WO1997038729A1 - In vivo gene transfer methods for wound healing - Google Patents
In vivo gene transfer methods for wound healing Download PDFInfo
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- WO1997038729A1 WO1997038729A1 PCT/US1997/007301 US9707301W WO9738729A1 WO 1997038729 A1 WO1997038729 A1 WO 1997038729A1 US 9707301 W US9707301 W US 9707301W WO 9738729 A1 WO9738729 A1 WO 9738729A1
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- A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
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- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
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- C07K14/575—Hormones
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
Definitions
- the present invention relates to a novel in vivo method 5 for the presentation and direct transfer of DNA encoding a therapeutic protein of interest into mammalian repair cells.
- the method involves implanting a matrix containing DNA of interest (referred to herein as a "gene activated matrix") into a fresh wound site.
- Repair cells which normally 10 originate in viable tissue surrounding the wound, proliferate and migrate into the gene activated matrix, wherein they encounter, take up and express the DNA.
- Transfected repair cells therefore act, as in situ bioreactors (localized within the wound site) which produce agents (DNA-encoded 15 RNAs, proteins, etc.) that heal the wound.
- the invention further relates to pharmaceutical compositions that may be used in the practice of the invention to transfer the DNA of interest.
- Such compositions include any suitable matrix in combination with the DNA of 20 interest.
- WOUND HEALING Currently available wound healing therapies involve the 25 administration of therapeutic proteins. Such therapeutic proteins may include regulatory factors involved in the normal healing process such as systemic hormones, cytokines, growth factors and other proteins that regulate proliferation and differentiation of cells. Growth factors, cytokines and 30 hormones reported to have such wound healing capacity include, for example, the transforming growth factor-/?
- TGF-0 TGF-0
- FGF acidic fibroblast growth factor
- M-CSF macrophage-colony stimulating factor
- PTH parathyroid hormone
- cytokines systemic delivery of therapeutic proteins, i.e. cytokines, can be associated with serious unwanted side effects in unwounded tissue. Due to inefficient delivery to specific cells and tissues in the body, administration of high doses of protein are required to ensure that sufficient amounts of the protein reach the appropriate tissue target. Because of the short half life in the body due to proteolytic degradation, the proteins must also be administered repeatedly which may give rise to an immune reaction to the therapeutic proteins. The circulation of high doses of therapeutic proteins is often toxic due to pleiotropic effects of the administered protein, and may give rise to serious side effects.
- ex vivo gene therapy e.g.. target cells are removed from the body, transfected or infected with vectors carrying recombinant genes, and re-implanted into the body
- Viral vectors are regarded as the most efficient system, and recombinant replication-defective viral vectors have been used to transduce (i.e. , infect) cells both ex vivo and in vivo .
- Such vectors have included retroviral, adenovirus and adeno-associated and herpes viral vectors.
- the present invention relates to a novel method for specific targeting and transfer of DNA into mammalian repair cells involved in wound healing in order to express therapeutic products at the wound site.
- the method of the invention involves administering a gene activated matrix into a fresh wound site in the body. In this setting, repair cells are localized to the wound site, where they become transfected and eventually produce DNA-encoded agents (RNAs, proteins, etc.) that enhance wound healing.
- the invention is based, in part, on the discovery that repair cells, active in the wound healing process, proliferate and migrate from surrounding tissue into the area of the wound and infiltrate the gene activated matrix.
- the matrix acts as a scaffolding that promotes cell ingrowth, and, in turn, gene transfer, through the local accumulation of repair cells near the DNA. While in the matrix, repair cells are surprisingly efficient at taking up the DNA and expressing it as translational products, i.e. , proteins, or transcriptional products, i.e.. antisense and ribozymes.
- the transfected repair cells then serve as local bioreactors amplifying the production of the gene product in vivo .
- DNA sequences While any number of DNA sequences can be used in the method, preferred DNA sequences are those that encode translational products (i.e. proteins) or transcriptional products (i.e. antisense or ribozymes) that (a) promote tissue repair; or (b) are capable of disrupting a disease process (thereby allowing normal tissue healing to take place) .
- the invention overcomes the shortcomings of procedures currently used for wound healing involving the administration of therapeutic proteins. First, DNA, which is both stable and non-toxic, can be safely administered in high doses in vivo . Second, repeated administration, while possible, is not required. The cells which take up and express the DNA provide a supply of gene product at the site of the wound. Third, the invention could be practiced in a way that addresses the temporal requirements of dosing.
- the DNA can be presented in vectors that integrate into the genome of the targeted cell.
- all daughter cells will contain and express the transferred DNA thereby acting as a continuous source for the therapeutic agent.
- non-integrating systems may be utilized wherein the DNA does not integrate into the genome and the gene is not passed on to daughter cells. In such an instance, when the wound healing process is completed and the gene product is no longer needed, the gene product will not be expressed.
- the invention is demonstrated by way of examples, which show that genes can be reproducibly transferred and expressed in a variety of wounded soft and hard tissues in vivo .
- the method of the invention overcomes the problems associated with currently available gene therapy protocols.
- the method of the invention provides gene transfer to a suitable number of repair cells to achieve functional effects, i.e. , in the absence of any further targeting or cellular identification by the practitioner.
- In vivo methods of gene therapy require some form of targeting which very often does not work.
- targeting is not a problem.
- the DNA acts much like "bait" in a "trap”: the DNA is encountered by unwitting repair cells that have proliferated and then migrated into the gene activated matrix. These cells, in turn, are surprisingly capable of taking up DNA and expressing it as a therapeutic agent.
- the method of the invention may be used as a drug delivery system through transfer of DNA into mammalian repair cells for the purpose of stimulating soft and hard tissue repair and tissue regeneration.
- the repair cells will be those cells that normally arrive at the area of the wound o be treated. Accordingly, there is no difficulty associated with the obtaining of suitable target cells to which the present therapeutic compositions should be applied. All that is required is the implantation of a gene activated matrix at the wound site. The nature of this biological environment is such that the appropriate repair cells will actively take up and express the "bait" DNA in the absence of any further targeting or cellular identification by the practitioner.
- the method of the invention may be used to transfer DNA into mammalian repair cells to stimulate skeletal regeneration.
- the method of the invention, using both biological and synthetic matrices may be used to transfer DNA into mammalian cells to stimulate ligament and tendon repair.
- the method of the invention may further be employed, using both biological and synthetic matrices to transfer DNA into mammalian repair cells to stimulate skeletal muscle repair and/or blood vessel repair.
- the DNA to be used in the practice of the invention may include any DNA encoding translational products (i.e. proteins) or transcriptional products (i.e. antisense or ribozymes) that promote tissue repair or are capable of disrupting a disease process.
- the DNA may comprise genes encoding therapeutically useful proteins such as growth factors, cytokines, hormones, etc.
- the DNA may encode anti-sense or ribozyme molecules that may inhibit the translation of mRNAs encoding proteins that inhibit wound healing or which induce inflammation.
- the DNA encoding the therapeutic product of interest is associated with, or impregnated within, a matrix to form a gene activated matrix. Once prepared, the gene activated matrix is placed within the mammal at the site of a wound.
- the invention is demonstrated by way of examples, wherein the efficient in vivo transfer and expression of genes into tissue undergoing repair and regeneration is demonstrated.
- a gene activated matrix is defined herein as any matrix material containing DNA encoding a therapeutic agent of interest.
- gene activated matrices are placed within wound sites in the body of a mammalian host to enhance wound healing.
- a repair cell is defined herein as any cell which is stimulated to migrate and proliferate in response to tissue injury.
- Repair cells are a component of the wound healing response.
- Such cells include fibrobiasts, capillary endothelial cells, capillary pericytes, mononuclear inflammatory cells, segmented inflammatory cells and granulation tissue cells.
- a wound site is defined as any location in the host that arises from traumatic tissue injury, or alternatively, from tissue damage either induced by, or resulting from, surgical procedures.
- FIG. IA Femoral Osteotomy Model of Fibrous Nonunion.
- a plain x-ray film showing a control rat femur immediately after surgery. The gap was stabilized by an external fixator consisting of a plate and 4 pins.
- FIG. IB A plain x-ray film showing a control rat femur osteotomy 9 weeks after surgery. Rounded surgical margins (arrows) are due to a reactive bone fo r mation and are consistent with the classical appearance of nonunion fracture.
- FIG. IC Histology section of gap tissue 3 weeks post- surgery showing proliferating repair fibrobiasts and capillaries embedded in an edematous extracellular matrix. Also present is a focal inflammatory infiltrate consisting of lymphocytes and macrophages.
- FIG. 2. Schematic diagram, of the pGAMl construct encoding mouse BMP-4. The position of the CMV promoter, BMP- 4 coding sequence, HA epitope, and bovine growth hormone polyadenylation signal are shown.
- FIG. 3A BMP-4 expression by repair fibrobiasts. Plasmid-encoded BMP-4 expression was detected in Bouins- fixed, demineralized, paraffin-embedded tissue sections using the anti-HA antibody and immunoperoxide method 4 weeks post- implantation of a gene activated matrix containing pGAMl plasmid DNA. Arrows point to examples of positive (red- brown) staining of fibroblast cytoplasm (micrograph on upper left) . These cells were identified as fibrobiasts based on spindled morphology, growth in fascicle, and positive immunostaining for type I procollagen (not shown) . Serial sections incubated with rabbit pre-immune serum or without the first antibody were negative.
- FIG. 3B Plain film radiographs of the animal (23 weeks post-operatively) .
- the arrow indicates the approximate position of the osteotomy gap, which is filled with radio-dense tissue. Note that the external fixator has been removed. As indicated by the variegated pattern, bone remodeling is taking place. Arrowheads point to defects in bone adjacent to the gap (a consequence of pin placement) . The two distal pin sites were completely healed at this time (not shown) .
- the whole mount photograph presents a Gomori trichrome-stained tissue section from the gap of the animal shown (following sacrifice) .
- Circular defects in marrow space result from placement of innermost fixator pins. Tissue disruption at bottom of micrograph is an artifact of specimen handling.
- FIG. 4A Schematic diagram of the pGAM2 construct encoding human PTH1-34. The position of an upstream long terminal repeat that drives PTH1-34 expression (arrow) , the PTH1-34 coding sequence, the SV40 promoter that drives neo expression (arrow) , the neo coding sequence, pBR sequences, and the downstream long terminal repeat are shown.
- FIG. 4B PTH1-34 gene transfer and expression drives new bone formation in vivo . Plain film radiograph showing new bone bridging of a 5 mm osteotomy gap 9 weeks post- implantation in an animal that received a gene activated matrix containing pGAM2 plasmid DNA. Arrows point to radio- dense tissue in the gap. Results shown here are representative of experiments with one additional animal.
- FIG. 5 New bone formation in vivo via a two-plasmid GAM.
- top Plain film radiograph showing new bone bridging of a 5mm gap 4 weeks post-implantation in an animal that received a gene activated matrix containing pGAMl plus pGAM2 plasmid DNA. Arrows point to radio-dense tissue in the gap (confirmed histologically to be bone) .
- bottom Plain film radiograph of the gap shown in photo at top following removal (5 weeks earlier; total of 17 weeks post-surgery) of the external fixator. Arrows indicate location of the gap, which is filled with radio-dense tissue except for a strip of undermineralized tissue near the proximal surgical margin.
- FIG. 6 Adenovirus-mediated Gene Transfer into Bone Repair/Regeneration Cells in vivo.
- the UltraFiberTM implant was soaked for 6 min. in a solution of the AdCMVlacZ virus (10 lo -10 n plaque forming units or PFU/ml) and then implanted into the osteotomy site.
- the defect was allowed to heal for 3 weeks, during which time the progress of the wound healing response was monitored by weekly radiographic examination. By three weeks, it was estimated that 40% of the defect was filled with callus tissue.
- the mammalian host was sacrificed and tissues were fixed in Bouins fixation and then demineralized for 7 days using standard formic acid solutions.
- Photomicrographs were taken from transverse sections of new bone (callus) that formed in the osteotomy site 3 weeks after surgery.
- Panel at top left Note the positive (red) jS-gal cytoplasmic staining of callus tissue cells from the UltraFiberTM adenovirus implant. This result indicates that cell surface receptors that mediate infection, and thus viral transduction, are expressed by (at least one population) callus cells during the fracture healing process.
- Panel at top left serial section negative control stained with the vehicle of the ⁇ -gal antibody plus cocktail of non- specific rabbit LgG antibodies.
- Panel at bottom note the positive (red) j ⁇ -gal nuclear staining of chondrocytes in the osteotomy site filled with UltraFiberTM and AdRSVntlacZ. This result demonstrates theaki specificity of the anti-/3- gal antibody, and conclusively demonstrates expression of the marker gene product in the osteotomy gap.
- FIG. 7 pGAM2 plasmid gene transfer to repair fibrobiasts results in new bone growth in the rat osteotomy model.
- Plain film radiograph showing new bone bridging of a 5 mm gap 6 weeks post-implantation in an animal that received a gene activated matrix containing pGAMl plus pGAM2 plasmid DNA. Arrows point to radio-dense tissue in the gap (confirmed histologically to be bone) . 5.
- DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an in vivo method for presentation and transfer of DNA into mammalian repair cells for the purpose of expressing therapeutic agents.
- the method of the invention involves implanting or placing gene activated matrices into a fresh wound site.
- Wound healing is usually a coordinated, stereotyped sequence of events that includes (a) tissue disruption and loss of normal tissue architecture; (b) cell necrosis and hemorrhage; hemostasis (clot formation) ; (c) infiltration of segmented and mononuclear inflammatory cells, with vascular congestion and tissue edema; (d) dissolution of the clot as well as damaged cells and tissues by mononuclear cells (macrophages) (e) formation of granulation tissue (fibroplasia and angiogenesis) .
- This sequence of cellular events has been observed in wounds from all tissues and organs generated in a large number of mammalian species (Gailet et al., 1994, Curr. Opin. Cell. Biol.
- the cellular sequence described above is a universal aspect of the repair of all mammalian tissues.
- the invention is based on the discovery that repair cells involved in the wound healing process will naturally proliferate and migrate to the site of tissue injury and infiltrate the gene activated matrix. Surprisingly, these repair cells, which are normally difficult to efficiently transfect, either in vitro or in vivo, are extremely efficient at taking up and expressing DNA when activated to proliferate by the wound healing process.
- the methods of the present invention are designed to efficiently transfer, one or more DNA molecules encoding therapeutic agents to the proliferating repair eel .
- the methods involve the administration of a gene activated matrix containing DNA encoding translational products (i.e. therapeutic proteins) or transcriptional products (i.e. antisense or ribozymes) within a mammalian host at the site of a wound.
- the wound may arise from traumatic tissue injury, or alternatively, from tissue damage either induced by, or resulting from, surgical procedures.
- the proliferating repair cells migrate into and contact a gene activated matrix, they take up and express the DNA of interest thereby amplifying the amount of the therapeutic agent, protein or RNA.
- the transfected repair cells thereby serve as local bioreactors producing therapeutic agents that influence the local repair environment. For example, growth factors or cytokines produced by the transfected repair cells, will bind and stimulate targeted effector cells that express cognate cell surface receptors, thereby stimulating and amplifying the cascade of physiological events normally associated with the wound healing process.
- the repair cells may take up and express DNA encoding proteins that inhibit the activity of antagonists of the wound healing process.
- the DNA may also encode antisense or ribozyme RNA molecules that may be used to inhibit translation of mRNAs encoding inflammatory proteins or other factors that inhibit wound healing or cause excessive fibrosis.
- the gene activated matrix of the invention can be transferred to the patient using a variety of techniques. For example, when stimulating wound healing and regeneration, the matrices are transferred directly to the site of the wound, i.e.. the fractured bone, injured connective tissue, etc. For use in skin repair, the matrices will be topically administered. For use in organ regeneration, the matrices will be surgically placed in a wound made in the organ. Since the method of the invention is based on the natural migration and proliferation of repair cells into a wound site, and infiltration into the gene activated matrix located at the wound site, followed by the uptake of DNA, it is understood that the matrices must be transferred into a site in the body where the wound healing process has been induced.
- the repair process may be engineered to result in either the formation of scar tissue and/or tissue regeneration.
- the overexpression of the therapeutic proteins at the site of the wound may result in regeneration of the injured tissue without the formation of scar tissue.
- such regeneration is desirable because scar tissue is not optimally designed to support normal mechanical function.
- around a suture it may be desirable to form scar tissue to hold inherently weak tissue together.
- the methods of invention may be used to stimulate wound healing either with, or without, the formation of scar tissue depending on the type and level of therapeutic protein expressed.
- Direct plasmid DNA transfer from a matrix to a mammalian repair cell, through stimulation of the wound healing process, offers a number of advantages.
- direct gene transfer may be an advantageous method of drug delivery for molecules that normally undergo complex biosynthetic processing or for receptors which must be properly positioned in the cellular membrane. These types of molecules would fail to work if exogenously delivered to cells.
- the present invention also relates to pharmaceutical compositions comprising matrices containing DNA for use in wound healing.
- the compositions of the invention are generally comprised of a biocompatible, or bone compatible, matrix material containing DNA encoding a therapeutic protein of interest.
- the invention overcomes shortcomings specifically associated with current recombinant protein therapies for wound healing applications.
- direct gene transfer is a rational strategy that allows transfected cells to (a) make physiological amounts of therapeutic protein, modified in a tissue- and context-specific manner, and (b) deliver this protein to the appropriate cell surface signaling receptor 5 under the appropriate circumstances. For reasons described above, exogenous delivery of such molecules is expected to be associated with significant dosing and delivery problems.
- Second, repeated administration, while possible, is not required with gene activated matrix technology: cell uptake
- DNA can be controlled precisely with well-established sustained release delivery technologies, or, alternatively, integration of transfected DNA can be associated with long term recombinant protein expression.
- the method of the invention can be universally applied
- biocompatible matrix material containing DNA encoding a therapeutic agent of interest such as a translational product, i.e. therapeutic proteins, or transcriptional products, i.e. antisense or ribozymes, can be formulated and used in accordance with the invention.
- the gene activated matrices of the invention may be derived from any biocompatible material. Such materials may include, but are not limited to, biodegradable or non- biodegradable materials formulated into scaffolds that support cell attachment and growth, powders or gels. Matrices may be derived from synthetic polymers or naturally occurring proteins such as collagen, other extracellular matrix proteins, or other structural macromolecules.
- the DNA incorporated into the matrix may encode any of a variety of therapeutic proteins depending on the envisioned therapeutic use. Such proteins may include growth factors, cytokines, hormones or any other proteins capable of regulating the growth, differentiation or physiological function of cells.
- the DNA may also encode antisense or ribozyme molecules which inhibit the translation of proteins that inhibit wound repair and/or induce inflammation.
- the transferred DNA need not be integrated into the genome of the target cell; indeed, the use of non-integrating DNA in the gene activated matrix is the preferred embodiment of the present invention. In this way, when the wound healing process is completed and the gene product is no longer needed, the gene product will not be expressed.
- kits containing a biocompatible matrix and DNA form another aspect of the invention.
- the kits will contain preformed gene activated matrices thereby allowing the physician, to directly administer the matrix within the body.
- the kits may contain the components necessary for formation of a gene activated matrix.
- the physician may combine the components to form the gene activated matrices which may then be used therapeutically by placement within the body.
- the matrices may be used to coat surgical devices such as suture materials or implants.
- gene activated matrices may include ready to use sponges, tubes, band-aids, lyophilized components, gels, patches or powders and telfa pads.
- compositions are prepared in which the DNA encoding the therapeutic agent of interest is associated with or impregnated within a matrix to form a gene activated matrix.
- the matrix compositions function (i) to facilitate ingrowth of repair cells (targeting) ; and (ii) to harbor DNA (delivery) .
- the type of matrix that may be used in the compositions, devices and methods of the invention is virtually limitless and may include both biological and synthetic matrices.
- the matrix will have all the features commonly associated with being “biocompatible", in that it is in a form that does not produce an adverse, allergic or other untoward reaction when administered to a mammalian host.
- Such matrices may be formed from both natural or synthetic materials.
- the matrices may be non-biodegradable in instances where it is desirable to leave permanent structures in the body; or biodegradable where the expression of the therapeutic protein is required only for a short duration of time.
- the matrices may take the form of sponges, implants, tubes, telfa pads, band-aids, bandages, pads, lyophilized components, gels, patches, powders or nanoparticles.
- matrices can be designed to allow for sustained release of the DNA over prolonged periods of time.
- matrix material will differ according to the particular circumstances and the site of the wound that is to be treated. Matrices such as those described in U.S. Patent 5,270,300, incorporated herein by reference, may be employed. Physical and chemical characteristics, such as, e.g. t biocompatibility, biodegradability, strength, rigidity, interface properties and even cosmetic appearance may be considered in choosing a matrix, as is well known to those of skill in the art. Appropriate matrices will both deliver the DNA molecule and also act as an in situ scaffolding through which mammalian repair cells may migrate.
- non-biodegradable matrices may be employed, such as sintered hydroxyapatite, bioglass, aluminates, other bioceramic materials and metal materials, particularly titanium.
- a suitable ceramic delivery system is that described in U.S. Patent 4,596,574, incorporated herein by reference.
- the bioceramics may be altered in composition, such as in calcium-aluminate-phosphate; and they may be processed to modify particular physical and chemical characteristics, such as pore size, particle size, particle shape, and biodegradability.
- Polymeric matrices may also be employed, including acrylic ester polymers and lactic acid polymers, as disclosed in U.S.
- Particular examples of useful polymers are those of orthoesters, anhydrides, propylene-cofumarates, or a polymer of one or more 7-hydroxy carboxylic acid monomers, e.g.. 7-hydroxy auric acid (glycolic acid) and/or 7-hydroxy propionic acid (lactic acid) .
- a particularly important aspect of the present invention is its use in connection with orthopaedic implants and interfaces and artificial joints, including implants themselves and functional parts of an implant, such as, e.g. , surgical screws, pins, and the like.
- an implant or a portion thereof such as a titanium surface
- the metal surface or surfaces of an implant or a portion thereof will be coated with a material that has an affinity for nucleic acids, most preferably, with hydroxyl apatite, and then the coated-metal will be further coated with the gene or nucleic acid that one wishes to transfer.
- biodegradable matrix is generally defined as one that is capable of being reabsorbed into the body.
- Potential biodegradable matrices for use in connection with the compositions, devices and methods of this invention include, for example, biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polyactic acid, polyanhidrides, matrices of purified proteins, and semi- purified extracellular matrix compositions.
- biocompatible biodegradable polymers that may be used are well known in the art and include, by way of example and not limitation, polyesters such as polyglycolides, polylactides and polylactic polyglycolic acid copolymers (“PLGA”) (Langer and Folkman, 1976, Nature 263:797-800); polyethers such as polycaprolactone (“PCL”) ; polyanhydrides; polyalkyl cyanoacrylates such as n-butyl cyanoacrylate and isopropyl cyanoacrylate; polyacrylamides; poly(orthoesters) ; polyphosphazenes; polypeptides; polyurethanes; and mixtures of such polymers. It is to be understood that virtually any polymer that is now known or that will be later developed suitable for the sustained or controlled release of nucleic acids may be employed in the present invention.
- the biocompatible biodegradable polymer is a copolymer of glycolic acid and lactic acid (“PLGA”) having a proportion between the lactic acid/glycolic acid units ranging from about 100/0 to about 25/75.
- the average molecular weight (“MW") of the polymer will typically range from about 6,000 to 700,000 and preferably from about 30,000 to 120,000, as determined by gel-permeation chromatography using commercially available polystyrene of standard molecular weight, and have an intrinsic viscosity ranging from 0.5 to 10.5.
- the length of the period of continuous sustained or controlled release of nucleic acids from the matrix according to the invention will depend in large part on the MW of the polymer and the composition ratiQ of lactic acid/glycolic acid. Generally, a higher ratio of lactic acid/glycolic acid, such as for example 75/25, will provide for a longer period of controlled of sustained release of the nucleic acids, whereas a lower ratio of lactic acid/glycolic acid will provide for more rapid release of the nucleic acids.
- the lactic acid/glycolic acid ratio is 50/50.
- the length of period of sustained or controlled release is also dependent on the MW of the polymer. Generally, a higher MW polymer will provide for a longer period of controlled or sustained release. In the case of preparing, for example, matrices providing controlled or sustained release for about three months, when the composition ratio of lactic acid/glycolic acid is 100/0, the preferable average MW of polymer ranges from about 7,000 to 25,000; when 90/10, from about 6,000 to 30,000; and when 80/20, from about 12,000 to 30,000.
- SIS small intestinal submucosa
- the SIS graft material may be prepared from a segment of jejunum of adult pigs. Isolation of tissue samples may be carried out using routine tissue culture techniques such as those described in Badybak et al., 1989, J. Surg. Res. 47:74-80. SIS material is prepared by removal of mesenteric tissue, inversion of the segment, followed by removal of the mucosa and superficial submucosa by a mechanical abrasion technique. After returning the segment to its original orientation, the serosa and muscle layers are rinsed and stored for further use.
- Matrices may also be prepared from tendon or dermal collagen, as may be obtained from a variety of commercial sources, such as, e.g. , Sigma and Collagen Corporation. Collagen matrices may also be prepared as described in U.S. Patents 4,394,370 and 4,975,527, each incorporated herein by reference..
- lattices made of collagen and glycosaminoglycan such as that described in Yannas & Burke, U.S. Patent 4,505,266, may be used in the practice of the invention.
- the collagen/GAG matrix may effectively serve as a support or "scaffolding" structure into which repair cells may migrate.
- Collagen matrix such as those disclosed in Bell, U.S. Patent No. 4,485,097, may also be used as a matrix material.
- the various collagenous materials may also be in the form of mineralized collagen.
- the fibrous collagen implant material termed UltraFiberTM as may be obtained from Norian Corp., (1025 Terra Bella Ave., Mountain View, CA, 94043) may be used for formation of matrices.
- UltraFiberTM the fibrous collagen implant material termed UltraFiberTM, as may be obtained from Norian Corp., (1025 Terra Bella Ave., Mountain View, CA, 94043) may be used for formation of matrices.
- U.S. Patent 5,231,169 incorporated herein by reference, describes the preparation of mineralized collagen through the formation of calcium phosphate mineral under mild agitation in situ in the presence of dispersed collagen fibrils. Such a formulation may be employed in the context of delivering a nucleic acid segment to a bone tissue site.
- Mineralized collagen may be employed, for example, as part of gene activated matrix therapeutic kit for fracture repair.
- collagen may be purified from hyaline cartilage, as isolated from diarthrodial joints or growth plates.
- Type II collagen purified from hyaline cartilage is commercially available and may be purchased from, e.g., Sigma Chemical Company, St. Louis.
- Type I collagen from rat tail tendon may be purchased from, e.g., Collagen Corporation.
- Any form of recombinant collagen may also be employed, as may be obtained from a collagen-expressing recombinant host cell, including bacterial yeast, mammalian, and insect cells.
- collagen it may be advantageous to remove what is referred to as the "telopeptide" which is located at the end of the collagen molecule and known to induce an inflammatory response.
- the collagen used in the invention may, if desired be supplemented with additional minerals, such as calcium, e.g., in the form of calcium phosphate.
- additional minerals such as calcium, e.g., in the form of calcium phosphate.
- Both native and recombinant type collagen may be supplemented by admixing, absorbing, or otherwise associating with, additional minerals in this manner.
- the present methods and compositions may employ a variety of different types of DNA molecules.
- the DNA molecules may include genomic, cDNAs, single stranded DNA, double stranded DNA, triple stranded DNA, oligonucleotides and Z-DNA.
- the DNA molecules may code for a variety of factors that promote wound healing including extracellular, cell surface, and intracellular RNAs and proteins. Examples of extracellular proteins include growth factors, cytokines therapeutic proteins, hormones and peptide fragments of hormones, inhibitors of cytokines, peptide growth and differentiation factors, interleukins, chemokines, interferons, colony stimulating factors and angiogenic factors.
- TGF-/3 molecules include the five TGF-/S isoforms and bone rnorphogenetic proteins (BMP) , latent TGF-/3 binding proteins, LTBP; keratinocyte growth factor (KGF) ; hepatocyte growth factor (HGF) ; platelet derived growth factor (PDGF) ; insulin-like growth factor (IGF); the basic fibroblast growth factors (FGF-l, FGF-2 etc.), vascular endothelial growth factor (VEGF); Factor VIII and Factor IX; erythropoietin (EPO) ; tissue plasminogen activator (TPA) ; activins and inhibins.
- BMP bone rnorphogenetic proteins
- Hormones which may be used in the practice of the invention include growth hormone (GH) and parathyroid hormone (PTH) .
- extracellular proteins also include the extracellular matrix proteins such as collagen, laminin, and fibronectin.
- cell surface proteins include the family of cell adhesion molecules (e.g., the integrins, selectins, lg family members such as N-CAM and Ll, and cadherin ⁇ ) ; cytokine signaling receptors such as the type I and type II TGF-jS receptors and the FGF receptor; and non-signaling co- receptors such as betaglycan and syndecan.
- RNAs and proteins examples include the family of signal transducing kinases, cytoskeletal proteins such as talin and vinculin, cytokine binding proteins such as the family of latent TGF-/3 binding proteins, and nuclear trans acting proteins such as transcription factors and enhancing factors.
- the DNA molecules may also code for proteins that block pathological processes, thereby allowing the natural wound healing process to occur unimpeded.
- blocking factors include ribozymes that destroy RNA function and DNAs that, for example, code for tissue inhibitors of enzymes that destroy tissue integrity, e.g.. inhibitors of metalloproteinases associated with arthritis.
- DNA segment encoding the protein of interest may be obtained using a variety of molecular biological techniques, generally known to those skilled in the art. For example, cDNA or genomic libraries may be screened using primers or probes with sequences based on the known nucleotide sequences. Polymerase chain reaction (PCR) may also be used to generate the DNA fragment encoding the protein of interest. Alternatively, the DNA fragment may be obtained from a commercial source.
- PCR Polymerase chain reaction
- Genes with sequences that vary from those described in the literature are also encompassed by the invention, so long as the altered or modified gene still encodes a protein that functions to stimulate wound healing in any direct or indirect manner.
- sequences include those caused by point mutations, those due to the degeneracies of the genetic code or naturally occurring allelic variants, and further modifications that have been introduced by genetic engineering, i.e., by the hand of man.
- the DNA encoding the translational or transcriptional products of interest may be recombinantly engineered into variety of vector systems that provide for replication of the DNA in large scale for the preparation of gene activated matrices. These vectors can be designed to contain the necessary elements for directing the transcription and/or translation of the DNA sequence taken up by the repair cells at the wound in vivo .
- Vectors that may be used include, but are not limited to those derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA.
- plasmid vectors such as pBR322, pUC 19/18, pUC 118, 119 and the M13 mp series of vectors may be used.
- Bacteriophage vectors may include ⁇ gtlO, ⁇ gtll, ⁇ gtl8-23, ⁇ ZAP/R and the EMBL series of bacteriophage vectors.
- Cosmid vectors that may be utilized include, but are not limited to, pJB8, pCV 103, pCV 107, pCV 108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16 and the charomid 9 series of vectors.
- Vectors that allow for the in vitro transcription of RNA such as SP6 vectors, may also be used to produce large quantities of RNA that may be incorporated into matrices.
- recombinant virus vectors including, but not limited to those derived from viruses such as herpes virus, retroviruses, vaccinia viruses, adenoviruses, adeno-associated viruses or bovine papilloma virus may be engineered. While integrating vectors may be used, non-integrating systems, which do not transmit the gene product to daughter cells for many generations are preferred for wound healing. In this way, the gene product is expressed during the wound healing process, and as the gene is diluted out in progeny generations, the amount of expressed gene product is diminished. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the protein coding sequence operatively associated with appropriate transcriptional/translational control signals.
- the genes encoding the proteins of interest may be operatively associated with a variety of different promoter/enhancer elements.
- the expression elements of these vectors may vary in their strength and specificities. Depending on the host/vector system utilized, any one of a number of suitable transcription and translation elements may be used.
- the promoter may be in the form of the promoter which is naturally associated with the gene of interest.
- the DNA may be positioned under the control of a recombinant or heterologous promoter, i.e. , a promoter that is not normally associated with that gene.
- tissue specific promoter/enhancer elements may be used to regulate the expression of the transferred DNA in specific cell types.
- transcriptional control regions that exhibit tissue specificity include but are not limited to: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:42S-51S); insulin gene control region which is active in pancreatic beta cells (Hanahan,
- beta-globin gene control region which is active in myeloid cells (Magram et al., 1985, Nature 315:338- 340; Kollias et al., 1986, Cell 46:89-94); myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al. , 1987, Cell 48:703-712) ; myosin light chain-2 gene control region which is active in skeletal muscle (Shani, 1985, Nature
- Promoters isolated from the genome of viruses that grow in mammalian cells may be used, as well as promoters produced by recombinant DNA or synthetic techniques.
- the promoter elements may be constitutive or inducible promoters and can be used under the appropriate conditions to direct high level or regulated expression of the gene of interest.
- Expression of genes under the control of constitutive promoters does not require the presence of a specific substrate to induce gene expression and will occur under all conditions of cell growth.
- expression of genes controlled by inducible promoters is responsive to the presence or absence of an inducing agent.
- Specific initiation signals are also required for sufficient translation of inserted protein coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where the entire coding sequence, including the initiation codon and adjacent sequences are inserted into the appropriate expression vectors, no additional translational control signals may be needed. However, in cases where only a portion of the coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon must be provided.
- initiation codon must be in phase with the reading frame of the protein coding sequences to ensure translation of the entire insert.
- exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency and control of expression may be enhanced by the inclusion of transcription attenuation sequences, enhancer elements, etc.
- ribozymes or antisense DNA molecules that may be transferred into the mammalian repair cells.
- ribozymes and antisense molecules may be used to inhibit the translation of RNA encoding proteins of genes that inhibit a disease process or the wound healing process thereby allowing tissue repair to take place.
- RNA molecules will act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation.
- ribozymes which are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA may also be used to block protein translation.
- the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage.
- engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences.
- RNA molecules may be generated by transcription of DNA sequences encoding the RNA molecule.
- genes combined on a single genetic construct under control of one or more promoters,, or prepared as separate constructs of the same or different types may be used.
- an almost endless combination of different genes and genetic constructs may be employed.
- Certain gene combinations may be designed to, or their use may otherwise result in, achieving synergistic effects on cell stimulation and regeneration, any and all such combinations are intended to fall within the scope of the present invention. Indeed, many synergistic effects have been described in the scientific literature, so that one of ordinary sill in the art would readily be able to identify likely synergistic gene combinations, or even gene- protein combinations.
- matrix or implant material is contacted with the DNA encoding a therapeutic product of interest by soaking the matrix material in a recombinant DNA stock solution.
- the amount of DNA, and the amount of contact time required for incorporation of the DNA into the matrix will depend on the type of matrix used and can be readily determined by one of ordinary skill in the art without undue experimentation.
- the DNA may be encapsulated within a matrix of synthetic polymers, such as, for example, block copolymers of polyactic-polyglycolic acid (See Langer and Folkman, 1976 Nature, 263:797-800 which is incorporated herein by reference) .
- the amount of DNA construct that is applied to the matrix will be determined considering various biological and medical factors. One would take into consideration the particular gene, the matrix, the site of the wound, the mammalian host's age, sex and diet and any further clinical factors that may effect wound healing such as the serum levels of various factors and hormones.
- compositions of both biological and synthetic matrices and DNA may be lyophilized together to form a dry pharmaceutical powder.
- the gene activated matrix may be,rehydrated prior to implantation in the body, or alternatively, the gene activated matrix may become naturally rehydrated when placed in the body.
- medical devices such as implants, sutures, wound dressings, etc. may be coated with the nucleic acid compositions of the invention using conventional coating techniques as are well known in the art. Such methods include, by way of example and not limitation, dipping the device in the nucleic acid composition, brushing the device with the nucleic acid composition and/or spraying the device with the aerosol nucleic acid compositions of the invention. The devide is then dried, either at room temperature or with the aid of a drying oven, optionally at reduced pressure. A preferred method for coating sutures is provided in the examples.
- a coating composition containing a total of about .01 to lOmg plasmid DNA and preferably about 1 to 5 mg plasmid DNA, to a 70 cm length of suture using about 5 to 100, preferably about 5 to 50, and more preferably about 15 to 30 coating applications yields a therapeutically effective and uniform coating.
- the invention provides coated sutures, especially sutures coated with a polymeric matrix containing nucleic acids encoding therapeutic proteins that stimulate wound healing in vivo .
- Sutures which may be coated in accordance with the methods and compositions of the present invention include any suture of natural or synthetic origin.
- Typical suture materials include, by way of example and not limitation, silk; cotton; linen; polyolefins such as polyethylene and polypropylene; polyesters such as polyethylene terephthalate; homopolymers and copolymers of hydroxycarboxylic acid esters; collagen (plain or chromicized) ; catgut (plain or chromicized) ; and suture-substitutes such as cyanoacrylates.
- the sutures may take any convenient form such as braids or twists, and may have a wide range of sizes as are commonly employed in the art.
- coated sutures especially sutures coated with a polymeric matrix containing nucleic acids encoding therapeutic proteins that stimulate wound healing cover virtually every field of surgical use in humans and animals.
- the invention is applicable to a wide variety of wound healing situations in human medicine. These include, but are not limited to, bone repair, tendon repair, ligament, repair, blood vessel repair, skeletal muscle repair, and skin repair.
- wound healing situations include, but are not limited to, bone repair, tendon repair, ligament, repair, blood vessel repair, skeletal muscle repair, and skin repair.
- cytokine growth factors produced by transfected repair cells will influence other cells in the wound, through binding of cell surface signaling receptors, thereby stimulating and amplifying the cascade of physiological events normally associated with the process of wound healing. The end result is the augmentation of tissue repair and regeneration.
- the method of the invention also is useful when the clinical goal is to block a disease process, thereby allowing natural tissue healing to take place, or when the goal is to replace a genetically defective protein function.
- Wounds may arise from traumatic injury, or alternatively, from tissue damage either induced by, or resulting from, a surgical procedure.
- the gene activated matrix of the invention can be transferred to the patient using various techniques. For example, matrices can be transferred directly to the site of the wound by the hand of the physician, either as a therapeutic implant or as a coated device (e.g., suture, stent, coated implant, etc.). Matrices can be topically administered, either as placed surgically in a normal tissue site in order to treat diseased tissue some distance away.
- the process of wound healing is a coordinated sequence of events which includes, hemorrhage, clot formation, dissolution of the clot with concurrent removal of damaged tissue, and deposition of granulation tissue as initial repair material.
- the granulation tissue is a mixture of fibrobiasts and capillary blood vessels.
- the wound healing process involves diverse cell populations including endothelial cells, stem cells, macrophages and fibrobiasts.
- the regulatory factors involved in wound repair are known to include systemic hormones, cytokines, growth factors, extracellular matrix proteins and other proteins that regulate growth and differentiation.
- the DNA transfer methods and matrix compositions of the present invention will have a wide range of applications as a drug delivery method for stimulating tissue repair and regeneration in a variety of different types of tissues. These include but are not limited to bone repair, skin repair, connective tissue repair, organ regeneration, or regulation of vasculogenesis and/or angiogenesis.
- the use of gene activated matrices may also be used to treat patients with impaired healing capacity resulting from, for example, the effects of aging or diabetes.
- the matrices may also be used for treatment of wounds that heal slowly due to natural reasons, e.g.. in the elderly, and those who do not respond to existing therapies, such as in those individuals with chronic skin wounds.
- the formation of scar tissue at the site of the wound may be regulated by the selective use of gene activated matrices.
- the formation of scar tissue may be regulated by controlling the levels of therapeutic protein expressed. In instances, such as the treatment of burns or connective tissue damage it is especially desirable to inhibit the formation of scar tissue.
- the methods of the present invention include the grafting or transplantation of the matrices containing the DNA of interest into the host. Procedures for transplanting the matrices may include surgical placement, or injection, of the matrices into the host. In instances where the matrices are to be injected, the matrices are drawn up into a syringe and injected into a patient at the site of the wound.
- the matrices may be surgically placed at the site of the wound.
- the amount of matrices needed to achieve the purpose of the present invention i.e. stimulation of wound repair and regeneration, is variable depending on the size, age and weight of the host.
- Bone has a substantial capacity to regenerate following fracture.
- the complex but ordered fracture repair sequence includes hemostasis, clot dissolution, granulation tissue ingrowth, formation of a callus, and remodeling of the callus to an optimized structure (A.W. Ham., 1930, J. Bone Joint Surg. 12, 827-844).
- Cells participating in this process include platelets, inflammatory cells, fibrobiasts, endothelial cells, pericytes, osteoclasts, and osteogenic progenitors.
- Recently, several peptide growth and differentiation factors have been identified that appear to control cellular events associated with bone formation and repair (Erlebacher, A., et al., 1995, Cell 80, 371-378).
- Bone rnorphogenetic proteins are soluble extracellular factors that control osteogenic cell fate: BMP genes are normally expressed by cultured fetal osteoblasts (Harris, S.E., et al., 1994, J. Bone Min. Res. 9, 389-394) and by osteoblasts during mouse embryo skeletogenesis (Lyons, K.M., et al., 1989, Genes Dev. 3., 1657-1668; Lyons, K.M.
- Osteogenic protein-1 a member of a family of molecules related to the BMPs (Ozkaynak, E. , et al., 1990, EMBO J. 9, 2085-2093), is capable of similar effects in vitro and in vivo (Sampath, T.K., et al., 1992, J. Biol. Chem. 267, 20352-20362; Cook, S.D., et al., (1994) J. Bone Joint Surg. 76-A, 827-838). TGF-/3 has also been shown to stimulate cartilage and bone formation in vivo (Centrella, M. , et al., 1994, Endocrine Rev.
- parathyroid hormone is an 84 amino acid hormone that raises the plasma and extracellular fluid Ca +2 concentration.
- PTH parathyroid hormone
- intermittent administration of a PTH fragment-possessing the structural requirements for biological activity produces a true anabolic effect: numerous in vivo and in vitro studies provide strong evidence that PTHl-34 administration in animals (including rats) results in uncoupled, high-quality bone formation due to a combined inhibitory effect on osteoclasts and stimulatory effect on osteogenic cells (Dempster, D.W., et al., 1993, Endocrine Rev.
- the PTH1-34 peptide is known to interact synergistically with BMP-4, which up-regulates the expression of functional cell surface PTH receptors in differentiating osteoblasts in vitro (Ahrens, M. , et al., 1993, J. Bone Min. Res. 12, 871-880) .
- peptide growth and differentiation factors such as BMP and TGF-/J1 represent promising therapeutic alternatives for fracture repair (Wozney, J.M. , 1992, Mol. Reprod. Dev. 32, 160-167; Reddi, A.H., 1994, Curr. Opin. Genet. Dev. 4, 737-744; Centrella, M. , et al., 1994, Endocrine Rev. 15, 27-38; Sumner, D.R., et al., 1995 J. Bone Joint Surg. 77-A, 1135-1147).
- relatively large doses are required to stimulate significant new bone formation in animals, raising the concern that future human therapies may be expensive and may possess an increased risk of toxicity.
- gene activated matrices are surgically implanted into a 5 mm osteomy site in the rat, a model of a complex, non-healing fracture in humans.
- the present inventors have found that gene transfer to repair cells in the osteotomy gap could be readily achieved.
- the present invention may be used to transfer a bone growth gene to promote fracture repair.
- Bone tissue is known to have the capacity for repair and regeneration and there is a certain understanding of the cellular and molecular basis of these processes.
- the initiation of new bone formation involves the commitment, clonal expansion, and differentiation of repair cells. Once initiated, bone formation is promoted by a variety of polypeptide growth factors. Newly formed bone is then maintained by a series of local and systemic growth and differentiation factors.
- BMPs as members of the transforming growth factor-/? (TGF- ⁇ ) superfamily based on DNA sequence homologies.
- TGF- ⁇ transforming growth factor-/?
- BMPs 2-8 are generally thought to be osteogenic while BMP-1 may be a more generalized morphogen; Shimell et al . , 1991, Cell, 67:469-481).
- BMP-3 is also called osteogen (Luyten et al . , 1989, J. Biol.
- TGFs and BMPs each act on cells via complex, tissue-specific interactions with families of cell surface receptors (Roberts & Sporn, 1989, M.B. Sporn and A.B. Roberts, Eds., Springer-Verlag, Heidelberg, 95 (Part 1); Aralkar et al., 1991).
- TGFs Transforming growth factors
- TGF- ⁇ l and TGF-j82 can initiate both chondrogenesis and osteogenesis (Joyce et al . , 1990, J. Cell Biol., 110:195-2007; Izumi et al . , 1992, J. Bone Min. Res., 7:115-11; Jingushi et al . , 1992, J. Orthop. Res., 8:364-371).
- fibroblast growth factor injected into a rat fracture site (Jingushi et al . , 1990) at multiple high doses (1.0. mg/50 ml) resulted in a significant increase in cartilage tissue in the fracture gap, while lower doses had no effect.
- Calcium regulating hormones such as parathyroid hormone (PTH) may also be used in one aspect of the invention.
- PTH is an 84 amino acid calcium-regulated hormone whose principal function is to raise Ca" concentration in plasma and extracellular fluid.
- Intact PTH was also shown to stimulate bone reabsorption in organ culture over 30 years ago, and the hormone is known to increase the number and activity of osteoclasts.
- Studies with the native hormone and with synthetic peptides have demonstrated that the amino terminus of the molecule (aa-1-34) contains the structural requirements for biological activity (Tregear et al., 1973; Hermann-Erlee et al . , 1976, Endocrine Research Communications, 3:21-35; Riond, 1993, Clin. Sci., 85:223- 228) .
- the gene activated matrices are surgically implanted into the site of the bone fracture.
- Such surgical procedures may include direct injection of a GAM preparation into the fracture site, the surgical repair of a complex fracture, or arthroscopic surgery.
- the mammalian repair cells will naturally migrate and proliferate at the site of bone damage.
- the present inventors have surprisingly found that gene transfer into repair cells in the regenerating tissue in the osteotomy gap could be readily achieved.
- the preferred methods for achieving gene transfer generally involve using a fibrous collagen implant material soaked in a solution of DNA shortly before being placed in the site in which one desires to promote bone growth or using a preparation of plasmid DNA encapsulated in a synthetic matrix such as a block copolymer of PLGA.
- the implant material facilitates the targeted uptake of exogenous plasmid constructs by cells in the osteotomy gap, which clearly participate in bone regeneration/repair.
- the transgenes following cellular uptake, direct the expression of recombinant polypeptides, as evidenced by the in vivo expression of functional marker gene products.
- a gene transfer vector coding for BMP-4 and a gene transfer vector encoding a fragment of human PTH1-34, alone and in combination will stimulate new bone formation.
- a gene transfer vector coding for a fragment of human parathyroid hormone, hPTHl-34 will stimulate new bone formulation in Sprague-Dawley rats, indicating that the human peptide can efficiently bind the PTH/PTHrP receptor on the rat osteoblast cell surface.
- the present invention may also be used to stimulate the growth or regeneration of soft tissues such as ligament, tendon, cartilage and skin. Skeletal connective tissue damage due to traumatic injury may be treated using matrices containing genes encoding a variety of growth factors. Connective tissue normally consists of cells and extracellular matrix organized in a characteristic tissue architecture. Tissue wounding can disrupt this architecture and stimulate a wound healing response.
- the methods of the present invention are particularly well suited for stimulation of growth and regeneration of connective tissue as it is important that the injured tissue regenerate without the formation of scar tissue as scar tissue can interfere the normal mechanical function of connective tissue.
- TGF- / 8 superfamily e.g., TGF-/3 itself
- EGF and PDGF vascular endothelial growth factor
- genes that may be used include (a) cytokines such as the peptide growth and differentiation factors, interleukines, chemokines, interferons, colony stimulating factors; (b) angiogenic factors such as FGF and VEGF; (c) extracellular matrix proteins such as collagen, laminin, and fibronectin; (d) the family of cell adhesion molecules (e.g., the integrins, selectins, lg family members such as N-CAM and Ll, and cadherins) ; (e) cell surface cytokine signaling receptors such as the type I and type II TGF-/3 receptors and the FGF receptors; (f) non-signaling co- receptors such as betaglycan and syndecan; (g) the family of signal transducing kinases; (h) cytoskeletal proteins such as talin and vinculin; (i) cytokine binding proteins such as the family of latent TGF- ⁇ binding proteins; and (j) nuclear trans acting proteins
- Such matrices may then be placed in the host mammal in the area of the connective tissue wound.
- the gene activated matrices may be injected directly into the area of connective tissue injury.
- surgical techniques such as arthroscopic surgery, may be used to deliver the matrices to the area of the connective tissue wound.
- ORGAN REGENERATION The present invention may also be used to stimulate the repair and regeneration of organ tissue. Organ damage due to traumatic injury, or surgery, may be treated using the methods of the present invention. In the case of liver, the liver may be damaged due to excessive alcohol consumption or due to infection with various types of infectious agents such as the hepatitis family of viruses.
- the kidney may likewise fail to function normally as a result of damage resulting from kidney disease.
- Mucous membranes of the esophagus, stomach or duodenum may contain ulcerations caused by acid and pepsin in gastric juices. The ulcerations may also arise from colonization of gastric mucosal cells with Helicobacter pylori bacteria.
- Matrices containing DNA encoding cytokines which stimulate proliferation and differentiation of cells, and/or regulate tissue morphogenesis may be transplanted to the appropriate organ site.
- factors may include but are not limited to, the transforming growth factor family of proteins, platelet derived growth factor (PDGF) , insulin like growth factor (IGF) and fibroblast growth factory (FGF) .
- PDGF platelet derived growth factor
- IGF insulin like growth factor
- FGF fibroblast growth factory
- hepatocyte growth factor may be expressed to stimulate the wound healing process in the liver. For treatment of ulcers, resulting from
- the gene activated matrices may contain DNA encoding anti-microbial proteins.
- the gene activated matrices of the invention can be surgically implanted into the organ that is to be treated. Alternatively, laproscopic surgical procedures may be utilized to transfer the gene activated matrices into the body. In cases where the treatment is in response to tissue injury, the natural wound healing process will stimulate the migration and proliferation of the repair cells to the transplanted matrices. Alternatively, where the gene activated matrices are transferred to organs which have not been injured, for example, where, matrices are implanted to express therapeutic proteins not involved in wound healing, the wound healing process can be stimulated by induction of tissue injury.
- the present invention may also be used to regulate the formation and spreading of blood vessels, or vasculogenesis and angiogenesis, respectively. Both these physiological processes play an important role in wound healing and organ regeneration.
- granulation tissue which is a mixture of collagen, matrix and blood vessels, is deposited and provides wound strength during tissue repair.
- the formation of new blood vessels involves the proliferation, migration and infiltration of vascular endothelial cells, and is known to be regulated by a variety of polypeptide growth factors.
- polypeptides with endothelial cell growth promoting activity have been identified, including acidic and basic fibroblastic growth factors (FGF) , vascular endothelial growth factor (VEGF) , and placental derived growth factor (PDGF) .
- FGF acidic and basic fibroblastic growth factors
- VEGF vascular endothelial growth factor
- PDGF placental derived growth factor
- DNA encoding such growth factors may be incorporated into matrices and these matrices may be implanted into the host. In some instances, it may be necessary to induce the wound healing process through tissue injury.
- Tumor angiogenesis may be inhibited through the transfer of DNA's encoding negative inhibitors of angiogenesis, such as thrombospondin or angiostatin.
- DNA encoding for example, thrombospondin or angiostatin, may be incorporated into a matrix followed by the implanting of the matrix into a patient at the site of the tumor.
- the present invention may also be used to stimulate the growth and repair of skin tissue.
- wounds which involve injury to areas of the skin, and particularly in the case of massive burns, it is important that the skin grow very rapidly in order to prevent infections, reduce fluid loss, and reduce the area of potential scarring.
- Skin damage resulting from burns, punctures, cuts and/or abrasions may be treated using the gene activated matrices of the present invention.
- Skin disorders such as psoriasis, atopic dermatitis or skin damage arising from fungal, bacterial and viral infections or treatment of skin cancers such as melanoma, may also be treated using the methods of the invention.
- Matrices containing DNA encoding cytokines which stimulate proliferation and differentiation of cells of the skin may be used to treat skin injuries and disorders.
- the gene activated matrices serve two functions, the protection of the wound from infection and dehydration and supplying the DNA for uptake by repair cells.
- the gene activated matrices of the invention may include dermal patches, cadaver skin, band- aids, gauze pads, collagen lattices such as those disclosed in U.S. Patent 4,505,266 or U.S. Patent 4,485,097, topical creams or gels.
- DNA to be incorporated into the matrices may encode a variety of different growth factors including keratinocyte-growth-factor (KGF) or epidermal growth factor (EGF) .
- KGF keratinocyte-growth-factor
- EGF epidermal growth factor
- DNA encoding IL-1 which has been shown to be a potent inducer of epithelial cell migration and proliferation as part of the healing process may also be incorporated into the matrices of the invention.
- Implant materials may be used for transferring genes into the site of bone repair and/or regeneration in vivo. These materials are soaked in a solution containing the DNA or gene that is to be transferred to the bone 0 regrowth site. Alternatively, DNA may be incorporated into the matrix as a preferred method of making.
- fibrous collagen which may be lyophilized following extraction and partial purification from tissue and then sterilized.
- Another particularly preferred collagen is type II collagen, with the most particularly preferred collagen being either recombinant type II collagen, or mineralized type II collagen.
- implant materials Prior to placement in osteotomy sites, implant materials are soaked in solutions of DNA (or virus) under 0 sterile conditions. The soaking may be for any appropriate and convenient period, e.g., from 6 minutes to over-night.
- the DNA (e.g., plasmid) solution will be a sterile aqueous solution, such as sterile water or an acceptable buffer, with the concentration generally being about 0.5 - 1.0 mg/ml.
- plasmids are those such as pGL2 (Promega), pSV40/3-gal, pAd.CMVlacZ, and pcDNA3.
- Endogenous peroxidase activity was 5 quenched by immersion of tissue .sections in 0.1% hydrogen peroxide (in 95% methanol) at room temperature for 10 min, and quenched sections were washed 3x in PBS. In some cases, sectioned calvariae were demineralized by immersion in 4% EDTA, 5% polyvinyl pyrrolidone, and 7% sucrose, pH 7.4, for
- Bacterial / 3-gal was also detected by substrate 0 utilization assays using commercially available kits (e.g., Promega) according to the manufacturers' instructions.
- Luciferase was detected by substrate utilization assays 5 using commercially available kits (e.g., Promega) according to the manufacturers' instructions.
- PTH TRANSGENES Recombinant PTH such as hPTHl-34 peptide was assayed 0 in homogenates of osteotomy gap tissue, for example, using two commercially available radioimmunoassay kits according to the manufacturer's protocols (Nichols Institute Diagnostics, San Juan Capistrano, CA) .
- kits are the Intact PTH-Parathyroid Hormone 100T Kit. 5
- This radioimmunoassay utilizes an antibody to the carboxy terminus of the intact hormone, and this is used to measure endogenous levels of hormone in gap osteotomy tissue. This assay may be used to establish a baseline value PTH expression in the rat osteotomy model.
- This kit uses affinity purified antibodies specific for the amino terminus of the intact rat hormone (PTH1-34) and thus will measure endogenous PTH production as well as the recombinant protein. Previous studies have shown that these antibodies cross-react with human PTH and this are able to recognize recombinant molecules in vivo.
- kit #1 antibody to the carboxy terminus
- kit #2 antibody to the amino terminus
- BMP TRANSGENE BMP proteins such as the murine BMP-4 transgene peptide product, were detected immunohistochemically using a specific antibody that recognizes the HA epitope (Majmudar et al., 1991, J. Bone and Min. Res. 6:869-881), such as the monoclonal antibody available from Boehringer-Mannheim. Antibodies to BMP proteins themselves may also be used. Such antibodies, along with various immunoassay methods, are described in U.S. Patent 4,857,456, incorporated herein by reference.
- Osteotomy tissue specimens were fixed in Bouins fixative, demineralized, and then split in half along the longitudinal plane. One-half of each specimen was embedded in paraffin for subsequent immunohistochemical identification of the recombinant murine BMP-4 molecule.
- STIMULATES BONE REGENERATION/REPAIR IN VIVO The following experiment was designed to investigate whether gene transfer could be employed to create transfected cells that constitutively express recombinant hPTHl-34 in vivo , and whether this transgene can stimulate bone formation.
- the rate of new bone formation was analyzed as follows. At necropsy the osteotomy site was carefully dissected for histomorphometric analysis. The A-P and M-L dimensions of the callus tissue -are measured using calipers. Specimens were then immersion fixed in Bouins fixative, washed in ethanol, and demineralized in buffered formic acid. Plastic embedding of decalcified material was used because of the superior dimensional stability of methacrylate during sample preparation and sectioning.
- Tissue blocks were dehydrated in increasing alcohol concentrations and embedded. 5 mm thick sections were cut in the coronal plane using a Reichert Polycot microtome. Sections were prepared from midway through the width of the marrow cavity to guard against a sampling bias. Sections for light microscopy were stained using a modified Goldner's trichrome stain, to differentiate bone, osteoid, cartilage, and fibrous tissue. Sections were cover-slipped using Eukitt's mounting medium (Calibrated Instruments, Ardsley, NY) . Histomorphometric analyses were performed under brightfield using a Nikon Optiphot Research microscope. Standard point count stereology techniques using a 10 mm x 10 mm eyepiece grid reticular.
- Total callus area was measured at 125x magnification as an index of the overall intensity of the healing reaction. Area fractions of bone, cartilage, and fibrous tissue were measured at 250x magnification to examine the relative contribution of each tissue to callus formation. Since the dimensions of the osteotomy gap reflect the baseline (time 0) , a measurement of bone area at subsequent time intervals was used to indicate the rate of bone infill. Statistical significance was assessed using analysis of variance, with post-hoc comparisons between groups conducted using Tukey's studentized range test. In the 5 mm rat osteotomy model described above, it was found that PTH transgene expression can stimulate bone regeneration/repair in live animals. This is a particularly important finding as it is known that hPTHl-34 is a more powerful anabolic agent when given intermittently as opposed to continuously, and it is the continuous-type delivery that results from the gene transfer methods used here.
- Gene activated matrices containing mammalian expression plasmid DNA were implanted into large segmental gaps created in the adult male femur. Implantation of gene- activated matrices containing beta-galactosidase or luciferase plasmids led to DNA uptake and functional enzyme expression by repair cells growing into the gap. Additionally, implantation of a gene activated matrix containing either a bone morphogenetic protein-4 plasmid or a plasmid coding for a fragment of parathyroid hormone (amino acids 1-34) resulting in a biological response of new bone filling the gap.
- IMMUNOHISTOCHEMISTRY Tissues were prepared for light microscopy and immunohistochemistry was performed as described (Wong et al., 1992, J. Biol. Chem. 267: 5592-5598). Histology sections were incubated with a commercially available anti-jS-gal antibody (1:200 dilution, 5 Prime >3 Prime) and with a commercially available anti-HA.11 polyclonal antibody (1:500 dilution, BAbCO) .
- Luciferase and 0-gal activity was determined using the Luciferase Assay System (Promega) and /3-galactosidase Enzyme Assay System (Promega) according to protocols supplied by the manufacturer.
- PGAMl EXPRESSION PLASMID To assemble pGAMl, mRNA was prepared from day 13.5 p. c. CD-I mouse embryos using kit reagents and protocols (Poly AT Tract mRNA Isolation System I, Promega) . An aliquot of mRNA was used to generate cDNA using commercial reagents (Reverse Transcriptase System, Promega) .
- a full length mouse BMP-4 cDNA coding sequence was generated by the polymerase chain reaction (PCR) using the following conditions: 94°C, 4 min., 1 cycle; 94°C, l min., 65°C, 1 min., 72°C, l min., 30 cycles; 72°C, 8 min., 1 cycle.
- the sequence of the PCR primers was based on the known mouse BMP-4 sequence (GenBank) : upstream primer-5• CCATGATTCCTGGTAACCGAATGCTG 3'; downstream primer-5' CTCAGCGGCATCCGCACCCCTC 3• .
- a single PCR product of the expected size (1.3kb) was purified by agarose gel electrophoresis and cloned into the TA cloning vector (Invitrogen).
- the 5' end of the BMP-4 insert was further modified (PCR) by addition of a 27 nucleotide sequence that codes for the HA epitope, and the BMP-4 insert was cloned into the pcDNA3 expression vector (InVitrogen) . Plasmid DNA was prepared and sequenced (both strands) to ensure the orientation and integrity of the BMP-4 insert.
- the pGAMl plasmid was expressed using an in vitro transcription and translation kit (TNT T7 Coupled Reticulocyte Lysate System, Promega) according to protocols supplied by the manufacturer. Protein radiolabeling, immunoprecipitation, sample preparation and SDS-PAGE, autoradiography, transient transfection, and Western analysis were performed as described (Yin et al., 1995, J. Biol. Chem. 270:10147-10160) .
- pGAM2 EXPRESSION PLASMID Human parathyroid hormone cDNA fragments encoding amino acids preprol-34 were generated by PCR.
- the sequence of the PCR primers was based on known human PTH sequence (GenBank) : upstream primer-5' GCGGATCCGCGATGATACCTGCAAAAGACATG 3 • ; downstream primer-5• GCGGATCCGCGTCAAAAATTGTGCACATCC 3' .
- This primer pair created BamHl sites at both ends of the PCR fragment.
- the fragment was digested with BamHl and ligated into a BamHl cloning site in the PLJ retrovirus vector (Wilson et al., 1992, Endocrinol. 130: 2947-2954).
- a clone with the insert in the coding orientation eventually was isolated and characterized by DNA sequence analysis.
- the ⁇ CRIP packaging cell line (Wilson, J.M. , et al., 1992, Endocrinology 130:2947- 2954) was transfected with lO ⁇ g of recombinant vector DNA using the calcium phosphate method. After an overnight incubation, culture medium (Dulbecco's Modified Eagle's Medium, supplemented with 10% fetal bovine serum, penicillin (100 units/ml) , and streptomycin ⁇ (100 mg/ml) (all reagents from Gibco-BRL Life Technologies, Inc.) containing retrovirion particles was harvested and applied to cultured Rat-l cells. Independent clones of successfully transduced Rat-1 cells were obtained by standard infection and selection procedures.
- culture medium Dulbecco's Modified Eagle's Medium, supplemented with 10% fetal bovine serum, penicillin (100 units/ml) , and streptomycin ⁇ (100 mg/ml) (all reagents from Gibco-BRL Life Technologies, Inc.
- Rat-1 cells were grown to confluence, split 1:10, and selected in G418 (1 mg/ml. Gibco- BRL Life Technologies, Inc.). In some instances, antibiotic- resistant colonies were pooled into a single culture. In other instances, single colonies of resistant cells were maintained. Similar methods were used to generated clones of Rat-1 cells transduced with the BAGT retrovirus, which encodes the bacterial b-gal enzyme.
- the hPTHl-34 concentration in cell culture media was estimated using a commercial radioimmunoassay kit (INS-PTH, Nichols) and according to the manufacturer's protocol.
- the biological activity of the peptide encoded by pGAM2 was evaluated as described (McCauley, et al., 1994, Mol. Cell. Endocrinol. 101: 331-336) .
- 25 expression plasmid DNA were successfully transferred in vivo .
- the goal was to standardize the gene activated matrix preparation protocol and post-operative time course.
- a GAM encoding luciferase was placed in the osteotomy gap of one rat and a gene activated matrix encoding /3-gal was placed in
- gap homogenates consisting a granulation tissue
- a granulation tissue was prepared after careful dissection of surrounding bone, cartilage, and skeletal muscle. Aliquots of each homogenate were evaluated for enzyme expression by substrate utilization assay.
- BMP-4 GENE TRANSFER Having demonstrated that gap cells express functional enzymes following uptake of plasm,id DNA from a matrix, we asked whether gene transfer could be used to modulate bone regeneration. We chose to overexpress BMP-4, an osteoinductive factor that normally is expressed by progenitor cells during fracture repair.
- a full length mouse BMP-4 CDNA was generated by PCR and subcloned into the pcDNA3 (Invitrogen) eukaryotic expression vector (Fig. 2) . To specifically detect recombinant proteins, the 3' end of the BMP-4 coding sequence was modified by addition of a hemagglutinin (HA) epitope.
- HA hemagglutinin
- Recombinant BMP-4 was expressed from this construct (pGAMI) using an in vitro transcription and translation protocol. Immunoprecipitation studies established the ability of the HA epitope to be recognized by an anti-HA polyclonal antibody. Biosynthesis of recombinant BMP-4 was evaluated following transient transfection of cultured 293T cells with PGAMI plasmid DNA. As demonstrated by immunoprecipitation, BMP-4 molecules were assembled into homodimers, secreted, and processed as expected. Taken together these results established that the HA-epitope was recognized by the anti-HA polyclonal antibody. Collagen sponges containing pGAMI DNA were placed in the gap of nine adult rats maintained for 4-24 weeks.
- Fig. 3A In seven mammalian hosts sacrificed 5-12 weeks post-surgery, the amount of radiographic new bone steadily increased (Fig. 3A) , even though BMP-4 encoded by the transgene was not detectable by immunohistochemistry. Bridging, defined as new bone extending from the surgical margins across the osteotomy gap, typically was observed by 9 weeks. A ninth mammalian host survived without complication for 24 weeks post-surgery. Sufficient new bone formed by 18 weeks to allow removal of the external fixator, and the mammalian host ambulated well for an additional 6 weeks (Fig. 3A) . At sacrifice, the gap was filled with new bone undergoing active remodeling, with the exception of a thin strip of radiolucent tissue near the distal margin of the gap.
- Bone regeneration normally is governed by multiple factors acting in a regulated sequence, and we knew, therefore, if the expression of several anabolic factors would stimulate bone formation more powerfully than a single factor alone.
- PTH parathyroid hormone
- ROS 17/2.8 cells possess PTH cell surface receptors, which belong to the G protein-coupled receptor superfamily (Dempster et al., 1993, Endocrin. Rev. 14:690- 709). Incubation of ROS 17/2.8 cells with aliquots of conditioned media from a stably transduced cell line 0 (secreting >2 pg/ml via radioimmunoassay) resulted in a 2.7- fold increase in CAMP response versus the control, a result that established that the secreted PTHl-34 peptide was biologically active.
- plasmid DNA could be delivered to cells in a sustained manner following encapsulation within a preparation of block co-polymers of polylactic-polyglycolic particles.
- the results demonstrate that cultured cells can be transfected by plasmid DNA released from polylactic- polyglycolic particles.
- Results also indicated that repair fibrobiasts (rat osteotomy model) in vivo will take up and express plasmid DNA released from block co-polymers of polylactic-polyglycolic particles.
- Figure 7 demonstrates that repair fibrobiasts (rat osteotomy model) in vivo will take up and express pGAM2 plasmid DNA following release from polylactic-polyglycolic particles. As shown in Figure 7, expression of plasmid-encoded PTHl-34 is associated with significant new bone formation in the osteotomy gap. Taken together, these studies show that the gene activated matrix technology does not depend on a collagenous matrix for success. Therefore, the technology is broad enough that it can be combined with both biological and synthetic matrices.
- a model system has been developed in which segmental defects in the Achilles' tendon is created and a novel biomaterial, small intestinal submucosa or SIS, is used as a tendon implant/molecular delivery agent.
- SIS small intestinal submucosa
- SIS graft material was prepared from a segment of jejunum of adult pigs (Badylak et al., 1989, J. Surg. Res. 47:74-80). At harvest, mesenteric tissues were removed, the segment was inverted, and the mucosa and superficial submucosa were removed by a mechanical abrasion technique. After returning the segment to its original orientation, the serosa and muscle layers were rinsed, sterilized by treatment with dilute peracetic acid, and stored at 4°C until use.
- Mongrel dogs (all studies) were anesthetized, intubated, placed in right-lateral recumbency upon a heating pad, and maintained with inhalant anesthesia. A lateral incision from the musculotendinous junction to the plantar fascia was used to expose the Achilles, tendon. A double thickness sheet of SIS was wrapped around a central portion of the tendon, both ends were sutured, a 1.5 cm segment of the tendon was removed through a lateral opening in the graft material, and the graft and surgical site were closed. The leg was immobilized for 6 weeks and then used freely for 6 weeks. Graft tissues were harvested at time points indicated below, fixed in Bouins solution, and embedded in paraffin. Tissue sections (8 ⁇ m) were cut and used for immunohistochemistry.
- SIS material alone (SIS-alone graft) engrafted and promoted the regeneration of Achilles, tendon following the creation of a segmental defect in mongrel dogs as long as 6 months post surgery.
- the remodeling process involved the rapid formation of granulation tissue and eventual -degradation of the graft. Scar tissue did not form, and evidence of immune-mediated rejection was not observed.
- a pSV / Sgal plasmid that employs simian virus 40 regulatory sequences to drive /3-galactosidase (/3-gal) activity was detectable by immuno ⁇ histochemistry using a specific antibody in 4/4 mammalian hosts. As a negative control, /3-gal activity was not detected in the unoperated Achilles, tendon and cruciate ligament of these mammalian hosts.
- slides were incubated with a well characterized anti-/3- galactosidase antibody (12:00 dilution, 5 Prime->3 Prime), washed in PBS, incubated with a biotinylated second antibody, washed, stained with the enzyme conjugate plus a substrate- chromogen mixture, and then counterstained with hematoxylin and eosin.
- An alternative method to achieve in vivo gene transfer into regenerating tissue is to utilize an adenovirus-mediated transfer event. Successful adenoviral gene transfer of a marker gene construct into bone repair cells in the rat osteotomy model has been achieved.
- Adenoviral vector pAd. CMVlacZ is an example of a replication-defective adenoviral vector which can replicate in permissive cells (Stratford-Perricaudet et al . , 1992, J. Clin. Invest. 90:626-630).
- the early enhancer/promoter of the cytomegalovirus (CMV) is used to drive transcription of lacZ with an SV40 polyadenylation sequence cloned downstream from the reporter gene (Davidson et al., 1993, Nature Genetics 3:219-223).
- pAd.RSV4 has essentially the same backbone as pAdCMVlacZ, however the CMV promoter and the single Bglll cloning site has been replaced in a cassette-like fashion with a Bglll fragment that consists of an RSV promoter, a multiple cloning site, and a poly(A + ) site.
- the greater flexibility of this vector is contemplated to be useful in subcloning osteotropic genes, such as the hPTHl-34 cDNA fragment, for use in further studies.
- An Ultra Fiber TM implant was soaked for 6 minutes in a solution of AdCMV lacZ virus (10 ,0 -10 ⁇ plaque forming units or PFU/ml) and then implanted into the osteotomy site.
- AdCMV lacZ virus (10 ,0 -10 ⁇ plaque forming units or PFU/ml)
- the defect was allowed to heal for 3 weeks, during which time the progress of the wound healing respon ⁇ e was monitored by weekly radiographic examination. By three weeks, it was estimated that 40% of the defect was filled with callus tissue.
- the mammalian host was sacrificed and tissues were fixed in Bouins fixation and then demineralized for 7 days using standard formic acid solutions.
- EXAMPLE TRANSFER OF GENES TO SKELETAL MUSCLE
- a model system has been developed in which incisions in adult rat skeletal muscle are made and a suture preparation coated with a preparation of sustained release PLGA particles and plasmid DNA is used as a skeletal muscle/gene delivery device.
- a surgical suture was coated with marker DNA (encoding human placental alkaline phosphatase) and used to suture rat muscle tissue. The experiment demonstrates successful transfer and expre ⁇ sion of DNA in the tissue repaired with the coated suture.
- a model sy ⁇ tem has been developed in which repair cells in large blood vessel ⁇ in rabbit are transfected with a preparation of sustained release PLGA particles and plasmid DNA. Repair cells are present because these rabbit blood vessels harbor a foam cell lesion that mimics clinical atherosclerosis in humans.
- the present example demonstrates the ability to deliver and express marker gene constructs into large blood vessel repair cells.
- a balloon angioplasty catheter (2.0 mm balloo ⁇ ) was introduced into iliac arteric segment ⁇ and balloon was dilated for 1-minute at 8 atm pre ⁇ ure. Following balloon dilatation, the angioplasty catheter was removed, 20 mg of heparin was injected intra-arterially to prevent distal thrombosis. Both ends of iliac artery were tightened with 10.0 silk, the 5 mg/ml DNA-Nanoparticle suspension was infused in each iliac artery over 3 minutes at 0.5 atm. The wound was sutured. Rabbits were sacrificed 2 weeks after the balloon angioplasty and nanoparticle delivery.
- both iliac arteries were isolated.
- a 2 cm segment of iliac artery was excised bilaterally.
- Carotid arteries from rabbit was taken as a control sample.
- the tissue was preserved in liquid nitrogen for alkaline phosphatase assay.
Abstract
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JP53746397A JP4114952B2 (en) | 1996-04-12 | 1997-04-11 | In vivo gene transfer method for wound healing |
AT97922578T ATE298253T1 (en) | 1996-04-12 | 1997-04-11 | IN VIVO GENE TRANSFER METHODS FOR WOUND HEALING |
CA2251655A CA2251655C (en) | 1996-04-12 | 1997-04-11 | In vivo gene transfer methods for wound healing |
AU28212/97A AU710386B2 (en) | 1996-04-12 | 1997-04-11 | In vivo gene transfer methods for wound healing |
DE69733605T DE69733605T2 (en) | 1996-04-12 | 1997-04-11 | IN VIVO GENTRANSFER METHODS FOR WOUND HEALING |
PL97343864A PL189174B1 (en) | 1996-04-12 | 1997-04-11 | Methods of in vivo transferring a gene for wound healing purposes |
NO19984729A NO323843B1 (en) | 1996-04-12 | 1998-10-09 | Use of a biocompatible matrix for the preparation of a drug for regulating angiogenesis such as the promotion or inhibition of blood vessel formation. |
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Also Published As
Publication number | Publication date |
---|---|
NO984729D0 (en) | 1998-10-09 |
PL343864A1 (en) | 2001-09-10 |
US5962427A (en) | 1999-10-05 |
CA2251655A1 (en) | 1997-10-23 |
JP2001519767A (en) | 2001-10-23 |
ATE298253T1 (en) | 2005-07-15 |
WO1997038729A8 (en) | 1999-04-22 |
PL189174B1 (en) | 2005-06-30 |
RU2170104C2 (en) | 2001-07-10 |
DE69733605D1 (en) | 2005-07-28 |
EP1510224A1 (en) | 2005-03-02 |
DE69733605T2 (en) | 2006-05-11 |
JP4114952B2 (en) | 2008-07-09 |
CN102274522A (en) | 2011-12-14 |
EP0892644A4 (en) | 1999-07-14 |
EP0892644B1 (en) | 2005-06-22 |
NO323843B1 (en) | 2007-07-09 |
CA2251655C (en) | 2010-09-21 |
EP0892644A1 (en) | 1999-01-27 |
CN1226835A (en) | 1999-08-25 |
AU710386B2 (en) | 1999-09-16 |
AU2821297A (en) | 1997-11-07 |
NO984729L (en) | 1998-12-14 |
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