WO2015035395A1 - Gene therapy for the regeneration of chondrocytes or cartilage type cells - Google Patents
Gene therapy for the regeneration of chondrocytes or cartilage type cells Download PDFInfo
- Publication number
- WO2015035395A1 WO2015035395A1 PCT/US2014/054804 US2014054804W WO2015035395A1 WO 2015035395 A1 WO2015035395 A1 WO 2015035395A1 US 2014054804 W US2014054804 W US 2014054804W WO 2015035395 A1 WO2015035395 A1 WO 2015035395A1
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- cells
- cartilage
- cell
- gene
- chondrocytes
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
- A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Definitions
- the present invention generally concerns at least the fields of medicine, surgery, anatomy, biology, cell biology, and/or molecular biology.
- osteoarthritis affects 33.6% (12.4 million) of adults age 65 and older in the U.S.
- AAOS American Academy of Orthopaedic Surgeons
- hyaline cartilage e.g., within diarthrodial joints
- fibrocartilage e.g., knee meniscus and TMJ disc
- elastic cartilage e.g., ear
- articular cartilage covering bone surfaces is a soft and specialized hyaline cartilage that exhibits superior lubrication, wear, and low friction properties; it also reduces stresses in the joint.
- Articular cartilage is composed of a small percentage of chondrocytes, but a dense extracellular matrix (ECM) prevents chondrocyte mobility.
- ECM extracellular matrix
- articular cartilage lacks vascular, neural, and lymphatic networks, as well as various local progenitor cells. It has also been described as having high levels of protease inhibitors, which may inhibit efficient tissue repair.
- tissue engineering may provide alternative solutions for articular cartilage repair and regeneration through developing biomimetic tissue substitutes.
- articular cartilage is a tissue that is not naturally regenerated once damaged.
- tissue engineering has raised tremendous attention.
- Tissue engineering involves the development of biocompatible materials capable of specifically interacting with biological tissues to produce functional tissue equivalents.
- Tissue engineering has a basic concept of collecting a desired tissue from a patient, isolating cells from the tissue specimen, proliferating cells, and re-introducing those cells back into the body.
- genes are utilized as therapeutic compositions for the regeneration of biological tissue.
- the genes Gata4, Mef2C and Tbx5 have been shown to produce cardiomyocytes in some patients. These genes have been researched to attract fibroblasts and induce their differentiation into cardiomyocytes.
- the heart and joint environments are completely different. Cardiomyocyte exist in heart tissue, which is vascular and has ready access to nutrient flow and oxygen. Cartilage, on the other hand, is considered avascular, aneural and has much less access to oxygen. For these reasons, the two environments are considerably drastically different and, as such, would require different biological approaches to regenerate the tissue.
- the present disclosure provides a solution for a long-felt need in the art of cartilage tissue repair or regeneration.
- Embodiments of the invention concern the regeneration of chondrocytes and cartilage-type cells.
- one or more genes are employed for the regeneration of chondrocytes and cartilage-type cells.
- one or more gene therapy regimens are employed for the regeneration of chondrocytes and cartilage-type cells.
- embodiments concern cartilage repair, such as articular cartilage repair. More particularly, embodiments for the disclosure concern using gene therapy for the attraction, generation and/or regeneration of chondrocytes or other cartilage-type cells and/or the generation and/or repair of cartilage tissue.
- gene therapy is provided that is capable of attracting and/or generating desired cells in vivo.
- gene therapy is employed for the attraction of fibroblasts that directly or indirectly are stimulated to differentiate into chondrocytes or chondrocyte-like cells or cartilage- type cells.
- fibroblasts in a joint or other type(s) of cells in a joint are present in the joint upon delivery of one or more gene therapy compositions to the joint and, following the delivery, the fibroblasts or one or more other types of cells differentiate into chondrocytes or chondrocyte-like cells or cartilage-type cells.
- the delivery of one or more gene therapy compositions in a joint act as a catalyst for differentiation of one or more types of cells in the joint to regenerate chondrocytes and/or cartilage-type cells or other cells that result at least in part in the same functionality that cartilage cells possess.
- the delivery of one or more gene therapy compositions in the presence of dying chondrocytes in a joint act as a catalyst for generation of connective tissue in the joint, including generation of cartilage tissue.
- one or more molecules from a dying chondrocyte are employed in the differentiation of one or more types of cells in a joint to chondrocytes, and such differentiation may or may not utilize gene products from one or more gene therapy composition(s).
- Embodiments of the invention concern all forms of cartilage and any other cells that may form and function in a location as if they were cartilage; in specific aspects, scar tissue is generated by methods of the invention. In particular cases wherein scar tissue is generated, that scar tissue acts as a cushion in the joint.
- gene therapy composition(s) are uptaken by one or more types of cells that already reside in vivo in a joint, although in other cases gene therapy composition(s) are uptaken by cells that are delivered to the joint and/or that already reside in the joint.
- the cells that are delivered to the joint may be differentiated prior to delivery to the joint or following delivery to the joint, and the gene therapy composition(s) may be exposed to the cells that are delivered to the joint prior to delivery to the joint or following delivery to the joint.
- Differentiation of cells into chondrocytes or chondrocyte-like cells may occur in any suitable manner in accordance with the disclosure, including, for example, differentiation of cells in vitro prior to implantation of the gene therapy into an individual or differentiation in vitro prior to implantation of the gene therapy into an individual and also in vivo following implantation.
- the cells that differentiate into chondrocytes or chondrocyte-like cells may be of any particular kind, but in specific embodiments the cells that differentiate into chondrocytes or chondrocyte-like cells are fibroblasts.
- fibroblasts, adipose cells, stem cells, stem cells derived from fibroblast and/or mesenchymal stem cells are employed in any method of the invention.
- the fibroblasts may differentiate into chondrocytes or chondrocyte-like cells prior to, during, and/or after exposure to the one or more gene therapy compositions.
- fibroblasts are exposed to mechanical strain in vivo or in vitro prior to in vivo delivery and are also exposed to one or more gene therapy compositions.
- pressure on the joint may act as mechanical strain to cause, at least in part, differentiation of cells, including stem cells and/or fibroblasts.
- chondrocytes or chondrocyte-like cells or cartilage -type are produced for the purpose of cartilage regeneration and/or repair. Any gene or combination of genes may be employed for the purposes of the disclosure.
- COL11A2, or others may be employed for chondrocyte generation, cartilage-type cell generation and/or cartilage regeneration and/or repair.
- TGF-beta and FGF-2 are utilized in cartilage regeneration.
- COL11A2, TGF-beta and/or FGF-2 are employed for in vivo cartilage generation from surrounding fibroblasts.
- other genes referred to herein are utilized in compositions and methods of the invention.
- Embodiments concern a therapeutic delivery (such as by injection, intravenous therapy (IV), oral ingestion, vascular placement, such as an angiogram, or even a topical approach) of certain gene(s), such as collagen-formation gene(s), including with or without a nutrient matrix and/or vessel. More specifically, but not exclusively, embodiments relate to methods and/or compositions for biological repair of cartilage using a delivery of cartilage-forming genes and/or a gene mixture that will attract surrounding fibroblasts to begin repair of the cartilage. This therapy acts as an in vivo workstation for cartilage restoration, in particular embodiments.
- cartilage generation initiated in vitro, such as by using autologous chondrocytes and/or allogenic chondrocytes and/or fibroblasts, such as dermal fibroblasts.
- This mixture may be delivered (such as by injection) into one or more joints or in the vicinity of one or more joints, and this will attract other non-captured fibroblasts (for example) into the cartilage regeneration process, in particular embodiments.
- a matrix or scaffold may be introduced and then seeded either in vitro or in vivo with the gene therapy composition, fibroblasts, and/or chondrocytes to provide structure to the cartilage regeneration process.
- the introduction of genes that aid in the production of cartilage attract fibroblasts or other suitable cells in the area of the cartilage undergoing degradation to differentiate into chondrocytes or chondrocyte-like cells for the purpose of replenishing and/or halting the cartilage degradation, in aspects of the disclosure.
- cartilage typically has low blood flow and access to nutrients, it is considered a difficult tissue in the body to regenerate.
- other elements besides the gene may be added to the therapeutic gene composition(s).
- regulatory elements suitable for activity in a joint may be employed in the invention.
- the composition comprises nutrients, and in particular aspects the composition Ahas an absorbable reservoir comprising oxygen and/or nutrients.
- proteins/amino acids, phosphates, calcium, sodium, lipids, iron, sugars/starches, and/or vitamins may be utilized in one or more methods of the invention.
- the gene therapy composition(s) comprises an expression vector.
- Any suitable expression vector may be utilized, but in some cases the expression vector is suitable for activity in a joint, including in avascular, aneural, and/or low oxygen environment.
- the expression vector comprises a promoter that is active in an avascular, aneural, and/or low oxygen environment.
- a or “an” may mean one or more.
- the words “a” or “an” when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
- another may mean at least a second or more.
- aspects of the invention may "consist essentially of or “consist of one or more sequences of the invention, for example.
- Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.
- cartilage-type cell refers to any cell that could exist and function in the same capacity as a cartilage cell.
- chondrocyte-like cells refers to cells that are not primary chondrocytes but are derived from stem cells (such as mesenchymal stem cells) or cells from other lineages (such as fibroblasts). These chondrocyte-like cells have a phenotype of chondrocytes (cells of cartilage).
- chondrocytes polygonal and/or rhomboidal cells, for example
- cartilage matrix components such as sulfated proteoglycan and type II collagen, for example.
- exemplary markers of chondrocyte-like cells include one or more of aggrecan, which is a chondroitin sulfate and keratan sulfate proteoglycan, type II collagen, Sox-9 protein, cartilage link protein, and perlecan, which is a heparan sulfate proteoglycan, for example.
- hypooxia refers to a deficiency in oxygen. In specific aspects, it refers to oxygen tension that is less than about 20%.
- joint refers to a region in the body wherein two bones of a skeleton join.
- regeneration is defined as a process that restores the normal functions of injured articular cartilage, and in at least certain cases also results in the formation of new tissue that is indistinguishable from the native cartilage.
- seeding refers to implanting cells in a scaffold. The cells will attach to the scaffold and then grow and differentiate in the scaffold.
- the term "scaffold” is a mechanical unit upon which a matrix of cells could form.
- matrix would be a geometric shape that would then provide strength and structure.
- the present invention is directed to systems, methods, and compositions for treatment of an individual in need thereof, including treatment of an individual in need of cartilage repair.
- the present invention concerns methods and compositions for biological repair of any kind of cartilage, including intervertebral and joint cartilage, for example.
- the present invention concerns the fields of cartilage repair, such as articular cartilage repair.
- the invention generates natural tissue in vivo (or in vitro or in vitro and in vivo), such as from fibroblasts, for example. More particularly, but not exclusively, the present invention relates to a method for growing and differentiating human fibroblasts into chondrocyte-like cells, for example.
- the cells may be autologous or allogeneic or a mixture thereof, in certain embodiments.
- the invention employs differentiation of certain cells into chondrocyte-like cells.
- human dermal fibroblasts for example, are differentiated into chondrocyte-like cells under particular conditions employing gene therapy with one or more genes. Differentiation of cells into chondrocytes or chondrocyte- like cells may occur in any suitable manner, including in vivo following implantation or in vivo with naturally-residing cells, including fibroblasts.
- the invention provides a method for in vivo regeneration of a joint, such as an intervertebral disc, elbow, knee, shoulder, hip, temporomandibular joint, ankle, metatarsal, metacarpal and so forth.
- a joint such as an intervertebral disc, elbow, knee, shoulder, hip, temporomandibular joint, ankle, metatarsal, metacarpal and so forth.
- a method to repair damaged disc there is provided a method to repair damaged disc.
- a method of repairing damaged cartilage in a joint (such as an intervertebral disc) of an individual comprising delivering fibroblasts subjected to gene therapy in accordance with the invention to the respective joint (such as intervertebral disc) of the individual or providing in vivo fibroblasts or other cells (including dying chondrocytes) with gene therapy.
- the gene therapy composition(s) or fibroblasts and the gene therapy composition(s) are delivered to a joint; in specific embodiments for the intervertebral disc, the gene therapy composition(s) or fibroblasts and the gene therapy composition(s) are delivered in the absence of removing part or all of the degenerated disk.
- an individual is provided another therapy in addition to the methods of the invention.
- the individual may receive one or more drugs.
- exemplary additional therapies include antibiotics, Non Steroidal Anti-Inflammatory Drugs (NSAIDs), simple pain killers (analgesics), muscle relaxants, and/or functional rehabilitation as needed.
- the individual may be provided one or more of an antibiotic, antifungal agent, antiviral agent, nutrient injection, IV, or oral tablet.
- the cells differentiate into chondrocyte cells or chondrocyte-like cells, such as wherein the chondrocyte cells or chondrocyte-like cells secrete a molecule selected from the group consisting of aggrecan, type II collagen, Sox-9 protein, cartilage link protein, perlecan, and combinations thereof.
- the cells are differentiated from fibroblast cells, and exemplary fibroblast cells include dermal fibroblasts, tendon fibroblasts, ligament fibroblasts, synovial fibroblasts, foreskin fibroblasts, or a mixture thereof.
- growth factors such as bone morphogenetic protein 2 (BMP-2), BMP-4, BMP-6, BMP-7, cartilage-derived morphogenetic protein (CDMP), transforming growth factor beta (TGF- ⁇ ), insulin growth factor one (IGF-I), fibroblast growth factors (FGFs), basic fibroblast growth factor (bFGF), FGF-2, platelet-derived growth factor (PDGF), and a mixture thereof.
- BMP-2 bone morphogenetic protein 2
- BMP-4 BMP-4, BMP-6, BMP-7
- CDMP cartilage-derived morphogenetic protein
- TGF- ⁇ transforming growth factor beta
- IGF-I insulin growth factor one
- FGFs fibroblast growth factors
- bFGF basic fibroblast growth factor
- FGF-2 platelet-derived growth factor
- PDGF platelet-derived growth factor
- kits comprising one or more gene therapy composition(s) and may also include certain cells, such as fibroblasts, wherein any component of the kit is housed in one or more suitable containers.
- the kit further comprises one or more reagents suitable for enhancing in vivo differentiation from fibroblasts to chondrocytes or chondrocyte-like cells.
- the kit of the invention includes one or more apparatuses for delivery of gene therapy composition(s) and/or fibroblasts to an individual.
- a site in vivo in an individual in need thereof is in vivo and in need of chondrocytes, including in need of cartilage.
- a site in need of chondrocytes includes joints, for example cartilaginous joints (e.g., vertebrae).
- the fibroblasts are obtained from the individual in need of cartilage.
- fibroblasts are delivered to at least one intervertebral disc in an individual.
- the gene therapy composition(s) are delivered to the joint.
- the gene therapy composition(s) are delivered between invertebral discs.
- the gene therapy composition(s) are delivered between or in nucleus pulposus and fissures in the inner annulus fibrosus.
- the gene therapy composition(s) may be delivered between invertebral discs, including nucleus pulposus and fissures in the inner annulus fibrosus, for example.
- one or more gene therapy composition(s) are delivered in vivo to a joint and resident cells in the joint uptake the composition(s).
- cells are provided to the joint before, during and/or after delivery of the one or more gene therapy composition(s).
- any cell may be employed so long as the cell is capable of differentiating into a chondrocyte or chondrocyte-like cell.
- the cell is a fibroblast cell, such as a dermal fibroblast, tendon fibroblast, ligament fibroblast, or synovial fibroblast, for example.
- Autologous cells may be utilized, although in alternative embodiments allogeneic cells are employed; in specific embodiments, the allogeneic cells have been assayed for disease and are considered suitable for human transmission.
- the cell or cells are autologous, although in alternative embodiments the cells are allogeneic.
- the cells may be processed by standard means in the art to remove potentially hazardous materials, pathogens, etc.
- one or more genes are used as therapeutic compositions.
- the compositions may be employed to facilitate differentiation of fibroblasts to chondrocytes or chondrocyte-like cells or may be employed to enhance the level of a gene product from the therapeutic composition once a fibroblast has differentiated to a chondrocyte or chondrocyte-like cell.
- the one or more provided genes work in conjunction with molecules present in an joint from dying chondrocytes to facilitate generation of chondrocytes or chondrocyte-type cells or cartilage tissue in the joint.
- polynucleotides encompassing the one or more therapeutic genes comprise the full-length genes, although in some aspects, a fragment of a gene is utilized, although the fragment still has therapeutic activity or encodes a peptide or polypeptide that has therapeutic activity.
- the genes may be mammalian in nature, including human, mouse, rat, and so forth.
- the one or more therapeutic genes are a collagen formation gene (including of any type), a cartilage formation gene, a connective tissue formation gene, a transcription factor, a cartilage matrix gene, a receptor gene, or a signaling molecule, for example.
- the one or more therapeutic genes are not a member of the transforming growth factor (TGF) beta superfamily.
- the gene therapy composition encodes one or more matrix molecules, such as collagen I, collagen II, proteoglycan, or a combination thereof.
- the collagen comprises type I and type II collagen.
- one of the proteoglycans is aggrecans.
- one or more of the following genes are employed in the invention: COL1A1, COL1A2, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL5A3, COL6A1, COL6A2, COL6A3, COL6A4, COL6A5, COL7A1, COL8A1, COL8A2, COL9A1, COL9A2, COL9A3, COL10A1, COLl lAl, COL11A2, COL12A1, COL13A1, COL14A1, COL15A1, COL16A1, COL17A1, COL18A1, COL19A1, COL20A1, COL21A1, COL22A1, COL23A1, COL24A1, COL25A1, COL26A1, COL27A1, and COL28A1.
- Exemplary genes include cartilage matrix genes, such as proteoglycans and COL2, -9, -10, and -11; receptor genes [fibroblast growth factor 2 (FGFR2); parathyroid hormone-related peptide receptor (PTHrP-R)]; one or more transcription factors (SOX5, -6, and - 9); SOX4; vascular endothelial growth factor (VEGF); matrix metalloproteinase 14 (MMP14); forkhead; CDIO; MMP13; collagens (such as COL3A1 and COL16A1); signaling molecule (WNT11); a homeobox homolog (BAPX1); a receptor (IL-1R1); an IGFs modulator (IGFBP5); and/or a mettaloproteinase (MMP16) (see Sekiya et al, 2002).
- cartilage matrix genes such as proteoglycans and COL2, -9, -10, and -11
- receptor genes [fibroblast growth factor 2 (FGFR2)
- genes may be employed: known markers of chondrogenic capacity (collagen II, FGFR3, BMP2, ALKl), anabolic growth factors (BMP5 and IGFl) or matrix-degrading enzymes (MMP13 and ADAMTS5) (see Hellingman et al, 2011).
- GREM1, BMP6, COL10A1, or MMP13 see Funari et al, 2007).
- BMC Genomics 2007, 8 165
- TGF ⁇ a member of the TGF ⁇ superfamily is not employed in the invention
- one or more members of the TGF beta superfamily is utilized including TGF ⁇ , TGF- 3, TGF- 2, TGF- 4, TGF- ⁇ , TGF- ⁇ 5 (Xenopus), BMP-2, BMP-4, Drosophila DPP, BMP-5, BMP-6, Vgrl, OP-l/BMP-7, Drosophila 60A, GDF-1, Xenopus Vgf, BMP-3, Inhibin- ⁇ , Inhibin- ⁇ , Inhibin- ⁇ , and MIS (see U.S. Patent No. 7,338,655, incorporated by reference herein in its entirety).
- part or all of a gene is utilized in embodiments of the methods and/or compositions. All or part of a coding region for a gene may be employed, and one or more regulatory regions for a particular gene may be utilized. In specific embodiments, one may utilize a fragment of a gene, including a fragment of a coding region, and the fragment will still retain therapeutic activity. In specific cases, a wild-type sequence of a particular gene is utilized, and the skilled artisan recognizes that the sequence for a gene may be identified in a variety of databases, such as the National Center for Biotechnology Information's GenBank® database.
- the sequence utilized in the polynucleotide compositions is not identical to a wild-type sequence but differs from the sequence in one or more nucleotides or in one or more amino acids of the encoded gene product.
- the sequence utilized in the composition may be 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, and so forth, identical to a corresponding wild-type DNA sequence or such percentage identical to the encoded gene product when compared to the polypeptide sequence of the encoded wild-type polypeptide.
- Embodiments include employing one or more polynucleotides capable of encoding a therapeutic gene product for chondrocyte regeneration, cartilage generation, and/or cartilage repair.
- the gene product is utilized by a fibroblast or other type of cell in a joint to facilitate differentiation of the fibroblast to chondrocytes or chondrocyte- like cells and/or is utilized by a chondrocyte or chondrocyte-like cell that differentiated from a fibroblast, stem cell, or adipose cell.
- the polynucleotide may or may not encode an entire gene product. In cases wherein less than the entire gene product is encoded, the fragment is nevertheless biologically active and capable of having activity for chondrocyte generation and/or cartilage repair and/or cartilage regeneration. In some cases, expression vectors that comprise the therapeutic gene product or biologically active fragment thereof comprise a selectable marker.
- a further embodiment of the present invention includes employing a polynucleotide capable of encoding a therapeutic gene product or a biologically active derivative or fragment thereof, and employing as the expression vector any vector known to one of ordinary skill in the art capable of stable maintenance within the targeted cell or tissue upon delivery, regardless of the method of delivery utilized.
- One such method is the direct delivery of the vector molecule, whether it be a viral or non- viral vector molecule, to the target cell or tissue.
- Another embodiment of this invention provides a method for introducing at least one gene encoding a product into at least one cell for use in treating the mammalian host.
- This method includes employing viral or non-viral means (including plasmids) for introducing the gene coding for the product into the desired cell. More specifically, certain methods include a liposome encapsulation, calcium phosphate coprecipitation, electroporation, or DEAE-dextran mediation, and includes employing as the gene a gene capable of encoding a member of a therapeutic gene product or biologically active derivative or fragment thereof, and optionally a selectable marker.
- Another embodiment of this invention provides a method for introducing at least one gene encoding a product into at least one desired cell for use in treating a mammalian host.
- This method includes employing the biologic means of utilizing a virus or other means to deliver a vector molecule to a target cell or tissue.
- the virus is a pseudo-virus, the genome having been altered such that the pseudovirus is capable only of delivery and stable maintenance within the target cell, but not retaining an ability to replicate within the target cell or tissue.
- the altered viral genome is further manipulated by recombinant DNA techniques such that the viral genome acts as a DNA vector molecule which contains the heterologous gene of interest to be expressed within the target cell or tissue.
- the viral vector may be retroviral vector, adenoviral vector, lentiviral vector, or adeno-associated viral vector, for example.
- An embodiment of the invention is a method of delivering a desired therapeutic gene product to a target joint space by delivering a polynucleotide that encodes the product to a desired location of a mammalian host.
- a DNA sequence of interest encoding a part or all of a therapeutic gene product is subcloned into a vector of choice, the recombinant vector is then used to infect in viiro-cultured desired cells, and the transduced connective tissue cells, such as autografted cells, are transplanted into a location of interest, such as by intra- articular injection; in alternative embodiments, the recombinant vector is delivered to a location of interest in vivo, wherein the vector infects in vivo cells, such as in vivo fibroblasts in a joint, for example.
- a method of the present invention involves direct in vivo delivery of a therapeutic gene to the desired tissue of a mammalian host through use of either an adenovirus vector, adeno-associated virus (AAV) vector or herpes- simplex virus (HSV) vector.
- AAV adeno-associated virus
- HSV herpes- simplex virus
- a DNA sequence of interest encoding a functional protein or protein fragment is subcloned into the respective viral vector.
- the gene product-containing viral vector is then grown to adequate titer and directed into the desired space, preferably by intra-articular injection.
- Methods of presenting the DNA molecule to target cells in a joint includes, but is not limited to, encapsulation of the DNA molecule into cationic liposomes, subcloning the DNA sequence of interest in a retroviral or plasmid vector, or the direct injection of the DNA molecule itself into the joint.
- the DNA molecule regardless of the form of presentation to the joint, is preferably presented as a DNA vector molecule, either as recombinant viral DNA vector molecule or a recombinant DNA plasmid vector molecule.
- Expression of the heterologous gene of interest is ensured by inserting a promoter fragment active in eukaryotic cells directly upstream of the coding region of the heterologous gene.
- One of ordinary skill in the art may utilize known strategies and techniques of vector construction to ensure appropriate levels of expression subsequent to entry of the DNA molecule into the connective tissue.
- fibroblasts recovered from a joint are cultured in vitro for subsequent utilization as a delivery system for gene therapy. It will be apparent that Applicants are not limited to the use of the specific connective tissue disclosed. It would be possible to utilize other tissue sources for in vitro culture techniques.
- the method of using the gene of this invention may be employed both prophylactically and in the therapeutic treatment. One may utilize the teachings herein either prophylactically or therapeutically to treat any susceptible or affected joint.
- a compound for parenteral administration to a patient in a therapeutically effective amount contains a gene encoding a TGF-.beta. superfamily protein and a suitable pharmaceutical carrier.
- Another embodiment of this invention provides for a compound for parenteral administration to a patient in a prophylactically effective amount that includes a gene encoding a TGF-beta superfamily protein and a suitable pharmaceutical carrier.
- a further embodiment of this invention includes the method as hereinbefore described including introducing the gene into the cell in vitro.
- This method also includes subsequently transplanting the infected cell into the mammalian host.
- This method includes after effecting the transfecting of the connective tissue cell but before the transplanting of the infected cell into the mammalian host, storing the transfected connective tissue cell.
- the infected connective tissue cell may be stored frozen in 10 percent DMSO in liquid nitrogen, for example.
- Embodiments include employing a method to substantially prevent the development of joint disease or injury in a mammalian host having a high susceptibility of developing joint disease or injury.
- Another embodiment of this invention includes a method of introducing at least one gene encoding a product into at least one cell of a connective tissue of a mammalian host for use in treating the mammalian host as hereinbefore described including effecting in vivo the infection of the cell by introducing the viral vector containing the gene coding for the product directly into the mammalian host.
- this method includes effecting the direct introduction into the mammalian host by intra- articular injection.
- Methods include employing the method on a mammalian host for therapeutic use. Further this method also includes employing the method to repair and regenerate the connective tissue as hereinbefore defined.
- the viral vectors employing a liposome are not limited by cell division as is required for the retroviruses to effect infection and integration of connective tissue cells.
- This method employing non-viral means as hereinbefore described includes employing as the gene a gene capable of encoding a member belonging to the TGF- ⁇ superfamily and a selectable marker gene, such as an antibiotic resistance gene.
- Another embodiment of the present invention is delivery of a DNA sequence encoding a member of the TGF- ⁇ superfamily to the connective tissue of a mammalian host by any of the methods disclosed within this specification so as to effect in vivo expression of collagen to regenerate connective tissue, such as cartilage.
- a DNA plasmid vector containing the TGF- ⁇ coding sequence was ligated downstream of the metallothionein promoter.
- vector is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated.
- a nucleic acid sequence can be "exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found.
- Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
- YACs artificial chromosomes
- expression vector refers to any type of genetic construct comprising a nucleic acid coding for a RNA capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes.
- Expression vectors can contain a variety of "control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra. a. Promoters and Enhancers
- a "promoter” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors, to initiate the specific transcription a nucleic acid sequence.
- the phrases "operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence.
- a promoter generally comprises a sequence that functions to position the start site for RNA synthesis.
- the best known example of this is the TATA box, but in some promoters lacking a TATA box, such as, for example, the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well.
- a promoter To bring a coding sequence "under the control of” a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame "downstream" of (i.e., 3' of) the chosen promoter.
- the "upstream” promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.
- the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
- the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
- individual elements can function either cooperatively or independently to activate transcription.
- a promoter may or may not be used in conjunction with an "enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
- a promoter may be one naturally associated with a nucleic acid sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous.”
- an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
- certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.
- a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment.
- Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression.
- promoters that are most commonly used in recombinant DNA construction include the ⁇ -lactamase (penicillinase), lactose and tryptophan (trp) promoter systems.
- sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCRTM, in connection with the compositions disclosed herein (see U.S. Patent Nos. 4,683,202 and 5,928,906, each incorporated herein by reference).
- control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
- promoter and/or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism chosen for expression.
- Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, (see, for example Sambrook et al. 1989, incorporated herein by reference).
- the promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides.
- the promoter may be heterologous or endogenous.
- any promoter/enhancer combination could also be used to drive expression.
- Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment.
- Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
- tissue-specific promoters or elements as well as assays to characterize their activity, is well known to those of skill in the art.
- Nonlimiting examples of such regions include the human LIMK2 gene (Nomoto et al. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), murine epididymal retinoic acid-binding gene (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen (Tsumaki, et al., 1998), D1A dopamine receptor gene (Lee, et ah, 1997), insulin-like growth factor II (Wu et ah, 1997), and human platelet endothelial cell adhesion molecule- 1 (Almendro et al., 1996).
- a specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.
- IRES elements are used to create multigene, or polycistronic, messages.
- IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988).
- IRES elements from two members of the picornavirus family polio and encephalomyocarditis have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991).
- IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages.
- each open reading frame is accessible to ribosomes for efficient translation.
- Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Patent Nos. 5,925,565 and 5,935,819, each herein incorporated by reference).
- Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector (see, for example, Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997, incorporated herein by reference.)
- MCS multiple cloning site
- Restriction enzyme digestion refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art.
- a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector.
- "Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology. d. Splicing Sites
- RNA molecules will undergo RNA splicing to remove introns from the primary transcripts.
- Vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression (see, for example, Chandler et ah, 1997, herein incorporated by reference.) e. Termination Signals
- the vectors or constructs of the present invention will generally comprise at least one termination signal.
- a “termination signal” or “terminator” is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase.
- a termination signal that ends the production of an RNA transcript is contemplated.
- a terminator may be necessary in vivo to achieve desirable message levels.
- the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site.
- RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently.
- terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message.
- the terminator and/or polyadenylation site elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
- Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator.
- the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.
- polyadenylation signal In expression, particularly eukaryotic expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript.
- the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed.
- Preferred embodiments include the SV40 polyadenylation signal or the bovine growth hormone polyadenylation signal, convenient and known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport. g. Origins of Replication
- a vector in a host cell may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated.
- ori origins of replication sites
- ARS autonomously replicating sequence
- cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by including a marker in the expression vector.
- markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector.
- a selectable marker is one that confers a property that allows for selection.
- a positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection.
- An example of a positive selectable marker is a drug resistance marker.
- a drug selection marker aids in the cloning and identification of transformants
- genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
- markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated.
- screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized.
- a plasmid vector is contemplated for use to transform a host cell.
- plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts.
- the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
- E. coli is often transformed using derivatives of pBR322, a plasmid derived from an E. coli species.
- pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
- the pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, for example, promoters which can be used by the microbial organism for expression of its own proteins.
- phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts.
- the phage lambda GEMTM- 11 may be utilized in making a recombinant phage vector which can be used to transform host cells, such as, for example, E. coli LE392.
- Further useful plasmid vectors include pIN vectors (Inouye et al., 1985); and pGEX vectors, for use in generating glutathione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage.
- GST glutathione S-transferase
- Other suitable fusion proteins are those with ⁇ -galactosidase, ubiquitin, and the like.
- Bacterial host cells for example, E. coli, comprising the expression vector, are grown in any of a number of suitable media, for example, LB.
- the expression of the recombinant protein in certain vectors may be induced, as would be understood by those of skill in the art, by contacting a host cell with an agent specific for certain promoters, e.g., by adding IPTG to the media or by switching incubation to a higher temperature. After culturing the bacteria for a further period, generally of between 2 and 24 h, the cells are collected by centrifugation and washed to remove residual media.
- an agent specific for certain promoters e.g., by adding IPTG to the media or by switching incubation to a higher temperature.
- the cells are collected by centrifugation and washed to remove residual media.
- viruses to infect cells or enter cells via receptor-mediated endocytosis, and to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign nucleic acids into cells (e.g., mammalian cells).
- cells e.g., mammalian cells.
- virus vectors that may be used to deliver a nucleic acid of the present invention are described below.
- a particular method for delivery of the nucleic acid involves the use of an adenovirus expression vector.
- adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors.
- "Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a tissue or cell-specific construct that has been cloned therein.
- Knowledge of the genetic organization or adenovirus, a 36 kb, linear, double- stranded DNA virus allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992).
- the nucleic acid may be introduced into the cell using adenovirus assisted transfection. Increased transfection efficiencies have been reported in cell systems using adenovirus coupled systems (Kelleher and Vos, 1994; Cotten et ah, 1992; Curiel, 1994).
- Adeno-associated virus (AAV) is an attractive vector system for use in embodiments of the present invention as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo.
- AAV has a broad host range for infectivity (Tratschin et ah, 1984; Laughlin et ah, 1986; Lebkowski et ah, 1988; McLaughlin et ah, 1988). Details concerning the generation and use of rAAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368, each incorporated herein by reference. 3. Retroviral Vectors
- Retroviruses have use as delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell-lines (Miller, 1992).
- a nucleic acid is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
- a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al., 1983).
- Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).
- Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known in the art (see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomer et al, 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples of lenti virus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 and the Simian Immunodeficiency Virus: SIV. Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe.
- Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences.
- recombinant lenti virus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference.
- One may target the recombinant virus by linkage of the envelope protein with an antibody or a particular ligand for targeting to a receptor of a particular cell-type.
- a sequence (including a regulatory region) of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target- specific.
- viral vectors may be employed as constructs in the present invention.
- Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), Sindbis virus, cytomegalovirus and herpes simplex virus may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990).
- a nucleic acid to be delivered may be housed within an infective virus that has been engineered to express a specific binding ligand.
- the virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell.
- a novel approach designed to allow specific targeting of retrovirus vectors was developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.
- Suitable methods for nucleic acid delivery for transformation of an organelle, a cell, a tissue or an organism for use with the current invention are believed to include virtually any method by which a nucleic acid ⁇ e.g., DNA) can be introduced into an organelle, a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art.
- Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection (Wilson et al., 1989, Nabel et al, 1989), by injection (U.S. Patent Nos.
- a nucleic acid may be delivered to an organelle, a cell, a tissue or an organism via one or more injections (i.e., a needle injection), such as, for example, subcutaneously, intradermally, intramuscularly, intravenously, intraperitoneally, etc.
- injections i.e., a needle injection
- Methods of injection of compositions are well known to those of ordinary skill in the art (e.g., injection of a composition comprising a saline solution).
- Further embodiments of the present invention include the introduction of a nucleic acid by direct microinjection. Direct microinjection has been used to introduce nucleic acid constructs into Xenopus oocytes (Harland and Weintraub, 1985). The amount of composition used may vary upon the nature of the gene product or gene as well as the organelle, cell, tissue or organism used.
- Electroporation i.e., Electroporation
- a nucleic acid is introduced into an organelle, a cell, a tissue or an organism via electroporation.
- Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge.
- certain cell wall-degrading enzymes such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells (U.S. Patent No. 5,384,253, incorporated herein by reference).
- recipient cells can be made more susceptible to transformation by mechanical wounding.
- a nucleic acid is introduced to the cells using calcium phosphate precipitation.
- Human KB cells have been transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique.
- mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et ah, 1990).
- DEAE-Dextran DEAE-Dextran
- a nucleic acid is delivered into a cell using DEAE-dextran followed by polyethylene glycol.
- reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985).
- Additional embodiments of the present invention include the introduction of a nucleic acid by direct sonic loading.
- LTK fibroblasts have been transfected with the thymidine kinase gene by sonication loading (Fechheimer et ah, 1987).
- a nucleic acid may be entrapped in a lipid complex such as, for example, a liposome.
- Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated is an nucleic acid complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen).
- a liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et ah, 1989).
- a liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et ah, 1991).
- HMG-1 nuclear non-histone chromosomal proteins
- a liposome may be complexed or employed in conjunction with both HVJ and HMG-1.
- a delivery vehicle may comprise a ligand and a liposome.
- a nucleic acid may be delivered to a target cell via receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in a target cell. In view of the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity to the present invention.
- Certain receptor-mediated gene targeting vehicles comprise a cell receptor- specific ligand and a nucleic acid-binding agent. Others comprise a cell receptor- specific ligand to which the nucleic acid to be delivered has been operatively attached.
- Several ligands have been used for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al, 1990; Perales et al, 1994; Myers, EPO 0273085), which establishes the operability of the technique. Specific delivery in the context of another mammalian cell type has been described (Wu and Wu, 1993; incorporated herein by reference).
- a ligand will be chosen to correspond to a receptor specifically expressed on the target cell population.
- a nucleic acid delivery vehicle component of a cell-specific nucleic acid targeting vehicle may comprise a specific binding ligand in combination with a liposome.
- the nucleic acid(s) to be delivered are housed within the liposome and the specific binding ligand is functionally incorporated into the liposome membrane.
- the liposome will thus specifically bind to the receptor(s) of a target cell and deliver the contents to a cell.
- Such systems have been shown to be functional using systems in which, for example, epidermal growth factor (EGF) is used in the receptor-mediated delivery of a nucleic acid to cells that exhibit upregulation of the EGF receptor.
- EGF epidermal growth factor
- the nucleic acid delivery vehicle component of a targeted delivery vehicle may be a liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding.
- lipids or glycoproteins that direct cell-specific binding.
- lactosyl-ceramide, a galactose-terminal asialganglioside have been incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes (Nicolau et al., 1987). It is contemplated that the tissue-specific transforming constructs of the present invention can be specifically delivered into a target cell in a similar manner. i. Microprojectile Bombardment
- Microprojectile bombardment techniques can be used to introduce a nucleic acid into at least one, organelle, cell, tissue or organism (U.S. Patent No. 5,550,318; U.S. Patent No. 5,538,880; U.S. Patent No. 5,610,042; and PCT Application WO 94/09699; each of which is incorporated herein by reference). This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., 1987). There are a wide variety of microprojectile bombardment techniques known in the art, many of which are applicable to the invention.
- Microprojectile bombardment may be used to transform various cell(s), tissue(s) or organism(s), such as for example any plant species.
- species which have been transformed by microprojectile bombardment include monocot species such as maize (PCT Application WO 95/06128), barley (Ritala et al, 1994; Hensgens et al, 1993), wheat (U.S. Patent No.
- one or more particles may be coated with at least one nucleic acid and delivered into cells by a propelling force.
- Several devices for accelerating small particles have been developed.
- One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., 1990).
- the microprojectiles used have consisted of biologically inert substances such as tungsten or gold particles or beads.
- Exemplary particles include those comprised of tungsten, platinum, and preferably, gold. It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment. However, it is contemplated that particles may contain DNA rather than be coated with DNA.
- DNA-coated particles may increase the level of DNA delivery via particle bombardment but are not, in and of themselves, necessary.
- cells in suspension are concentrated on filters or solid culture medium.
- immature embryos or other target cells may be arranged on solid culture medium.
- the cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate.
- An illustrative embodiment of a method for delivering DNA into a cell (e.g., a plant cell) by acceleration is the Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with cells, such as for example, a monocot plant cells cultured in suspension.
- the screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectiles aggregate and may contribute to a higher frequency of transformation by reducing the damage inflicted on the recipient cells by projectiles that are too large.
- host cells are employed in generation of one or more gene therapy compositions.
- the terms "cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.
- “host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector.
- a host cell can, and has been, used as a recipient for vectors.
- a host cell may be "transfected” or “transformed,” which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
- a transformed cell includes the primary subject cell and its progeny.
- the terms “engineered” and “recombinant” cells or host cells are intended to refer to a cell into which an exogenous nucleic acid sequence, such as, for example, a vector, has been introduced. Therefore, recombinant cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced nucleic acid.
- RNAs or proteinaceous sequences may be co-expressed with other selected RNAs or proteinaceous sequences in the same host cell. Co-expression may be achieved by co-transfecting the host cell with two or more distinct recombinant vectors. Alternatively, a single recombinant vector may be constructed to include multiple distinct coding regions for RNAs, which could then be expressed in host cells transfected with the single vector.
- a tissue may comprise a host cell or cells to be transformed with a composition of the invention.
- the tissue may be part or separated from an organism.
- a tissue may comprise, but is not limited to, adipocytes, alveolar, ameloblasts, axon, basal cells, blood (e.g., lymphocytes), blood vessel, bone, bone marrow, brain, breast, cartilage, cervix, colon, cornea, embryonic, endometrium, endothelial, epithelial, esophagus, facia, fibroblast, follicular, ganglion cells, glial cells, goblet cells, kidney, liver, lung, lymph node, muscle, neuron, ovaries, pancreas, peripheral blood, prostate, skin, skin, small intestine, spleen, stem cells, stomach, testes, anthers, ascite tissue, cobs, ears, flowers, husks, kernels, leaves, meristematic cells, poll
- the host cell or tissue may be comprised in at least one organism.
- the organism may be, but is not limited to, a prokayote (e.g., a eubacteria, an archaea) or an eukaryote, as would be understood by one of ordinary skill in the art (see, for example, webpage http://phylogeny.arizona.edu/tree/phylogeny.html).
- a plasmid or cosmid for example, can be introduced into a prokaryote host cell for replication of many vectors.
- Cell types available for vector replication and/or expression include, but are not limited to, bacteria, such as E. coli (e.g., E. coli strain RR1, E. coli LE392, E. coli , E. coli X 1776 (ATCC No.
- E. coli W3110 F-, lambda-, prototrophic, ATCC No. 273325
- DH5cc DH5cc
- JM109 DH5cc
- JM109 DH5cc
- JM109 DH5cc
- JM109 DH5cc
- JM109 DH5cc
- JM109 DH5cc
- JM109 DH5cc
- JM109 DH5cc
- KC8 bacilli
- enterobacteriaceae such as Salmonella typhimurium, Serratia marcescens, various Pseudomonas specie
- SURE ® Competent Cells SOLOPACKTM Gold Cells
- ST ATAGENE ® La Jolla
- bacterial cells such as E. coli LE392 are particularly contemplated as host cells for phage viruses.
- Examples of eukaryotic host cells for replication and/or expression of a vector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC 12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.
- Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
- control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
- One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.
- compositions discussed above Numerous expression systems exist that comprise at least a part or all of the compositions discussed above.
- Prokaryote- and/or eukaryote -based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.
- the insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patent No. 5,871 ,986, 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC ® 2.0 from INVITROGEN ® and BACPACKTM BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH ® .
- expression systems include STRATAGENE ® ' S COMPLETE CONTROLTM Inducible Mammalian Expression System, which involves a synthetic ecdysone- inducible receptor, or its pET Expression System, an E. coli expression system.
- INVITROGEN ® Another example of an inducible expression system is available from INVITROGEN ® , which carries the T- PvEXTM (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter.
- INVITROGEN ® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica.
- a vector such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.
- proteins, polypeptides or peptides produced by the methods of the invention may be "overexpressed", i.e., expressed in increased levels relative to its natural expression in cells.
- overexpression may be assessed by a variety of methods, including radio-labeling and/or protein purification.
- simple and direct methods are preferred, for example, those involving SDS/PAGE and protein staining or western blotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot.
- a specific increase in the level of the recombinant protein, polypeptide or peptide in comparison to the level in natural cells is indicative of overexpression, as is a relative abundance of the specific protein, polypeptides or peptides in relation to the other proteins produced by the host cell and, e.g., visible on a gel.
- the expressed proteinaceous sequence forms an inclusion body in the host cell
- the host cells are lysed, for example, by disruption in a cell homogenizer, washed and/or centrifuged to separate the dense inclusion bodies and cell membranes from the soluble cell components. This centrifugation can be performed under conditions whereby the dense inclusion bodies are selectively enriched by incorporation of sugars, such as sucrose, into the buffer and centrifugation at a selective speed.
- Inclusion bodies may be solubilized in solutions containing high concentrations of urea (e.g.
- chao tropic agents such as guanidine hydrochloride
- reducing agents such as ⁇ -mercaptoethanol or DTT (dithiothreitol)
- refolded into a more desirable conformation as would be known to one of ordinary skill in the art.
- An individual in need of regeneration of chondrocytes or cartilage-type cells is the subject of one or more methods of the disclosure and/or exposed to one or more compositions of the disclosure.
- the individual may be at risk for being in need of regeneration of chondrocytes or cartilage type cells, or the individual may be diagnosed as needing regeneration of chondrocytes or cartilage type cells.
- An individual being at risk for needing regeneration of chondrocytes or cartilage type cells may be at risk for any reason, including by being or having been an athlete, by having a joint injury, obese individuals, those whose occupations or lifestyle require physical labor, including excessive lifting, or by being susceptible to having a medical condition that deleteriously affects joints or cartilage (such as with a family history or having one or more known markers and/or risk factors for susceptibility to the medical condition, for example), those who exercise for health reasons, or as a result of trauma.
- An individual known to need regeneration of chondrocytes or cartilage type cells includes an individual with a medical condition that deleteriously affects joints or cartilage. Exemplary medical conditions include intevertebral disc disease, chondrodystrophies, including osteoarthritis, achondroplasia, costochondritis, spinal disc herniation, and so forth.
- a joint of an individual in need of repair or prevention of repair is provided one or more gene therapy composition(s).
- the gene therapy composition(s) may be provided to the joint of the individual with or without a second component, such as a plurality of cells, for example, and/or reservoir (such as an absorbable reservoir) comprising oxygen and/or nutrients; one or more drugs may also be provided.
- a second component such as a plurality of cells, for example, and/or reservoir (such as an absorbable reservoir) comprising oxygen and/or nutrients; one or more drugs may also be provided.
- scaffolding laced with gene therapy and nutrients is provided.
- chondrocytes or chondrocyte-like cells or cartilage-type cells are generated in the respective joint, in some cases.
- cartilage tissue is regenerated, in some cases.
- the mechanism by which such results are produced from the gene therapy delivery may be from one or more of a variety of reasons, including 1) attraction of fibroblasts or other non-chondrocyte cells to a joint that then differentiate into chondrocytes or chondrocyte-like cells or cartilage-type cells; 2) differentiation of fibroblasts or other non-chondrocyte cells (including those residing in the joint) into chondrocytes or chondrocyte-like cells or cartilage-type cells; 3) a combination of one or more gene therapy composition(s) with one or more molecules from a chondrocyte that is dying or present in dying tissue or tissue in need of repair; and/or 4) the resulting production of advantageous or mechanically sufficient scar tissue or fibrous scar tissue.
- hWJCs Human Wharton's jelly cells
- hWJCs Human Wharton's jelly cells
- hWJCs were isolated from Wharton's jelly of human umbilical (Devarajan et al., 2013) cords collected with informed (KU-IRB #15402) following our previously published protocol.
- hWJCs were cultured in traditional hWJC medium (10% fetal bovine serum (FBS-MSC Qualified) and 1% Penicillin-Streptomycin in low glucose DMEM (Life Technologies, Carlsbad, CA)).
- hWJCs medium was changed three times per week, and hWJCs were maintained at 37°C with 5% C0 2 in a cell culture grade incubator.
- hWJCs were flash frozen at passage 2 (P2) until needed for experiments.
- hWJCs were thawed and expanded from P2 to P5, then used for experiments. All experiments were performed in triplicate for each cord.
- the Sox9 gene (NCBI GenBank ID: NC_000017.11) was synthesized by Blue Heron Biotech LLC. (Bothel, WA), and cloned into a Dendra2-C plasmid (Clontech, Mountain View, CA) at the carboxyl end of the Dendra2 sequence.
- Dendra2 is a green-to-red photo-convertible fluorescent protein.
- the Dendra2 plasmid contains a cytomegalovirus (CMV) promoter to drive transcription of Dendra2 and any sequence fused to Dednra2.
- the Dendra2 plasmid additionally contains a Simian virus 40 (SV40) promoter to drive transcription of a kanamycin resistance cassette for bacterial selection and a neomycin resistance cassette for eukaryotic selection.
- CMV cytomegalovirus
- SV40 Simian virus 40
- hWJCs were treated with ROCK Inhibitor and transfected via a 4D-Nucleofector using program FF-104 according to previous published protocol. (Mellott et al., 2014) hWJCs were transfected at a concentration of 5xl0 5 per reaction, and were transfected with 5 ⁇ g of pDNA.
- hWJCs were transferred to a 6-well plate (BD Biosciences, San Jose, CA) or NuncTM Lab-TekTM 8-well chambered coverglass slides (Thermo Scientific, Waltham, MA) pre-coated with Fibronectin (BD Biosciences) containing 1.5 mL or 0.5 mL, respectively, of pre-warmed 37°C traditional hWJC medium with 10 ⁇ of Y-27632 ROCK Inhibitor, and placed into a cell culture grade incubator at 37°C with 5% C0 2 .
- transfected cells and untreated controls were collected and harvested for gene expression analysis via real time quantitative polymerase chain reaction (RT-qPCR).
- RT-qPCR real time quantitative polymerase chain reaction
- Cells were analyzed for the chondrogenic genes, Sox9 and Collagen type II.
- Cycle threshold (Ct) values were recorded and analyzed via the Delta-Delta-Ct method. Values were normalized to day 0 untreated control samples and the endogenous controls. Three technical replicates from each umbilical cord were taken for gene expression analysis at 1 and 7 d post transfection.
- Live cell fluorescent imaging hWJCs were collected and stained with Hoechst 33342 dye (Life Technologies) for live cell imaging 24 h after transfection.
- hWJCs were imaged using a an Olympus ⁇ 81 inverted spinning disc confocal microscope base (Olympus America, Center Valley, PA). Images were captured using the acquisition and analysis software, SlideBook (Intelligent Imaging Innovations (3i), Denver, CO).
- a mercury arc lamp was used with the following excitation filters (Excitation/Emission) for image collection: Hoechst (387 + 11 nm/447 + 60 nm), GFP (494 + 20 nm/531 +22 nm), and RFP (575 + 25 nm/624 + 40 nm).
- a montage was generated from 49 (seven by seven arrangement) neighboring fields of view that were aligned together to generate one comprehensive composite image of the sample.
- transfected cells and controls were collected for immunocytochemistry.
- Cells were fixed by first washing cells in 37°C PBS, followed by fixation with 4% formaldehyde in PBS for 15 min. Cells were then washed and incubated for 5 min with PBS three times. Afterward, cells were permeabilized with 0.25% Triton X-100 in PBS, then washed three times in PBS for 5 min. Cells were blocked with 4% bovine serum albumin (BSA) and 10% normal serum (from secondary antibody host) in PBS for 60 min. Afterward, cells were incubated with Sox9 (Abeam, Cambridge, MA) and Collagen type II (Abeam) primary antibodies overnight.
- Sox9 Abeam, Cambridge, MA
- Collagen type II Abeam
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Application Number | Priority Date | Filing Date | Title |
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AU2014317861A AU2014317861B2 (en) | 2013-09-09 | 2014-09-09 | Gene therapy for the regeneration of chondrocytes or cartilage type cells |
EP14841529.2A EP3043825A4 (en) | 2013-09-09 | 2014-09-09 | Gene therapy for the regeneration of chondrocytes or cartilage type cells |
CN201480057130.7A CN105636614A (en) | 2013-09-09 | 2014-09-09 | Gene therapy for the regeneration of chondrocytes or cartilage type cells |
CA2923857A CA2923857A1 (en) | 2013-09-09 | 2014-09-09 | Gene therapy for the regeneration of chondrocytes or cartilage type cells |
JP2016542044A JP2016531147A (en) | 2013-09-09 | 2014-09-09 | Gene therapy for chondrocyte or chondrocyte regeneration |
US14/917,560 US11819555B2 (en) | 2013-09-09 | 2014-09-09 | Gene therapy for the regeneration of chondrocytes or cartilage type cells |
HK16112454.6A HK1224188A1 (en) | 2013-09-09 | 2016-10-28 | Gene therapy for the regeneration of chondrocytes or cartilage type cells |
AU2019268095A AU2019268095A1 (en) | 2013-09-09 | 2019-11-20 | Gene therapy for the regeneration of chondrocytes or cartilage type cells |
AU2021273570A AU2021273570A1 (en) | 2013-09-09 | 2021-11-24 | Gene therapy for the regeneration of chondrocytes or cartilage type cells |
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Cited By (8)
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JP2017123840A (en) * | 2016-01-07 | 2017-07-20 | 学校法人自治医科大学 | Adeno-associated virus vector for expression of glucose transporter 1 |
WO2017123951A1 (en) * | 2016-01-14 | 2017-07-20 | Spinacyte, Llc | Cellular blend for the regeneration of chondrocytes or cartilage type cells |
WO2018013013A1 (en) * | 2016-07-14 | 2018-01-18 | Limited Liability Company Biochemical Agent | Fusion protein, polynucleotide, genetic construct, producer, preparation for regeneration of cartilage (variants) |
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AU2014317861B2 (en) | 2019-11-28 |
EP3043825A1 (en) | 2016-07-20 |
CN105636614A (en) | 2016-06-01 |
US11819555B2 (en) | 2023-11-21 |
EP3043825A4 (en) | 2017-05-03 |
AU2019268095A1 (en) | 2019-12-19 |
AU2014317861A1 (en) | 2016-03-31 |
CA2923857A1 (en) | 2015-03-12 |
AU2021273570A1 (en) | 2022-01-06 |
US20160220699A1 (en) | 2016-08-04 |
JP2016531147A (en) | 2016-10-06 |
JP2020100661A (en) | 2020-07-02 |
HK1224188A1 (en) | 2017-08-18 |
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