mg / kg, about 0.3-3.0 mg / kg, about 0.5-3.0 mg / kg, or about 1.0-3.0 mg / kg, for example, about 0.1 mg / kg, about 0.3 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 3.0 mg / kg, or about 5.0 mg / kg. In some embodiments, the first effective amount is 0.3 mg / kg or 3.0 mg / kg. In some embodiments, the second effective amount is about 0.1-5.0 mg / kg, about 0.3-5.0 mg / kg, about 0.5-5.0 mg / kg, about 1.0-5.0 mg / kg, about 0.1-3.0 mg / kg, about 0.3-3.0 mg / kg, about 0.5-3.0 mg / kg, or about 1.0-3.0 mg / kg, for example, about 0.1 mg / kg, about 0.3 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 3.0 mg / kg, or about 5.0 mg / kg. In some embodiments, the second effective amount is 0.3 mg / kg or 3.0 mg / kg. In some embodiments, the first effective amount and the second effective amount are the same. In some embodiments, the first effective amount and the second effective amount are both 3.0 mg / kg. In some embodiments, the subject has RDEB. In some embodiments, one or more parameters of the second dosing regimen (e.g., duration, second frequency, second effective amount) are determined, at least in part, based on evaluation of one or more clinical parameters of the subject during or after completion of the first dosing regimen.

[0125] Assessment of treatment effectiveness As discussed herein, treatment regimen can be personalized with respect to dose frequency, dosage amount, and duration according to subject's response, side effects, and treatment effectiveness.In some embodiments, the subject being treated is assessed and evaluated for treatment effectiveness (e.g., between a first dosage regimen and a second dosage regimen); such evaluation can be used to individualize treatment schedule.

[0126] DEB patients are represented by active and chronic wounds and may have different degrees of disease severity (e.g., mild, moderate, or severe) per skin lesion based on percentage of body surface area (BSA) involvement as supported by the classification system described by Fine et al. (J Am Acad Dermatol 2008, 58(6), 931-502), the contents of which are incorporated herein by reference in their entirety. Changes from a patient's baseline (pre-treatment measurements) may be assessed during and / or after rhCol7 protein therapy. In some instances, one or more target wounds are selected for treatment efficacy evaluation. Wounds targeted for evaluation are between about 3-100 cm2. 2 The wounds may be of a size of 100 mm or less. Wounds may be assessed using images obtained using medical photography and / or by a physician's assessment. Each wound may be assessed individually one or more times, for example, at each dose after the first dose of Col7 material. Wound assessment may use a 7-point scale (Table 1) for change from baseline.

[0127] [Table 1]

[0128] Skin wounds can also be assessed using imaging methods for quantitative wound surface area (WSA), including whole body and wound reference medical imaging. In some embodiments, WSA can be measured as a primary parameter of treatment. As used herein, WSA is defined as the percentage of wound-affected body surface area (BSA), which is the calculated measure of WSA divided by estimated BSA. WSA is defined by wound borders and other aspects of wound severity for a patient population. Wounds can include acute wounds, such as blisters, erosions, and crusted erosions, as well as chronic wounds. WSA methods may be based on the nine body area rule by Wallace (The Lancet, 1951), the contents of which are incorporated herein by reference in their entirety, and / or based on the Psoriasis Area and Severity Index (PASI) (Berth-Jones et al., 2006; Langley, 2004). In some instances, WSA may be measured by wound-based quantitative medical imaging with a handheld three-dimensional camera.

[0129] In addition, the Global Assessment (GA) can be used as the basis for clinical assessment of the mean overall severity of the wound (Table 2).

[0130] [Table 2]

[0131] During the course of rCol7 replacement treatment (e.g., as described herein), biopsies (e.g., punch skin biopsies) can be obtained at intervals, e.g., between doses of the first dosing regimen (e.g., loading dosing phase) and / or the second dosing regimen (e.g., maintenance dosing phase), to directly measure Col7 protein deposition in the DEJ of the skin. Measurements can be performed with immunofluorescence (DIF) staining using anti-Col7 antibodies. Immunoelectron microscopy (IEM) can also be performed to assess whether functional anchoring fibrils form in the tissue and whether dermal-epidermal separation is reversed following rhCol7 protein therapy.

[0132] In some embodiments, suction blister time (SBT) can be determined to measure epidermal-dermal skin adhesion and thereby assess treatment efficacy and modify dosing regimens, as RDEB skin blister more easily than normal skin.

[0133] In some embodiments, other co-morbidities and symptoms associated with the DEB patient population are evaluated, such as gastrointestinal-related clinical symptoms and other non-cutaneous systemic symptoms, dysphagia (difficulty swallowing), eye erosions, nail dysplasia, alopecia, and mitten-like deformities of the hands and feet, and skin cancer (see, e.g., Fine et al., J Am Acad Dermatol. 2014, 70:1103-1126). In other embodiments, a general evaluation of skin conditions, including multiple areas of skin involvement, such as psoriasis or atopic dermatitis, is performed.

[0134] In some embodiments, subjects receiving rCol7 replacement therapy are assessed for overall daily activities using the Epidermolysis Bullosa Disease Activity and Scarring Index (EBDASI) activity subscale, a measure used to characterize disease severity and response. Subjects may be assessed prior to treatment to provide a baseline status of the patient's overall EB severity (e.g., Global Impression of Severity, GIS) that considers the skin and systemic symptoms of DEB. The GIS may not be rated, for example, as mild, moderate, or severe. After treatment begins, subjects may be assessed after each dose to provide a global status change that considers the skin and systemic symptoms of DEB. Status change is referred to as the Global Impression of Change (GIC). Change scores range from 1 to 7, as shown in Table 1.

[0135] Combination Treatment In one embodiment, the Col7 material (e.g., rhCol7 material) can be used in combination with one or more other treatments for DEB, e.g., one or more treatments for skin wounds associated with DEB, e.g., RDEB, in a patient.

[0136] Other treatments that may be used in combination with the systemic rhCol7 replacement therapy provided herein include, but are not limited to, topical therapies (e.g., topical creams containing Col7 as described in U.S. Patent Application Publication No. 2015 / 313967, the contents of each of which are incorporated herein by reference in their entireties, or topical therapies containing Coenzyme Q10 as described in U.S. Patent Application Publication No. 2018 / 369164, the contents of each of which are incorporated herein by reference in their entireties); cell-based delivery of Col7 (e.g., topical creams containing Col7 as described in U.S. Patent Application Publication No. 2015 / 313967, the contents of each of which are incorporated herein by reference in their entireties); skin keratinocyte-mediated Col7 delivery in International Publication No. WO 2017 / 120147; viral vector-mediated delivery of Col7 (e.g., herpes simplex virus (HSV)-mediated delivery of Col7 in PCT application publication WO 2017 / 176336, the contents of which are incorporated herein by reference in their entirety); aminoglycoside agents that induce read-through of the COL7A1 premature stop codon (PTC) mutation (e.g., Atanasova et al., Journal of Investigative Dermatology, 2014). Invest Dermatol. 2017, 137(9), pp. 1842-1849, e.g., gentamicin and amlexanox; RNA editing therapies (e.g., Bremer et al., Mol Ther Nucleic Acids. 2016, vol. 5, e379); and / or physical interventions including bandages, wraps, sutures, stitches, braces, patches, sealing films and glues (e.g., bandages that are initially applied as a liquid), staples, clips, and the like.

[0137] In some embodiments, systemic rhCol7 protein therapy is used in combination with an anti-infective agent (e.g., an antibiotic), an antioxidant (e.g., vitamin B3, vitamin A, or vitamin C), a growth factor (e.g., FGFP), an anti-inflammatory agent, an analgesic, and / or another skin healing agent, such as an antibiotic preparation, an antiseptic, a hydrogel, a hydrocolloid, an alginate, petrolatum, aloe vera, a cleanser, a detergent, a moisturizer, or a physical intervention (e.g., those described herein).

[0138] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments according to the disclosure set forth herein. The scope of the present disclosure is not intended to be limited to the above detailed description, but is as set forth in the appended claims.

[0139] In the claims, articles such as "a," "an," and "the" may mean one or more, unless indicated to the contrary or otherwise clear from the context. A claim or detailed description containing "or" between one or more members of a group is deemed to be satisfied if one, more than one, or all of the group members are present, employed, or otherwise relevant in a given product or process, unless indicated to the contrary or otherwise clear from the context. The present disclosure includes embodiments in which exactly one member of a group is present, employed, or otherwise relevant in a given product or process. The present disclosure includes embodiments in which more than one group member, or the entire group member, is present, employed, or otherwise relevant in a given product or process.

[0140] It should also be noted that the term "comprising" is intended to be open-ended, allowing but not requiring the inclusion of additional elements or steps. Thus, when the term "comprising" is used herein, the term "consisting of" is also included and disclosed.

[0141] When ranges are recited, the endpoints are included. Furthermore, unless otherwise indicated or otherwise clear from the context and the understanding of one of ordinary skill in the art, it should be understood that values ​​expressed as ranges can assume any specific value or subrange within the ranges described in different embodiments of this disclosure, down to the tenth of the unit of the lower limit of that range, unless the context clearly indicates otherwise.

[0142] In addition, it should be understood that any particular embodiment of the present disclosure that falls within the scope of the prior art may be expressly excluded from any one or more of the claims. Such embodiments may be excluded even if the exclusion is not expressly described herein, since they are deemed to be known to those skilled in the art. Any particular embodiment of the composition of the present disclosure (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use, etc.) may be excluded from any one or more of the claims for any reason, whether related to the existence of prior art or not.

[0143] It is to be understood that the words that have been used are words of description rather than of limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the disclosure in its broader aspects.

[0144] While the present disclosure has been described at some length and with some specificity with respect to certain described embodiments, it is not intended to be limited to any such specifics or embodiments or to any particular embodiment, but should be construed with reference to the appended claims so as to provide the broadest possible interpretation of such claims in light of the prior art and thus effectively encompass the intended scope of the present disclosure. EXAMPLES

[0145] Example 1: In vitro pharmacokinetic studies Platelet aggregation The platelet aggregation and hemolytic activities can be caused by the homology of the N-terminal non-collagenous domain 1 (NC-1) of the C7α chain with von Willebrand factor (vWF) (Leineweber S et al., FEBS Lett. 2011, 585(12), 1748-52).

[0146] In this study, rhCol7 material was tested for in vitro platelet aggregation potential in monkey and human blood samples at concentrations up to 200 micrograms per milliliter (μg / mL). Citrated whole blood samples were collected from six human subjects and six cynomolgus monkeys. Platelet-rich plasma (PRP) samples were prepared, with a platelet aggregation potential of 244 × 10 per microliter in human PRP. 3 ~267×10 3 cells (cells / μL) and 210 × 10 in monkey PRP 3 ~255×10 3 Platelet concentrations were standardized to reach 100 cells / μL. Samples were loaded in duplicate into the aggregometer in cuvettes pre-warmed for 2 min at 37° C. and then spiked with 0.9% NaCl (negative control); ReoPro® (inhibition control); collagen (positive control); reference material (buffer); rhCol7 composition at final concentrations of 2, 50, 100 and 200 μg / mL in human PRP; or rhCol7 composition at final concentrations of 2, 25, 62 and 126 μg / mL in monkey PRP. Immediately after spiking, platelet aggregation was monitored for 6 min. Results show that no activating effect was observed after in vitro treatment of human PRP with rhCol7 at 2, 50, 100 and 200 μg / mL. rhCol7 also showed no biologically significant activating effect after in vitro treatment of monkey PRP with rhCol7 at 2, 25, 62 and 126 μg / mL. A small dose-dependent increase was observed in monkey PRP, but this increase was not biologically significant compared to the collagen positive control.

[0147] There is no biologically significant increase in platelet activation and aggregation observed in monkey platelet-rich plasma (PRP) compared to collagen positive control. No platelet activation effect was observed following in vitro treatment of human PRP with up to 200 μg / mL of rhCol7 material.

[0148] hemolysis The hemolytic potential of the rhCol7 composition was investigated in vitro in blood from rats, cynomolgus monkeys, and humans. The rhCol7 material formulated in vehicle (10 mM sodium phosphate, 5 mM sodium citrate, 70 mM NaCl, 100 mM L-arginine, 1.7% sucrose, and 0.05% (v / v) polysorbate 20, pH 7.2) was tested at concentrations of 40, 95, and 185 μg / mL in rat whole blood samples from Crl:CD (SD) rats; at concentrations of 25, 62.5, and 125 μg / mL in non-human primate (cynomolgus monkey) whole blood; and at concentrations of 50, 100, and 200 μg / mL in human whole blood. Three concurrent control tests were performed using the control vehicle; positive (deionized water) and negative (0.9% sodium chloride for injection) controls were tested for each blood type. Blood and treatment volumes per sample were 0.5 mL each (total 1.0 mL) for all tests, and five replicates were used for each test. Each incubation was performed for approximately 60 minutes at approximately 37°C. Percent hemolysis for each sample was calculated according to a formula modified from Makroo et al. (Asian Journal of Transfusion Science, 2011, Vol. 5, p. 15-17).

[0149] In rat, non-human primate, and human blood, hemolysis was detected in the positive control group with mean percent hemolysis values ​​ranging from 34.6% to 37.0%. No hemolysis was detected at all exposure levels in each of the three blood matrices. Mean percent hemolysis values ​​for the treatment groups ranged from 0.3% to 0.5%. No hemolysis was detected in the negative or vehicle control groups. Mean percent hemolysis values ​​ranged from 0.3% to 1.2% for the negative controls and from 0.3% to 0.6% for the vehicle controls. According to these results, hemolysis was not detected up to a concentration of 185 μg / mL rhCol7 in rat blood, up to a concentration of 125 μg / mL rhCol7 in non-human primate blood, and up to a concentration of 200 μg / mL rhCol7 in human blood.

[0150] Example 2: Preclinical efficacy study of rhCol7 composition in the mouse DEB model (in vivo primary pharmacokinetic study) A mouse model of DEB in which the COL7A1 gene was inactivated by targeted injection (Col7a1 - / - ) (Heinonen et al., J Cell Sci. 1999, 112, 3641-3648) are born with no detectable Col7 in the dermal-epidermal BMZ, completely lacking anchoring fibrils, with extensive skin blisters on their ventral surface and hemorrhagic blisters on their limbs and neck, and typically die within the first week of life. This DEB mouse model (Col7a1 - / - ) results in severe blister formation that mimics the clinical, histological, and ultrastructural presentation of severe human recessive DEB (RDEB). Using this mouse model, non-clinical efficacy studies were conducted on dose-dependent response and efficacy after intravenous administration of rhCol7 drug product to test anchoring fibril formation, distribution and localization of rhCol7, and histological and survival effects after rhCol7 intravenous dosing. The study design is summarized in Table 3. In Table 3, doses of 16 μg, 28 μg, and 40 μg are equivalent to approximately 8, 14, and 20 mg / kg, respectively, on a 2 g body weight basis.

[0151] [Table 3-1]

[0152] [Table 3-2]

[0153] [Table 3-3]

[0154] In studies 1 to 3, neonatal DEB Col7a1 - / - Mice were administered a single iv bolus of phosphate buffered saline (PBS) and formulation vehicle control, rhCol7 composition (batches 1, 2, and 3) or reference Col7 (fibroblast-derived wild-type Col7; FB-Col7) via the superficial temporal vein. Anchoring fibril formation was assessed after a single dose of rhCol7 (iv; 16 μg (8 mg / kg)) (Study 3) and after 7 days of once-daily intradermal (id) dosing (16 μg (8 mg / kg)) (Study 2).

[0155] Distribution of rhCol7 and histological and survival endpoints were assessed following administration of a single dose of rhCol7 (28 μg (14 mg / kg), 40 μg (20 mg / kg)) (Studies 1, 6, and 7). Indirect immunofluorescence (IF) staining of tongue tissue from DEB Col7a1- / - mice following iv administration of rhCol7 composition, FB-rCol7, or control PBS.

[0156] Results demonstrated that rhCol7 composition was detected in the dermal-epidermal BMZ in the tongue and skin in 3 of 5 rhCol7 composition-treated mice and 2 of 6 FB-rCol7-treated mice that survived beyond 90 days. Both rhCol7 composition and FB-rCol7 were distributed to the appropriate location within the dermal-epidermal junction. rhCol7 composition was also detected in the esophagus of 2 mice that survived up to 18 and 90 days. Results suggest that both rhCol7 and reference collagen 7 (FB-rCol7) were distributed to the dermal-epidermal BMZ in the tongue and skin after a single iv dose (16 μg (8 mg / kg)), while no detectable rhCol7 was found in vehicle control DEB mice.

[0157] Transmission electron microscopy of the tongue revealed the formation of anchoring fibrils just below the lamina densa of the dermal-epidermal BMZ after a single iv dose (28 or 40 μg) of rhCol7 composition and FB-rCol7, whereas no anchoring fibrils were present in vehicle control mice. The rhCol7 composition administered id (16 μg) in the dorsal skin for 7 days demonstrated the formation of horizontal stripes of anchoring fibrils projecting vertically from the dermal-epidermal BMZ (Study 2).

[0158] A single dose of rhCol7 composition or FB-rCol7 intravenously administered to Col7a1- / -DEB mice corrected the separation between the epidermis and dermis in tongue tissue. In contrast, PBS-injected mice showed severe separation of the epidermis from the dermis. The percentage of mice with complete closure between the epidermis and dermis in the tongue was 48% (12 / 25 mice), 67% (12 / 18 mice), and 0% (0 / 12 mice) for rhCol7 composition, FB-rCol7, and vehicle control, respectively. The percentage of complete closure was statistically different in rhCol7 composition and FB-rCol7 treated mice compared to vehicle control mice.

[0159] Studies have demonstrated that iv administration of rhCol7 restores functional Col7 to the dermal-epidermal BMZ, which is predicted to promote the assembly of normal anchoring fibrils to provide stability to dermal-epidermal adhesion at the stratum densa / upper papillary dermis interface and correct blister abnormalities and complications in patients with DEB. rhCol7 formed anchoring fibrils and corrected separation of the epidermis from the dermis in DEB mice.

[0160] A single iv dose of 16 μg (8 mg / kg) of rhCol7 composition or FB-rCol7 resulted in a statistically significant improvement in survival compared to vehicle control. Median survival (interquartile range) was 4, 8, and 16.5 days for Col7a1− / − mice receiving vehicle control, rhCol7 composition, and FB-rCol7, respectively. The proportion of mice surviving beyond 30 days was 0% (0 / 13), 19% (5 / 27), and 35% (7 / 20) for mice receiving vehicle control, rhCol7 composition, and FB-rCol, respectively.

[0161] Separate single dose response (5, 16 or 28 μg; approximately equivalent to 2.5, 8 and 14 mg / kg, respectively) and repeat dose response (dosing at 0.5, 1 and 5 mg / kg on days 1, 3 and 5) studies in the same mouse model of DEB were conducted (Studies 4 and 5) (Table 3). Intravenous administration of rhCol7 composition resulted in dose-dependent deposition in the dermal-epidermal BMZ in target tissues after single doses of 2.5, 8 and 14 mg / kg (tissue harvested at death) and repeat doses of 0.5, 1 and 5 mg / kg on days 1, 3 and 5 (tissue harvested on day 6) (Figure 1).

[0162] Semiquantification of IF signals from repeated-dose dose-response studies also showed that rhCol7 distribution to the dermal-epidermal junction was dose-dependent; The minimal effective dose was deemed to be 5 mg / kg based on distribution to the dermal-epidermal BMZ in both the tongue and skin. A single dose of the rhCol7 composition produced a dose-dependent correction of dermal-epidermal separation in the tongue.

[0163] Dose-dependent correction of dermal-epidermal separation in neonatal Col7a1- / - mice was also observed after a single intravenous administration of the rhCol7 composition (as demonstrated in Figure 2A). Quantitation of the number of mice showing complete closure of the dermal-epidermal separation is shown in Figure 2B.

[0164] Similar distribution results were observed in studies 6 and 7 for the efficacy of the rhCol7 composition. After a single dose (8 mg / kg (16 μg)) administered intravenously, rhCol7 was distributed to the dermal-epidermal BMZ in the tongue in all rhCol7-treated mice, and to skin obtained from the abdomen, back, and forelimbs in assessed roCol7-treated mice. No rhCol7 was detected in any of the vehicle control mice.

[0165] Administration of rhCol7 composition (Batch 1 and 2) also resulted in the correction of dermal-epidermal separation in Col7a1- / - mice. The percentage of mice with complete closure between the epidermis and dermis in the tongue was 79% (15 / 19 mice), 71% (10 / 14 mice), and 27% (3 / 11 mice) for mice in Study 6 administered rhCol7 composition (Batch 1), rhCol7 composition (Batch 2), and vehicle control, respectively. The percentage of mice with reversal of dermal-epidermal separation was significantly higher in the rhCol7 composition treatment group compared to the vehicle control. In contrast, the percentage of mice with reversal of dermal-epidermal separation was similar between both treatment groups. A statistically significant improvement in survival time was observed in the rhCol7 treatment group compared to the vehicle control. Median survival times (interquartile range) were 3, 4.5, and 11 days for mice receiving vehicle control, rhCol7(batch 1), and rhCol7(batch 2), respectively.

[0166] Similar results are observed in Study 7. For example, rhCol7 composition (both batches) distributed to the dermal-epidermal BMZ in tongue and forelimb skin, whereas rhCol7 was not detected in any vehicle control mice. Correction of dermal-epidermal separation was observed in rhCol7 composition treated animals. The percentage of mice with complete closure of the dermal-epidermal separation was higher in rhCol7 composition treated mice (46% (12 / 26) (Batch 3) and 58% (15 / 26) (Batch 4)) than in vehicle controls (10% (1 / 10). A statistically significant improvement in survival time of PTR-01 treated animals compared to vehicle controls was observed. Median survival times were 2.5, 9, and 14 days for Col7a1- / - mice administered vehicle control, rhCol7 (Batch 3), and rhCol7 (Batch 4), respectively. Correlation between histology and survival time is observed.

[0167] These results and observations suggest that a single intravenous dose of rhCol7 composition can distribute and localize to the appropriate location within the dermal-epidermal BMZ, form anchoring fibrils, correct dermal-epidermal separation, and result in a statistically significant improvement in survival compared to vehicle control. Results are similar for mice administered the positive control, recombinant human rhCol7 purified from human dermal fibroblasts (FB-rhC7).

[0168] Dose-dependent responses were observed in both dose-escalation studies utilizing a single iv administration of the rhCol7 composition and in dose-dependent studies with repeated administration of the rhCol7 composition; responses included dose-dependent distribution of the rhCol7 composition to the dermal-epidermal BMZ in the target tissue and dose-dependent reversal of dermal-epidermal separation. Observations suggest that the minimal effective dose is 5 mg / kg based on distribution to the dermal-epidermal BMZ in both the tongue and skin.

[0169] Example 3: In vivo pharmacokinetics and toxicokinetics of rhCol7 compositions The pharmacokinetic (PK) / toxicokinetic (TK) characteristics of rhCol7 were evaluated in mice, rats, and monkeys, including single dose by intravenous (iv) route and 28-day weekly repeat dose studies. Characterization included PK / TK assessment in repeat dose studies in rats and monkeys, tissue distribution using quantitative whole body autoradiography (QWBA) in rats, and ADA analysis in repeat dose toxicity. Absorption, distribution, and excretion of rhCol7 compositions were examined in rats, monkeys, and dogs. The studies for these purposes are listed in Table 4.

[0170] [Table 4]

[0171] Single-dose absorption in rats and monkeys Male Sprague-Dawley Crl:CD rats (12 males / group) were administered 0.5, 2, and 5 mg / kg after a single iv slow push (1-3 min) dose (Study 8). Blood samples were collected pre-dose and at approximately 2, 5, 15, 30, and 45 min, 1, 2, 4, 8, 12, and 24 h after dose administration on Day 0. Serum samples were analyzed by electrochemiluminescence (ECL). Exposure to the rhCol7 composition was assessed by area under the curve from zero to infinity (AUC inf ) was greater than dose-proportional, and C max However, AUC inf The increase in exposure to C was dose-proportional from 2 to 5 mg / kg. A 10-fold increase in dose was max and AUC inf Peak serum concentrations of rhCol7 were observed at the first sampling point (2 min) after administration at all dose levels. Cl was low, ranging from 83.7 to 276 mL / h / kg, and the volume of distribution at steady state (V ssThe terminal half-lives (T 1 / 2 ) were short, ranging from 0.108 to 0.293 hours. Cl appeared to decrease slightly from 0.5 to 2 mg / kg and remained similar at 2 and 5 mg / kg. At 5 mg / kg, the maximum serum concentration (C max ) was 105,000 ng / mL, and AUC 0-24 is 59,800ng * The mean plasma glucose concentration was 1.25 mg / mL.

[0172] Similarly, female cynomolgus monkeys (3 females / group) were administered a single iv slow push (1-2 min) dose of 0.5, 2, and 5 mg / kg rhCol7 in a dose volume of 10 mL / kg (Study 9). Blood samples were collected pre-dose and at approximately 2, 5, 15, 30, and 45 min and 1, 2, 4, 8, 12, and 24 h after dose administration on Day 0. Serum samples were analyzed by ECL. rhCol7 serum concentrations were measurable in at least one of three animals up to 4, 12, and 24 h after dose administration at 0.5, 2, and 5 mg / kg, respectively. Peak serum concentrations of rhCol7 were observed at the first sampling point (2 min) after dosing at all dose levels. Serum concentrations appeared to decrease in a biphasic manner. Exposure to rhCol7 decreased with increasing rhCol7 doses from 0.5 to 5 mg / kg, with AUC inf The effect was greater than dose proportional for C max The AUC inf The increase in exposure to β-lactamase was approximately dose-proportional from 2 to 5 mg / kg. Plasma clearance (Cl) was low, ranging from 43.7 to 163 mL / h / kg, and V ss The t 1 / 2 ranged from 0.347 to 5.59 hours. At 5 mg / kg, C max was 135,000 ng / mL, and AUC 0-24 is 116,000ng * The mean plasma glucose concentration was 1.25 mg / mL.

[0173] Repeated dose absorption in rats and monkeys Sprague-Dawley Crl:CD rats (n=12 / dose) were administered repeated iv bolus injections of 2, 5, and 10 mg / kg / week in a dose volume of 10 mL / kg once per week (total of 5 doses over a 28 day dosing period) (Study 10). A concurrent control group (n=8) received vehicle in the same regimen. Blood samples were collected approximately 5 and 15 minutes after dose administration on days 0 and 28 for the control group, and pre-dose, approximately 5 and 15 minutes after dose administration and approximately 1, 2, 4, 8, 12, and 24 hours after dose administration on days 0 and 28. Serum samples were analyzed by ECL.

[0174] rhCol7 serum concentrations were below the level of quantification (BLQ) in all samples prior to dosing with the rhCol7 composition and in samples collected from control animals on days 0 and 28. In general, exposure was similar on days 0 and 28 for all dose groups, and no accumulation of rhCol7 composition was observed after dosing once weekly for 28 days. Accumulation ratios ranged from 0.516 to 1.27. Clearance was low (approximately 1.07 to 3.64 mL / min / kg for combined male and female data) and V ss The mean terminal t 1 / 2 No gender differences were observed. The TK parameters of rhCol7 are summarized in Table 5.

[0175] [Table 5]

[0176] Two studies were conducted in monkeys to investigate the toxicokinetic profile of the rhCol7 composition. In Study 11, cynomolgus monkeys were administered repeated iv slow push (1-2 min) injections of 2, 5, and 10 mg / kg / week at a dose volume of 10 mL / kg once per week (total of 5 doses over a 28 day dosing period). Blood samples were collected during acclimation, prior to the first dose, prior to dosing on day 28, and once at approximately 5 and 15 min and 1, 2, 8, 12, and 24 h after dose administration on days 0 and 28. Serum samples were analyzed by ECL. There were no measurable rhCol7 concentrations in serum samples collected from control animals. Overall, rhCol7 (AUC and C max ) exposure was similar in males and females, with no consistent gender differences.

[0177] After iv bolus administration of the rhCol7 composition at 2 mg / kg / week, rhCol7 serum concentrations were measurable from 0.083 hours post-dose until 2, 4, or 8 hours post-dose on both days. In the 5 and 10 mg / kg / week dose groups, rhCol7 serum concentrations were measurable from 0.083 hours post-dose until 4, 8, 12, or 24 hours post-dose on both days.

[0178] Accumulation ratios ranged from 0.738 to 1.50. Clearance was low (<5 mL / min / kg), and V ss The mean time t was small (<0.105 L / kg) and ranged from 0.304 to 1.36 hours for the combined male and female data. 1 / 2 Male and female toxicokinetic parameters for rhCol7 are summarized in Table 6.

[0179] [Table 6]

[0180] In Study 12, cynomolgus monkeys were administered repeated iv infusions (1 h) of 4 and 8 mg / kg / week at an infusion rate of 6.6 mL / kg / h once per week (total of 5 doses over a 28 day dosing period). Blood samples were collected pre-dose, 30 min after the start of the infusion, and 5 and 15 min and 1, 2, 4, 8, 12, and 24 h after the end of the infusion. Serum samples were analyzed by ECL. Exposure to rhCol7 (AUC 0-Tlast , AUC inf and C max The AUC and C values ​​(based on mean ± SD) generally increased in a dose-dependent and slightly to moderately greater than dose-proportional manner. In general, there were no significant sex-related differences. max The accumulation ratios (day 29 / day 1) ranged from 0.9 to 1.7, indicating that rhCol7 did not accumulate when administered to cynomolgus monkeys as an iv infusion over a period of 1 h / day once per week for a total of 5 doses over 4 weeks.

[0181] mean estimate t 1 / 2 ranged from 0.359 to 0.857 hours on day 1 and from 0.427 to 1.40 hours on day 29. rhCol7 was cleared at mean rates ranging from 43.8 to 71.9 mL / hour / kg on day 1 and from 27.1 to 54.4 mL / hour / kg on day 29. The mean volume of distribution (V z ) ranged from 31.0 to 52.4 mL / kg on day 1 and 31.3 to 47.8 mL / kg on day 29, suggesting that rhCol7 remains in the circulation to a significant extent. TK parameters for rhCol7 are summarized in Table 7.

[0182] [Table 7]

[0183] distribution The half-life (T 1 / 2) was determined in a DEB mouse model (Col7a1- / 1) ​​following a single iv administration of rhCol7 composition (8 mg / kg (16 μg) (Study 13). Tongues (n=25) and skin from the front and hind paws (n=27) were collected and subjected to IF staining for rhCol7. Tissue T 1 / 2 was specifically estimated using semi-quantitative IF signal in the dermal-epidermal BMZ. Tissue t1 / 2 in the tongue and skin from the limbs was estimated to be 26.4 and 32.6 days, respectively. The distribution of rhCol7 in the tongue, esophagus and skin (intact and wounded) was further determined in a tamoxifen-induced Col7 knockout DEB mouse model after iv administration (Study 14). After a small wound on the back of C7-inducible knockout mice was incised, two iv injections of 100 μg rhCol7 (8 mg / kg (total 200 μg; n=5) or vehicle (n=4) were administered. Two weeks after rhCol7 placebo injection, mice were sacrificed and samples of tongue, esophagus, and skin (intact and wounded) were collected for rhCol7 detection at the dermal-epidermal junction. rhCol7 was distributed to the dermal-epidermal BMZ in wounded skin of tamoxifen-inducible Col7 knockout mice after iv administration. rhCol7 was not detected in the tongue, esophagus, or non-wounded skin.

[0184] Following a single iv dose of 2 mg / kg to eight male Sprague-Dawley rats, 111 The distribution of [In]-PTR-01 was evaluated (Study 15). One animal per time point was sacrificed at 0.5, 2, 6, 24, 72, 168, 216, and 336 hours after administration. 111Following administration of [In]-PTR-01, Cmax in blood and plasma was observed at 0.5 hours post-dose. Radioactivity concentrations in plasma were approximately 2-fold higher than in blood. Levels of radioactivity in both matrices dropped rapidly, approximately 5-fold lower by 2 hours post-dose, but were still detectable at 336 hours post-dose. The highest radioactivity concentrations and dose / g ratios were observed in bone marrow, kidney, liver, and spleen. The majority of tissues had tissue:plasma concentration ratios >1 at later time points, indicating that drug-associated radioactivity was retained in the tissues. rhCol7 was detected in the skin (non-pigmented) at each time point, including 336 hours post-dose; the skin:plasma ratio at 336 hours post-dose was 4.61.

[0185] The distribution of rhCol7 in the lips and skin (ear) was determined in one female RDEB dog after iv injection of 4 mg / kg (Study 16). The RDEB dog model is a spontaneous model with a homozygous glycine mutation (G1906S) in the collagen-like domain. rhCol7 was faintly detected in the dermal-epidermal BMZ in the skin (ear) 3 days after administration. A very weak IF signal in the dermal-epidermal BMZ in the skin was also detected 25 days after administration. In contrast, IF staining without the primary antibody (immunoglobulin G (IgG) control) showed no staining in the dermal-epidermal BMZ. rhCol7 was faintly detected in the skin (ear) but not in the lips. There were no changes in the anchoring fibrils and suction blister examination in the one treated animal.

[0186] excretion rhCol7 tissue distribution and excretion 111 Indium (In)]-rhCol7-treated rats were tested (Study 15). Male Sprague-Dawley rats were treated with [ 111Indium (In)]-rhCol7 was administered at a dose level of 2 mg / kg by a single iv injection. Urine was collected from animals 0-6 and 6-24 hours after dosing and at 24-hour intervals thereafter until 168 hours after dosing. Feces were collected at 24-hour intervals until 168 hours after dosing. The concentration of total radioactivity was determined in the dose formulations using instant thin-layer chromatography (iTLC) and solid scintillation counting (SSC), as well as quantitative whole-body autoradiography (QWBA). Autoradiography images were analyzed to determine tissue concentrations of radioactivity.

[0187] Blood, plasma, urine, feces, and carcasses were analyzed by SSC to determine radioactivity concentrations. 111 Following iv administration of [In]-rhCol7, the highest recovery of drug-associated radioactivity was found in the urine, averaging 56.8% over the 0-168 h recovery period. Drug-associated radioactivity was also present in the feces. Approximately 30% of the administered dose remained in the carcass, with total recovery (urine, feces, and carcass) averaging 95.9 ± 1.04%.

[0188] In summary, the AUC and C max There were no general sex differences in systemic exposure as measured by t 1 / 2was often less than 30 min in all tested species. In both rodents and non-human primates, rhCol7 half-life in plasma increased with dose. Exposure data support allometric scaling for human equivalent dose (HED) calculations. In a tamoxifen-induced Col7 knockout mouse model, rhCol7 was distributed to the dermal-epidermal BMZ in wounded skin, further supporting specific homing to collagen VII. This is similar to the tissue half-life of endogenous mouse Col7 in target tissues (skin, tongue, and esophagus) estimated in a tamoxifen-induced Col7 knockout mouse model (Kuhl et al., J Invest Dermatol. 2016, 136:1116-1123). rhCol7 was widely distributed throughout the entire body of rats after iv administration. The highest amounts of rhCol7 were observed in bone marrow, kidney, liver, and spleen. In Sprague-Dawley rats, the blood and plasma 111 Levels of [In]-rhCol7 fell rapidly, approximately 5-fold lower by 2 hours post-dose, but were still detectable 336 hours post-dose after iv administration. rhCol7 was faintly detectable in the dermal-epidermal BMZ in skin 3 days after a single iv injection.

[0189] Example 4: Patient Selection Before rhCol7 Drug Treatment At least one >2cm 2 Recessive dystrophic epidermolysis bullosa (RDEB) patients with chronic (e.g., at least 6 weeks) lesions are selected. Patients diagnosed with RDEB undergo genetic analysis prior to enrollment for rhCol7 protein treatment. Blood samples are collected from each patient for Col7A1 genotyping. Only patients who exhibit a mutation(s) in the COL7A1 gene consistent with a recessive inheritance pattern are enrolled. Biopsy samples are also obtained from patients prior to enrollment for IF staining. Patients are selected who have some Col7 present but lack Col7 staining at the dermal-epidermal junction (DEJ) by IF staining.

[0190] Example 5: Efficacy of rCol7 supplementation in human patients with RDEB This study was conducted to evaluate the efficacy of rCol7 supplementation in treating humans with RDEB. Adult patients with recessive dystrophic epidermolysis bullosa (RDEB) were selected as described in Table 4. Twelve patients with a diagnosis of RDEB and a history of at least one chronic lesion were enrolled and divided into four dosing cohorts. Patients received three doses of PTR-01 at doses ranging from 0.1 to 3.0 mg / kg. Specifically, the four cohorts were dosed at 0.1 mg / kg (active drug), 0.3 mg / kg (active drug), 1.0 mg / kg (active drug), and 3.0 mg / kg (active drug), respectively.

[0191] Efficacy assessments were performed prior to the first dose of therapy and repeated at days 15, 29, 43, 57, 71, 85, and 127 during the treatment and follow-up periods. C max , T max , AUC, clearance, and t 1 / 2 Pharmacokinetic parameter estimates including were measured. Skin samples were obtained and subjected to IF staining for Col7 staining at the dermal-epidermal junction (DEJ). Electron microscopy (EMS) was also used to evaluate anchoring fibrils in the skin biopsies. Persistence of rhCol7 in tissues was measured in the skin biopsies.

[0192] Changes in skin blistering, rash size, and wound healing were assessed, as well as measurements of biomedical markers including albumin, iron / TIBC, hemoglobin, and hematocrit. Additionally, patient-reported outcomes such as pruritus and pain scales, overall quality of life, and DEB activity were included as part of the evaluation.

[0193] Interim efficacy data showed a mean 200% increase in C7 deposition at the dermal-epidermal junction (DEJ) by direct immunofluorescence (DIF) was seen at the 1 mg / kg dose. A dose-dependent increase in C7 deposition at the DEJ was seen at C7 levels of 3 mg / kg, approaching that seen in asymptomatic heterozygous and normal human skin.

[0194] A trend towards improved wound healing in patients was observed. max The dose-dependent increase in levels approached that seen at the minimally effective dose in animals. In addition, the increase in C7 DIF was maintained for approximately one month after cessation of dosing.

[0195] Example 6: Dosing Frequency Study in Patients with RDEB This Phase 2 open-label study determines the dose frequency and efficacy on uptake of rCol7 in the skin in patients with RDEB. The treatment plan for RDEB patients was divided into three parts, including two dosing regimens (Parts 1 and 2, alternatively referred to as "loading" and "maintenance" phases) and a three-month observation period (Part 3). In Part 1, patients receive a dose of 3.0 mg / kg of rCol7 every week for a total of four doses. In Part 2, which follows Part I, patients receive a dose of 3.0 mg / kg of rCol7 every two weeks for a total of seven doses. Efficacy evaluations are performed at the end of each dosing period. During Part 3, patients are assessed at 1 and 3 months after completion of dosing to examine the persistence of wound healing and other efficacy parameters. All doses were administered by intravenous infusion. Figure 4 illustrates the study design diagrammatically.

[0196] Dosing will be scheduled as follows: for patients 16 years of age or older, PTR-01 will be administered IV at a dose of 3.0 mg / kg weekly for 4 doses, then every 2 weeks for 7 doses; for patients under 16 years of age, PTR-01 will be administered IV at a dose of 120 mg / m 2 is given IV at a dose of 10 mg once every week for 4 doses, then every 2 weeks for 7 doses.

[0197] The primary endpoints for this study included improvement in the majority of target lesions by at least 2 levels using a 7-point Global Impression of Change (GIC) measure, as well as treatment-emergent adverse events (TEAEs), infusion-related reactions (IARs), and immunogenicity.Secondary and exploratory endpoints included delivery of PTR-01 to the skin; formation of new anchoring fibrils measured by electron microscopy; wound area of ​​target lesions by imaging; Investigator GIC (IGIC) assessment; total body wound surface area; pain severity and impact of pain on quality of life (modified PROMIS subscales and iscorEB); Global Impression of Severity and Change (IGIS&C, PGIS&C); wound care burden; patient interviews and case reports; and markers of skin fibrosis.

[0198] Table 8 summarizes the details of the patients enrolled in the study. Six patients were enrolled and five completed the study. Patients 202-001, 202-002, and 202-003 participated in the Phase 1 study described in Example 5. The median patient age was 19.0 years.

[0199] [Table 8]

[0200] Table 9 summarizes wound response at day 120 compared to baseline by wound-specific 7-point scale.In this table, m is the number of wounds that meet the response criteria; M is the total number of wounds; Responders are patients with a wound-specific 7-point scale increase of ≥ 2 points in ≥ 50% of wounds; Total wound response is the total number of wounds (all patients) with a wound-specific 7-point scale increase of ≥ 2 points for all patients.The total number of patients evaluated was 5 instead of 6 enrolled in the study because one patient discontinued after day 36 due to lack of efficacy.

[0201] [Table 9]

[0202] Table 5 shows a comparison of wound assessments at Day 120 versus baseline. As shown in Table 9 and Figure 5, treatment with PTR-01 resulted in rapid, consistent and sustained wound healing. By Day 15, 15 / 26 wounds (57.7%) met response criteria of > 2 point increase on a wound-specific 7-point rating scale, and by Day 120, 18 / 26 wounds (69.2%) did so. Based on these criteria, 4 / 6 patients (66.7%) were responders.

[0203] Figure 6 shows wound response by percent reduction in wound surface area by Canfield imaging. Wounds showed a rapid response to treatment with the majority (80%) reaching >50% closure by day 68. At the end of treatment, day 120, over 80% of wounds were >50% closed compared to baseline. Persistence of treatment continued for one month after the last dose with treatment effect beginning to wane by day 204.

[0204] Figure 7 shows the wound closure observed in chronic and recurrent wounds. PTR-01 affects wound closure in chronic wounds, which are the main cause of morbidity. The analysis was based on 14 wounds characterized as "recurrent" and 8 wounds characterized as "chronic"; efficacy was observed in small and large wounds alike. Of the 26 wounds analyzed in the study, 4 were labeled as "physician choice" by protocol, and therefore no clinical wound history was provided, and these wounds were removed from the analysis. A robust wound healing response was observed across different wound types: small, large, chronic and recurrent wounds. The majority of wounds achieved >50 and >75% healing at day 120.

[0205] FIG. 8 shows example wound images demonstrating a clear reduction in wound surface area following PTR-01 treatment across all wound types and sizes. 9A-9B show the individual wound changes from baseline and median wound surface area. When examining wound size over time versus baseline using area under the curve (AUCi) analysis, there was a greater reduction in wound size over time at day 43 in patients receiving PTR-01 than was observed in the existing Phase 1 PTR-01 study for patients receiving placebo (53.6% vs. 75%).

[0206] 10A-10B show improvements in pain, disease impact, activities of daily living, mood, and essential function as measured by iscorEB-P in patients in a Phase 2 study. Significant mean and median reductions from baseline to day 204 were observed in iscorEB scores for these metrics.

[0207] Figure 11 shows investigator and patient Global Impression of Change (GIC) scores by patient and time point. At days 22, 78, 120, and 148, both IGIC and PGIC scores improved, with good correlation between investigator and patient assessments.

[0208] FIG. 3 shows immunofluorescence data showing deposition of C7 at the dermal-epidermal junction (DEJ) after administration of PTR-01. The top panel shows deposition for patients already treated in the Phase 1 study described in Example 5, and the bottom panel shows deposition for patients not treated in the pre-study. As shown in FIG. 3, deposition of C7 at the DEJ in patients receiving PTR-01 at 0.3 mg / kg and 3 mg / kg was dose-dependent. Rapid deposition of C7 at the DEJ was observed during the loading phase, achieving levels predicted to confer a therapeutic effect (35% of normal). These levels were maintained throughout treatment and for one month after completion of treatment. All patients showed continued deposition of C7 in the DEJ compartment ranging from 1 to 3 months after their last dose of PTR-01 (e.g., at days 148 and 204). These data support the possibility of monthly or possibly longer maintenance dosing.

[0209] Figure 12 shows reduction in dermal pro-fibrosis biomarker staining with PTR-01 administration. Fibrosis biomarkers decreased over the course of treatment from an elevated baseline and remained reduced after treatment was completed.

[0210] In general, PTR-01 was well tolerated when given once weekly for 4 weeks and then every 2 weeks for 14 weeks. Table 10 summarizes the TEAEs observed in the study. Twenty AEs were reported for 4 patients, all of which resolved. No deaths, serious adverse events, or unexpected AEs were observed, and no AEs led to treatment discontinuation. All AEs were mild or moderate, except for a single AE of anemia that was deemed unrelated to the study drug. One patient had an infusion reaction that responded to supportive care and resolved within a few hours. Three patients had detectable low-titer ADAs observed at least once during the study. These observations were not associated with clinical symptoms or laboratory findings. One patient had a high-titer ADA. This patient had a mild infusion reaction and was eventually withdrawn from the study due to lack of efficacy.

[0211] [Table 10]

[0212] In summary, weekly infusions of PTR-01 at 3.0 mg / kg per week for 4 weeks followed by infusions every 2 weeks (qow) for 14 weeks were well tolerated and resulted in rapid and sustained improvements in measures of wound healing including the proportion of patients with at least a 2-point improvement in the majority of wounds and the proportion of total patient wounds with a ≥50% reduction in surface area; reduction in iscorEB symptom scores; deposition of rC7 at the DEJ; and reduction in pro-fibrotic biomarkers in the skin. Investigator and patient global assessments of change were concordant and reflected overall improvement in disease. The results of this study support further testing of PTR-01 administration for the treatment of DEB.

Claims

1. 1. A pharmaceutical composition for treating dystrophic epidermolysis bullosa (DEB) in a subject in need thereof by intravenous administration according to a first dosing regimen and a second dosing regimen, comprising: 1.2 milligrams per milliliter (mg / mL) recombinant human type VII collagen (rhCol7) material, 10 millimolar (mM) sodium phosphate, 5 mM sodium citrate, 100 mM L-arginine, 1.7% sucrose (weight / volume), 70 mM sodium chloride, and 0.05% (vol / vol) polysorbate 20 (pH 7.2); i) the first dosing regimen comprises administering a first therapeutically effective amount of the agent weekly; ii) the second dosing regimen comprises administering a second therapeutically effective amount of the agent every two weeks; wherein the first therapeutically effective amount and the second therapeutically effective amount are independently about 0.1 milligrams per kilogram (mg / kg), 0.3 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, or 3.0 mg / kg of the subject, and wherein administration according to the first dosing regimen occurs before administration according to the second dosing regimen.

2. 1. A pharmaceutical composition for alleviating, alleviating, reducing, ameliorating, delaying the onset of, slowing the progression of, eliminating, and / or curing symptoms or complications of dystrophic epidermolysis bullosa (DEB) in a subject in need thereof by intravenous administration according to a first dosing regimen and a second dosing regimen, comprising: 1.2 milligrams per milliliter (mg / mL) recombinant human type VII collagen (rhCol7) material, 10 millimolar (mM) sodium phosphate, 5 mM sodium citrate, 100 mM L-arginine, 1.7% sucrose (weight / volume), 70 mM sodium chloride, and 0.05% (vol / vol) polysorbate 20 (pH 7.2); i) the first dosing regimen comprises administering a first therapeutically effective amount of the agent weekly; ii) the second dosing regimen comprises administering a second therapeutically effective amount of the agent every two weeks; wherein the first therapeutically effective amount and the second therapeutically effective amount are independently about 0.1 milligrams per kilogram (mg / kg), 0.3 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, or 3.0 mg / kg of the subject, and wherein administration according to the first dosing regimen occurs before administration according to the second dosing regimen.

3. The symptoms or complications of DEB include skin wounds including blister formation, itching, pain, skin erosion, scar formation, fragile skin, gangrene, hypoplasia or hypoplasia of the skin, hypopigmentation of the skin, milia, skin infections, cheilitis, dystrophic fingernails or toenails, abnormal dental enamel, pseudosyndactyly or camptodactyly of the fingers or toes, syndactyly of the fingers or toes, flexion contractures of the toes, dental caries, dysphagia, fissured or fissured tongue, and esophageal stricture. , pharyngeal stenosis, growth disorder, dilated cardiomyopathy, pulmonary interstitial dysplasia, acute constipation, hearing or visual impairment, eczema, glomerulopathy, immunological hypersensitivity, nasolacrimal duct obstruction, anemia, chronic ear infection, corneal erosion, corneal abrasion and scarring, oral erosion, microstomia, osteopenia, ectropion, immunological hypersensitivity, nephrotic syndrome, phimosis, renal failure, urinary retention, ureteral stenosis, stroke, squamous cell carcinoma, or a combination thereof.

4. 4. The pharmaceutical composition of claim 3, wherein the symptom or complication of DEB is skin wounds, including blister formation.

5. 3. The pharmaceutical composition of claim 2, which relieves, alleviates, reduces, improves, or eliminates one or more skin blemishes.

6. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the first dosing regimen lasts for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks.

7. 7. The pharmaceutical composition of claim 6, wherein the first dosing regimen lasts for at least four weeks.

8. The pharmaceutical composition of any one of claims 1 to 5, wherein the second dosing regimen lasts for at least 4 weeks.

9. The pharmaceutical composition of any one of claims 1 to 5, wherein the first therapeutically effective amount is the same as the second therapeutically effective amount.

10. 10. The pharmaceutical composition of claim 9, wherein the first therapeutically effective amount and the second therapeutically effective amount are about 0.3 milligrams per kilogram of subject (mg / kg).

11. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the first dosing regimen continues for at least 4 weeks and the first therapeutically effective amount is about 0.3 mg / kg.

12. 12. The pharmaceutical composition of claim 11, wherein the second dosing regimen lasts for at least 7 weeks.

13. The pharmaceutical composition according to any one of claims 1 to 5, wherein the intravenous administration is by intravenous infusion or slow push injection.

14. The pharmaceutical composition of any one of claims 1 to 5, wherein the subject has recessive dystrophic epidermolysis bullosa (RDEB) or dominant dystrophic epidermolysis bullosa (DDEB).

15. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the subject has a mutation in the COL7A1 gene consistent with a recessive inheritance pattern.

16. The pharmaceutical composition according to any one of claims 1 to 5, wherein the subject is a human.

17. The pharmaceutical composition according to any one of claims 1 to 5, wherein the subject is under 18 years of age.

18. 18. The pharmaceutical composition of claim 17, wherein the subject is at least 6 years old, at least 2 years old, or less than 2 years old.

19. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein one or more clinical parameters are assessed between the first and second dosing regimens.

20. 20. The pharmaceutical composition of claim 19, wherein the one or more clinical parameters are selected from wound surface area, wound healing, time to chronic wound healing, and time to reblistering.

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