WO2012025746A1 - Medical use of heparin-binding egf-like growth factor (hb-egf) - Google Patents

Abstract

The present invention provides an agent comprising heparin-binding EGF-like growth factor (HB-EGF), or a functional fragment, variant or derivative thereof, for use in the prevention and/or treatment of skin blistering. Also provided are compositions comprising the agent, nucleic acids and expression vectors encoding the agent, and a method of treating skin blistering using the agent.

Description

MEDICAL USE OF HEPARIN-BINDING EGF-LIKE

GROWTH FACTOR (HB-EGF)

FIELD ^v '

The present invention relates to the field of the prevention and treatment of skin blistering, particularly in skin blistering diseases such as epidermolysis bullosa.

BACKGROUND

Skin blistering refers to the formation of vesicle-like lesions, often filled with fluid, beneath the epidermis. Blistering may be caused, for example, by localized trauma such as burns or friction, as well as chemical irritation, infection or skin diseases. A number of inherited and acquired conditions are associated with skin blistering.

Epidermolysis bullosa (EB) is typically an inherited condition which results in skin blistering and extreme fragility of the epidermis. Various forms of the disease have been described according to the location within the skin at which blistering typically occurs. In epidermolysis bullosa simplex, blistering is at the basal cell level or above, whereas in the junctional form blistering occurs at the lamina lucida level. In dystrophic epidermolysis bullosa, blistering occurs below the lamina densa level.

These different forms of epidermolysis bullosa are typically associated with specific molecular defects. Dystrophic epidermolysis bullosa, in either its recessive or dominant forms, is associated with mutations of the COL7A1 gene coding for type VII collagen, whereas individuals with dominant and recessive forms of epidermolysis bullosa simplex have mutations in keratin genes 5 and 14 or plectin. In junctional epidermolysis bullosa, mutations in genes encoding one of the three subunits of laminin-5 are commonly found, although mutations in the type XVII collagen gene or oc6 or β4 integrin subunit genes may also occur.

Epidermolysis bullosa simplex (EBS) is a rare autosomal dominant disease in which the epidermis loses its integrity following trivial mechanical trauma. The disease is characterized by extreme fragility of the keratinocytes, and skin blistering, resulting from missense mutations in the gene that encodes keratin 5 or keratin 14. Keratins 5 and 14 are abundant cellular proteins which normally co-polymerize to form an intricate network of 10-12 nm- wide intermediate-sized filaments in basal keratinocytes of epidermis and related epithelia. EBS may manifest itself as a relatively mild blistering condition involving the hands and feet (EBS, Weber-Cockayne type), or as a generalized blistering condition, sometimes associated with mucosal blistering that involves the oropharynx, the oesophagus and ocular mucosa, and which can be fatal (e.g., EBS, Dowling-Meara type).

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disease (OMIM 226600) caused by loss-of- function mutations in the COL7A1 gene, which encodes for type VII collagen (C7), the major component of anchoring fibrils at the dermal-epidermal junction (DEJ) (Fine et al., 2008). In RDEB, there is typically reduced expression of C7 at the DEJ and anchoring fibrils are reduced in number and rudimentary (McGrath et al., 1993). A lack of anchoring fibrils leads to skin fragility beneath the lamina densa and clinically there is widespread trauma-induced muco-cutaneous blistering (Fine and Mellerio, 2009).

Several of the structural skin proteins that are mutated in the different forms of epidermolysis bullosa may also serve as target antigens in patients with acquired blistering skin diseases such as bullous pemphigoid or pemphigus. For example, autoantibodies against type VII collagen lead to sub-epidermal blistering and a clinical disease known as epidermolysis bullosa acquisita.

Currently, there is no effective long-term therapy for EB, although strenuous efforts are being made to develop gene, cell, protein and drug-based treatment strategies (Uitto et al., 2010). Current therapies focus on dressing blisters, preventing and treating skin infections with antibiotics and nutritional management. Acquired blistering diseases are commonly treated using immune suppressants such as systemic corticosteroids, cyclosporine, cyclophosphamide and azathioprine. Tetracycline, nicotinamide and dapsone have also been used. All the treatments for acquired blistering diseases are systemic and there are currently no local therapies capable of reducing duration of blisters or speeding healing of blistered skin.

For inherited blistering skin diseases, it has been demonstrated that allogeneic fibroblast therapy may have therapeutic value in patients with recessive dystrophic epidermolysis bullosa (RDEB) (Wong et al., 2008). A single intradermal injection of fibroblasts from an unrelated donor could, in some individuals with RDEB, increase C7 expression at the DEJ for at least 3 months (Wong et al., 2008). However, this cell-based therapeutic study did not disclose molecular targets which might be useful in the treatment of EB. Thus there is still a need for improved therapies for skin blistering, for example in conditions such as epidermolysis bullosa.

SUMMARY

Accordingly, in one aspect the present invention provides an agent comprising heparin- binding EGF-like growth factor (HB-EGF), or a functional fragment, variant or derivative thereof, for use in the treatment of skin blistering.

Preferably the agent is used to prevent and/or treat epidermolysis bullosa, especially for the prevention and/or treatment of skin blistering associated with epidermolysis bullosa. More preferably the skin blistering is associated with dystrophic epidermolysis bullosa, e.g. recessive dystrophic epidermolysis bullosa.

In one embodiment, the agent comprises a polypeptide having the sequence of SEQ ID NO:2, or a functional fragment, variant or derivative thereof. For example, the agent may comprise a polypeptide having at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%) or at least 99% sequence identity with SEQ ID NO:2, preferably over at least 20, at least 50 or at least 100 amino acids of SEQ ID NO:2.

In a further aspect, the present invention provides a nucleic acid encoding an agent as defined above, for use in the prevention and/or treatment of skin blistering. Preferably the nucleic acid has at least at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%) or at least 99% sequence identity with SEQ ID NO: l, preferably over at least 30, at least 100 or at least 300 nucleotides of SEQ ID NO: 1.

In another aspect, the present invention provides an expression vector comprising a nucleic acid as defined above, for use in the prevention and/or treatment of skin blistering.

In another aspect, the present invention provides a composition comprising a therapeutically effective amount of an agent, nucleic acid or expression vector as defined above, and a pharmaceutically acceptable carrier, for use in the prevention and/or treatment of skin blistering. Preferably the composition is a topical pharmaceutical composition, i.e. the composition is suitable for topical application or for use in topical application to the skin.

In a further aspect, the present invention provides a method for the prevention and/or treatment of skin blistering in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent comprising heparin-binding EGF-like growth factor (HB-EGF), or a functional fragment, variant or derivative thereof, or a nucleic acid encoding the agent.

In a further aspect, the present invention provides use of an agent comprising heparin-binding EGF-like growth factor (HB-EGF), or a functional fragment, variant or derivative thereof, or a nucleic acid encoding the agent, for the preparation of a medicament for preventing and/or treating skin blistering.

In a further aspect, the present invention provides an agent comprising heparin-binding EGF- like growth factor (HB-EGF), or a functional fragment, variant or derivative thereof, or a nucleic acid encoding the agent, for use in promoting type VII collagen production in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Immunofluorescence microscopy shows increased C7 labelling at the DEJ and within basal keratinocytes following allogeneic fibroblast injection and, to a lesser extent, following intradermal saline injection, (a) normal control skin; (b-f) RDEB patient biopsies at the specified time-points up to 360 days following a single injection of allogeneic fibroblasts; (g, h) RDEB patient biopsies at different time-points following a single intradermal injection of saline. Numerical values represent mean fluorescence intensities +/- standard error. Bars = 50μιη.

Figure 2. qRT-PCR reveals increased expression of COL7A1 for up to 90 days following a single injection of allogeneic fibroblasts and a smaller, more transient increase following saline injection, (a) COL7A1 gene expression using primers designed to amplify exons 54-55 reveals increased expression 15 days following fibroblast injection which is sustained for 90 days but not 180 days or thereafter. The day 15 post-saline injection sample also shows a slight increase in COL7A1 expression but this is not present at 90 days, (b) COL7A1 gene expression using primers designed to amplify exons 86-90 which also allows for measurement of the RDEB patient's mutant COL7A1 allele (in-frame exon skipping) shows a similar profile of gene expression changes to the upstream amplification in (a) with clear evidence of increased expression of the mutant allele. (Col = control subject 1; Co2 = control subject 2; D = day; S = saline; numbers refer to days post-injection). Figure 3. q T-PCR for HB-EGF expression reveals a similar pattern to COL7A1 gene expression at the different time-points. Expression of HB-EGF is shown in the different biopsy samples following fibroblast or saline injection. (Col = control subject 1; Co2 = control subject 2; D = day; S = saline; numbers refer to days post-injection).

Figure 4. Keratinocytes and fibroblasts treated with recombinant HB-EGF show increased expression of COL7A1, JUN and FOS. (a) COL7A1 expression; (b) JUN expression; (c) FOS expression. The 4 grouped bars represent different cells types, control and RDEB, as shown in the key.

Figure 5. Injection of allogeneic fibroblasts into RDEB wound margins promotes rapid re-epi th eli aliza tion . Treatment of 2 chronic erosions is illustrated. The arrows indicate injection points.

Figure 6. Nucleotide sequence of cDNA encoding human pro-HB-EGF precursor. Figure 7. Amino acid sequence of human pro-HB-EGF precursor protein.

DETAILED DESCRIPTION Agent

The agent of the present invention comprises heparin-binding EGF-like growth factor (HB- EGF), or a functional fragment, variant or derivative thereof.

HB-EGF is a member of the epidermal growth factor (EGF) family and binds to the EGF receptor (Iwamoto and Mekeda, 2000). It is synthesized as a membrane-anchored protein (pro-HB-EGF) (Higashiyama et al, 1991), but is also cleaved by proteases to yield a soluble mature growth factor (HB-EGF), which is similar to other EGFR ligands (Goishi et al, 1995; Massague and Pandiella, 1993). Both the membrane precursor form and the soluble cleaved form may have complex and diverse functions (Iwamoto and Mekeda, 2000). HB-EGF contributes to skin wound healing, cardiac development/maintenance, eyelid formation, blastocyst implantation, atherosclerosis progression, and tumor formation (Higashiyama et al, 1992 and 1993).

Biological activity of HB-EGF is regulated by receptor-ligand interactions between HB-EGF and epidermal growth factor receptor (EGFR = ErbBl : erythroblastic leukaemia viral oncogene homolog 1) or erythroblastic leukaemia viral oncogene homologue 4 (ErbB4) and 3 main signal transduction pathways are activated: the RAS (ERK) pathway, the PI3 pathway and the JAK/STAT pathway (Meng et al, 2005; Smith et al, 2006). hi the skin, HB-EGF is a potent mitogen for keratinocytes and fibroblasts (Sorensen et al, 2006). HB-EGF is produced and secreted by keratinocytes and acts as an autocrine growth factor (Hashimoto et al, 1994). It has also been shown to be involved in epithelialization in skin wounding in vivo (Shirakata et al, 2005). However, HB-EGF has not been previously shown to regulate expression of COL7A1 gene expression, nor has it been indicated for the prevention or treatment of skin blistering conditions.

The nucleotide and amino acid sequences of human pro-HB-EGF precursor are disclosed in NCBI database accession numbers NM 001945.2 and NP_001936.1 respectively, and are shown in Figures 6 and 7 (SEQ ID NO:s 1 and 2).

The agent may comprise HB-EGF in any functional form, including the precursor or mature soluble forms. Thus the term "HB-EGF" as used herein is understood to include the pro-HB- EGF precursor. For example, the agent may comprise a fragment, variant or derivative of HB-EGF or its precursor, provided that the agent retains one or more biological properties of HB-EGF (e.g. binding to EGFR and/or stimulation of COL7A1 gene expression in fibroblasts and/or keratinocytes). Functional activity may be determined using methods described herein and disclosed in the art, e.g. using known assays for EGFR binding or a method for detecting COL7A1 gene expression as described in the Examples.

Preferably the agent is a functional fragment of SEQ ID NO:2, e.g. a polypeptide comprising at least 10, 20, 30, 50, 80, 100, 150 or 200 amino acid residues of SEQ ID NO:2 having HB- EGF-like activity. Fragments include proteolytic fragments and deletion fragments. In one embodiment, the agent comprises residues 1 to 148 of SEQ ID NO:2, e.g. the agent comprises the mature soluble form of HB-EGF. In another embodiment, the agent comprises residues 1 to 208 of SEQ ID NO:2, e.g. the agent comprises the full-length pro-HB-EGF precursor.

Functional variants of HB-EGF may also be employed, e.g. the agent may comprise a polypeptide in which one or more (e.g. 1 to 5, 1 to 10, 1 to 20 or 1 to 50) residues are substituted, deleted from or inserted into the sequence of SEQ ID NO:2. By "functional" it is meant that the variant retains HB-EGF biological activity. Typically such variants show homology to the polypeptide sequence of HB-EGF and/or its precursor, e.g. at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:2, e.g. over a region of at least 10, 20, 30, 50, 80, 100, 150 or 200 residues, or over the entire length of the sequence.

The percentage of sequence identity may be determined by analysis with a sequence comparison algorithm or by a visual inspection. In one aspect, the sequence comparison algorithm is a BLAST algorithm, e.g., a BLAST version 2.2.2 algorithm.

Variants may occur naturally or be non-naturally occurring. Non-naturally occurring variants may be produced using known mutagenesis techniques. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions.

The agent may also comprise a modified or derivatized HB-EGF polypeptide. Derivatives of HB-EGF include polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. For example, the polypeptide may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. The polypeptide may be modified naturally or artificially, for example by disulphide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labelling component. An HB-EGF derivative may comprise a polypeptide having one or more residues chemically derivatized by reaction of a functional side group. The agent may comprise one or more amino acid analogues, (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. For example, 4-hydroxyproline may be substituted for proline; 5- hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine. In one embodiment, the agent is a fusion protein comprising HB-EGF or a functional fragment or variant thereof fused to an additional polypeptide sequence.

Composition

The present invention also provides pharmaceutical compositions. Such compositions typically comprise a therapeutically effective amount of the agent, and a pharmaceutically acceptable carrier. The term "carrier" typically refers to a diluent, adjuvant, excipient, or vehicle with which the agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

The compositions may take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. In one embodiment, the agent may be delivered in a vesicle, in particular a liposome or a controlled release system (see Langer, Science 249:1527-1533 (1990)).

Preferably the agent is formulated into a pharmaceutical composition with suitable pharmaceutically acceptable excipients for topical administration to mammals. Such excipients are well known in the art. Topical administration includes administration to the skin or mucosa, including surfaces of the lung and eye. Other forms of localized administration may comprise application in conjunction with a wound dressing, by injection, by means of a catheter, by means of a suppository, or by means of an implant.

Preferred dosage forms for topical administration include, but are not limited to, ointments, creams, emulsions, lotions and gels and agents that favour penetration within the epidermis. In one embodiment, the composition is in the form of topical ointment.

The agent of the invention may be administered alone or in combination with additional active agents, including pharmaceutical, biological and/or molecular biological active agents in the context of combination or adjuvant therapy. The compositions can also contain adjuvants such as, but not limited to, solubilizers, skin permeation enhancers, preservatives, wetting agents, moisturizers, gelling agents, buffering agents, surfactants, emulsifying agents, emollients, thickening agents, stabilizers, humectants and dispersing agents.

Moisturizers include carriers that allow the agent to penetrate into the epidermis. This may be achieved by varying the formulation according to several factors affecting human skin, including the age of the subject being treated and the body site. Examples of moisturizers include, but are not limited to, jojoba oil and evening primrose oil.

Suitable skin permeation enhancers include lower alkanols, such as methanol ethanol and 2- propanol; alkyl methyl sulfoxides such as dimethylsulfoxide (DMSO), decylmethylsulfoxide and tetradecylmethyl sulfoxide, pyrrolidones, urea; N,N-diethyl-m-toluamide; C2-C6 alkanediols; dimethyl formamide (DMF), Ν,Ν-dimethylacetamide (DMA) and telxahydromrfuryl alcohol.

Examples of solubilizers include, but are not limited to, hydrophilic ethers such as diethylene glycol monoethyl ether (ethoxydiglycol) and diethylene glycol monoethyl ether oleate; polyoxy 35 castor oil, polyoxy 40 hydrogenated castor oil, polyethylene glycol (PEG), particularly low molecular weight PEGs, such as PEG 300 and PEG 400, and polyethylene glycol derivatives such as PEG-8 caprylic/capric glycerides; alkyl methyl sulfoxides, such as DMSO; pyrrolidones, DMA, and mixtures thereof.

Prevention and/or treatment of infections can be achieved by the inclusion of antibiotics, as well as various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like, in the composition.

In another embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anaesthetic such as lignocaine to ease pain at the site of the injection. A suitable pharmaceutical composition for injection may comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), and optionally a stabilizer agent (e.g. human albumin). Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The agent can be formulated as a neutral or salt form. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.

Dosage

The amount of the agent which will be effective in the treatment, inhibition and prevention of skin blistering can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose- response curves derived from in vitro or animal model test systems.

For example, a dosage of the agent topically administered to the patient may be from about 1 ng/cm² to about 10 mg/ cm², e.g lOOng to 100μg/cm². The composition may be applied directly on the skin over relevant portions of the body of the patient as required, e.g. daily or two or three times a week, so as to prevent or minimize blistering resulting from frictional trauma. A dosage of the agent administered intravenously to a patient may be about 0.01 mg/kg to about 100 mg/kg of the patient's body weight, more preferably about 1 mg/kg to about 10 mg/kg of the patient's body weight.

Nucleic acids

In another embodiment, a nucleic acid comprising a sequence encoding HB-EGF, or a functional fragment, variant or derivative thereof, may be used to prevent or treat skin blistering, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.

Any known gene therapy method can be used according to the present invention. For general reviews of the methods of gene therapy, see e.g. Goldspiel et al., Clinical Pharmacy 12:488- 505 (1993); and Morgan and Anderson, Arm. Rev. Biochem. 62: 191-217 (1993). In a preferred embodiment, a nucleic acid sequence encoding the agent is comprised in an expression vector suitable for expressing the agent in a desired host subject. The expression vector may comprise one or more promoter sequences operably linked to the region coding for the agent, said promoter being inducible or constitutive, and, optionally, tissue-specific. In one embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription. In another specific embodiment, nucleic acid molecules are used in which the agent coding sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the agent- encoding nucleic acids (Roller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989).

Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. Expression vectors may be administered, for example, by infection using defective or attenuated retro virals or other viral vectors including adenoviruses (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules. In some embodiments, an expression vector may be transferred to cells (e.g. fibroblasts or keratinocytes) in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection, followed by delivery of cells to the patient (e.g. as described in the example below or Wong et al, 2008). In one embodiment, the cell used for gene therapy is autologous to the patient. In one embodiment, the nucleic acid encoding the agent comprises the sequence of SEQ ID NO:l , or a fragment or variant thereof encoding a polypeptide having HB-EGF activity. Preferably the nucleic acid comprises at least 30, 50, 100, 300, 500, 1000, 1500 or 2000 continuous nucleotides of SEQ ID NO: l, or the full-length sequence of SEQ ID NO:l , encoding a functional HB-EGF polypeptide, e.g. encoding the mature soluble form of HB- EGF or the full-length pro-HB-EGF precursor. In another embodiment, the nucleic acid comprises a variant of SEQ ID NO:l, e.g. a nucleic acid in which one or more (e.g. 1 to 10, 1 to 30, 1 to 50, 1 to 100 or 1 to 500) residues are substituted, deleted from or inserted into the sequence of SEQ ID NO:l . Preferably the variant nucleic acid shows at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:l, e.g. over a region of at least 30, 50, 100, 300, 500, 1000 or 2000 residues, or over the entire length of the sequence.

Treatment of skin blistering

The agents and compositions disclosed herein, as well as nucleic acids and expression vectors encoding such agents, may be used for the prevention and/or treatment of skin blistering. In preferred embodiments, the agents and other products of the invention are used to prevent and/or treat a skin blistering disease, e.g. an inherited or acquired condition associated with the spontaneous formation of skin blisters. The products may be used for prophylactic treatment, e.g. before blisters appear on the skin of the subject.

In one embodiment, the skin blistering disease is epidermolysis bullosa (EB). The products described herein may be used to prevent or treat various forms of EB, including dystrophic epidermolysis bullosa, epidermolysis bullosa simplex, junctional epidermolysis bullosa and epidermolysis bullosa aquisita.

It is particularly preferred that the blistering disease to be treated is associated with defective or abnormal expression of collagen, e.g. type VII collagen (C7) derived from the COL7A1 gene. Preferably the blistering disease is dystrophic epidermolysis bullosa, more preferably recessive dystrophic epidermolysis bullosa.

In another embodiment, the disease is pemphigus or pemphigoid, e.g. pemphigus vulgaris or bullous pemphigoid, or epidermolysis bullosa acquisita. In alternative embodiments, the skin blistering is associated with burns, friction, or chemical irritation. In a further general aspect, the agents, compositions, nucleic acids and expression vectors described herein may be used to promote type VII collagen (C7) production or expression in a subject. Typically, the products are used to promote C7 expression in the skin of the subject. Such uses include the prevention and/or treatment of any condition in which it is desirable to increase C7 expression (typically in the skin). For instance the products may be used to increase the strength, suppleness, and/or mechanical resilience of the skin, reduce inflammation or accelerate wound healing, e.g. where the skin is damaged or prone to damage. Preferably the products are used to promote C7 expression in a condition associated with abnormal or defective C7 production in the skin, e.g. dystrophic epidermolysis bullosa.

Preferably the subject is a mammal, more preferably a human.

The invention will now be described with reference to the following non-limiting examples. EXAMPLES

It is known that type VIII collagen (C7) is synthesized by keratinocytes and fibroblasts (Stanley et al., 1985). Intradermal injection of fibroblasts increases C7 expression at the dermal-epidermal junction (DEJ) (Wong et al., 2008). However, since the molecular mechanism of this cell-based therapy is unknown, alternative therapies for increasing C7 expression based on protein or peptide mediators have not been described. Moreover, it is unknown whether the new C7 at the DEJ that follows allogeneic fibroblast injections is derived directly from the donor cells or indirectly through enhanced synthesis of mutant C7 by the recipients" own keratinocytes and/or fibroblasts.

In the current study, we have examined duration of response and mechanism of action of intradermal injection of allogeneic fibroblasts. In a single patient study, we analyzed serial skin biopsies for C7 protein and COL7A1 gene expression (including mutant allele specific assessment) for up to one year and also undertook microarray profiling on whole skin- extracted RNA to search for altered cytokine responses which lead to increased C7 expression. In addition, the effect of HB-EGF on expression of COL7A1 in keratinocytes and fibroblasts was studied. Finally, we observed whether intradermally injected allogeneic fibroblasts can accelerate wound healing in RDEB.

Materials and methods Allogeneic fibroblast injections and skin biopsies

Commercially available neonatal foreskin fibroblasts (ICX-RHY, Valveta®, Intercytex; Manchester, UK) were used. Following informed consent and in accordance with the Declaration of Helsinki principles, the cells were injected at a density of 5 xlO⁶ per cm² (equates to 0.25ml per cm ). Under local anaesthetic using 2% lidocaine, skin biopsies were obtained at day 0, 7, 15, 90, 180, 27 and 360. We also injected similar volumes of 0.9% physiological saline as a control and took biopsies using the same local anaesthetic at days 15 and 90.

Immunofluorescence microscopy for C7 and quantification of fluorescence intensity

Skin sections measuring 5μηι were air-dried, fixed, and mitially blocked with normal goat serum (Sigma-Aldrich, Dorset, UK) and then incubated with mouse monoclonal anti-C7 antibody (clone LH 7.2; Sigma-Aldrich) diluted 1 : 1,000 in phosphate buffered saline (PBS), followed by detection with FITC-labelled goat anti-mouse secondary antibody (Invitrogen, Paisley, UK). All immunostaining experiments were repeated twice. Negative controls omitting the primary antibody were performed for each set of labelling experiments. All sections were photographed using the same camera and identical exposure times (3 seconds). Mean fluorescence intensity was calculated for each sample using Image J (Rasband WS, National Institutes of Health, Bethesda MD; http://rsb.info.nih.gov/ij/, 1997-2007). Briefly, 10 measurements were taken at regular intervals using an area of 8x8 pixels, and measuring every 50 pixels along the DEJ, starting from the outermost edge of the image. The mean average values and standard errors were calculated for each image.

Cell cultures

Primary keratinocyte and fibroblast cultures were established from the skin biopsies taken from the individual with RDEB patients and an age and sex-matched healthy control following standard procedures (Rheinwald, 1989). The keratinocytes were cultured in EpiLife medium supplemented with defined growth supplement (EDGS; Invitrogen). To establish fibroblast cultures, the cells were cultured in DMEM supplemented with 10% fetal calf serum. Passages two to five were used for the experiments. Statistical significances of differences between the samples of the same cell lines collected at different time points were assessed by variance analysis: one-way ANOVA for independent or correlated samples (VassarStats: Web Site for Statistical Computation; http://faculty.vassar.edu/lowry/VassarStats.html). Similarity between the expression profiles of different genes was calculated by means of the basic linear correlation test. All statistical analyses were carried out through the use of (VassarStats).

Whole genome gene expression microarray and data analysis

Gene expression microarray experiment was performed using total RNA extracted from baseline (day 0), fibroblast injected sites at days 7, 15 and 90, and the saline injected site at day 15. Whole genome gene expression microarray in each extracted RNA sample was performed using Sentrix Human-6 Whole Genome Expression Beadchips (Illumina Inc, San Diego, CA, USA). The data were normalized using a cubic spline function of Beadstudio software version 3.0 (Illumina Inc). Differential gene expression was calculated based on the expression difference score (DiffScore) of >13 or <-13, which takes into account the background noise and sample variability, and a differential fold change of two or greater. In addition, the average signal intensity for each probe was considered significant if its detection p value was <0.05. Any probe with a signal intensity p value of >0.05 was excluded from the analysis. Average signal intensity for each probe was calculated for both subjects (S) and controls (N). Fold change was calculated according to the formula S(average probe signal intensity) / N(average probe signal intensity).

Quantitative real-time PCR

RNA was isolated using the RNeasy Fibrous Tissue Mini Kit (Qiagen, Crawley, UK), then first strand cDNA synthesis was performed with 1 μg of total RNA using Superscript II Reverse Transcriptase (Invitrogen, Paisley, UK). After cDNA was generated, quantitative, real-time, reverse-transcriptase PCRs (qRT-PCR) was carried out using J8S internal control and COL7A1 TaqMan® Gene Expression Assays (18S Hs03003631_gl, COL7A1 Hs01574745_gl, Applied Biosystems; Foster City, CA, USA). The COL7A1 TaqMan assay measured the amplification of the exon 54-55 fragment of the COL7A1 cDNA. The exon skipped allele of the investigated RDEB patient was separately amplified and quantified using qRT-PCR with a SYBR Green protocol (Applied Biosystems) and the following primers: COL7A1 cDNA exon 86 forward 5 " -GTGCCAGTGG AAAAGATGG A-3 " (SEQ ID NO:3), COL7A1 cDNA exon 87 forward 5 -CGGACCTAAAGG AG AACCTG-3 ^Λ (SEQ ID NO:4) and COL7A1 cDNA exon 90 reverse 5^,-AGTCCTCGGTCACCTTTGG-3^, (SEQ ID NO:5). The microarray data were confirmed with validated Taqman assays (FOS Hs00170630_ml, JUN Hs01103582_sl, IL1R2 Hs01030385_ml, LAMC2 Hs01043707_ml, GAL Hs01032385_ml, STAT1 Hs01014002_ml , TNFSF13B Hs00198106_ml, IRF1 Hs00971960_ml, VCAM1 Hs01003372 ml, COL1A1 Hs01076756_gl , COL4A1 Hs01007434_gl, COL7A1, COL17A1 Hs00166711_ml, CCL18 Hs00268113_ml , HB-EGF Hs00181813_ml) purchased from Applied Biosystems (Foster City, CA, USA) using qRT- PCR). PCR reactions were performed in an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Each PCR reaction consisted of 0.5 μΐ of cDNA, 10.75 μΐ of H₂0, 12.5 μΐ of Taqman MasterMix (Applied Biosystems) and 1.25 μΐ of Taqman assay, making up a total volume of 25 μΐ. Forty cycles of PCR amplification were used. The gene expression was normalized against the expression of 18S internal control and expressed relative to the average of control skin samples.

In vitro HB-EGF studies

For experiments examining added recombinant HB-EGF (Cat No.: 259-HE; R&D Systems; Abingdon, UK) and transforming growth factor beta 1 (TGFpl ; Cat No.: 240-B; R&D Systems) cells were fed with fresh DMEM medium without FBS supplementation and were cultured for 2 hours before growth factor addition. 10 ng/ml of TGFpl and 100 ng/ml of HB- EGF were added to the cells (Higashiyama et al, 1993; Calonge et al, 2004). Samples were collected 15, 90 and 180 minutes after the application of HB-EGF or TGF i. RNA isolation from cells was performed using TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's protocol.

Results

A single intradermal injection of allogeneic fibroblasts into RDEB skin can increase C7 at the DEJ for more than 9 months

The clinicopathological details of the patient selected for this study have been published previously as Case 5 in the report by Wong et al. (2008). We selected this patient for the current study because in the previous trial she showed an increase in C7 labelling at the DEJ at the 3 -month end-point and was therefore deemed suitable to determine duration of response in a longer follow-up assessment. We injected 5 x 10 6 per cm 2 normal control neonatal allogeneic fibroblasts over a 9 cm area of non-blistered skin and took serial biopsies from within this area at day 7, 15, 30, 90, 180 and 360. A baseline biopsy (day 0) adjacent to the fibroblast treated area was also sampled. As noted previously, a single injection of allogeneic fibroblasts resulted in increased fluorescent staining for C7 at the DEJ and also within basal keratinocytes, although more prolonged follow up was performed in this study.

Details of the fluorescence patterns and intensities at the different time-points are presented in Fig. 1. Of note, C7 labelling intensity increased over the first 2 weeks and persisted at similar levels for the 90, 180 and 270 day biopsies. At day 360, however, labelling was comparable to baseline. Interestingly, for the day 15 biopsy, we noted that the saline injection resulted in a slight increase in labelling at the DEJ and within basal keratinocytes but the pattern and intensity had returned to baseline levels in the day 90 saline-injected skin biopsy. No biopsies were available for the saline injections between days 15 and 90 to comment further on the time course of this transient increase in C7 within basal keratinocytes and at the DEJ. Thus following allogeneic fibroblast injection, the increase in C7 labelling noted after 2 weeks is maintained for at least 9 months but returns to baseline levels by 12 months.

A single intradermal injection of allogeneic fibroblasts into RDEB skin can increase COL7A1 gene expression for at least 3 months

We assessed changes in the COL7A1 mRNA levels using a TaqMan assay that amplifies the exon 54-55 part of the 118 exon gene. Details of the COL7A1 gene expression levels at different time-points are illustrated in Fig. 2a. At baseline, COL7A1 expression in the patient's skin was -25% of the control skin findings. At day 7 there was no significant change in COL7A1 gene expression but at day 15, expression levels had increased more than 20-fold and were ~4-5 times more than normal skin control COL7A1 gene expression. Similar levels of increased COL7A1 gene expression were also noted in the patient^'s skin at day 90; however, levels at 180 days and thereafter resembled those at baseline.

In addition, we noted that the control saline injection resulted in a ~5-fold increase in patient skin COL7A1 gene expression to a level similar or slightly higher than that seen in control skin. In contrast to the fibroblast injected skin, however, no increase in the saline site COL7A1 gene expression was seen in the day 90 biopsy. This study shows that both fibroblasts and saline can lead to increased COL7A1 gene expression in RDEB skin but that the size and duration of the increase is greater following an injection of fibroblasts. A single injection of fibroblasts injected into RDEB skin can increase COL7A1 gene expression for at least 3 months but not for 6 months or longer.

Allogeneic fibroblast cell therapy in RDEB increases mutant COL7A1 gene expression

The patient we studied is a compound heterozygote for a nonsense and splice site combination of mutations in COL7A1: C.2044OT (p.Arg682X) and IYS87+4A>G. This donor splice site mutation creates a leaky splice site which leads to m-frame skipping of exon 87 (69-bp). The patient's father is heterozygous for this splice site mutation and has a phenotype of mild dominant DEB.

We assessed day 0 skin in the patient and performed qRT-PCR spanning exons 86-90 (Fig. 2b). We observed that the ratio of wild-type to in-frame exon skip of exon 87 transcripts was -2:3. We then examined expression of this region of COL7A1 in the other time-point biopsies and found a similar increase in total COL7A1 levels to qRT-PCR for exons 54-55, but that the ratio of wild-type exon 86-90 transcript to exon 87 skipped transcript remained ~2:3. This finding demonstrates that the patient's own mutant COL7A1 allele(s) is/are being expressed at increased levels at days 15 and 90 after fibroblast injection.

Moreover, quantification of the exon-skipped allele and the ratio compared to wild-type sequence indicates that the major, if not the sole, reason for the increased COL7A1 gene expression is increased expression of the patient's own allele(s). This interpretation is supported by previous studies showing that the allogeneic fibroblasts are no longer detectable just 2 weeks after injection (Wong et ah, 2008), so any increased COL7A1 gene expression 3 months after the injection is very likely to be the recipient's alone. In addition, assessment of the saline-injected site showed a similar proportional increase in differential COL7A1 transcripts, albeit at a total level ~5-fold less.

Microarray and qRT-PCR data identify HB-EGF is a factor relevant to increased COL7A1 gene expression following allogeneic fibroblast injection

To investigate the nature of the response initiated by the allogeneic fibroblast injection that leads to increased COL7A1 expression, we performed gene expression profiling comparing the baseline, fibroblast-injected and saline-injected skin at different time-points. Comparison of the gene expression profiles of the skin samples taken at day 0 and day 7 after the fibroblast injections showed more than 2-fold changes in the expression of 19 cytokines, chemokines and growth factors but no differences in the gene expression level for COL7A1. Most of these encoded cytokines and chemokines are involved in inflammation and may be synthesized by keratinocytes, fibroblasts or inflammatory cells.

Comparing the gene expression profiles of the skin samples before for baseline and day 15 day after the fibroblast injections, we noted further differences in the expression of cytokines, chemokines and growth factors. Given that the day 15 time-point was when increased expression of COL7A1 (and other collagens, including COL1A1, COL4A1 and COL17A1) was noted, by both microarray and qRT-PCR, differences in cytokine expression at this time-point are likely to be relevant to understanding how fibroblast cell-therapy increases COL7A1 gene expression. No differences were noted in gene expression for cytokines already known to increase COL7A1, including those encoding transforming growth factor-beta (TGF ), interleukin-1 (IL-1) or tumor necrosis factor-alpha (TNFa).

However, we noted a >3-fold increase in expression of the gene for heparin binding- epidermal growth factor-like growth factor (HB-EGF). Increased expression of HB-EGF was also noted in the day 90 microarray data analysis, when COL7A1 gene expression was also increased. We then assessed HB-EGF expression by qRT-PCR and noted a similar pattern to the COL7A1 qRT-PCR data for all time-points and for the fibroblast injected sites (linear correlation r=0.978, pO.0001) (Fig. 3).

It has been previously demonstrated that HB-EGF can increase expression of COL1A1 in lung fibrosis (Ingram et αί, 2003), but a putative association with the regulation of COL7A1 has not been demonstrated previously. We also noted that expression profiles of FOS (linear correlation r=0.864, p<0.0006) and JUN (linear correlation r=0.945, pO.0001) were also highly similar to the pattern of increased COL7A1 expression at the different time-points. JUN and FOS form the AP-1 transcription complex which can bind to the COL7A1 promoter and enhance gene expression (Nakano et αί, 2001).

HB-EGF stimulation of normal control and RDEB keratinocytes and fibroblasts increases JUN, FOS and COL7A1 gene expression

To investigate whether the up-regulation of HB-EGF might be related to the increased expression of COL7A1 or just a temporal association, sub-confluent cultured keratinocytes and fibroblasts, both normal control and RDEB, were treated with 100 ng/ml recombinant HB-EGF protein and changes in COL7A1 expression levels were monitored after 15, 90 and 180 minutes (dose and time course, as determined elsewhere; Higashiyama et al, 1993) and normalized using 18S internal control. For each cell population, the COL7A1 expression levels in the treated cells were compared to the levels of the same untreated cells. Stimulation with HB-EGF was repeated at least twice in each cell sub-type. In addition to HB-EGF treatment, other experiments using 10 ng/ml TGFpi stimulation were also performed as positive controls (data not shown) since TGF i is a well-known up-regulator of COL7A1 (Calonge et al, 2004).

HB-EGF treatment led to up-regulation in COL7A1 mRNA in keratinocytes and fibroblasts in both normal control and RDEB cells. In keratinocytes, the highest COL7A1 expression levels (relative to baseline) were detected at the 90 minute time-point, whereas in fibroblasts the peak increases were noted in the 15 minute measurements. Maximal relative increases were ~3-5-fold in keratinocytes and ~2-3-fold in fibroblasts (both control and RDEB cells).

To investigate whether HB-EGF specifically increases expression of COL7A1 alone, we also measured COL1A1 and COL17A1 gene expression under similar conditions and noted that HB-EGF treatment of cells resulted in a ~2 fold increase in COL1A1 in normal control and RDEB keratinocytes and fibroblasts, as well as up-regulation of COL17A1 in keratinocytes but not fibroblasts (data not shown). We also measured JUN and FOS expression levels after similar exposure to HB-EGF. There was up-regulation in both genes and in all cells at the 15 minute measurement point, but no significant relative differences from baseline were noted at the later time-points (Figs 4b, c).

Intradermal injection of allogeneic fibroblasts into the margins of chronic erosions in RDEB induces rapid re-epithelialization.

We also assessed the effects of injecting allogeneic fibroblasts on wound healing. In the same RDEB individual, we selected two non-healing erosions (present for more than 6 weeks) and injected fibroblasts into the dermis around the erosion margins. At baseline, both erosions measured ~3 x 2 cm and ~40 x 10⁶ fibroblasts were injected to each wound (equivalent to ~5 x 10⁶ cells per cm of wound edge). Within 7 days, the wound surface area had decreased and by 15 days there was complete re-epithelialization (Fig. 5). The treated site remained healed with no recurrent blistering in 6 months follow-up observation. Moreover, the patient reported subjective changes of softer, less inflamed skin over this period. Discussion

This study shows that a single treatment with allogeneic fibroblasts can increase COL7A1 gene expression for 3-6 months and C7 protein expression for 9-12 months when injected into non-blistered skin. Our findings also implicate an "indirect mechanism" for this response, with increased expression of the patient's own mutant COL7A1 gene sequence. Significantly, the results indicate that the beneficial effects of fibroblast treatment are mediated by the growth factor HB-EGF. HB-EGF and COL7A gene expression are both upregulated after fibroblast treatment and HB-EGF stimulates COL7A1 expression in fibroblasts and keratinocytes.

The present findings indicate that HB-EGF contributes to the changes in C7 labelling at the DEJ following injection of allogeneic fibroblasts into intact RDEB skin. The source of the HB-EGF is likely to be the RDEB keratinocytes, since keratinocytes are the principal source of HB-EGF in skin and expression of HB-EGF increased after the time-point at which allogeneic fibroblasts have been shown to no longer be present.

The HB-EGF response occurs more significantly following fibroblast injection rather than the non-specific inflammatory response triggered by saline injection. We did not detect increases in expression of any other cytokine or growth factor that is known to upregulate COL7A1 gene expression. A role for HB-EGF in increasing C7 in RDEB skin was also supported by our in vitro observations of increased JUN and FOS gene expression following HB-EGF stimulation, since AP-1 transcription factor is known to bind the COL7A1 gene promoter.

Collectively, our in vivo and in vitro data suggest that increased expression of HB-EGF by RDEB keratinocytes is a mechanism for increasing expression of mutant C7 in RDEB skin after allogeneic fibroblast injection. Accordingly, HB-EGF may be used as an alternative treatment to cell-based therapies in blistering diseases such as EB, particularly where it is desirable to promote C7 expression.

Each of the applications and patents mentioned in this document, and each document cited or referenced in each of the above applications and patents, and any manufacturer's instructions or catalogues for any products cited or mentioned in each of the applications and patents and in any of the application cited documents, are hereby incorporated herein by reference. Furthermore, all documents cited in this text, and all documents cited or referenced in documents cited in this text, and any manufacturer's instructions or catalogues for any products cited or mentioned in this text, are hereby incorporated herein by reference.

Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments and that many modifications and additions thereto may be made within the scope of the invention. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the claims. Furthermore, various combinations of the features of the following dependent claims can be made with the features of the independent claims without departing from the scope of the present invention.

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Claims

1. An agent comprising heparin-binding EGF-like growth factor (HB-EGF), or a functional fragment, variant or derivative thereof, for use in the prevention and/or treatment of skin blistering.

2. An agent according to claim 1, for use in the prevention and/or treatment of skin blistering associated with epidermolysis bullosa.

3. An agent according to claim 2, for use in the prevention and/or treatment of skin blistering associated with dystrophic epidermolysis bullosa.

4. An agent according to any preceding claim, wherein the agent comprises a polypeptide having the sequence of SEQ ID NO:2, or a functional fragment, variant or derivative thereof.

5. An agent according to claim 4, wherein the agent comprises a polypeptide having at least 90% sequence identity to at least 50 amino acids of SEQ ID NO:2.

6. A nucleic acid encoding an agent as defined in any preceding claim, for use in the prevention and/or treatment of skin blistering.

7. A nucleic acid according to claim 6, wherein the nucleic acid has at least 90% sequence identity to at least 100 nucleotides of SEQ ID NO:l .

8. An expression vector comprising a nucleic acid as defined in claim 6 or claim 7, for use in the prevention and/or treatment of skin blistering.

9. A composition comprising a therapeutically effective amount of an agent, nucleic acid or expression vector as defined in any preceding claim, and a pharmaceutically acceptable carrier, for use in the prevention and/or treatment of skin blistering.

10. A composition according to claim 9, for topical application to the skin.

11. A method for the prevention and/or treatment of skin blistering in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent comprising heparin-binding EGF-like growth factor (HB-EGF), or a functional fragment, variant or derivative thereof, or a nucleic acid encoding the agent.

12. Use of an agent comprising heparin-binding EGF-like growth factor (HB-EGF), or a functional fragment, variant or derivative thereof, or a nucleic acid encoding the agent, for the preparation of a medicament for preventing and/or treating skin blistering.

13. An agent comprising heparin-binding EGF-like growth factor (HB-EGF), or a functional fragment, variant or derivative thereof, or a nucleic acid encoding the agent, for use in promoting type VII collagen production in a subject.