Patent Application
20200375868
Gene therapy has shown great promise to prevent, treat and cure a variety of skin diseases and conditions in human and animals. Skin is the largest and one of the most complex organs in the human body. It performs a diverse set of functions, ranging from protection, sensation, heat regulation, absorption of gases, excretion of sweat, control of evaporation and water resistance. Skin's structure and function gradually deteriorate with age (intrinsic aging) and in response to varying environmental conditions (extrinsic aging) such as exposure to solar radiation and a variety of chemicals becoming prone to common benign and malignant skin lesions such as Seborrheic keratosis, Actinic keratosis and non-melanoma skin cancers. Furthermore, skin's health gradually declines in response to chronic conditions including HIV, diabetes, atherosclerosis, and even inadequate nutrition. As a result, skin accumulates high mutational loads evinced in altered translation of key proteins maintaining skin homeostasis. At tissue level, the stratum corneum loses its ability to barrier function, regeneration and wound healing; the epidermis becomes prone to errors in metabolic reprogramming and the rete ridges lose surface area; the dermis becomes thinner and less elastic; the sebaceous and eccrine glands contract and secrete less oils and sweat; and the Langerhans immune cells decline in number and function.
As a superficial organ, the skin is an easily accessible target for gene therapy. To perform gene therapy, recombinant viral vectors have been developed as attractive alternatives to non-viral vectors to deliver genes and nucleic acid molecules of interest to the skin. These recombinant viral vectors include recombinant retrovirus, adenovirus, adeno-associated virus (AAV), vaccinia virus and herpes simplex virus. However, the efficacy of skin gene therapy is hampered by low level of transgene expression, due to difficulty of viral permeation in the skin tissue. There is a continuing need in the art to improve the efficacy of skin gene therapy by enhancing viral permeation in the skin tissue.
Aspects of the present disclosure are directed to methods of non-invasive delivery of nucleic acid molecules including genes via recombinant viral vectors to skin tissue in vivo and in vitro. In certain embodiments, the delivery method comprises electroporation such as by applying short high voltage pulses to the skin, heat (between about 32° C.-39° C.), needleless injections such as by firing liquid at supersonic speed through the stratum corneum, pressure waves generated by laser radiation, fraction laser, or radiofrequency (about 100 kHz), magnetophoresis by external magnetic field, iontophoresis, chemical peels, abrasion techniques such as diamond or sand paper abrasion, tape stripping, and the like. In an exemplary embodiment, ultrasonic pre-treatment of skin tissue is used to enable increased tissue permeation before administering the recombinant viruses to the treated skin tissue. In one embodiment, recombinant adeno-associated viruses are used to deliver nucleic acid molecules of interest to skin tissue/cells to modify target gene expression. The methods disclosed herein are suitable for simultaneously modifying the expression of sets of target genes involved in maintaining skin homeostasis and health. Aspects of the present disclosure are directed to methods of introducing nucleic acid molecules comprising nucleic acid sequences for expression in skin cells. The nucleic acid sequences encode RNA and polypeptides that function to activate or repress target gene expression. The nucleic acid sequences can also integrate into the cell's genome and modulate target gene expression. Recombinant viral vectors are employed to package and deliver the nucleic acid molecules. For example, nucleic acid molecules are packaged in recombinant adeno-associated viral (rAAV) vectors. The methods of the present disclosure have demonstrated long-term transgene expression and modulated protein translation from rAAV vectors in animal (in vivo) and human (ex vivo and in vitro) experimental models. In some embodiments, the methods of the present disclosure include optimal tissue specificity and efficiency of gene transfer based on rAAV vector serotypes such that these vectors selectively target one, more, or all skin tissue layers and structures (i.e. stratum corneum, epidermis, basement membrane, dermis, hair follicles, and sebaceous and eccrine glands). The methods of the present disclosure improve skin gene therapies and are well-suited to enable reversal of skin aging phenotypes and phenotypes resulting from complex disease- and age-associated skin pathologies.
According to one aspect, a method of delivering a recombinant virus to a skin tissue is provided. In one embodiment, the method includes applying ultrasound to the skin tissue, and administering the recombinant virus to the skin tissue. In one embodiment, the recombinant virus is delivered to the skin tissue of a subject in vivo. In some embodiments, the skin tissue comprises native and autogeneic, isogeneic, xenogeneic and allogeneic skin tissue. In another embodiment, the recombinant virus is delivered to the skin tissue in vitro. In some embodiments, the skin tissue comprises skin explants and artificial skin tissues. In one embodiment, the ultrasound is applied prior to administering the recombinant virus. In another embodiment, the ultrasound is stopped prior to administering the recombinant virus. In some embodiments, the ultrasound is applied at a frequency between about 20 kHz and about 100 kHz. In other embodiments, the ultrasound is applied at a frequency of about 10 kHz, 20 kHz, 30 kHz, 40 kHz, 50 kHz, 60 kHz, 70 kHz, 80 kHz, 90 kHz and 100 kHz. In some embodiments, the ultrasound is applied at an intensity of about 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 W/cm2. In other embodiments, the ultrasound is applied at an intensity between about 1 W/cm2 and about 10 W/cm2. In some embodiments, the ultrasound is applied for a duration between about one minute to about 10 minutes. In other embodiments, the ultrasound is applied for a duration of about 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 minutes. In some embodiments, the ultrasound is applied at duty cycles in the range between about 10% and 100%. In some embodiments, the ultrasound is applied at duty cycles in the range between about 10%, 25%, 50%, 75% and 100%. In certain embodiments, the ultrasound is applied topically or intra-dermally. In other embodiments, the methods further include delivering the recombinant virus to the skin tissue via electroporation, heat, needleless injections, pressure waves generated by laser radiation, fraction laser, or radiofrequency (100 kHz), magnetophoresis by external magnetic field, iontophoresis, chemical peels, abrasion techniques including diamond or sand paper abrasion, tape stripping, and the like. In some embodiments, the recombinant virus is selected from retrovirus, adenovirus, adeno-associated virus (AAV), vaccinia virus and herpes simplex virus. In other embodiments, the recombinant AAV includes serotypes 1-9. In one embodiment, the recombinant virus comprises a heterologous nucleic acid sequence. In another embodiment, the nucleic acid sequence encodes a gene which is expressible in the skin tissue. In one embodiment, expression of the gene effects treatment of a skin disease or condition. In certain embodiments, the gene is selected from COL1A1, COL3A1, TIMP1, TIMP2, SMAD2, SMAD3, CTGF, TGF-beta1, KRT6A, NOTCH1(icd), TET2, TET3, Sirt1, Sirt6, Pck1, Pparg, and Cisd2, MDH1, MDH2, Aco1, Aco2, IDH1, IDH2, IDH3, ENO1, GOT1, GOT2, MUC1, and MCU. In one embodiment, the gene encodes a green fluorescent protein (GFP). In some embodiments, the skin disease or condition includes Epidermolysis Bullosa, Recessive Dystrophic Epidermolysis Bullosa, Junctional Epidermolysis Bullosa, Epidermolysis Bullosa Simplex, Pachyonychia Congenita, Melanoma, non-melanoma skin cancer, Ichthyosis, Harlequin Ichthyosis, Sjogren-Larsson Syndrome, Xeroderma Pigmentosum, Wound Healing, Netherton Syndrome, age-associated skin pathologies, benign and malignant skin lesions, inflammatory and autoimmune skin disorders. In other embodiments, the recombinant virus is delivered to keratinocytes, epidermal stem cells, fibroblast cells, mesenchymal stem cells, immune cells, melanocytes, vascular endothelial cells, adipocytes, Merkel cells and peripheral neural cells of the skin tissue. In some embodiments, the recombinant virus is delivered to skin tissue layers and structures including stratum corneum, epidermis, basement membrane, dermis, hair follicles, blood vessels and sebaceous and eccrine glands. In certain embodiments, multiple recombinant viruses comprising multiple genes are delivered to the skin tissue. In some embodiments, the subject is human or non-human mammal. In other embodiment, the non-human mammal is selected from a mouse, rat, cow, pig, sheep, goat, and horse.
According to another aspect, a recombinant virus comprising a heterologous nucleic acid sequence is provided. In one embodiment, the nucleic acid sequence encodes a gene which is expressible in a skin tissue. In some embodiments, expression of the gene effects treatment of a skin disease or condition. In certain embodiments, the gene is selected from COL1A1, COL3A1, TIMP1, TIMP2, SMAD2, SMAD3, CTGF, TGF-beta1, KRT6A, NOTCH1(icd), TET2, TET3, Sirt1, Sirt6, Pck1, Pparg, and Cisd2, MDH1, MDH2, Aco1, Aco2, IDH1, IDH2, IDH3, ENO1, GOT1, GOT2, MUC1, and MCU. In one embodiment, the gene encodes a green fluorescent protein (GFP). In some embodiments, the recombinant virus is selected from retrovirus, adenovirus, adeno-associated virus (AAV), vaccinia virus and herpes simplex virus. In other embodiments, the recombinant AAV includes serotypes 1-9.
According to yet another aspect, a method of delivering a polypeptide to a skin tissue is provided. In one embodiment, the method includes applying ultrasound to the skin tissue, and administering a nucleic acid sequence encoding the polypeptide to the skin tissue. In another embodiment, the nucleic acid sequence encoding the polypeptide is delivered to the skin tissue of a subject in vivo. In one embodiment, the skin tissue comprises native and autogeneic, isogeneic, xenogeneic and allogeneic skin tissue. In another embodiment, the nucleic acid sequence encoding the polypeptide is delivered to the skin tissue in vitro. In one embodiment, the skin tissue comprises skin explants and artificial skin tissues. In another embodiment, the ultrasound is applied prior to administering the nucleic acid sequence encoding the polypeptide. In one embodiment, the ultrasound is stopped prior to administering the nucleic acid sequence encoding the polypeptide. In some embodiments, the ultrasound is applied at a frequency between about 20 kHz and about 100 kHz. In other embodiments, the ultrasound is applied at a frequency of about 10 kHz, 20 kHz, 30 kHz, 40 kHz, 50 kHz, 60 kHz, 70 kHz, 80 kHz, 90 kHz and 100 kHz. In some embodiments, the ultrasound is applied at an intensity of about 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 W/cm2. In other embodiments, the ultrasound is applied at an intensity between about 1 W/cm2 and about 10 W/cm2. In some embodiments, the ultrasound is applied for a duration between about one minute to about 10 minutes. In other embodiments, the ultrasound is applied for a duration of about 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 minutes. In some embodiments, the ultrasound is applied at duty cycles in the range between about 10% and 100%. In some embodiments, the ultrasound is applied at duty cycles in the range between about 10%, 25%, 50%, 75% and 100%. In certain embodiments, the ultrasound is applied topically or intra-dermally. In certain embodiments, the nucleic acid sequence encoding the polypeptide is DNA or RNA. In one embodiment, wherein the polypeptide is expressible in the skin tissue. In another embodiment, expression of the polypeptide effects treatment of a skin disease or condition. In some embodiments, the nucleic acid sequence encodes a gene selected from COL1A1, COL3A1, TIMP1, TIMP2, SMAD2, SMAD3, CTGF, TGF-beta1, Anti-MMP1, anti-MMP2, KRT6A, NOTCH1(icd), TET2, TET3, Sirt1, Sirt6, HIF-1a, Pten, Pck1, Pparg, and Cisd2, MDH1/2, Aco1/2, IDH1/2/3, Enolase, GOT1/2, MUC1, and MCU. In one embodiment, the nucleic acid sequence encodes a green fluorescent protein (GFP). In some embodiments, the skin disease or condition includes Epidermolysis Bullosa, Recessive Dystrophic Epidermolysis Bullosa, Junctional Epidermolysis Bullosa, Epidermolysis Bullosa Simplex, Pachyonychia Congenita, Melanoma, non-melanoma skin cancer, Ichthyosis, Harlequin Ichthyosis, Sjogren-Larsson Syndrome, Xeroderma Pigmentosum, Wound Healing, Netherton Syndrome, age-associated skin pathologies, benign and malignant skin lesions, inflammatory and autoimmune skin disorders. In some embodiments, the nucleic acid sequence encoding the polypeptide is delivered to keratinocytes, epidermal stem cells, fibroblast cells, mesenchymal stem cells, immune cells, melanocytes, vascular endothelial cells, adipocytes, Merkel cells and peripheral neural cells of the skin tissue. In other embodiments, the nucleic acid sequence encoding the polypeptide is delivered to skin tissue layers and structures including stratum corneum, epidermis, basement membrane, dermis, hair follicles, blood vessels and sebaceous and eccrine glands. In some embodiments, multiple nucleic acid sequences encoding multiple polypeptides are delivered to the skin tissue. In some embodiments, native polypeptide is delivered to the skin tissue.
According to one aspect, a heterologous nucleic acid sequence encoding a gene which is expressible in a skin tissue is provided. In one embodiment, expression of the gene effects treatment of a skin disease or condition. In some embodiments, the heterologous nucleic acid encodes a gene selected from COL1A1, COL3A1, TIMP1, TIMP2, SMAD2, SMAD3, CTGF, TGF-beta1, Anti-MMP1, anti-MMP2, KRT6A, NOTCH1(icd), TET2, TET3, Sirt1, Sirt6, HIF-1a, Pten, Pck1, Pparg, Cisd2, MDH1/2, Aco1/2, IDH1/2/3, Enolase, GOT1/2, MUC1, and MCU. In one embodiment, the heterologous nucleic acid sequence encodes a green fluorescent protein (GFP).
Further features and advantages of certain embodiments of the present invention will become more fully apparent in the following description of embodiments and drawings thereof, and from the claims.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The foregoing and other features and advantages of the present embodiments will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:
The present disclosure describes a method of systemic delivery of a polypeptide to a subject including genetically modifying target skin cells within skin of a subject using an engineered virus or nucleic acid sequences. The engineered virus includes one or more genomic nucleic acid sequences and one or more foreign nucleic acid sequences encoding one or more target polypeptides. The one or more genomic nucleic acid sequences and the one or more nucleic acid sequences encoding one or more target polypeptides are introduced into the target skin cells to produce genetically modified target skin cells. The genetically modified target skin cells produce the one or more target polypeptides.
According to one aspect, an engineered virus is administered to the skin of the subject in a manner to direct the engineered virus to the target skin cells. Various administration methods are contemplated including electroporation, heat, needleless injections, pressure waves generated by laser radiation, fraction laser, or radiofrequency (about 100 kHz), magnetophoresis by external magnetic field, iontophoresis, chemical peels, abrasion techniques including diamond or sand paper abrasion, tape stripping, and the like.
According to one aspect, the skin of the subject may be treated so as to permeabilize the stratum corneum to the presence of the engineered virus or nucleic acid sequences or otherwise improve efficiency of the engineered virus or nucleic acid sequences to traverse the stratum corneum to the target skin cells. After treating the skin surface, the engineered virus or nucleic acid sequences may be topically administered to the skin surface and the engineered virus or nucleic acid sequences may passively diffuse to the target skin cells whereupon the engineered virus infects the target cells to include the one or more nucleic acid sequences encoding one or more target polypeptides, or whereupon the nucleic acid sequences encoding one or more target polypeptides transduce the target cells. The one or more target polypeptides are produced by the genetically modified target cells. In some embodiments, the one or more target polypeptides are excreted from the genetically modified target cells and into the blood stream of the subject. According to one aspect, the one or more target polypeptides are excreted from the genetically modified target cells in a manner to provide a prolonged release of the one or more target polypeptides into the bloodstream of the subject.
According to one aspect, a delivery platform is provided that utilizes human skin to enable a single-step, extended production, such as year-long production of biologics wherein gene-encoded vectors are topically administered to skin in a non-invasive manner so as to treat or prevent a disease. Skin cells are provided with non-integrative viral vectors which, according to one embodiment, may lack specific cytotoxicity and pathogenicity. Delivery of the viral vectors is achieved by “needleless” methods leveraging breakage of the stratum corneum. The genetic modification of skin cells to include the gene-encoded vectors provides for long-lived and efficient translation of a polypeptide, such as a therapeutic agent in vivo to provide a safe and effective gene transfer for treatment or prevention. According to one aspect, skin is pretreated using noninvasive technology, such as ultrasound or microdermabrasion, to premeabilize or score or remove the stratum corneum. The engineered virus, such as a gene-encoding adeno-associated virus (“AAV particles”) is topically administered and delivered to the pretreated skin, which may be a section of skin near active lymph nodes. According to one aspect, target cells, such as dermal fibroblasts, endosome the AAV particles and the AAV particles release the DNA contained therein into the fibroblast cell nucleus. The fibroblast cells translate and secrete the one or more polypeptides to the intercellular matrix of the skin tissue or blood stream. The polypeptides are present within the intercellular matrix of the skin tissue or blood system for therapy or prevention. For example, the one or more polypeptides may be broadly neutralizing antibodies present within the intercellular matrix of the skin tissue or the blood system to prevent infection. In this manner, the skin may be transformed into an in vivo bioreactor for the production of biologics, such as antibodies, for transfer into the blood stream.
Embodiments of the present disclosure are directed to methods of delivering nucleic acid molecules of interest via recombinant viruses to a skin tissue. In addition to gene therapies correcting for one gene as in rare genetic diseases, the disclosed method also includes delivery of multiple sets of genes along key aging and disease signaling pathways affecting skin tissues so as to globally restore healthy and youthful transcriptional and translational profiles of skin cells and tissues. In exemplary embodiments, the method includes two major steps. In step one, ultrasound is applied to a skin tissue to increase tissue permeation. In step two, recombinant viruses carrying foreign nucleic acid molecule(s)/gene(s) of interests are delivered to the skin tissue.
Ultrasound treatment of skin has been known. A skilled in the art can choose the appropriate ultrasound device according to an application. To increase skin tissue permeation, ultrasound is applied to the skin tissue. A skilled in the art can determine the frequency, intensity and duration of ultrasound application that is effective for a specific purpose. In an exemplary embodiment, a treatment with ultrasound at 20 kHz frequency is applied at an intensity of less than 8 W/cm2 for up to one minute at 50% duty cycle. The ultrasonic pre-treatment of skin tissue improves tissue diffusivity by increasing its effective diffusion coefficient. This process is enabled by the disruption of skin's stratum corneum.
Alternatively, other delivery methods can be used to deliver the recombinant viruses to the skin. These delivery methods comprise electroporation such as by applying short high voltage pulses to the skin, heat (between about 32° C. and 39° C.), needleless injections such as by firing liquid at supersonic speed through the stratum corneum, pressure waves generated by laser radiation, fraction laser, or radiofrequency (about 100 kHz), magnetophoresis by external magnetic field, iontophoresis, chemical peels, abrasion techniques such as diamond or sand paper abrasion, tape stripping, and the like. A skilled in the art can choose the appropriate delivery method according to an application. These methods can be used in combination with the method of ultrasound pre-treatment of skin and administering of the recombinant viruses as disclosed herein.
According to one aspect, viral vectors may be selected based on the ability to target cell types in a specific manner. Such viral vectors may be identified by multiplexed screening of hybrid capsid variations of adeno-associated viruses (“AAVs”). Hybrid AAV constructs typically exhibit less immunogenicity than the wild-type AAV, and have greater tissue specificity.
A large set of existing viral serotypes is optimized, synthesized and tested in human organotypic cultures. Human abdominal skin is cultured ex vivo, using native fluorescence of reporter genes, FACS, and in situ screening approaches. The method is high-throughput, allows for combinatorial optimization, and accounts for donor-to-donor variability related to immune response and metabolic state. According to one aspect, a human skin explant model is utilized that preserves the physiological complexity, the proliferative capacity and the structural integrity of all skin components for up to 28 days. Viable explants are utilized with a surface area of 15-20 mm to enable topical treatment with test agents and compositions.
According to one aspect, rAAV vector serotypes exhibit tissue specificity and efficiency of gene transfer. To establish delivery efficiency, the native fluorescence was studied of a reporter gene (rAAV: EGFP) distributed over a large surface area in full thickness human (breast) skin tissues (16 mm×2 mm in cross-sectional area) maintained in a culture dish for 24 hours, post-treatment. To enable quantification, the native fluorescence was studied of a reporter gene distributed over a large surface area in full thickness human (breast) skin tissues cultured for 24 hours post-treatment. The signal of EGFP in frozen samples (16 mm×1 mm×20 μm) was analyzed and quantified using a custom MatLab code for image post-processing. This algorithm executes flat-field and background corrections and creates a logical mask of the image. According to one aspect, the skin of the subject may be treated prior to topical application of the engineered virus so as to permeabilize the stratum corneum or otherwise The use of recombinant RNA or DNA viral based vector systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the skin tissue and trafficking the viral payload to the nucleus. According to certain embodiments, recombinant viral vectors can be administered directly to the skin of a subject (in vivo) or they can be administered to skin tissues or cells in vitro, and skin tissues or cells that were modified by the recombinant viruses may optionally be grafted or administered back to the subject (ex vivo). Conventional recombinant viral based vector systems can include retroviral, lentivirus, adenoviral, adeno-associated virus (AAV), vaccinia virus and herpes simplex virus vectors for gene transfer. Of these viral vectors, recombinant AAV is thought to be the safest due to its lack of pathogenicity. Integration in the host genome is possible with the retrovirus and lentivirus vector transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies using these recombinant viruses have been observed in many different cell types and target tissues. In certain embodiments, following ultrasound treatment of the skin, rAAV vectors containing genes of interest are topically applied to the skin tissue and let passively diffuse to reach skin cells in both epidermal and dermal skin layers. The tropism of an AAV can be altered by different capsid proteins. A skilled in the art can select appropriate rAAV serotype, including serotypes 1-9 based on the tropism for a particular cell type. Table 1 shows a list of non-limiting target genes and their functions for skin gene therapy according to certain embodiments of the disclosure.
Embodiments of the present disclosure contemplate delivery of nucleic acids encoding genes producing extracellular matrix proteins including but not limited to COL1A1, COL3A1, TIMP1, TIMP2, SMAD2, SMAD3, CTGF, TGF-beta1) (Table 1, 1). The disclosed methods contemplate combating the age-related alterations of the dermis; the largest portion of the skin. The bulk of the epidermis is composed of collagenous extracellular matrix, which confers mechanical strength, elasticity and resilience to the skin. These functions are failing in both chronologically aged and photo aged skin due to alterations in the expression levels of extracellular proteins. The disclosed methods contemplate restoring “youthfull” levels of the extracellular matrix to counteract aging defects.
Embodiments of the present disclosure further contemplate delivery of nucleic acids encoding genes controlling the proper epidermal differentiation and renewal (Table 1, 2).
Embodiments of the present disclosure also contemplate delivery of nucleic acids encoding genes including but not limited to KRT6A, NOTCH1(icd), TET2, and TET3 (Table 1, 3). Photoaged skin sustains more numerous than any other tissue insults to its DNA. As a consequence, skin undergoes “extrinsic aging”, which at molecular level is caused by the high mutational loads evinced in the epidermis of all healthy individuals as early as at the age of 40. The continued degradation (i.e. aging) of the skin is the major factor leading to easily observable changes in skin appearance and pigmentation, and on the other end of the spectrum to onsets of benign and malignant skin lesions such as seborrheic keratoses, actinic keratoses and non-melanoma skin cancer. The disclosed methods contemplate restoring the proper epidermal homeostasis in photoaged skin by delivering genes encoding wild type (not mutated) determinants of epidermal differentiation (Notch) and stem cell renewal (Krt6A, TET2/3).
Embodiments of the present disclosure also contemplate methods for reversing age related alterations in the skin (Table 1, 4 & 5). The disclosure provides for a gene therapy method for the delivery of nucleic acids encoding Sirt1, Sirt6, Pck1, Pparg, and Cisd2, MDH1/2, Aco1/2, IDH1/2/3, Enolase, GOT1/2, MUC1, and MCU. The metabolic state of epidermal progenitors is emerging as an important determinant of skin age. In the epidermis, stem cell's commitment to differentiation, triggered by an increase in intracellular calcium, corresponds to a critical metabolic switch from cytosolic glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). Alterations in mitochondrial OXPHOS is associated with failure to maintain functioning “youth” epidermis. Importantly, the capacity to elevate mitochondrial respiration fails in aging epidermal stem cells simultaneously with decreased expression of rate-limiting mitochondrial enzymes. Thus, to combat the failure in the switch to mitochondrial OXPHOS at the onset of commitment to differentiation during skin aging, the disclosed methods contemplate delivering multiple genes affecting key metabolic pathways to reverse the effects of aging.
Different skin layers, structures and cells can be targeted for gene delivery according to certain embodiments of the disclosed methods. The skin is composed of diverse cells derived from three distinct embryonic origins: neurectoderm, mesoderm, and neural crest. Recombinant viral vectors can be delivered to one or more of the three layers of the skin: the epidermis, dermis, and hypodermis. The epidermis, the outermost layer, is primarily composed of stratified squamous epithelium of keratinocytes, which is derived from neurectoderm and comprises over ninety percent of epidermal cells. The stratified squamous epithelium is further divided into four layers, starting with the outermost layer: stratum corneum (SC), stratum granulosum (SG), stratum spinosum (SS), and stratum basale (SB). Cells of the epidermis including keratinocytes which are responsible for the cohesion of the epidermal structure and the barrier function, pigment-containing melanocytes, antigen-processing Langerhans cells, and pressure-sensing Merkel cells can be targeted by the viral vectors.
The dermis is a connective tissue that is responsible for the mechanical properties of the skin. It is composed of fibroblasts of mesoderm origin, which lie within an extracellular specialized matrix. Collagens are interwoven with elastin, proteoglycans, fibronectin, and other components. The epidermis and dermis are connected by a basement membrane that is composed of various integrins, laminins, collagens, and other proteins that play important roles in regulating epithelial-mesenchymal cross-talk. The superficial papillary dermis is arranged in ridge-like structures called the dermal papillae, which contains microvascular and neural networks and extends the surface area for these epithelial-mesenchymal interactions. Sebaceous glands, eccrine glands, apocrine glands and hair follicles are of neurectoderm origin and develop as downgrowths of the epidermis into the dermis. Outer root sheath of the hair follicle is contiguous with the basal epidermal layer. In addition, the dermis also contains blood vessels and lymphatic vessels of mesoderm origin, and sensory nerve endings of neural crest origin. The hypodermis, which is deep to the dermis, is composed primarily of adipose tissue of mesoderm origin, and separates the dermis from the underlying muscular fascia. Viral vectors can also target these cells, glands, and structures of the dermis and hypodermis.
Recombinant viral vectors can also target skin-specific stem cells which possess the ability for skin tissue to self-renew. Multipotent or unipotent skin stem cells are slowly-cycling cells that reside in at least five distinct niches in the skin: basal (innermost) layer of epidermis, hair follicle bulge, base of sebaceous gland, dermal papillae, and dermis. Not only are these stem cells critical for the long-term maintenance of the skin tissue but also are activated by wounding to proliferate and regenerate the tissue. Skin-specific stem cells include hair follicle stem cells for hair follicle and continual hair regeneration, melanocyte stem cells giving rise to the melanocytes in both the hair matrix and epidermis, stem cells at the base of the sebaceous gland for continually generating terminally differentiated sebocytes, which degenerate to release lipids and sebum through the hair canal and lubricate the skin surface, mesenchymal stem cells that giving rise to fibroblasts, nerves and adipocytes, and a skin-derived precursor stem cell (SKP) distinct from mesenchymal stem cells.
It is to be understood that the basic concepts of the present disclosure described herein are not limited by cell type. Further, cells include any in which it would be beneficial or desirable to regulate a target nucleic acid. Such cells may include those which are deficient in expression of a particular protein leading to a disease or detrimental condition of the skin. Such diseases or detrimental conditions are readily known to those of skill in the art. According to the present disclosure, the nucleic acid responsible for expressing the particular protein may be targeted by the methods described herein and a transcriptional activator resulting in upregulation of the target nucleic acid and corresponding expression of the particular protein. In this manner, the methods described herein provide therapeutic treatment. Such cells may include those which over express a particular protein leading to a disease or detrimental condition. Such diseases or detrimental conditions are readily known to those of skill in the art. According to the present disclosure, the nucleic acid responsible for expressing the particular protein may be targeted by the methods described herein and a transcriptional repressor resulting in downregulation of the target nucleic acid and corresponding expression of the particular protein. In this manner, the methods described herein provide therapeutic treatment.
According to one aspect, the cell is a mammalian cell. According to one aspect, the cell is a human cell. According to one aspect, the cell is a stem cell whether adult or embryonic. According to one aspect, the cell is a pluripotent stem cell. According to one aspect, the cell is an induced pluripotent stem cell. According to one aspect, the cell is a human induced pluripotent stem cell. According to one aspect, the skin cell is in vitro, in vivo or ex vivo.
According to certain aspects, the skin tissue is in vivo, ex vivo, or in vitro. According to certain aspects, the skin tissue includes skin grafts, explants, artificial skin tissues and skin substitutes.
The skin tissues and cells can derive from a subject of a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
Complex signaling pathways that control self-renewal, proliferation, and differentiation are critical for maintaining skin homeostasis and regeneration. The methods of the present disclosure are amenable for skin gene therapy and genome editing therapy that are feasible for modulate gene expression and genome editing of target molecules in the signaling pathways related to maintaining skin homeostasis and regeneration. According to certain aspects, recombinant viral vectors can be designed to combine with the CRISPR system for delivery of nucleic acid molecules that alter target genome and modulate target gene expression of skin cells. For example, the CRISPR type II system is a recent development that has been utilized for genome editing in a broad spectrum of species. See Friedland, A. E., et al., Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat Methods, 2013. 10(8): p. 741-3, Mali, P., et al., RNA-guided human genome engineering via Cas9. Science, 2013. 339(6121): p. 823-6, Hwang, W. Y., et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol, 2013, Jiang, W., et al., RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol, 2013, Jinek, M., et al., RNA-programmed genome editing in human cells. eLife, 2013. 2: p. e00471, Cong, L., et al., Multiplex genome engineering using CRISPR/Cas systems. Science, 2013. 339(6121): p. 819-23, Yin, H., et al., Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol, 2014. 32(6): p. 551-3. CRISPR is particularly customizable because the active form consists of an invariant Cas9 protein and an easily programmable guide RNA (gRNA). See Jinek, M., et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012. 337(6096): p. 816-21. Of the various CRISPR orthologs, the Streptococcus pyogenes (Sp) CRISPR is the most well-characterized and widely used. The Cas9-gRNA complex first probes DNA for the protospacer-adjacent motif (PAM) sequence (−NGG for Sp Cas9), after which Watson-Crick base-pairing between the gRNA and target DNA proceeds in a ratchet mechanism to form an R-loop. Following formation of a ternary complex of Cas9, gRNA, and target DNA, the Cas9 protein generates two nicks in the target DNA, creating a double-strand break (DSB) that is predominantly repaired by the non-homologous end joining (NHEJ) pathway or, to a lesser extent, template-directed homologous recombination (HR). CRISPR methods are disclosed in U.S. Pat. Nos. 9,023,649 and 8,697,359. See also, Fu et al., Nature Biotechnology, Vol. 32, Number 3, pp. 279-284 (2014). Additional references describing CRISPR-Cas9 systems including nuclease null variants (dCas9) and nuclease null variants functionalized with effector domains such as transcriptional activation domains or repression domains include J. D. Sander and J. K. Joung, Nature biotechnology 32 (4), 347 (2014); P. D. Hsu, E. S. Lander, and F. Zhang, Cell 157 (6), 1262 (2014); L. S. Qi, M. H. Larson, L. A. Gilbert et al., Cell 152 (5), 1173 (2013); P. Mali, J. Aach, P. B. Stranges et al., Nature biotechnology 31 (9), 833 (2013); M. L. Maeder, S. J. Linder, V. M. Cascio et al., Nature methods 10 (10), 977 (2013); P. Perez-Pinera, D. D. Kocak, C. M. Vockley et al., Nature methods 10 (10), 973 (2013); L. A. Gilbert, M. H. Larson, L. Morsut et al., Cell 154 (2), 442 (2013); P. Mali, K. M. Esvelt, and G. M. Church, Nature methods 10 (10), 957 (2013); and K. M. Esvelt, P. Mali, J. L. Braff et al., Nature methods 10 (11), 1116 (2013).
The practice of the disclosed methods employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).
Skin diseases and conditions may be characterized by abnormal loss of expression or underexpression of a particular protein or abnormal gain or overexpression of a particular protein. Such skin diseases or conditions can be treated by upregulation or down regulation of the particular protein. Accordingly, methods of treating a skin disease or condition are provided where delivery of nucleic acid sequences via recombinant viruses to skin cells results in up- or down-regulation of expression of the target nucleic acid. One of skill in the art will readily identify such diseases and conditions based on the present disclosure. Examples of target proteins/polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide. Examples of target polynucleotides include a disease associated gene or polynucleotide. A “disease-associated” gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected skin tissues compared with skin tissues or cells of a non disease control. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. A disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease. The transcribed or translated products may be known or unknown, and may be at a normal or abnormal level. Examples of disease-associated genes and polynucleotides of skin are available from McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), available on the World Wide Web. Mutations in these genes and pathways can result in production of improper proteins or proteins in improper amounts which affect skin function. Such genes, proteins and pathways may be the target polynucleotide of the disclosed methods.
Embodiments of the present disclosure provide methods for delivering foreign or heterologous nucleic acids (i.e. those which are not part of a cell's natural nucleic acid composition) encoding genes of interest into a cell of the skin tissue. In one embodiment, the skin tissue is pre-treated with ultrasound prior to deliver of foreign or heterologous nucleic acids. Alternative methods for introducing foreign or heterologous nucleic acids into cells can be used in combination with the delivery methods disclosed herein. These alternative methods are known to those skilled in the art including transfection, transduction, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment, transformation, conjugation and the like. One of skill in the art will readily understand and adapt such methods using readily identifiable literature sources.
Embodiments of the present disclosure provide methods for delivering vectors encoding genes of interest into a cell of the skin tissue. The term “vector” includes a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors used to deliver the nucleic acids to cells as described herein include vectors known to those of skill in the art and used for such purposes. Certain exemplary vectors may be plasmids, lentiviruses or adeno-associated viruses known to those of skill in the art. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, doublestranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, lentiviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” or “operatively linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). Vectors according to the present disclosure include those known in the art as being useful in delivering genetic material into a cell and would include regulators, promoters, nuclear localization signals (NLS), start codons, stop codons, a transgene etc., and any other genetic elements useful for integration and expression, as are known to those of skill in the art.
According to certain aspect, the present disclosure provides viral vectors for use in gene therapy methods disclosed herein and these viral vectors are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
According to certain aspects, the present disclosure provides methods of non-viral delivery for use in gene therapy methods disclosed herein. Methods for non-viral delivery of nucleic acids or native DNA binding protein, native guide RNA or other native species include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration). The term native includes the protein, enzyme or guide RNA species itself and not the nucleic acid encoding the species.
According to some aspects, the present disclosure provides nucleic acid sequences encoding gene of interest including regulatory elements for optimum expression of the gene of interest in target cell or target tissue. The term “regulatory element” is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g. liver, pancreas), or particular cell types (e.g. lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In some embodiments, a vector may comprise one or more pol III promoter (e.g. 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g. 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the 0-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter and Pol II promoters described herein. Also encompassed by the term “regulatory element” are enhancer elements, such as WPRE; CMV enhancers; the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. A vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., clustered regularly interspersed short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).
Aspects of the methods described herein may make use of terminator sequences. A terminator sequence includes a section of nucleic acid sequence that marks the end of a gene or operon in genomic DNA during transcription. This sequence mediates transcriptional termination by providing signals in the newly synthesized mRNA that trigger processes which release the mRNA from the transcriptional complex. These processes include the direct interaction of the mRNA secondary structure with the complex and/or the indirect activities of recruited termination factors. Release of the transcriptional complex frees RNA polymerase and related transcriptional machinery to begin transcription of new mRNAs. Terminator sequences include those known in the art and identified and described herein.
Aspects of the methods described herein may make use of epitope tags and reporter gene sequences. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, betaglucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP).
The following examples are set forth as being representative of the present disclosure. These examples are not to be construed as limiting the scope of the present disclosure as these and other equivalent embodiments will be apparent in view of the present disclosure, figures and accompanying claims.
Recombinant AAV (rAVV) Vector A “recombinant parvoviral” or “AAV vector” or “rAAV vector” herein refers to a vector comprising one or more polynucleotide sequences of interest, genes of interest or “transgenes” that are flanked by at least one parvoviral or AAV inverted terminal repeat sequences (ITRs). Such rAAV vectors can be replicated and packaged into infectious viral particles when present in an insect host cell that is expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When an rAAV vector is incorporated into a larger nucleic acid construct (e.g. in a chromosome or in another vector such as a plasmid or baculovirus used for cloning or transfection), then the rAAV vector is typically referred to as a “pro-vector” which can be “rescued” by replication and encapsidation in the presence of AAV packaging functions and necessary helper functions. Thus, in a further aspect the invention relates to a nucleic acid construct comprising a nucleotide sequence encoding a porphobilinogen deaminase as herein defined above, wherein the nucleic acid construct is a recombinant parvoviral or AAV vector and thus comprises at least one parvoviral or AAV ITR. Preferably, in the nucleic acid construct the nucleotide sequence encoding the porphobilinogen deaminase is flanked by parvoviral or AAV ITRs on either side.
The AAV VP proteins are known to determine the cellular tropicity of the AAV virion. The VP protein-encoding sequences are significantly less conserved than Rep proteins and genes among different AAV scrotypes. The ability of Rep and ITR sequences to cross-complement corresponding sequences of other serotypes allows for the production of pseudotyped rAAV particles comprising the capsid proteins of one serotype (e.g., AAV5) and the Rep and/or ITR sequences of another AAV serotype (e.g., AAV2). Such pseudotyped rAAV particles are a part of the present invention. Herein, a pseudotyped rAAV particle or hybrid rAAV may be referred to as being of the type “x/y”, where “x” indicates the source of ITRs and “y” indicates the serotype of capsid, for example a 2/5 rAAV particle has ITRs from AAV2 and a capsid from AAV5. Modified “AAV” sequences also can be used in the context of the present disclosure, e.g. for the production of rAAV vectors in insect cells. Such modified sequences e.g. include sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more nucleotide and/or amino acid. sequence identity (e.g., a sequence having from about 75% to about 99% nucleotide sequence identity) to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 ITR, Rep, or VP can be used in place of wild-type AAV ITR, Rep, or VP sequences. Preferred adenoviral vectors are modified to reduce the host response. See, e.g., Russell (2000) J. Gen. Virol. 81:2573-2604; US patent publication no. 20080008690; and Zaldumbide et al. (2008) Gene Therapy 15(4):239-46; all publications incorporated herein by reference.
The schematic of the backbone vector is as follows:
were used to amplify COL3A1 gene sequence. The bold and italicized part of the forward primer is the Kozak sequence. The bold and italicized part of the reverse primer is the stop codon sequence. The two parts were combined and used in PCR to amplify the COL3A1 sequence. The underlined sequences in both the forward and reverse primers are overhangs attached during PCR to create a fusion COL3A1 sequence for a total length of 3526 base pairs. After restriction digest using unique restriction enzyme site overhangs NotI and NheI, the backbone vector and gene were ligated together.
The method of AAV production and titer quantification was carried out according to Lock, M. 2010 Human gene therapy; Kwon, O. et al., (2010) J Histochem Cytochem. 58(8):687-694. Briefly, Hek293 cells were triple co-transfected at 75% confluency in one 10 layer Nunc™ Cell Factory™ System from Thermo Scientific (Rockford, Ill.) using PEI transfection reagent following manufacturer's instructions. Cells and supernatant were harvested separately after 72 hours post transfection. The cells were spun down and lysed with 3 freeze-thaw cycles and incubated with Benzonase (E1015-25KU, Sigma). They were then clarified by spinning at 10,500×G for 20 min and the supernatant was added to the rest of the media supernatant. Everything was filtered through a 0.2 uM filter and was then concentrated using lab scale TFF system (EMD Chemicals, Gibbstown, N.J.) down to 15 ml. We used a Pellicon XL 100 kDa filter and followed manufactures instructions (EMD Chemicals, Gibbstown, N.J.). The concentrated prep was re-clarified by centrifugation at 10,500 Řg and 15° C. for 20 min and the supernatant was carefully removed to a new tube. Six iodixanol step gradients were formed according to the method of Zolotukhin and colleagues. See Zolotukhin S., (1999) Gene Ther. 6:973-85, with some modifications as follows: Increasingly dense iodixanol (OptiPrep; Sigma-Aldrich, St Louis, Mo.) solutions in phosphate-buffered saline (PBS) containing 10 mM magnesium chloride and 25 mM potassium were successively underlaid in 39 ml of 62 Quick-Seal centrifuge tubes (Beckman Instruments, Palo Alto, Calif.). The steps of the gradient were 4 ml of 15%, 9 ml of 25%, 9 ml of 40%, and 5 ml of 54% iodixanol. Fourteen milliliters of the clarified feedstock was then overlaid onto the gradient and the tube was sealed. The tubes were centrifuged for 70 min at 242,000 Řg in a VTi 50 rotor (Beckman Instruments) at 18° C. and the 40% gradient was collected through an 18-gauge needle inserted horizontally at the 54%/40% interface. The virus containing iodixanol was diafiltered using Amicon 15-Ultra and washed 5 times with final formulation buffer (PBS-35 mM NaCl), and concentrated to 1 ml.
DNase I-resistant vector genomes were titered by TaqMan PCR amplification (AppliedBiosystems, Foster City, Calif.), using primers and probes directed against the WPRE3 poly Adenylation signal encoded in the transgene cassette. The purity of gradient fractions and final vector lots were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the proteins were visualized by SYPRO ruby staining (Invitrogen) and UV excitation.
Skin samples were mounted in custom diffusion chambers. Immediately before administration, the donor chamber was filled with 1.5 mL of phosphate buffered saline (PBS). 20 kHz ultrasound was utilized to maximize transient cavitation events, which have previously been shown to be the primary mechanism of enhancement. 20 kHz ultrasound was generated with a 12-element probe (probes 9 mm diameter) driven by a VCX 500 (Sonics and Materials, Inc., Newtown, Conn.). For all applications, the ultrasound probe tip was placed 3 mm away from the surface of the tissue. Ultrasound intensities were calibrated by calorimetry to 5 W/cm2. Ultrasound was applied using a 50% duty cycle (5 s on, 5 s off). After administration, the PBS was removed. A solution (10 μl) of AAV was the applied topically on the skin and incubated at 32° C. for 60 minutes in the skin explant experiments, and 5 minutes in the hairless mice experiments. The effective dose range for small animals (mice), skin explants, and reconstructed human skin, following skin permeation by ultrasound, is between 5×108 and 1×1012 genome copy (gc)/cm2. 5×109 and 5×1010 were used for low and high dose, respectively.
The signal of EGFP in frozen samples from skin explants (16 mm×1 mm×20 μm) were analyzed and quantified using a custom MatLab scripts for image post-processing. The algorithm executes flat-field and background corrections and creates a logical mask of the image. Based on the different emission spectra of tissue auto-fluorescence and signal due to the expression of EGFP, the algorithm performs linear un-mixing of the total fluorescence intensity. Finally, it identified the areas of these unmixed signals of background autofluorescence and GFP signal.
Mouse or human skin was harvested and fixed in 10% formalin or 4% paraformaldehyde overnight for frozen sections, respectively. Frozen sections were used for Hematoxylin and Eosin (H&E) staining and histological analysis. H&E staining was carried out following the standard protocol (http://www.ihcworkd.com). Slides were mounted in Entellan New rapid mounting media (Electron Microscopy Sciences). Frozen sections (mounted in OCT embedding compound and frozen at −80 C) were used for immunofluorescence staining: primary antibodies were incubated for 3 hours, and second antibodies were incubated for 1 hour at room temperature in 5% BSA/PBST. Nuclei were stained with DAPI (Invitrogen), and the slides were mounted in Prolong Gold Antifade Mount (Invitrogen). Primary antibodies were used at 1:200 dilution, while secondary antibodies at 1:1000.
Discarded human skin samples from abdominoplasty and/or breast reduction procedures were obtained from Massachusetts General Hospital (Boston, Mass.) under patients' agreement and institutional approval. Skin samples, sterilized in 70% ethanol and cut, after removal of subcutaneous fat, into 1.6 cm diameter pieces, were placed in keratinocyte serum-free medium (KSF, GIBCO-BRL) supplemented with epidermal growth factor (EGF) and bovine pituitary extract (BPE), in 0.25% agar (Sigma). The epidermis was maintained at the air-medium interface. For RNA and protein collection, skin samples were chilled (on ice) and homogenized using PRO200 BIO-GEN tissue homogenizer (Pro Scientific Inc., Oxford, Conn.).
Protein from all tissues was isolated with RIPA (radioimmunoprecipitation assay) buffer containing protease and phosphatase inhibitors (all reagents purchased from Boston BioProducts, Ashland, Mass.). All specimens were chopped in small pieces and disrupted by PRO200 BIO-GEN tissue homogenizer (Pro Scientific Inc., Oxford, Conn.). Protein concentration in the clear lysates after centrifugation was measured with the Pierce BCA Protein Assay (Pierce Biotechnology, Grand Island, N.Y.). Western blots were quantified using the Fiji image processing software (open-source tool by ImageJ, https://imagej.nih.gov/ij/).
One-step TaqMan (AppliedBiosystems, Foster City, Calif.) RT-qPCR were used with primers and probes directed against human COL3A1 encoded in the transgene cassette to perform quantification for gene expression. ACTB gene was used to quantify reference levels in the RNA samples. Equal amounts (as quantified by Agilent's bioanalyzer) of total RNA were used as input for all gene expression measurements.
Skin is immediately harvested after euthanasia. Part of it was snap frozen in dry ice for qPCR/qRT-PCR analysis and RNA/DNA-sequencing and the other part of each organ was then PFA-fixed for 3-24 hours depending on size and frozen in OCT buffer in liquid nitrogen bath for sectioning and analysis.
Animals are euthanized by the slow fill method of CO2 administration according to the equipment available in the facility. Typically, animals are euthanized in the home cage out of view from other animals. A regulator is used to ensure the proper flow rate. Animals should lose consciousness rapidly ˜30 sec. At the cessation of breathing (several minutes) animals will undergo a secondary physical method of euthanasia.
Most of the presently existing delivery methods for skin gene therapies rely on systemic delivery, electroporation or μ-needle injections. The delivery methods for skin gene therapy disclosed herein contemplate intra-dermal or topical delivery of recombinant viruses in a highly targeted and completely non-invasive manner. The ultrasound pre-treatment described herein is recognized to result in no pain or distress.
To determine delivery efficiency, the native fluorescence of a reporter gene distributed over a large surface area in full thickness human (breast) skin tissues cultured for 24 hours post-ultrasound treatment was studied. The reporter gene is enhanced GFP, and it is packaged in rAAV. The signal of EGFP in frozen samples (16 mm×1 mm×20 μm) was analyzed and quantified using a custom MatLab code for image post-processing. Briefly, the algorithm executes flat-field and background corrections and creates a logical mask of the image. Based on the different emission spectra of tissue auto-fluorescence and signal due to the expression of EGFP, the algorithm performs linear un-mixing of the total fluorescence intensity. Finally, it identified the areas of these unmixed signals. In
Based on the results described in
Because wild type AAV2 shows best gene delivery across all skin major cell types, rAAV2/2 was adopted to perform another set of tests in human skin explants (ex vivo) taken from the forehead skin of a 60-year-old donor. For cell tropism analysis, these tissues were stained for Vimentin (a fibroblast marker), anti-EGFP (a marker for the reporter gene), and Keratin 15 (a marker for epithelial stem cells) (
Next, to determine prolonged gene expression and stable protein modulation in vivo, hairless mice model was used. For these proof-of-concept experiments, we chose to deliver a single precursor gene to human collagen III (alpha domain), packaged in rAAV2 (rAAV2/2: COL3A1). Type III collagen is a human gene which encodes for collagen III fibrils which serve as a major component of the skin extracellular matrix, thus being an important target for the purposes of rebuilding aged- and diseased-skin dermis. Protein analysis and quantification using Western blot showed up to 5.4-fold over expression of collagen III, levels comparable to those in human skin (
Similar to the in vivo mouse experiments, gene and protein modulation of collagen III was determined in vitro in human artificial skin (EpiDermFT tissues commercially made available by MatTek, Inc.). These artificial skin tissues contain primary human dermal fibroblasts and epidermal keratinocytes that are cultured to form a full thickness multilayer model of human skin. The tissue layers are metabolically and mitotically active and mimic in vivo characteristics. We delivered viral vectors of rAAV2/2: EGFP and rAAV2/2: COL3A1, and measured 40,000-fold and 2.8-fold increase in the gene expression of EGFP and COL3A1 via RT-qPCR. The reported gene expression fold increase is relative to untreated (no-AAV, no ultrasound) control samples. All gene expression levels are normalized to the expression of ACTB. Measured by Western blotting, collagen levels showed 2.3-fold enhancement relative to untreated negative control tissue. As another end-point, we confirmed collagen accumulation by histological analysis using Picro-sirius red and trichrome staining which quantify total collagen content (
AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAA
ATGATGAGCTTTGTGCAAAAGGGGAGC
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Chronic exposure to UV irradiation causes an aged phenotype (photo-aging) that is superimposed with chronological aging of the skin. As a consequence, nearly every aspect of skin biology is affected by aging. The self-renewing capability of the epidermis, which provides vital barrier function, is diminished with age and results in numerous clinical presentations, ranging from benign but potentially excruciating disorders like pruritus and defective wound healing to the more threatening carcinomas and melanomas. Yet our current knowledge of the molecular determinants of declining epidermal function in the elderly population is quite limited. Several genome-wide studies have attempted to analyze the transcriptome and epigenome of the aging skin but failed to identify robust drivers of cellular aging in the skin epidermis (Haustead, D. J., Stevenson, A., Saxena, V., Marriage, F., Firth, M., Silla, R., Martin, L., Adcroft, K. F., Rea, S., Day, P. J., Melton, P., Wood, F. M. & Fear, M. W. Transcriptome analysis of human ageing in male skin shows mid-life period of variability and central role of NF-kappaB. Sci Rep 6, 26846 (2016); Makrantonaki, E., Brink, T. C., Zampeli, V., Elewa, R. M., Mlody, B., Hossini, A. M., Hermes, B., Krause, U., Knolle, J., Abdallah, M., Adjaye, J. & Zouboulis, C. C. Identification of biomarkers of human skin ageing in both genders. Wnt signalling—a label of skin ageing? PLoS One 7, e50393 (2012); Raddatz, G., Hagemann, S., Aran, D., Sohle, J., Kulkarni, P. P., Kaderali, L., Hellman, A., Winnefeld, M. & Lyko, F. Aging is associated with highly defined epigenetic changes in the human epidermis. Epigenetics Chromatin 6, 36 (2013)), and reported that the mammalian epidermis appears to resist the aging process (Racila, D. & Bickenbach, J. R. Are epidermal stem cells unique with respect to aging? Aging (Albany N.Y.) 1, 746-50 (2009)). It is widely accepted that besides transcriptional and epigenetic changes, cellular aging is characterized also by profound metabolic alterations. Nonetheless, recent non-targeted metabolomics analysis of full thickness human skin indicated that only a minimal fraction (less than 10%) of detectable metabolites significantly drifted during aging [Kuehne, A., Hildebrand, J., Soehle, J., Wenck, H., Terstegen, L., Gallinat, S., Knott, A., Winnefeld, M. & Zamboni, N. An integrative metabolomics and transcriptomics study to identify metabolic alterations in aged skin of humans in vivo. BMC Genomics 18, 169 (2017); Randhawa, M., Sangar, V., Tucker-Samaras, S. & Southall, M. Metabolic signature of sun exposed skin suggests catabolic pathway overweighs anabolic pathway. PLoS One 9, e90367 (2014)). To the best of our knowledge, all reports on omics studies of transcriptome, epigenome, and metabolome of aging human skin use bulk analysis performed on whole tissue lysates, which very often fails to detect profound changes in isolated small cellular populations (such as stem cells) which drive homeostatic processes.
Adult organs are maintained through a balance of proliferation, differentiation, and self-renewal of stem cells that take place during normal tissue homeostasis or tissue repair. The epidermis relies on a population of stem cells and proliferating progenitors to continuously maintain its barrier-protective function. It is composed of different cellular lineages: the interfollicular epidermis and its appendages; and the sebaceous glands and the hair follicles. Human epidermis is regenerated approximately every 4 weeks, a process driven by commitment of progenitor cells located within the basal membrane which develop into more differentiated populations. Initial models of epidermal maintenance proposed that the basal layer is composed of two populations of stem cells: slow cycling stem cells and their transiently amplifying progenitors. However, recent advances in linage tracing and live imaging techniques combined with genetic manipulations have now established a simple model of epidermal homeostasis in which basal keratinocytes are born as equally uncommitted stem cells making random choices to divide or differentiate. This process allows both for continuous renewal of the proliferating basal layer and departure of committed cells away from the basal membrane towards the differentiated upper layers of the epidermis. The ultimate goal of this homeostatic behavior is the generation of a solid cornified envelope as a barrier to the outside insults.
primary cultures of human keratinocytes from donors of different ages (ranging from Age 18 to Age 72) WERE isolated and grown in strictly progenitor conditions as described in (Roshan, A., Murai, K., Fowler, J., Simons, B. D., Nikolaidou-Neokosmidou, V. & Jones, P. H. Human keratinocytes have two interconvertible modes of proliferation. Nat Cell Biol 18, 145-56 (2016)), after which half of the population were prompted to commit to differentiation by exogenous calcium. To investigate the metabolic changes occurring during the process of differentiation, we subjected both populations (progenitors and committed cells) from young, medium, and old ages to polar steady-state metabolomics analysis by liquid chromatography-based tandem mass spectrometry (LC-MS/MS), and determined 296 metabolite profiles for each sample. Experiments with progenitor and committed primary cultures were run in triplicates and values for every measured metabolite were compared across all samples. Normalization was performed based on cell number in each individual sample. Alterations in multiple classes of metabolites were observed by hierarchical clustering in the mature keratinocyte population (but not the progenitor population) pointing to age-related functional metabolic deteriorations of progenitors' ability to build a young epidermis.
For each sample, total RNA was extracted using RNeasy mini kit (Qiagen) and treated with on-column RNase-free DNase I (Qiagen) following manufacturer's instructions. 1 ug of RNA from each sample was used for library preparation. RNA-seq libraries were constructed using TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold (Illumina) designed for cytoplasmic and mitochondrial rRNA depletion. All coding RNA and certain forms of non-coding RNA were isolated using bead-based rRNA depletion, followed by cDNA synthesis, and PCR amplification as per manufacturer's protocol. Final libraries were analyzed on Bioanalyzer (Agilent), quantified with qPCR, pooled together, and run on one lane of an Illumina HiSeq 2500 using 2×100-bp paired-end reads. The Illumina paired-end adapter sequences were removed from the raw reads using Cutadapt v1.8.1. The TruSeq adaptor sequence 5′-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3′ was used for read 1, and its reverse complement, 3′-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT-5′ was used for read 2.
Next, RNA libraries were processed using a pipeline which includes STAR-HtSeq-GFOLD for alignment, count generation, and gene expression. Briefly, STAR aligner (v. 2.4.0j) was used to map the reads to hg19, and HtSeq was used to generate gene expression counts. Since each donor is considered an N of 1 (i.e. donors are not grouped in replicates), GFOLD and custom R scripts were used to determine gene and differential expression.
To identify functional modules of genes that reflect age-related changes, gene-gene interaction (protein-protein interaction, PPI) network were integrated with expression data by a computational algorithm. This method is called Network Propagation, which propagates the expression values by the topology of the network. In literature, network propagation is typically employed on mutation data to classify cancer informative subtypes by clustering patients with mutations in similar network regions (Vanunu O, Magger O, Ruppin E, Shlomi T, Sharan R (2010) Associating genes and protein complexes with disease via network propagation. PLoS Comput Biol 6: e1000641). In this analysis, a network graph was first created by using the STRING database, such that nodes correspond to genes and edges correspond to interactions between genes. For each gene (node), the value was mapped with the deviation of gene expression in specific sample (age18, age46, age64, or age72) and the mean value of the four samples. Next, the implementation of network propagation processed a random work on a network with the function: Ft+1=αFtA+(1−α)F0, where F0 is a comparison-by-gene matrix, A is a degree-normalized adjacency matrix derived from the topology of the network, α is a tuning parameter governing the amount of signal that was passed to the neighboring nodes of the network during signal propagation. Based on the function, the propagation occurred by iteration during which a certain ratio of the node value was spread to its neighbors. After several iterations, each gene gets a propagated score. Genes with high propagated scores were regarded as candidates associated with aging functions. In different age samples, a common module with higher propagated values (1,306 genes) was detected by clustering the results from the propagation.
Next a gene set highly correlated with metabolites across different ages was identified using the O2-PLS regression method which is based on partial least squares and orthogonal signal correction (OSC) filter (Johan Trygg, Svante Wold, O2-PLS, a two-block (X-Y) latent variable regression (LVR) method with integral OSC filter, J. Chemometrics; 17: 53-64 (2003)). Broadly, the algorithm separates structured noise from transcript and metabolite matrices, X and Y respectively, and identifies a joint covariation to use in a predictive model. To identify highly correlated genetic and metabolic markers of aging, we used the model on metabolic data of progenitor and committed to differentiation primary cells, and transcriptomic data on progenitor cells only. As a measure of stem cell function, i.e. capacity to commit to differentiation and form proper epidermis, we calculated a metabolic score matrix based on the difference of metabolite levels in the differentiated cell population relative to that of their progenitors for ages ranging from young to old. We then combined these metabolic scores with the transcript data across different ages to identify trends of common variation. Briefly, the algorithm consists of: 1. Decomposition of the covariance Y T X matrix into orthogonal score matrix C, singular value matrix D, and orthonormal loading matrix W; 2. Calculation of X score matrix T where T=XW, and respective removal of structured noise; 3. Calculation of Y score matrix U where U=YC, and respective removal structured noise; and 4. Predictions of U and T with least squares. We determined significance level of (1−α), with α=0.05/n for the transcript data with n=number of genes, and α=0.05/m for the metabolite data with m=number of metabolites. We then performed randomization by reshuffling the original data sets X and Y, 1000 times, and identified thresholds for lower and upper α/2 quantiles for transcript and metabolite correlation loadings. We determined a list of 176 significant genes which we included in network propagation for further processing as part of the network.
Global network-similarity measures were adopted to better capitalize on biological relationships between selected genes and identify master regulators of skin stem and progenitor cells aging. A gene association network was first constructed by mapping STRING network to each gene level. The network is an undirected graph in which nodes represent genes and edges between two nodes denote an association between two corresponding genes. Weights are used on the edges to represent the probability that such an association exists. After constructing the gene network graph, a random walk graph kernel method was used to capture global relationships within the graph. A graph kernel is a kernel function that computes the probability of reaching one node after a random walk starting from another node, and the computed probability is used for global similarity of two nodes (genes). The resulting graph creates a global distance network where the edge between two nodes (genes) represents the global distance in this network instead of a direct interaction. Laplacian Exponential Diffusion Kernel was used as the kernel function, i.e.:
where L is an undirected graph Laplacian matrix, and β is the diffusion parameter that determines the degree of diffusion. eβL is a random walk that starts from a node to its neighboring node with the probability β. Since eβL is positive definite for a Laplacian matrix, it can be used as a kernel matrix. The resulting kernel matrix is a connected network, which detected not only the direct interaction from the original gene association network, but also all indirect interactions via other genes. As a result, the distances among all genes in the network are determined. Next, these distances are used to distinguish highly expressed neighborhoods with a certain distance from a candidate gene, even in cases when the genes may not directly interact. However, the above equation cannot be solved directly because of computation complexity (O(n3)). We therefore further applied a dimension reduction method, called Cholesky decomposition, to trim the Laplacian matrix L by decomposing it into the product of a lower triangular matrix. Cholesky decomposition is to transform a matrix A into a product of a lower triangular matrix P of rank n (P=(pij) with pij=0 if i<j and pij>0, where i=1, 2, . . . , n) and its transpose, PT: A=P·PT. To reduce the dimensionalities of kernel matrices, we applied the Incomplete Cholesky Decomposition (ICD) with pivoting in order to reduce the dimensionalities by approximating a lower rank matrix (m<<n), such that A≈{tilde over (P)}·{tilde over (P)}T, where A∈Rn×m, m<<n. We then obtained a lower triangular matrix {tilde over (P)} of rank m. The overall complexity was O(m2n) and the storage requirement was O(mn). Previously, similar approach has been implemented by Nitch et. al in C++ for the study on disease-causing genes in monogenic genetic diseases [Nitsch D, Tranchevent L C, Thienpont B, Thorrez L, Van Esch H, et al. (2009) Network analysis of differential expression for the identification of disease-causing genes. PLoS One 4: e5526.]. In our study, we constructed the network in R environment.
The gene expression profiles were mapped to the distance network obtained above. More specifically, the fold changes between the two conditions (two ages) were computed to obtain the differential expression level for the genes in genome. It was considered whether the gene was highly differentially expressed or not, hence, the absolute value of the fold-change was relevant for our method. All differential expression levels, without threshold to distinguish between highly and lowly differentially expressed genes, were used to compute the scores. Since our method computes the scores with all differential expression, there is no threshold used to distinguish between highly and lowly differentially expressed genes. The score of the candidate biomarker gene was calculated by measuring the differential expression levels of its neighborhood. First, the differential expression level of all neighbors in the distance network were ordered by their distance to the candidate gene. The rank of the diffusion distance was then taken as the new distance measure. Second, the new differential expression levels were generated by multiplying the gene expression (fold change value here) with a weighting function (w=e−β≠γ, where γ is the rank, β is the parameter of neighborhood size) to consider the expression of both close and far neighbors. β is the scale parameter to determine how quickly the weight decreased as a function of distance, and it was set to 0.5 to reach sufficiently far away genes in the network for the candidate gene. Lastly, we randomly shuffled original expression values over the network, and then defined the gene score for a candidate gene was by select the maximum deviation between the new differential expression values (weighted) and the randomized expression. Hence, the gene score was related to the level of differential expression level of close neighbors. To estimate the significance of the signal of the actual candidates, we defined the distribution of the scores by randomly distributing the expression data on the network and repeating 3,000 times. By comparing the score of each candidate gene, an empirical p-value for each candidate gene was determined. The score of a candidate gene was considered significant if the score was greater than 95% (α=0.05).
Although previous analyses of aging human skin revealed only non-significant changes in transcriptome, epigenome and metabolome and failed to define molecular drivers of altered epidermal function in the elderly, our model for selection of aging biomarker genes indicates that the processes associated with skin aging hinder the ability of epidermal progenitors to effectively differentiate to mature keratinocytes during commitment to differentiation, resulting in thinner aged epidermis. In
One of the hallmarks of skin stem cell aging is found that and deterioration in differentiation capacity is characterized with altered Carbon metabolism and TCA cycle, and aim to validate the functional properties of a herein reported regulator gene—Maleate dehydrogenase 2 (MDH2). MDH2 is a metabolic enzyme which is involved in processes associated with oxidation of malate to oxaloacetate by utilizing NAD/NADH cofactors in the Citrate cycle (TCA cycle), and affects energy consumption and metabolism between the mitochondria and cytosol. In
According to certain embodiments, genes relating to skin aging as disclosed herein represent skin aging biomarkers and their expression can be modulated by the methods disclosed herein to promote skin function and health. According to certain embodiments, the disclosed method comprises delivery of genes comprising sequences of SEQ ID NOS 1-122 to the skin or delivery of genes that modulate the expression of the genes comprising sequences of SEQ ID NOS 1-122.
To create an optimal framework for delivery of transgenes to skin cells, fluorescent enhanced GFP reporter transgene was cloned in an AAV vector containing AAV2-derived inverted terminal repeat 1 (ITR1) in the flip direction and an inverted terminal repeat 2 (ITR2) in the flop direction. The ITR1 element is annealed to human hEF1a (human elongation factor-1 alpha) promoter, while ITR2 element is annealed to 134b-long SV40 late polyadenylation (truncated SV40 late poly(A)) element and 248b-long WPRE3 (truncated) element.
To evaluate the efficacy of gene transfer to human skin cells, a variety of AAV capsids were used to make hybrid AAV viral serotypes. A typical workflow is shown on
To determine the expression potential of a panel of viral promoters in human skin tissue, a group of ubiquitous and tissue-specific promoters was tested in human skin explants as represented in the workflow of
To confirm dose-dependency within the range of use, AAV2/8-hEF1a-EGFP was administered to human skin explants at the doses of 5E+10, 1E+11, 2E+11, and 5E+11 GC. The strength of cell expression was evaluated by the gene expression of reporter gene, EGFP both in terms of relative (to negative control) expression (
To establish delivery efficiency selectively to dermal skin cells, the native fluorescence of a reporter gene, EGFP was measured over a large surface area in full thickness human breast skin tissues (16 mm×2 mm in cross-sectional area) maintained in culture conditions post-treatment. Human skin explants were harvested 24 hours after the treatment, and embedded in OCT. To determine the total signal over the cross-sectional area of the dermis (16 mm×1 mm×20 μm), native GFP fluorescence was quantified using a custom image post-processing pipeline in MatLab. The algorithm executes flat-field and background corrections, creates a logical mask of the image, and performs linear un-mixing of the total fluorescence intensity based upon different emission spectra of tissue auto-fluorescence and signal due to the expression of EGFP. The process is shown in
The infectivity of the CMV promoter in human dermal cells shows a transient response over time and is the highest during the first week of infection. To quantify longer term expression response in the human dermis, skin explants (from human donor id=4 of medium age) were AAV2-infected with CMV, CASI, and hEF1a promoters and harvested at Day 12. To separate epidermis from dermis, explants were treated with a protease (dispase II at 5U/ml, overnight) to facilitate peeling off the epidermis. A population of dermal cells (predominantly skin fibroblasts) was then dissociated using Collagenase I at 1 mg/ml in DMEM/Serum (20%) solution at 37 C. The isolated cells were stained with anti-EGFP and anti-Cytokeratin 15 antibodies for processing with FACS. A population of ˜30,000 cells was analyzed. As shown on
The expression potential of recombinant AAV virus to infect and deliver genes to human epidermis was quantified by flow cytometry. Whole skin was permeabilized using topical ultrasonic treatment, after which it was spot-treated with therapy. In one instance, skin explants (from human donor id=4 of medium age) were AAV-treated with hybrid serotypes of AAV2/2, AAV2/5, AAV2/6.2, AAV2/8, AAV2/9, and AAV2/10 at a dose of 2E+11 GC per explant, and cultured for 12 days. All vectors were driven by the hEF1a promoter. The epidermis of the explants was separated from the dermis using an overnight protease treatment (dispase II at 5U/ml), and keratinocyte cells were dissociated with Trypsin-EDTA (0.25%) for 15 min at 37 C. The dissociated cells were stained with an anti-EGFP antibody and quantified for expression of GFP. As shown in
Skin is maintained through a balance of proliferation, differentiation, and self-renewal of stem cells that take place during normal tissue homeostasis or tissue repair. The epidermis relies on a population of stem cells and proliferating progenitors to continuously maintain its barrier-protective function. While the epidermal differentiated population (mature keratinocytes) has a lifespan of ˜4 weeks, the stem and progenitor cell populations have a nearly life-long span.
To achieve long-term expression of genes in skin tissues, gene transfer to the populations of stem cells located within the basal membrane (slow cycling stem cells and their transiently amplifying progenitors) was optimized. As shown in
Across all examined viral vectors, the ones with the highest infectivity capacity in the epidermal progenitor and stem cell populations expressing K15 and a6-integrin were AAV2/2-hEF1a, AAV2/2-CASI, AAV2/2-CMV, AAV2/5-hEF1a, and AAV2/8-hEF1a. As shown in
Recombinant AAV2/2 virus that expresses human collagen III (alpha domain) driven by a truncated hEF1a promoter was administered to human skin explants at a dose of 2E+11 GC per sample. Ultrasound-mediated gene delivery was executed in a single step, and the process of skin permeabilization required ultrasonic initiation of vibrating cavitational bubbles, active oscillation, instability and bursting of bubbles followed by topical, passive-diffusion delivery of a single therapy dose (
To determine the robustness of dermal matrix remodeling and age-associated thinning of the dermis by modulation of collagen III, three human explant from another donor were treated with the same rAAV virus encoding collagen III protein. rAAV2/2 virus was administered to human skin which was cultured for 8 days and analyzed for levels of collagen III using Western blot. The highest amount of collagen III expression reached 3.2-fold overproduction compared to the native levels in the control tissue.
This example shows that the recombinant AAV virus expressing collagen III can be effectively used to provide consistent protein overexpression with the human dermis.
This example describes modulation of 4 age-related genes—(mouse) KRT6A, (human) TET3, (mouse) TGFb1, and (human) COL3A1 in SKH-1E hairless mice. KRT6 Å and TET3 target the epidermis and improve functions related to stem and progenitor cell renewal and DNA de-methylation, while TGFb1 and COL3A1 target the dermis and modulate production of extracellular proteins (as shown in
To determine expression capabilities of rAAV in hairless mice as a function of time, rAAV vector expressing collagen III was administered at a dose of 2E+11 GC per animal.
This example shows that the recombinant AAV virus disclosed here can be used to drive effective and robust expression of proteins over long periods of time.
HEK293T Cells (ATCC) were expanded in DMEM (Corning) with 10% FBS (Genessee) and 1% Pen/Strep (Life) and cultured on 5-Layer Flasks (Corning) until 70-80% confluent. On Day 0, helper plasmid, capsid plasmid, and transgene ITR plasmid were combined with PEI Max (Polysciences) in a triple plasmid transfection. On Day 3, additional complete media was added to the culture (50% of original volume). On Day 6, NaCl was added to each flask to a final concentration of 0.5M and incubated for 2 hours. Lysed and dissociated cells were then collected and stored overnight at 4 C. On Day 7, the supernatant of the cell lysate was collected and 0.22 μM sterile filtered before addition of PEG 8000 (Calbiochem) to a final concentration of 8% and left at 4 C overnight on a stirplate. On Day 8, PEG mixture was centrifuged at 4000G for 20 minutes. Supernatant was discarded, and pellet resuspended with PBS to a final volume of 8 mL. Benzonase (Millipore) was added and incubated at 37 C for 45 minutes. An ultracentrifuge gradient in optiseal tubes (Beckman-Coulter) was then created by layering resuspended PEG pellet, then 15%, 25%, 40%, and 60% iodixanol (Sigma) from the bottom-up. Samples were then balanced before ultracentrifugation at 240,000G for 1 hour. Tubes were then punctured on the bottom before collecting 500 μL fractions, stopping at the 40-25% interface. Samples were run on a protein gel and fractions with high VP protein purity (
Ex Vivo Human Immune Response to rAAV
This example illustrates the therapeutic use of genetically engineered skin patch for in vivo immunomodulation, immunoprophylaxis, and passive delivery of therapies for diseases such as cancers, autoimmune diseases, metabolic disorders and viral infections. The virtual therapeutic skin patch is primed with transgenes serving preventative or therapeutic use by non-invasive delivery of recombinant AAV virus whose penetration to the epidermal and dermal layers is enabled by topical cavitational, low-frequency ultrasound method. Ultrasonic disruption of the stratum corneum is reversible and facilitates rAAV transport into the epidermis, the papillary and reticulous dermis avoiding injury and inflammation of the treated and surrounding tissues.
The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference in their entirety for all purposes.
Other embodiments will be evident to those of skill in the art. It should be understood that the foregoing description is provided for clarity only and is merely exemplary. The spirit and scope of the present invention are not limited to the above examples, but are encompassed by the following claims. All publications and patent applications cited above are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent application were specifically and individually indicated to be so incorporated by reference.
This application is a National Stage Application under 35 U.S.C. 371 of co-pending PCT application PCT/US18/32597 designating the United States and filed May 14, 2018; which claims the benefit of U.S. provisional application No. 62/505,359 filed on May 12, 2017 each of which are hereby incorporated by reference in their entireties.
This invention was made with government support under Grant Nos. HG008525, MH113279, and EB000244 from the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US18/32597 | 5/14/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62505359 | May 2017 | US |
