WO2015138502A1 - In-situ skin regeneration for epidermolysis bullosa and other skin disorders - Google Patents

In-situ skin regeneration for epidermolysis bullosa and other skin disorders Download PDF

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WO2015138502A1
WO2015138502A1 PCT/US2015/019774 US2015019774W WO2015138502A1 WO 2015138502 A1 WO2015138502 A1 WO 2015138502A1 US 2015019774 W US2015019774 W US 2015019774W WO 2015138502 A1 WO2015138502 A1 WO 2015138502A1
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peptide
skin
method
col7
col4
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PCT/US2015/019774
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French (fr)
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Gary Cleary
Richard WALDRON
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Gary Cleary
Waldron Richard
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/70Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A61K9/7007Drug-containing films, membranes or sheets
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088Compounds having three or more nucleosides or nucleotides
    • A61K31/7105Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0002Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0009Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61MDEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0092Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin using ultrasonic, sonic or infrasonic vibrations, e.g. phonophoresis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61NELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00Electrotherapy; Circuits therefor
    • A61N1/18Applying electric currents by contact electrodes
    • A61N1/20Applying electric currents by contact electrodes continuous direct currents
    • A61N1/30Apparatus for iontophoresis, i.e. transfer of media in ionic state by an electromotoric force into the body, or cataphoresis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61NELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00Electrotherapy; Circuits therefor
    • A61N1/18Applying electric currents by contact electrodes
    • A61N1/32Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/325Applying electric currents by contact electrodes alternating or intermittent currents for iontophoresis, i.e. transfer of media in ionic state by an electromotoric force into the body
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61NELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00Electrotherapy; Circuits therefor
    • A61N1/18Applying electric currents by contact electrodes
    • A61N1/32Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/328Applying electric currents by contact electrodes alternating or intermittent currents for improving the appearance of the skin, e.g. facial toning or wrinkle treatment

Abstract

The present invention discloses a device for transdermal delivery of a bioactive agent for a skin disorder such as epidermolysis bullosa (EB) or a related disorder or complication. The present invention also discloses methods of making and using the device.

Description

IN-SITU SKIN REGENERATION FOR EPIDERMOLYSIS BULLOSA AND OTHER

SKIN DISORDERS

Gary W. Cleary

Richard A. Waldron

CROSS-REFERENCE OF RELATED APPLICATION

The present application claims the benefit to U.S. provisional application U No. 61/951,391, filed on March 11, 2014, the teaching of which is incorporated herein in its entirety by reference.

BACKGROUND OF THE INVENTION

The present invention generally relates to transdermal delivery of a bioactive agent. More specifically, the present invention relates to transdermal delivery of bioactive agent for a skin disorder such as epidermolysis bullosa (EB).

There are more than 30 kinds of skin disorders of genetic origin. Disorders such as Epidermolysis Bullosa (EB) and other skin disorders have small to large areas of stratum corneum and epidermal layers lift away for the dermis layer leaving the skin to form blisters or to have only the dermis layer exposed to the environment without the protection of the stratum corneum and the vital epidermis layers. Here, the skin has an abnormal structure and loses its normal functions and durability. This often leads to a continuous state of injury that often leads to an early death or a life of itching and painful areas of hands, arms legs, throat, teeth and other areas of the body throughout a person's life.

Current therapy consists of layering of cotton gauze soaked in petrolatum, adhesive tape, staples and other types of wound products and antibacterial solutions to protect the wound from further damage and infection. Using the current methodologies there is no skin regeneration that takes place.

The present invention addresses the above issues and problems. Embodiments of the present invention provide a transdermal delivery device for treating EB wounds in-situ to deliver therapeutic genes, cells, proteins that will regenerate the missing layers of skin cells found in EB and other skin disorders with new skin regenerated. The present invention provides a patient friendly method to rejoin the dermis to the epidermal layer in-situ and provide skin correction for the patient directly rather than using non- regenerative protective materials to the EB wound followed by sheets of skin cells that are produced elsewhere outside the patient's wound areas and achieve no regeneration of the patient's skin.

SUMMARY OF THE INVENTION

In one aspect of the present invention, it is provided a device for transdermal delivery of a biologically active agent effective for treating or ameliorating a skin condition, which device comprising

a skin layer comprising sheets of film comprising one or more layers that contain a biodegradable polymer that serves as a scaffold containing a bioactive agent, and

a backing layer formed of an ultrathin polymer film that provides conduction of a stimulus wherein the backing layer has a thickness of one or more microns,

wherein the stimulus, upon application to the device, causes the bioactive agent to have an enhanced delivery into the cells in the skin area.

In some embodiments of the invention device, optionally in combination in any or all of the various embodiments of the present invention, the stimulus is electricity, ultrasound, heat, magnetic field, light, or mechanical based energy, e.g., force, distance, time, the stimulus is electricity, ultrasound, heat, magnetic field, or light. Other stimulus could include mechanical or electromagnetic energy spectrum technologies, e.g., iontophoresis, ultracound (low frequency).

In some embodiments of the invention device, optionally in combination in any or all of the various embodiments of the present invention, the bioactive agent is selected from the group consisting of genes, cells, proteins, RNA, and other biologically active compounds.

In some embodiments of the invention device, optionally in combination in any or all of the various embodiments of the present invention, the skin disorder is epidermolysis bullosa (EB) or an EB related disorder.

In some embodiments of the invention device, optionally in combination in any or all of the various embodiments of the present invention, the biologically active agent is selected from: a) type VII collagen (COL7), Type XVII collagen al (COL17al), type IV collagen (COL4), COL17al;

b) a gene expressing COL7, Col7al, COL4, or COL17al;

c) a cell expressing proteins or R As of COL7, COL7al, COL4, or COL17al;

d) a peptide comprising the function domain of COL7 (COL7 peptide), a peptide comprising the function domain of COL7al (COL7al peptide), a peptide comprising the function domain of COL4 (COL4 peptide), or a peptide comprising the function domain of COL17al;

e) a peptide mimetics of COL7 peptide, COL7al peptide, a peptide mimetics of COL4 peptide, or a peptide mimetics of COL17al peptide; or

f) a combination of any of a)-e).

In some embodiments of the invention device, optionally in combination in any or all of the various embodiments of the present invention, the skin disorder is a disorder listed in Table 3.

In some embodiments of the invention device, optionally in combination in any or all of the various embodiments of the present invention, the biologically active agent is a gene listed in Table 3, a gene product of a gene listed in Table 3, or a cell expressing a gene listed in Table 3.

In some embodiments of the invention device, optionally in combination in any or all of the various embodiments of the present invention, the biodegradable polymer dissolves in a wound or diseased skin area after delivery of the bioactive agent is completed or substantially completed.

In some embodiments of the invention device, optionally in combination in any or all of the various embodiments of the present invention, the stimulus is ultrasound.

In some embodiments of the invention device, optionally in combination in any or all of the various embodiments of the present invention, the device is a patch or a roll which is to be placed or wrapped in situ over the hands, fingers, arms, legs, elbows, knees, foot toes and other parts of a patient's affected skin.

In some embodiments of the invention device, optionally in combination in any or all of the various embodiments of the present invention, the device is made into a glove to aid a patient with fingers and/or toes that have become webbed or to prevent the fingers or toes to become deformed, webbed or "mittens" in various stages of EB or an EB related disorder. In some embodiments of the invention device, optionally in combination in any or all of the various embodiments of the present invention, the biodegradable polymer is poly( - amino esters).

In a second aspect of the present invention, it is provided a method of fabricating a device for transdermal delivery of a biologically active agent effective for treating or ameliorating a skin condition, which method comprising:

providing a skin layer comprising sheets of film comprising one or more layers that contain a biodegradable polymer that serves as a scaffold containing a bioactive agent, and providing a backing layer formed of an ultrathin polymer film that provides conduction of a stimulus wherein the backing layer has a thickness of one or more microns, wherein the stimulus, upon application to the device, causes the bioactive agent to have an enhanced delivery into the cells in the skin area.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the stimulus is electricity, ultrasound, heat, magnetic field, light, or mechanical based energy, e.g., force, distance, time.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the bioactive agent is selected from the group consisting of genes, cells, proteins, R A, and other biologically active compounds.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the skin disorder is epidermolysis bullosa (EB) or an EB related disorder.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the biologically active agent is selected from:

a) type VII collagen (COL7), Type XVII collagen al (COL17al), type IV collagen (COL4), COL17al;

b) a gene expressing COL7, Col7al, COL4, or COL17al;

c) a cell expressing proteins or R As of COL7, COL7al, COL4, or COL17al;

d) a peptide comprising the function domain of COL7 (COL7 peptide), a peptide comprising the function domain of COL7al (COL7al peptide), a peptide comprising the function domain of COL4 (COL4 peptide), or a peptide comprising the function domain of COL17al; e) a peptide mimetics of COL7 peptide, COL7al peptide, a peptide mimetics of COL4 peptide, or a peptide mimetics of COL17al peptide; or

f) a combination of any of a)-e).

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the skin disorder is a disorder listed in Table 3.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the biologically active agent is a gene listed in Table 3, a gene product of a gene listed in Table 3, or a cell expressing a gene listed in Table 3.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the biodegradable polymer dissolves in a wound or diseased skin area after delivery of the bioactive agent is completed or substantially completed.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the stimulus is ultrasound.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the device is a patch or a roll which is to be placed or wrapped in situ over the hands, fingers, arms, legs, elbows, knees, foot toes and other parts of a patient's affected skin.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the device is made into a glove to aid a patient with fingers and/or toes that have become webbed or to prevent the fingers or toes to become deformed, webbed or "mittens" in various stages of EB or an EB related disorder.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the biodegradable polymer is poly( - amino esters).

In a further aspect of the present invention, it is provided a method of treating skin condition, comprising applying a device to a patient to effect transdermal delivery of a biologically active agent effective for treating or ameliorating a skin condition, wherein the device comprises a skin layer comprising sheets of film comprising one or more layers that contain a biodegradable polymer that serves as a scaffold containing a bioactive agent, and

a backing layer formed of an ultrathin polymer film that provides conduction of a stimulus wherein the backing layer has a thickness of one or more microns,

wherein the stimulus, upon application to the device, causes the bioactive agent to have an enhanced delivery into the cells in the skin area.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the stimulus is electricity, ultrasound, heat, magnetic field, light, or mechanical based energy, e.g., force, distance, time.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the bioactive agent is selected from the group consisting of genes, cells, proteins, R A, and other biologically active compounds.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the skin disorder is epidermolysis bullosa (EB) or an EB related disorder.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the biologically active agent is selected from:

a) type VII collagen (COL7), Type XVII collagen al (COL17al), type IV collagen (COL4), COL17al;

b) a gene expressing COL7, Col7al, COL4, or COL17al;

c) a cell expressing proteins or R As of COL7, COL7al, COL4, or COL17al;

d) a peptide comprising the function domain of COL7 (COL7 peptide), a peptide comprising the function domain of COL7al (COL7al peptide), a peptide comprising the function domain of COL4 (COL4 peptide), or a peptide comprising the function domain of COL17al;

e) a peptide mimetics of COL7 peptide, COL7al peptide, a peptide mimetics of COL4 peptide, or a peptide mimetics of COL17al peptide; or

f) a combination of any of a)-e).

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the skin disorder is a disorder listed in Table 3. In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the biologically active agent is a gene listed in Table 3, a gene product of a gene listed in Table 3, or a cell expressing a gene listed in Table 3.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the biodegradable polymer dissolves in a wound or diseased skin area after delivery of the bioactive agent is completed or substantially completed.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the stimulus is ultrasound.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the device is a patch or a roll which is to be placed or wrapped in situ over the hands, fingers, arms, legs, elbows, knees, foot toes and other parts of a patient's affected skin.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the device is made into a glove to aid a patient with fingers and/or toes that have become webbed or to prevent the fingers or toes to become deformed, webbed or "mittens" in various stages of EB or an EB related disorder, such as the ones described in Tables 2 and 3. The covered area is based on size and shape of blister's area.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the biodegradable polymer is poly( - amino esters).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the schematic structure of an embodiment of the invention device.

Figure 2 illustrates an embodiment method of using an embodiment of the invention device. DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the term device refers to a transdermal delivery patch for delivery of a biologically active agent to a patient. As used herein, the term treating refers to a treatment process or clinic management regimen on a patient that generates clinically significant result.

As used herein, the term biodegradable refers to the attributes of a material useful herein capable of degradation into fragment or molecular entities forming the material under physiological conditions. In this connection, the term biodegradable shall be construed to mean that the material useful herein is also "biocompatible" and the term shall encompass "degradable", "bioerodible", "biodissolvable" and other terms that referring to breaking down of a material under physiologic conditions. Biodegradation of a material generally proceeds via two mechanisms: hydrolysis - that is, breaking down of a material effected by hydrolysis of the material ~ and enzymolysis, which refers to the breaking down of a material via an enzyme catalyzed process.

Transdermal delivery device

In one aspect of the present invention, it is provided a device for transdermal delivery of a biologically active agent effective for treating or ameliorating a skin condition. The device comprises

a skin layer comprising sheets of film comprising one or more layers that contain a biodegradable polymer that serves as a scaffold containing a bioactive agent, and

a backing layer formed of an ultrathin polymer film that provides conduction of a stimulus wherein the backing layer has a thickness of one or more microns,

wherein the stimulus, upon application to the device, causes the bioactive agent to have an enhanced delivery into the cells in the skin area.

In some embodiments of the invention device, optionally in combination with any or all the various embodiments described herein, the stimulus is electricity, ultrasound, heat, magnetic field, light, or mechanical based energy, e.g., force, distance, time.

In some embodiments of the invention device, optionally in combination with any or all the various embodiments described herein, the bioactive agent is selected from the group consisting of genes, cells, proteins, R A, and other biologically active compounds.

In some embodiments of the invention device, optionally in combination with any or all the various embodiments described herein, the skin disorder is epidermolysis bullosa (EB) or an EB related disorder (Table 2). In some embodiments of the invention device, optionally in combination with any or all the various embodiments described herein, the biologically active agent is type VII collagen (COL7), a gene expressing COL7 (Col7al), or a cell expressing COL7.

In some embodiments of the invention device, optionally in combination with any or all the various embodiments described herein, the skin disorder is a disorder listed in Table 3.

In some embodiments of the invention device, optionally in combination with any or all the various embodiments described herein, the biologically active agent is a gene listed in Table 3, a gene product of a gene listed in Table 3, or a cell expressing a gene listed in Table 3.

In some embodiments of the invention device, optionally in combination with any or all the various embodiments described herein, the biodegradable polymer dissolves in a wound or diseased skin area after delivery of the bioactive agent is completed or substantially completed.

In some embodiments of the invention device, optionally in combination with any or all the various embodiments described herein, the stimulus is ultrasound.

In some embodiments of the invention device, optionally in combination with any or all the various embodiments described herein, the device is a patch or a roll which is to be placed or wrapped in situ over the arms, legs, elbows, knees, fee toes and other parts of a patient's affected skin.

In some embodiments of the invention device, optionally in combination with any or all the various embodiments described herein, the device is made into a glove to aid a patient with fingers and/or toes that have become webbed or to prevent the fingers or toes to become deformed, webbed or "mittens" in various stages of EB or an EB related disorder (Table 2).

In some embodiments of the invention device, optionally in combination in any or all of the various embodiments of the present invention, the biodegradable polymer is poly( - amino esters).

Figure 1 shows an embodiment of the invention device. In Figure 1, the invention device includes an ultrasound source & transducer layer, an e-skin protective layer, a scaffold layer, which is bioerodible and includes a biologically active agent such as a proteins, genes, cells, and/or other agents, and a release liner. As used herein, the term bioerodible refers to the attributes of a material being biodegradable, bioerodible, and/or biodissolvable, the terms biodegradable, bioerodible, and/or biodissolvable can be used interchangeably.

As further embodiments of the invention device, the ultra-sound layer from the capacitive micro-machined ultrasound transducer (CMUT) enables the gene to have a greater transfection into the skin cells delivered or are already in the patient. The scaffold has two goals to perform. It serves as a matrix that holds the genes in place to await delivery into the EB wound area. Its second priority is to increase the uptake of the gene into the cells and transfection on its own even without the ultrasound. The scaffold will automatically dissolve at a particular rate once it reaches the liquid material in the EB wound delivering the genes. In this manner this layer does not have to be removed from the wound area and the Backing layer continues to serve as a protective layer in-situ. Ultimately the backing is removed from its original sight, leaving the newly regenerated skin in-tact.

As further embodiments of the invention device, the invention of device having a total of 3 to 4 layers of film can have a thickness that makes the film very flexible and can also be stretchable. This film can be made in the size of a "patch" or in large rolls of the 3 or 4 layers that can be used to wrap the area of need for skin regeneration. The film can be placed or wrapped over the arms, legs, elbows, knees, foot toes and other parts of the patient's affected skin. This film can be applied in-situ directly at the EB site rather than in sheets of skin cells that is prepared elsewhere using the patient's own cells that takes several weeks to

manufacture.

As further embodiments of the invention device, the device of invention can take a form of the film that can be made into a glove to aid the patient with fingers and/or toes that have become webbed or to prevent the fingers or toes to become deformed, webbed or "mittens" in various stages of EB or an EB related disorder (Table 2). A new feature of this new approach allows gene and cell therapy to be placed directly on the EB wound bed, the arm, elbow, legs, fingers and other areas of open wounds.

As used herein, by "in situ" it is meant to place the film (example: a wound) at the EB wound side directly and exactly in place where it occurs, e.g., wounds on the arm or leg (i.e. rather than moving it to some special medium, e.g., a petri dish with agar gel ). In some embodiments of the present invention, the delivery system is placed directly on the EB blister or wound site typically on an arm or leg directly and not on an agar gel in a petri dish). Basement membrane and basal lamina

The basement membrane (BM) lies between the epidermis and the dermis, keeping the outside layer tightly connected to the inside layer. BM is often referred to as basal lamina (BL).

The basal lamina is a layer of extracellular matrix secreted by the epithelial cells, on which the epithelium sits. It is often confused with the basement membrane, and sometimes used inconsistently in the literature, see below.

BM and/or BL is a structure that is visible only with the electron microscope, where it appears as an electron-dense layer, 20-100 nm thick (with exceptions such as the basal laminae that compose the 100-200 nanometer thick glomerular basement membrane). The layers of BL and BM are described below in Table 1 :

Table 1

Figure imgf000012_0001

The three above layers of the basal lamina typically sit on top of the reticular lamina, which is synthesized by cells from the underlying connective tissue and contains fibronectin. The exception is when two epithelial layers abut one another as in the alveoli of the lungs and glomeruli of the kidneys, in which the basal lamina of one epithelial layer fuses with that of the other.

Anchoring fibrils composed of type VII collagen extend from the basal lamina into the underlying reticular lamina and loop around collagen bundles. Although found beneath all basal laminae, they are especially numerous in stratified squamous cells of the skin.

The distinction between BM and BL The basement membrane is visible under light microscopy. Electron microscopy shows that the basement membrane consists of three layers: the lamina lucida (electron- lucent), lamina densa (electron-dense), and lamina fibroreticularis (electron-lucent).

The lamina densa was formerly known as the basal lamina. The terms basal lamina and basement membrane were often used interchangeably, until it was realised that all three layers seen with the electron microscope represent the single layer seen with the light microscope. This has led to considerable terminological confusion and, if used, the term basal lamina should be confined to its meaning as lamina densa.

Some theorize that the lamina lucida is an artifact created when preparing the tissue, and that the basement membrane is therefore equal to the lamina densa in vivo.

The term "basal lamina" is usually used with electron microscopy, while the term "basement membrane" is usually used with light microscopy.

Examples of basement membranes include: basilar membrane, bruch's membrane, descemet's membrane, and glomerular basement membrane.

Epidermolysis bullosa

Epidermolysis bullosa refers to a group of inherited disorders that involve the formation of blisters following trivial trauma. Over 30 mutations have been identified in this condition. They have been classified into the following types:

Epidermolysis bullosa simplex: Epidermolysis bullosa simplex is a form of epidermolysis bullosa that causes blisters at the site of rubbing. It typically affects the hands and feet, and is typically inherited in an autosomal dominant manner, affecting the keratin genes K T5 and K T14.

Junctional epidermolysis bullosa: Junctional epidermolysis bullosa is an inherited disease affecting laminin and collagen. This disease is characterised by blister formation within the lamina lucida of the basement membrane zone, and is inherited in an autosomal recessive manner. It also presents with blisters at the site of friction, especially on the hands and feet, and has variants that can occur in children and adults. Less than one per million people are estimated to have this form of epidemolysis bullosa.

Dystrophic epidermolysis bullosa: Dystrophic epidermolysis bullosa is an inherited variant affecting the skin and other organs. "Butterfly children" is the term given to those born with the disease, as their skin is seen to be as delicate and fragile as that of a butterfly wing. Dystrophic epidermolysis bullosa is caused by genetic defects (or mutations) within the human COL7A1 gene encoding the protein type VII collagen (collagen VII). [11] DEB- causing mutations can be either autosomal dominant or autosomal recessive.

Pathophysiology: The human skin consist of two layers: an outermost layer called the epidermis and a layer underneath called the dermis. In individuals with healthy skin, there are protein anchors between these two layers that prevent them from moving independently from one another (shearing). In people born with EB, the two skin layers lack the protein anchors that hold them together, resulting in extremely fragile skin— even only minor mechanical friction (like rubbing or pressure) or trauma will separate the layers of the skin and form blisters and painful sores. Sufferers of EB have been compared to the sores from third-degree burns. [12] Furthermore, as a complication of the chronic skin damage, people suffering from EB have an increased risk of malignancies (such as cancers) of the skin.

Treatment of the epidermolysis bullosa by transplantation of laminin5 modified stem cells

Recent research has focused on changing the mixture of keratins produced in the skin. There are 54 known keratin genes— of which 28 belong to the type I intermediate filament genes and 26 to type II— which work as heterodimers. Many of these genes share substantial structural and functional similarity, but they are specialized to cell type and/or conditions under which they are normally produced. If the balance of production could be shifted away from the mutated, dysfunctional keratin gene toward an intact keratin gene, symptoms could be reduced. For example, sulforaphane, a compound found in broccoli, was found to reduce blistering in a mouse model to the point where affected pups could not be identified visually, when injected into pregnant mice (5 μιηοΐ/day = 0.9 mg) and applied topically to newborns (1 μιηοΐ/day = 0.2 mg in jojoba oil). [13]

Current clinical research at the University of Minnesota has included a bone marrow transplant to a 2-year-old child who is one of 2 brothers with EB. The procedure was successful, strongly suggesting that a cure may have been found. A second transplant has also been performed on the child's older brother, and a third transplant is scheduled for a

California baby. The clinical trial will ultimately include transplants to 30 subjects. [14] However, the severe immunosuppression that bone marrow transplantation requires causes a significant risk of serious infections in patients with large scale blisters and skin erosions. Indeed, at least four patients have died in the course of either preparation for or institution of bone marrow transplantation for epidermolysis bullosa, out of only a small group of patients treated so far.

Biologically active agent

The biologically active agent of the invention device refers to a bioactive agent effective for treating, preventing, or ameliorating a skin disorder. In some embodiments, the bioactive agent is one of the following:

a) type VII collagen (COL7), Type XVII collagen al (COL17al), type IV collagen (COL4), COL17al;

b) a gene expressing COL7, Col7al, COL4, or COL17al;

c) a cell expressing proteins or R As of COL7, COL7al, COL4, or COL17al;

d) a peptide comprising the function domain of COL7 (COL7 peptide), a peptide comprising the function domain of COL7al (COL7al peptide), a peptide comprising the function domain of COL4 (COL4 peptide), or a peptide comprising the function domain of COL17al;

e) a peptide mimetics of COL7 peptide, COL7al peptide, a peptide mimetics of COL4 peptide, or a peptide mimetics of COL17al peptide; or

f) a combination of any of a)-e) (for example, combination of COL7 and COL4).

In some embodiments, the biologically active agent can be a cell expressing COL7, COL7al, COL4, and/or COL17al . An example of such a cell is a stem cell or induced pluripotent stem (iPS) cell. In some embodiments, the stem cell or iPS cell are genetically engineered to have an enhanced expression of expressing COL7, COL7al, COL4, and/or COL17al .

In some other embodiments, the bioactive agent can be any protein or peptide that helps joining of the stratum corneum and epidermal layers with the dermis layer of skin.

In some embodiments, the bioactive agent can include a second agent or therapeutics for a skin condition. Such second agent or therapeutics include, for example, antiviral agents, antibacterial agents, antifungal agents, anti-inflammatory agents, vitamins, and/or

antioxidants.

In some embodiments, the biologically active agent of the invention device can specifically exclude any of the above agents or combination of agents. Scaffolds

A skin layer of the invention device refers to a layer of polymer of the invention device that serves as the scaffold of the biologically active agent of invention device (see embodiments shown in Figures 2-3, the scaffold), which is placed underneath the backing layer of invention device (see embodiments shown in Figures 2-3, the e-skin). The skin layer is formed of a biodegradable polymer or biosoluble polymer, which, upon application a wounded or diseased skin area, degrades or dissolves completely. In some embodiments, the dissolved skin layer gels on the wounded or diseased skin area. Polymers having a degradation rate or dissolution rate that meet such degradation or dissolution requirement are well documented and readily available.

Scaffolds of the invention device can include a carrier, which can be biodegradable, such as degradable by enzymatic or hydro lytic mechanisms. Examples of carriers include, but are not limited to synthetic absorbable polymers such as such as but not limited to poly(alpha- hydroxy acids) such as poly (L-lactide) (PLLA), poly (D, L-lactide) (PDLLA), polyglycolide (PGA), poly (lactide-co-glycolide (PLGA), poly (-caprolactone), poly (trimethylene carbonate), poly (p-dioxanone), poly (-caprolactone-co-glycolide), poly (glycolide-co- trimethylene carbonate) poly (D, L-lactide-co-trimethylene carbonate), polyarylates, polyhydroxybutyrate (PHB), polyanhydrides, poly (anhydride-co-imide), propylene-co- fumarates, polylactones, polyesters, polycarbonates, polyanionic polymers, polyanhydrides, poly(ester-amides), poly(amino acids), homopolypeptides, poly(phosphazenes), poly

(glaxanone), polysaccharides, and poly(orthoesters), polyglactin, polyglactic acid, polyaldonic acid, polyacrylic acids, polyalkanoates; copolymers and admixtures thereof, and any derivatives and modifications. See for example, U.S. Pat. No. 4,563,489, and PCT Int. Appl. # WO/03024316, herein incorporated by reference. Other examples of carriers include cellulosic polymers such as, but not limited to alkylcellulose, hydroxyalkylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,

hydroxypropyl-methylcellulose, carboxymethylcellulose, and their cationic salts. Other examples of carriers include synthetic and natural bioceramics such as, but not limited to calcium carbonates, calcium phosphates, apatites, bioactive glass materials, and coral-derived apatites. Further examples of the scaffold polymer useful herein include, but are not limited to, poly(vinylpyrrolidone), poly( vinyl alcohol), alkylene diol based polymers such as poly(ethylene glycol) (PEG), or poly(propyl glycol), or a copolymer thereof. A still further example of the scaffold polymer useful herein is a PVP/PEG, which is water soluble, pressure sensitive adhesive. A preferred example of the scaffold polymer useful herein is poly( - amino esters) (Anderson et al, Molecular Therapy 11(3), pp426-434 (2005); U.S. Patent No. 6,998,115 to Langer et al.), poly(ester amide) elastomers or a combination thereof. Some other useful materials useful include lipid- like materials described by Akin et al., in Nature Biotechnology, 26(5), 561-569 (2008) or by Love, et al, in PNAS, 107(5), 1864-1869 (2010). The teachings of these references referred to here are incorporated herein by reference in their entirety.

In one embodiment, the carrier can be coated compositions, including bioglass and or apatites derived from sol-gel techniques, or from immersion techniques such as, but not limited to simulated body fluids with calcium and phosphate concentrations ranging from about 1.5 to 7-fold the natural serum concentration and adjusted by various means to solutions with pH range of about 2.8-7.8 at temperature from about 15-65 degrees C. Other examples of carriers include collagen (e.g. Collastat, Helistat collagen sponges), hyaluronan, fibrin, chitosan, alginate, and gelatin, or a mixture thereof.

In one embodiment, the carrier may include heparin-binding agents; including but not limited to heparin-like polymers e.g. dextran sulfate, chondroitin sulfate, heparin sulfate, fucan, alginate, or their derivatives; and peptide fragments with amino acid modifications to increase heparin affinity. See for example, Journal of Biological Chemistry (2003), 278(44), p. 43229-43235, the teachings of which are incorporated herein by reference.

In one embodiment, the scaffold may be in the form of a liquid, solid or gel.

In one embodiment, the scaffold can be a carrier that is in the form of a flowable gel. The gel may be selected so as to be injectable, such as via a syringe at the site where bone formation is desired. The gel may be a chemical gel or a film of PEG and PVP which may be a chemical gel formed by primary bonds, and controlled by pH, ionic groups, and/or solvent concentration. The gel may also be a physical gel which may be formed by secondary bonds and controlled by temperature and viscosity. Examples of gels include, but are not limited to, pluronics, gelatin, hyaluronan, collagen, polylactide-polyethylene glycol solutions and conjugates, chitosan, chitosan & b-glycerophosphate (BST-gel), alginates, agarose, hydroxypropyl cellulose, methyl cellulose, polyethylene oxide, polylactides/glycolides in N- methyl-2-pyrrolidone. See for example, Anatomical Record (2001), 263(4), 342-349, the teachings of which are incorporated herein by reference.

In one embodiment of the scaffold, the carrier may be photopolymerizable, such as by electromagnetic radiation with wavelength of at least about 250 nm. Example of

photopolymerizable polymers include polyethylene (PEG) acrylate derivatives, PEG methacrylate derivatives, propylene fumarate-co-ethylene glycol, polyvinyl alcohol derivatives, PEG-co-poly(-hydroxy acid) diacrylate macromers, and modified polysaccharides such as hyaluronic acid derivatives and dextran methacrylate.

In one embodiment, the scaffold may include a carrier that is temperature sensitive. Examples include carriers made from N-isopropylacrylamide (NiPAM), or modified NiPAM with lowered lower critical solution temperature (LCST) and enhanced peptide (e.g. NELLl) binding by incorporation of ethyl methacrylate and N-acryloxysuccinimide; or alkyl methacrylates such as butylmethacrylate, hexylmethacrylate and dodecylmethacrylate (PCT Int. Appl. WO/2001070288; U.S. Pat. No. 5,124,151, the teachings of which are incorporated herein by reference).

In one embodiment of the scaffold, where the carrier may have a surface that is decorated and/or immobilized with cell adhesion molecules, adhesion peptides, and adhesion peptide analogs which may promote cell-matrix attachment via receptor mediated

mechanisms, and/or molecular moieties which may promote adhesion via non-receptor mediated mechanisms binding such as, but not limited to polycationic polyamino-acid- peptides (e.g. poly-lysine), polyanionic polyamino-acid-peptides, Mefp-class adhesive molecules and other DOPA-rich peptides (e.g. poly-lysine-DOPA), polysaccharides, and proteoglycans. See for example, PCT Int. Appl. WO/2004005421; WO/2003008376;

WO/9734016, the teachings of which are incorporated herein by reference.

In one embodiment of the scaffold, the carrier may be comprised of sequestering agents such as, but not limited to, collagen, gelatin, hyaluronic acid, alginate, poly(ethylene glycol), alkylcellulose (including hydroxyalkylcellulose), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl- methylcellulose, and carboxymethylcellulose, blood, fibrin, polyoxyethylene oxide, calcium sulfate hemihydrate, apatites, carboxyvinyl polymer, and poly( vinyl alcohol). See for example, U.S. Pat. No. 6,620,406, herein incorporated by reference. In one embodiment of the scaffold, the carrier may include buffering agents such as, but not limited to glycine, glutamic acid hydrochloride, sodium chloride, guanidine, heparin, glutamic acid hydrochloride, acetic acid, succinic acid, polysorbate, dextran sulfate, sucrose, and amino acids. See for example, U.S. Pat. No. 5,385,887, herein incorporated by reference. In one embodiment, the carrier may include a combination of materials such as those listed above. By way of example, the carrier may be a PLGA/collagen carrier membrane.

In one embodiment, the scaffold can be an implant of the various embodiments described herein.

In some embodiments, the scaffold of invention device can include a stimulus sensitive component. Such stimulus sensitive component is sensitive to a stimulus such that it can be triggered by the stimulus to cause the biologically active agent to have an enhanced delivery to a patient. Examples of such stimulus are mechanical and/or electromagnetic energies (Table 1 a) such as electricity, ultrasound, heat, electromagnetic field, laser light, laser radio frequency, or change of temperature, e.g., heating, change of physiological conditions such as pH or ionic field or strength.

In some embodiments, the scaffold can include microspheres including one or a combination of the genes expressing COL4, COL7, or COL17al, cells and/or biodegradable polymer such as poly( -amino esters) and/poly(ester amides). The cells can be genetically altered to express proteins or RNAs of COL4, COL7, and/or COL17al . The microspheres can have different degradation rate under different physiological conditions. For example, poly( -amino esters) and/or poly(ester amides) degrade at a slower rate at pH = 5 and degrade faster at pH=7. As a person of ordinary skill in the art would recognize, the pH of the physiological condition of a human being is about 5-7.

In the above described embodiments, the invention device can be a single emulsion device or double emulsion device.

Table la. Examples of mechanical or electromagnetic energies

1. Mechanical based energy/Electromagnetic Energy

a. Needle & Syringe

b. Microneedles

c. Ultrasound (Sonophoresis)

d. Lasers e. Others such as abrasion and alternative particle delivery, microscission, jet delivery, or ballistic delivery

2. Electromagnetic Energy

a. Iontophoresis

b. Sonophoresis

c. Radio frequency

d. Others such as electric current, electroporation, or laser

Preferably, the stimulus sensitive component is an ultrasound conductive component such as nanoparticles or microparticles of metals, metal oxides, or other materials. Useful nanoparticles of metal or metal oxides include, but are not limited to, nanoparticles of titanium, silver, gold, magnesium, iron, aluminum, zirconium, or an oxide thereof Other ultrasound conductive materials include piezoelectric material such as piezoelectric ceramics or polymer. Example of such piezoelectric polymer are polyvinylidene fluoride (PVDF), polyurethane, and/or poly(ethylene-vinyl acetate)

Backing layer

A backing layer of the invention device refers to a flexible film made of a sheeted material. Such sheeted material can be, for example, an e-skin as shown in Figures 2-3. As used herein, the term e-skin refers to a thin electronic material that mimics human skin in one or more ways. Various design of flexible e-skins are developed by researchers of the

University of California, Berkeley, Stanford University, and Tokyo University, see, e.g., Siegfried Bauer, Nature Materials 12, 871-872 (2013); C. Wang, et al, Nature Materials 12, 899-904 (2013); G. Schwartz, B. C-K. Tee, J. Mei, A. L. Appleton, D H. Kim, H. Wang, Z. Bao, Nature Comm., 4, Article number: 1859, 2013; Mallory L. Hammock, et al., 25th Anniversary Article: The Evolution of Electronic Skin (E-Skin): A Brief History, Design Considerations, and Recent Progress, Adv. Mater. 2013, 25, 5997-6038. The teachings in these references are incorporated herein in their entirety by reference.

Transdermal delivery using ultrasound

Ultrasound has long been documented to have the capability of enhancing transdermal delivery of a bioactive agent or therapeutics. Published review articles documented the art of drug delivery enhanced by ultrasound (see, e.g., Pitt et al, Expert Opin Drug Deliv. 2004 November; 1(1): 37-56 (Review); Mitragotri , Samir, Nature Reviews Drug Discovery 4, 255-260 (March 2005) (Review)).

Ultrasound is also documented to enhance the efficacy of gene therapy. See, e.g., C M H Newman and T Bettinger, "Gene therapy progress and prospects: Ultrasound for gene transfer" in Gene Therapy (2007) 14, 465-475.

The ultrasound can be administered or applied as a stimulus to enhance the delivery of the biologically active agent of invention device in any manner as is commonly practiced in the art. Preferably, the ultrasound can be administered via capacitive micromachined ultrasonic transducers (CMUTs). An example of such CMUTs is described in Wygant, I.O., et al, IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control, vol. 56 No. 1, January 2009, pp 193-197 (Wygant, 2009). The CMUTs can be incorporated in the backing layer of the device of invention. In some embodiments, low frequency ultrasound can also be used.

Method of Fabrication

In a second aspect of the present invention, it is provided a method of fabricating a device for transdermal delivery of a biologically active agent effective for treating or ameliorating a skin condition, which method comprising:

providing a skin layer comprising sheets of film comprising one or more layers that contain a biodegradable polymer that serves as a scaffold containing a bioactive agent, and providing a backing layer formed of an ultrathin polymer film that provides conduction of a stimulus wherein the backing layer has a thickness of one or more microns, wherein the stimulus, upon application to the device, causes the bioactive agent to have an enhanced delivery into the cells in the skin area.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the stimulus is electricity, ultrasound, heat, magnetic field, light, or mechanical based energy, e.g., force, distance, time.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the bioactive agent is selected from the group consisting of genes, cells, proteins, RNA, and other biologically active compounds. In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the skin disorder is epidermolysis bullosa (EB) or an EB related disorder.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the biologically active agent is selected from:

a) type VII collagen (COL7), Type XVII collagen al (COL17al), type IV collagen (COL4), COL17al;

b) a gene expressing COL7, Col7al, COL4, or COL17al;

c) a cell expressing proteins or R As of COL7, COL7al, COL4, or COL17al;

d) a peptide comprising the function domain of COL7 (COL7 peptide), a peptide comprising the function domain of COL7al (COL7al peptide), a peptide comprising the function domain of COL4 (COL4 peptide), or a peptide comprising the function domain of COL17al;

e) a peptide mimetics of COL7 peptide, COL7al peptide, a peptide mimetics of COL4 peptide, or a peptide mimetics of COL17al peptide; or

f) a combination of any of a)-e).

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the skin disorder is a disorder listed in Table 3.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the biologically active agent is a gene listed in Table 3, a gene product of a gene listed in Table 3, or a cell expressing a gene listed in Table 3.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the biodegradable polymer dissolves in a wound or diseased skin area after delivery of the bioactive agent is completed or substantially completed.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the stimulus is ultrasound.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the device is a patch or a roll which is to be placed or wrapped in situ over the hands, fingers, arms, legs, elbows, knees, foot toes and other parts of a patient's affected skin.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the device is made into a glove to aid a patient with fingers and/or toes that have become webbed or to prevent the fingers or toes to become deformed, webbed or "mittens" in various stages of EB or an EB related disorder (see, Table 2).

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the biodegradable polymer is poly( - amino esters).

Various elements used in the invention method are either commercially available or can be readily constructed by a person of ordinary skill in the art. For example, the backing layer can be an e-skin and readily made according to documented methods, see, e.g., Siegfried Bauer, Nature Materials 12, 871-872 (2013); C. Wang, et al, Nature Materials 12, 899-904 (2013); G. Schwartz, B. C-K. Tee, J. Mei, A. L. Appleton, D H. Kim, H. Wang, Z. Bao, Nature Comm., 4, Article number: 1859, 2013. The teachings in these references are incorporated herein in their entirety by reference.

In some embodiments, where the stimulus is ultrasound, the ultrasound transducer layer can include a CMUTs, which can be readily made according to the micro fabrication method described by the Wygant reference (Wygant, 2009, supra), the teaching of which is incorporated herein in its entirety by reference.

Method of Use

In a further aspect of the present invention, it is provided a method of treating skin condition, comprising applying a device to a patient to effect transdermal delivery of a biologically active agent effective for treating or ameliorating a skin condition, wherein the device comprises

a skin layer comprising sheets of film comprising one or more layers that contain a biodegradable polymer that serves as a scaffold containing a bioactive agent, and

a backing layer formed of an ultrathin polymer film that provides conduction of a stimulus wherein the backing layer has a thickness of one or more microns, wherein the stimulus, upon application to the device, causes the bioactive agent to have an enhanced delivery into the cells in the skin area.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the stimulus is electricity, ultrasound, heat, magnetic field, light, or mechanical based energy, e.g., force, distance, time.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the bioactive agent is selected from the group consisting of genes, cells, proteins, R A, and other biologically active compounds.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the skin disorder is epidermolysis bullosa (EB) or an EB related disorder.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the biologically active agent is selected from:

a) type VII collagen (COL7), Type XVII collagen al (COL17al), type IV collagen (COL4), COL17al;

b) a gene expressing COL7, Col7al, COL4, or COL17al;

c) a cell expressing proteins or R As of COL7, COL7al, COL4, or COL17al;

d) a peptide comprising the function domain of COL7 (COL7 peptide), a peptide comprising the function domain of COL7al (COL7al peptide), a peptide comprising the function domain of COL4 (COL4 peptide), or a peptide comprising the function domain of COL17al;

e) a peptide mimetics of COL7 peptide, COL7al peptide, a peptide mimetics of COL4 peptide, or a peptide mimetics of COL17al peptide; or

f) a combination of any of a)-e).

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the skin disorder is a disorder listed in Table 3.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the biologically active agent is a gene listed in Table 3, a gene product of a gene listed in Table 3, or a cell expressing a gene listed in Table 3. In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the biodegradable polymer dissolves in a wound or diseased skin area after delivery of the bioactive agent is completed or substantially completed.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the stimulus is ultrasound.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the device is a patch or a roll which is to be placed or wrapped in situ over the hands, fingers, arms, legs, elbows, knees, foot toes and other parts of a patient's affected skin.

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the device is made into a glove to aid a patient with fingers and/or toes that have become webbed or to prevent the fingers or toes to become deformed, webbed or "mittens" in various stages of EB or an EB related disorder (see Table 2 below).

In some embodiments of the invention method, optionally in combination in any or all of the various embodiments of the present invention, the biodegradable polymer is poly( - amino esters).

Table 2. List of EB related complications and disorders

1. Ocular problems, i.e. cornea blisters and erosions; corneal scarring, pannus formation, limbal broadening, conjunctival blisters and erosions, symblepharon, eyelid blisters and scars, ectropion, lacrimal duct obstructions.

2. Ear, nose and throat complications, i.e. upper respiratory tract, etc.

3. Gastrointestinal complications

4. Musculoskeletal deformities

5. Cardiac Complications

6. Renal Complications

7. Genitorurinary tract

8. Osteoporosis and osteropenia

9. Gynecological considerations

10. Premature death (from failure to thrive, sepsis, respiratory and renal failure, squamous cell carcinoma

11. Psychological and Sociological aspects Some further (27) embodiments are where the skin disorder is a genetic or locus heterogeneity in genodermatoses listed in Table 3 (Liv Kraemer and Angela M. Christiano, "Basic Principles of Genetics and Gene Therapy" in Therapy of Skin Diseases, Thomas Krieg, David R. Bickers, Yoshiki Miyachi, Editors, Springer Publication, 2010, Tablel .5.1, (p. 42)).

Table 3. Examples of genetic or locus heterogeneity in genodermatoses

Disorder ( lene S\ mli l Gene or ye no proilucl

Epidermolytic hyperkeratosis KRT1 Keratin 1

KRT10 Keratin 10

Erythrokeratoderma variabilis Cx31 Connexin 31

(Mendes da Costa)

Cx30.3 Connexin 30.3

Lamellar ichthyosis TGM1 Transglutaminase 1

ICR2B

LI2

Nonbullous congenital ichthyosi- TGM1 Transglutaminase I

form erythroderma ALOXE3 Arachidonate Lipoxygenase 3

ALOX12B Arachidonate 12-Lipoxygenase, R Type E 3

Palmoplantar keratoderma, KRT1 Keratin 1

epidermolytic (Voerner) KRT9 Keratin 9

Palmoplantar keratoderma, KRT1 Keratin 1

nonepidermolytic (Unna-Thost) KRT16 Keratin 16

Palmoplantar keratoderma MTTSI Mitochondrial tRNA-serine I

with deafness CX26, GJB2 Connexin 26

Striate palmoplantar keratoderma DSP Desmoplakin

(Bruenauer-Fuhs-Siemens) DSG1 Desmoglein 1

Naxos disease JUP Plakoglobin

DSP Desmoplakin

Monilethrix KRTHB1 Hair keratin, basic 1

KRTHB3 Hair keratin, basic 3

KRTHB6 Hair keratin, basic 6

White sponge nevus KRT4 Keratin 4

KRT13 Keratin 13

EBS Weber-Cockayne variant KRT5 Keratin 5

KRT14 Keratin 14

EBS Dowling-Meara variant KRT5 Keratin 5

KRT14 Keratin 14

EBS Koebner KRT5 Keratin 5

KRT14 Keratin 14 JEB Herlitz LAMC2 Laminin-5 polypeptide subunit a2

LAMB3 Laminin-5 polypeptide subunit β3

LAMA 3 Laminin-5 polypeptide subunit a3

JEB Non-Herlitz COL17A1 CollagenXVII

Figure imgf000027_0001

LAMC2 Laminin-5 polypeptide subunit a2

LAM3B Laminin-5 polypeptide subunit β3

LAMA 3 Laminin-5 polypeptide subunit a3

EEC syndrome EEC1 p63

EEC2

TP63

Hypohidrotic ED EDAR Ectodysplasin receptor

EDARADD EDAR-associated death domain

Griscelli syndrome MY05A Myosin a

RAB27A RAS-associated protein

Hermansky-Pudlak syndrome HPS1 Transmembrane protein

HPS2 Adaptor-related protein complex 3

HPS3 HPS3

HPS4 HPS4

Phenylketonuria PAH Phenylalanine hydroxylase

DHPR Dihydropteridine reductase

PTS 6-Pyruvoyltetra-hydroxylase

Tuberous sclerosis TSC1 Hamartin

TSC2 Tuberin

Epidermodysplasia verruciformis EV1 EVER1 (Integral membrane protein of the

endoplasmic reticulum 1)

EV2 EVER2

Muir- Torre-syndrome hMSH2 MutS, E.coli, homolog

hMLHl MutL, E.coli, homolog

Cockayne syndrome ERCC6 or CSB Excision-repair coss-complementing, group 6

ERCC8 or CSA Excision-repair coss-complementing, group 8

Noonan syndrome PTPN11 Nonreceptor protein tyrosine phosphatase

SOS1 SHP2

KRAS Son of sevenless, drosophila, homolog 1

V-Ki-Ras2 Kirsten rat sarcoma viral

NS oncogene homolog

Neurofibromin gene

Omenn syndrome RAG I Rag-1

RAG2 Rag-2

Figure 2 illustrates using an embodiment of the invention device to generate new skin. The invention device includes ultrasound source, an e-skin protective layer, and a scaffold layer. The scaffold layer includes a biologically active agent such as intermediate particulates (LP.), proteins, genes, cells, and/or other agents. Underneath the scaffold, an epidermal layer is regenerated, which includes cells, I. P., and genes, upon the basement membrane zone (BMZ). The BMZ lies between the regenerated epidermal layer and the dermal layer of the skin, so as to provide a cure to EB.

Those skilled in the art will know, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These and all other equivalents are intended to be encompassed by the following claims.

Claims

We claim
1. A device for transdermal delivery of a biologically active agent effective for treating or ameliorating a skin condition, comprising
a skin layer comprising sheets of film comprising one or more layers that contain a biodegradable polymer that serves as a scaffold containing a bioactive agent, and
a backing layer formed of an ultrathin polymer film that provides conduction of a stimulus wherein the backing layer has a thickness of one or more microns,
wherein the stimulus, upon application to the device, causes the bioactive agent to have an enhanced delivery into the cells in the skin area.
2. The device of claim 1, wherein the stimulus is electricity, ultrasound, heat, magnetic field, light, or mechanical based energy.
3. The device of claim 1, wherein the bioactive agent is selected from the group consisting of genes, cells, proteins, R A, and other biologically active compounds.
4. The device of claim 1, wherein the skin disorder is epidermolysis bullosa (EB) or an EB related disorder.
5. The device of claim 4, wherein the biologically active agent is selected from:
a) type VII collagen (COL7), Type XVII collagen al (COL17al), type IV collagen (COL4), COL17al;
b) a gene expressing COL7, Col7al, COL4, or COL17al;
c) a cell expressing proteins or R As of COL7, COL7al, COL4, or COL17al;
d) a peptide comprising the function domain of COL7 (COL7 peptide), a peptide comprising the function domain of COL7al (COL7al peptide), a peptide comprising the function domain of COL4 (COL4 peptide), or a peptide comprising the function domain of COL17al;
e) a peptide mimetics of COL7 peptide, COL7al peptide, a peptide mimetics of COL4 peptide, or a peptide mimetics of COL17al peptide; or
f) a combination of any of a)-e).
6. The device of claim 1, wherein the skin disorder is a disorder listed in Table 3.
7. The device of claim 6, wherein the biologically active agent is a gene listed in Table 3, a gene product of a gene listed in Table 3, or a cell expressing a gene listed in Table 3.
8. The device of claim 1 , wherein the biodegradable polymer dissolves in a wound or diseased skin area after delivery of the bioactive agent is completed or substantially completed.
9. The device of claim 1, wherein the stimulus is ultrasound.
10. The device of claim 1, which is a patch or a roll which is to be placed or wrapped in situ over the hands, fingers, arms, legs, elbows, knees, foot toes and other parts of a patient's affected skin.
11. The device of claim 1 , which is made into a glove to aid a patient with fingers and/or toes that have become webbed or to prevent the fingers or toes to become deformed, webbed or "mittens" in various stages of EB or an EB related disorder.
12. The device of claim 1, wherein the biodegradable polymer is poly( -amino esters).
13. A method of fabricating a device for transdermal delivery of a biologically active agent effective for treating or ameliorating a skin condition, comprising:
providing a skin layer comprising sheets of film comprising one or more layers that contain a biodegradable polymer that serves as a scaffold containing a bioactive agent, and providing a backing layer formed of an ultrathin polymer film that provides conduction of a stimulus wherein the backing layer has a thickness of one or more microns, wherein the stimulus, upon application to the device, causes the bioactive agent to have an enhanced delivery into the cells in the skin area.
14. The method of claim 13, wherein the stimulus is electricity, ultrasound, heat, magnetic field, light, or mechanical based energy, e.g., force, distance, time.
15. The method of claim 13, wherein the bioactive agent is selected from the group consisting of genes, cells, proteins, R A, and other biologically active compounds.
16. The method of claim 13, wherein the skin disorder is epidermolysis bullosa (EB) or an EB related disorder.
17. The method of claim 13, wherein the biologically active agent is selected from:
a) type VII collagen (COL7), Type XVII collagen al (COL17al), type IV collagen (COL4), COL17al;
b) a gene expressing COL7, Col7al, COL4, or COL17al;
c) a cell expressing proteins or R As of COL7, COL7al, COL4, or COL17al; d) a peptide comprising the function domain of COL7 (COL7 peptide), a peptide comprising the function domain of COL7al (COL7al peptide), a peptide comprising the function domain of COL4 (COL4 peptide), or a peptide comprising the function domain of COL17al;
e) a peptide mimetics of COL7 peptide, COL7al peptide, a peptide mimetics of COL4 peptide, or a peptide mimetics of COL17al peptide; or
f) a combination of any of a)-e).
18. The method of claim 13, wherein the skin disorder is a disorder listed in Table 3.
19. The method of claim 18, wherein the biologically active agent is a gene listed in Table 3, a gene product of a gene listed in Table 3, or a cell expressing a gene listed in Table 3.
20. The method of claim 13, wherein the biodegradable polymer dissolves in a wound or diseased skin area after delivery of the bioactive agent is completed or substantially completed.
21. The method of claim 13, wherein the stimulus is ultrasound.
22. The method of claim 13, which is a patch or a roll which is to be placed or wrapped in situ over the hands, fingers, arms, legs, elbows, knees, foot toes and other parts of a patient's affected skin.
23. The method of claim 13, which is made into a glove to aid a patient with fingers and/or toes that have become webbed or to prevent the fingers or toes to become deformed, webbed or "mittens" in various stages of EB or an EB related disorder.
24. The method of claim 13, wherein the biodegradable polymer is poly( -amino esters).
25. A method of treating a skin condition, comprising applying a device to a patient to effect transdermal delivery of a biologically active agent effective for treating or ameliorating a skin condition, wherein the device comprises
a skin layer comprising sheets of film comprising one or more layers that contain a biodegradable polymer that serves as a scaffold containing a bioactive agent, and
a backing layer formed of an ultrathin polymer film that provides conduction of a stimulus wherein the backing layer has a thickness of one or more microns,
wherein the stimulus, upon application to the device, causes the bioactive agent to have an enhanced delivery into the cells in the skin area.
26. The method of claim 25, wherein the stimulus is electricity, ultrasound, heat, magnetic field, light, or mechanical based energy, e.g., force, distance, time.
27. The method of claim 25, wherein the bioactive agent is selected from the group consisting of genes, cells, proteins, R A, and other biologically active compounds.
28. The method of claim 25, wherein the skin disorder is epidermolysis bullosa (EB) or an EB related disorder.
29. The method of claim 28, wherein the biologically active agent is selected from:
a) type VII collagen (COL7), Type XVII collagen al (COL17al), type IV collagen (COL4), COL17al;
b) a gene expressing COL7, Col7al, COL4, or COL17al;
c) a cell expressing proteins or R As of COL7, COL7al, COL4, or COL17al;
d) a peptide comprising the function domain of COL7 (COL7 peptide), a peptide comprising the function domain of COL7al (COL7al peptide), a peptide comprising the function domain of COL4 (COL4 peptide), or a peptide comprising the function domain of COL17al;
e) a peptide mimetics of COL7 peptide, COL7al peptide, a peptide mimetics of COL4 peptide, or a peptide mimetics of COL17al peptide; or
f) a combination of any of a)-e).
30. The method of claim 25, wherein the skin disorder is a disorder listed in Table 3.
31. The method of claim 31 , wherein the biologically active agent is a gene listed in Table 3, a gene product of a gene listed in Table 3, or a cell expressing a gene listed in Table 3.
32. The method of claim 25, wherein the biodegradable polymer dissolves in a wound or diseased skin area after delivery of the bioactive agent is completed or substantially completed.
33. The method of claim 25, wherein the stimulus is ultrasound.
34. The method of claim 25, which is a patch or a roll which is to be placed or wrapped in situ over the hands, fingers, arms, legs, elbows, knees, foot toes and other parts of a patient's affected skin.
35. The method of claim 25, which is made into a glove to aid a patient with fingers and/or toes that have become webbed or to prevent the fingers or toes to become deformed, webbed or "mittens" in various stages of EB or an EB related disorder.
36. The method of claim 25, wherein the biodegradable polymer is poly( -amino esters).
PCT/US2015/019774 2014-03-11 2015-03-10 In-situ skin regeneration for epidermolysis bullosa and other skin disorders WO2015138502A1 (en)

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US20090076479A1 (en) * 2003-06-30 2009-03-19 Ying Sun Device for treatment of barrier membranes
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