Methods of in vivo transferring a gene for wound healing purposes

The subject of the invention is the use of a composition comprising a biocompatible matrix containing one or more DNA molecules encoding polypeptides with vascular growth stimulating activity, antisense or ribozyme molecules for the preparation of a medicament for promoting blood vessel formation, wherein the matrix acts as a scaffold promoting cell penetration.

In the invention, preferably at least one DNA molecule encodes a growth factor or cytokine. Also preferably, at least one DNA molecule encodes fibroblast growth factor (FGF) -1, (FGF) -2, endothelial vascular cell growth factor (VEGF) or platelet-derived growth factor (PDGF). In another preferred embodiment of the invention, the matrix is ​​a collagen, metal, biodegradable or chemically defined calcium sulfate composition, calcium triphosphate, hydroxyapatite, polylactic acid, polyanhydride, biological glass, linate, bioceramic material, metal material, biocompatable and biodegradable polymer, purified proteins or semi-purified proteins extracellular.

Preferably, said cells are vascular endothelial cells in the invention. Also preferably, the cells are fibroblasts, capillary endothelial cells, pericytes, mononuclear inflammatory cells, segmented inflammatory cells, or granular tissue cells.

Another object of the invention is the use of a composition comprising a biocompatible matrix containing one or more DNA molecules encoding polypeptides

189 174 with angiogenesis inhibitory activity, antisense or ribozyme molecules for the preparation of a medicament for inhibiting the formation of blood vessels, which matrix acts as a scaffold promoting cell penetration.

Preferably, in this invention at least one DNA molecule encodes an anti-angiogenic factor.

Preferably, the anti-angiogenic agent may be encoded by the DNA molecule thrombospodine, TGF-β or angiostatin.

Preferably, the matrix may be a collagen, metal, biodegradable or chemically defined calcium sulfate composition, calcium triphosphate, hydroxyapatite, polylactic acid, polyanhydride, biological glass, aluminate, bioceramic material, metal material, biocompatable and biodegradable polymer, purified proteins or semi-purified matrices .

Preferably, the cells may be vascular endothelial cells. More preferably, the cells may be fibroblasts, capillary endothelial cells, pericytes, mononuclear inflammatory cells, segmented inflammatory cells, or granular tissue cells.

To stimulate hard and soft tissue repair and tissue regeneration, DNA transfer to mammalian repair cells via systemic delivery can be used.

The repair cells will be those cells that normally arrive at the area of ​​injury being treated. Accordingly, there are no difficulties in obtaining appropriate target cells to which the present therapeutic compositions should be used. All that is required is the implantation of a gene activated matrix into the wound site. The nature of this biological environment is that the respective repair cells will actively absorb and express "bait" DNA in the absence of any other cell targeting or identification by the practitioner.

Using both biological and synthetic arrays, DNA transfer to mammalian repair cells can be made to stimulate skeletal regeneration, as well as to connect and repair the tendon, and to stimulate skeletal muscle repair and / or repair of blood vessels.

DNA used in the practice of the invention may include any DNA encoding translation products (i.e. proteins) or transcription products (i.e. nonsense or ribozyme) that stimulate tissue repair or are capable of interrupting the disease process. For example, DNA may include genes that encode therapeutically useful proteins such as growth factors, cytokines, hormones, etc. In addition, DNA can encode nonsense or ribozyme molecules that can inhibit RNA translation, encoding proteins that inhibit wound healing, or that induce inflammation.

The DNA encoding the therapeutic product in question is bound or impregnated with the matrix to form a gene activated matrix. Once created, the gene activated matrix is ​​inside the mammal at the site of injury.

The invention is shown by way of examples showing the efficiency of in vivo transfer and gene expression during tissue repair and regeneration.

3.1 Definitions

As used herein, the following terms shall have the meanings indicated below.

The gene activated matrix (GAM) is here defined as any matrix material containing DNA encoding the therapeutic agent in question. For example, gene activated arrays are located inside the wound sites in the host organism, which is a mammal, promoting wound healing.

The repair cell is defined herein as any cell stimulated to migrate and proliferate in response to tissue damage. Repair cells are part of the wound healing response. Such cells include fibroblasts, capillary endothelial cells, capillary pericytes, inflammatory mononuclear cells, divided inflammatory cells, and tissue laminating cells.

The site of injury is defined here as any site in the host that results from traumatic tissue injury, or alternatively, tissue damage either induced by or resulting from surgical procedures.

189 174

4. Description of drawings

Figure 1A. Femoral incision model of non-fibrous connectivity. A 5 mm surgical incision was made in adult separated male Sprague-Dawley rats. The intervals shown here are representative of the entire control group, where the mammalian host receives either the bone incision itself (n = 3), bone incision plus collagen sponge (n = 10) or incision plus collagen sponge containing control plasmid DNA (marker gene) (n = 23). Plain x-ray film showing the femur of the control rat immediately after surgery. The fissure was stabilized by external fixation consisting of a plate and 4 pins. The skin incision was closed with a metal clip.

Figure 1B. Plain x-ray film, showing the incision of the femur of the control rat 9 weeks after surgery. The rounded edges of the surgical wound (arrows) caused reactive bone formation and are consistent with the classic disclosure of a non-connective fracture.

Figure 1C. Histological cross-section of the fissure tissue 3 weeks after surgery, showing proliferative repair fibroblasts and capillaries embedded in the edema extracellular matrix. Focal infiltration, consisting of lymphocytes and macrophages, is also present.

Figure 1D. Histological cross-section of a 9-week control slit showing dense fibrous tissue. 1 cm = 20 μ (C and D).

Figure 2. Schematic diagram of the construction of pGAM1 encoding BMP-4 mice. The CMV promoter positions, BMP-4 coding sequence, HA epitope and bovine growth hormone polyadenylation signal are shown.

Figure 3A. Expression of BMP-4 by repair fibroblasts. BMP-4 expression encoded by plasmid was detected in Bouins-fixed, demineralized, paraffin-embedded tissue sections, using anti-HA antibody and immuno peroxide method 4 weeks after implantation of the gene activated matrix containing pGAM1 plasmid DNA. Arrows indicate examples of positive (red-brown) staining of fibroblast cytoplasm (upper left micrograph). These cells were identified as fibroblasts based on spindle morphology, bundle growth, and type I positive immunostaining (not shown). Serial sections incubated with or without pre-residual rabbit serum were negative. Negative results were also obtained for sham operated controls (collagen sponge alone) incubated with anti-HA antibody. 11 (micrograph in the upper right part). False positive staining of macrophages, osteoclasts and osteoblasts was also observed in control sections incubated with HA-11 antibody. Islet of newly formed bone 3 weeks after pGAM1 transfer is shown in the micrograph in the lower left. The new bone is associated with the formation of granulation tissue. A view of the high strength of the newly formed bone appears in the micrograph on the bottom right. Arrows indicate presumed osteoblasts on the surface of the new bone trabeculae. The gap tissues were stained with hemotoxylin and eosin (upper micrograph) or the Gomori three-color method (collagen rich tissues appear as green, lower micrographs). 1 cm = 20 mm (top micrographs).

Figure 3B. Distinct x-ray of the animal (23 weeks after surgery). In ordinary radiography (left), arrows indicate the approximate position of the incision gap, filled with radiological density tissue. Note that the outer stiffener has been removed. As indicated by the colored patterns, secondary bone modeling takes place. The arrow heads indicate defects in the bone adjacent to the slit (consequence of pin placement). The two distal pin places healed completely during this time (not shown). The entire upper part of the photograph (on the right) shows a tissue section stained with the three-colored Gomori method, from the aperture of the animal shown (after killing). The arrows indicate the cleft, which is now covered by well-integrated cortical bone. Circular defects in the marrow space (both gap spaces) result from the placement of internal fixation pins. The tissue break at the bottom of the micrograph is caused by the sample holder.

Figure 4A. Schematic diagram of the pGAM2 structure, coding for human PTH1-34. The position of the long term upward repeat that shows the expression of PTH1-34 (arrow), the PTH1-34 coding sequence, the SV40 promoter which directs

189 174 with new expression (arrow), new coding sequence, pBR sequence and downstream repeat.

Figure 4B. PTH1-34 gene transfer and expression drives new bone formation in vivo. Plain x-ray film showing 5 mm incision fusion with new bone 9 weeks after implantation into the animal that received the gene activated matrix containing plasmid DNA pGAM2. Arrows indicate tissue with radiological density in the gap. The results shown here are representative of experiments with an additional animal.

Figure 5. New bone formation in vivo by dual plasmid GAM. (top) Normal membrane radiography, showing fusion with a new bone gap of 5 mm, 4 weeks after implantation in an animal that received the gene activated matrix containing plasmid DNA pGAM1 plus pGAM2. Arrows indicate tissue with radiological density in the fissure (histologically confirmed to be bone), (bottom) Radiography with the usual fissure membrane shown in the photo at the top, after removal (5 weeks before; total 17 weeks after surgery) of the external stiffening. The arrows indicate the location of the fissure, which is filled with radiological density tissue except for the non-demineralized tissue band next to the proximal wound edge. As indicated by the multicolored patterns, the secondary external modeling response takes place. The results shown here are representative of the experiments with one additional animal.

Figure 6. Adenovirus mediated gene transfer to bone repair / regenerative cells in vivo. The UltraFiber ™ implant was saturated for 6 minutes in the AdCMVlazZ virus solution (10^AND°-10^{1 1} plaque forming units (or PFU / ml) and then implanted into the bone incision site. The defect was allowed to heal for 3 weeks, during which the progress of the wound healing response was monitored by weekly radiography. After three weeks, it was estimated that 40% of the defect was filled with scar tissue. The mammalian host was sacrificed and the tissues fixed by Bouins fixation and then demineralized for 7 days using standard formic acid solutions. Photomicrographs were obtained from cross sections of the new bone (scar) that formed at the incision site 3 weeks after surgery. Upper left panel: Note the positive (red) cytoplasmic staining of β-gal scar tissue cells from the UltraFiber ™ adenovirus implant. This result indicates that cell surface receptors mediating infection, and thus viral transduction, are expressed by scar cells (at least one population) during the healing of the incision. Upper left table: serial control sections negatively stained with β-gal antibody carrier plus a mixture of non-specific LgG rabbit antibodies. Table below: positive (red) staining of β-gal chondrocyte nuclei at the site of the bone incision filled with UltraFiber ™ and AdRSVlacZ should be noted. This result shows the excellent specificity of the anti - ['S-gal antibody and shows the expression of the marker gene product in the bone incision gap.

Figure 7. Transfer of the pGAM2 plasmid gene to repair fibroblasts results in new bone growth in a rat bone incision model. Plain membrane radiography showing new bone jointing at a 5 mm gap, 6 weeks after implantation, in an animal that received a gene activated matrix containing plasmid DNA pGAMl plus pGAM2. Arrows indicate tissue with radiological density in the crack (confirmed histologically that it is a bone).

5. Detailed description of the invention

The present invention relates to an in vivo method for the presentation and transfer of DNA to mammalian repair cells to express therapeutic agents. The method of the invention involves implanting or placing a gene activated matrix at the site of fresh injury.

Wound healing is usually a coordinated, stereotyped sequence of events that includes (a) tissue disruption and loss of normal tissue formation; (b) cell necrosis and bleeding; stopping bleeding (clot formation); (c) penetration of inflammatory mononuclear and divided cells; with vascular congestion and tissue edema; (d) dissolution of the clot as well as damaged cells and tissues by mononuclear cells

189 174 (macrophages) (e) granulation tissue formation (non-woven tissue growth and angiogenesis). This order of cellular events was observed in the wounds of all tissues and organs formed in a large number of mammalian species (Gailet et al., 1994, Cum Opin. Cell. Biol. 6: 717-725). Thus, the sequence of events described above is a universal aspect of repairing all mammalian tissues.

The invention is based on the observation that the repair cells associated with the wound healing process will naturally proliferate and migrate to the tissue damage site and penetrate the gene activated matrix. Surprisingly, these repair cells, which are usually difficult to successfully transfect both ex vivo and in vivo, are extremely effective in absorbing and expressing DNA when activated for proliferation through wound healing.

Because of this advantage, one or more DNA molecules encoding therapeutic agents can be efficiently transferred to proliferating repair cells. Administration of a gene-activated matrix containing DNA encoding translation products (i.e. therapeutic proteins) or transcription products (i.e. nonsense or ribozyme) within the host mammal at the site of injury. The wound may arise as a result of traumatic tissue injury or, alternatively, due to tissue damage induced by or resulting from surgical procedures.

As the proliferative repair cells migrate and contact the gene activated matrix, they absorb and express the DNA of interest, thereby increasing the amount of therapeutic agent, protein or RNA. The transfected repair cells thus serve as local bioreactors, producing therapeutic agents that affect the local repair environment. For example, growth factors or cytokines produced by transfected repair cells will bind to and stimulate targeted effector cells that express related cell surface receptors, thereby stimulating and enhancing the cascade of physiological events normally associated with wound healing.

Alternatively, the repair cells may absorb and express DNA encoding proteins that inhibit the activity of wound healing antagonists. DNA can also encode nonsense or ribozyme RNA molecules that can be used to inhibit mRNA translation, encoding inflammatory proteins or other factors that inhibit wound healing or cause excessive fibrosis.

The gene activated matrix can be transferred to a patient using various techniques. For example, when stimulating wound healing and regeneration, matrices are transferred directly to the wound site, i.e. broken bone, damaged connective tissue, etc. For use in skin repair, the matrix will be administered locally. For use in organ regeneration, arrays will be surgically placed in a wounded organ.

Since the use of the compositions of the invention is based on the natural migration and proliferation of repair cells to the wound site and penetration into the gene activated matrix located at the wound site followed by DNA absorption, it is understood that the matrices must be transferred to the body site where the healing process the wound was induced.

One particularly important feature of the present invention is that the repair process can be constructed to obtain either scar tissue formation and / or tissue regeneration. For example, overexpression of therapeutic proteins at the site of injury may result in regeneration of damaged tissue without scar formation.

In many cases, e.g., such as bone repair, such regeneration is desirable because the scar tissue is not intended to maintain normal mechanical function. Alternatively, scar tissue formation may be desirable around the suture to hold inherently weak tissue together. Thus, these methods can be used to stimulate wound healing either with or without scar tissue formation depending on the type and level of expression of the therapeutic protein.

Direct transfer of plasmid DNA from the matrix to mammalian repair cells by stimulating wound healing offers numerous benefits. First of all, the ease of producing and purifying DNA construction is in favor of the cost of traditional protein production methods. Secondly, arrays can act as structural constructions,

189 174 which, as such, stimulate cell growth and proliferation. Thus, they facilitate targeting of repair cells for the gene transfer. Thirdly, direct gene transfer can be the preferred method of drug delivery for molecules that normally undergo a complex of biosynthetic processes, or for receptors that must be properly aligned in the cell membrane. These types of molecules will not work properly for external delivery to cells.

Pharmaceutical compositions, including matrices, containing DNA can be used for wound healing. These compositions generally consist of a biocompatible or bone compatible matrix material containing DNA encoding the therapeutic proteins in question.

The invention bypasses the drawbacks specifically associated with current recombinant protein treatments for use in wound healing. First, direct gene transfer is a rational strategy that allows transfected cells to (a) modify the physiological amounts of therapeutic proteins in a tissue or context-specific manner, and (b) deliver this protein to the appropriate signaling receptor on the cell surface under appropriate circumstances. For the reasons described above, exogenous delivery of such molecules is expected to involve significant dosing and delivery problems. Secondly, reproducible administration, although possible, is not necessary with gene activated matrix technology: DNA uptake by the cell can be accurately controlled using well-developed sustained release delivery technologies or, alternatively, the integration of transfected DNA may be associated with long-term expression of recombinant protein.

These methods can be widely used in wounds that involve many different cells, tissues and organs; repair cells of the iamin tissue (Gailet et al., 1994, Curr. Opin. Cell. Biol. 6: 717-725). The invention is presented here in three animal models (canine, rat and rabbit) and five tissues (bone, tendon, connective, blood vessels and skeletal muscles), using three marker genes ((β-galactosidase, luciferase and alkaline phosphatase), three promoter systems (CMV, RSV, LTR and SV40), two types of matrix (biological and synthetic). In all cases, the repair cells that migrated into the gene activated matrix were successfully transfected. In particular, a functional result (bone growth) following gene transfer to repair fibroblasts of plasmid, coding or BMP-4 constructs that acts as a signal transducing switch for osteoblast differentiation and growth is shown (Wozney 1992, Mol. Reprod. Dev. 32 : 160-167; Reddi, 1994, Curr. Opin. Genet. Deve. 4: 737-744) or PTH1-34, which stimulates bone parent cells (Orloff, et al., 1992, Endocrinology 131: 1603-16111 Dempster et al., 1995 Endocrin Rev. 4: 247-250).

5.1 Gene activated matrix

Any biocompatible matrix material containing DNA encoding the therapeutic agent in question, such as a translation product i.e. therapeutic protein or transcription product i.e. nonsense or ribosomal, can be stacked and used in accordance with the invention.

The gene activated arrays of the invention can be derived from any biocompatible material. Such materials may include, but are not limited to, biodegradable or non-biodegradable materials that are part of structures that support cell adhesion and growth, powders or gels. Arrays can be obtained from synthetic polymers or naturally occurring proteins such as collagen, other extracellular matrix proteins or other structural macromolecules.

DNA incorporated into the matrix can encode each of the different therapeutic proteins depending on the intended therapeutic application. Such proteins may include growth factors, cytokines, hormones or all other proteins capable of regulating cell growth, differentiation or physiological function. DNA can also encode nonsense or ribozyme molecules that inhibit protein translation, inhibit wound repair and / or induce inflammation.

The transferred DNA does not have to integrate into the genome of the target cell; indeed, the use of DNA that does not integrate in a gene activated matrix is ​​the preferred form of implementation

189 174 of the present invention. In this way, after the wound healing process is over and when the gene product is no longer needed, the gene product will not be expressed.

Therapy kits containing a biocompatible matrix and DNA will contain pre-formed gene activated matrices, thereby allowing doctors to directly administer the matrix within the body. Alternatively, the kits may contain the components necessary to form a gene activated matrix. In these cases, your doctor may combine the ingredients to form gene activated matrices, which you can then use therapeutically by placing inside the body. Matrices can be used to coat surgical instruments such as sewing materials or implants. The gene activated matrix may contain ready-to-use sponges, tubes, bands, freeze-dried ingredients, gels, patches or powders, and telfa tampons.

5.1.1 Matrix materials

Compositions are made in which DNA, encoding the therapeutic agent of interest, binds or saturates the matrix to form a gene activated matrix. The matrix compositions work (i) to facilitate repair cell ingrowth (targeting); and (ii) to include DNA (delivery). After the gene activated matrix is ​​created, it is stored for future use or placed directly at the site of injury.

The type of matrix that can be used in the compositions, devices and methods of the invention is in fact unlimited and can include both biological and synthetic matrices. The matrix will have all the features usually associated with its "biocompatibility", i.e. it will occur in a form that does not cause adverse, allergic or other non-targeted reactions after administration to the host. Such matrices can be created from both natural and synthetic materials. Matrices may not be biodegradable in cases where it is desirable to leave solid structures in the body; or biodegrade when it is desired to express the therapeutic protein only for a short period of time. Matrices can take the form of sponges, implants, tubes, telfa tampons, bands, bandages, tampons, freeze-dried ingredients, gels, patches, powders or nanoparticles. In addition, arrays can be used for prolonged DNA release over extended periods of time.

The choice of matrix material will vary depending on the particular circumstances and the site of injury being treated. Arrays such as those described in US Patent Application may be used. 5 270 300, incorporated herein by reference. Physical and chemical characteristics, such as biocompatibility, biodegradability, strength, stiffness, coupling properties and even cosmetic characteristics can be considered when selecting the matrix as they are well known to those skilled in the art. Appropriate matrices will both provide the DNA molecule and also act as an in situ construct through which mammalian repair cells can migrate.

If the matrices are to be retained for a long time, non-biodegradable matrices, such as sintered hydroxyapatite, biological glass, clay, other ceramics and metal materials, especially titanium, may be used. A suitable ceramic delivery system is described in patent application 4 596 574, incorporated herein by reference. Biological ceramic materials can be changed in the composition, such as calcium aluminate phosphate; and they can be developed to modify particular physical and chemical characteristics such as pore size, particle size, particle shape and biodegradability. Polymer matrices may also be used, including acrylic ester polymers and lactic acid polymers as described in US Patent Applications 4,521,909 and 4,563,489, each of which is incorporated herein by reference. Particular examples of useful polymers are orthoester, anhydride, propylene co-fumarate polymers or a polymer of one or more γ-hydroxycarboxylic acid monomers, e.g. γ-hydroxyzylic acid (glycolic acid) and / or γ-hydroxypropionic acid (lactic acid).

The biologically compatible matrix can be used in conjunction with orthopedic implants and coupling devices as well as artificial connectors, including implants themselves and functional parts of the implants, such as, for example, surgical threads, pins and the like. It is recommended that the implant's surface or metal surfaces or parts as well as the surface

189 174 titanium coated with a material that has an affinity for nucleic acids, most preferably hydroxyapatite, and then coated metal additionally with the gene or nucleic acid that you want to transfer. The available chemical group of absorption materials, such as hydroxyapatite, can be easily manipulated to control its affinity for nucleic acids, as known by those skilled in the art.

In the preferred embodiments, it is believed that the biodegradable matrix will be almost the most useful. A biodegradable matrix is ​​generally referred to as such, the body can absorb. Potential biodegradable matrices for use in combination with the compositions, devices and methods of this invention include, e.g. biodegradable and chemically defined calcium sulfate, calcium triphosphate, hydroxyapatite, polylactic acid, polyanhydrides, matrices of purified proteins, and semi-purified extracellular matrix compositions.

Other biocompatible and biodegradable polymers that can be used are well known and include, by way of example and not limitation, polyesters such as polyglycolides, polylactides and polylactic polyglycol ("PLGA") copolymers (Langer and Folkman, 1976, Natura 263: 797-800 ); polyethers such as polycaprolactone ("PCL"); polyanhydrides; polyalkyl cyanoacrylates such as n-butyl cyanoacrylate and isopropyl cyanoacrylate; polyacrylamides; poly (orthoesters); polyphosphazenes; polypeptide; polyurethanes and mixtures of such polymers.

It should be understood that, in fact, any polymer that is currently known or will be used later that is suitable for sustained or controlled release of nucleic acids can be used in the present invention.

In preferred embodiments, the biocompatible biodegradable polymer is a copolymer of glycolic acid and lactic acid ("PLGA"), with a ratio between lactic acid / glycolic acid units, from about 100/0 to about 25/75. The average molecular weight ("MW") of the polymer will usually be 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 standard molecular weight polystyrene and internal viscosity is 0.5-10.5.

The length of the period of continuous, sustained or controlled release of nucleic acids from the matrix according to the invention will largely depend on the MW of the polymer and the ratio in the lactic acid / glycolic acid composition. Generally, a higher lactic acid / glycolic acid ratio, such as e.g. 75/25 will provide a longer period of controlled or prolonged release of nucleic acids, while a lower lactic acid / glycolic acid ratio will provide faster release of nucleic acids. Preferably, the lactic acid / glycolic acid ratio is 50/50.

The length of the sustained or controlled release period also depends on the MW of the polymer. Generally, a higher MW of polymer will provide a longer controlled or sustained release period. In the case of matrices providing controlled or sustained release for about three months at a 100/0 lactic acid / glycolic acid ratio, the recommended average MW of polymer is from about 7,000 to 25,000; at 90/10 from about 6,000 to 30,000; and at 80/20 from about 12,000 to 30,000.

Another type of biological material that can be used is small intestinal submucosa (SIS). SIS strain material can be prepared from the jejunum fragment of an adult pig. Isolation and tissue samples can be performed using routine tissue culture techniques such as those described by Badybak et al., 1989 J. Surg. Res. 47 "74-80. SIS material is prepared by removing mesenteric tissue, reversing the fragment, followed by removal of the mucosa and surface submucosa by mechanical abrasion. After the fragment is inverted to its initial orientation, the serosa and muscle layer are rinsed and stored for further use.

Another particular example of a suitable material is fibrous collagen, which can be lyophilized after extraction and partial purification from the tissue, and then sterilized. Arrays can also be made of tendon or skin collagen, which can be obtained from various commercial sources, such as Sigma and Collagen Corporation. Collagen arrays as described in patent applications 4 394 370 and 4 975 527, each of which is incorporated herein by reference, can also be used as matrix material.

189 174

Various collagen materials may also be in the form of mineralized collagen. For example, a fiber collagen implant material called UltraFiber ™, which can be obtained from Norian Corp., (1025 Terra Bella Ave., Mountain View, CA, 94043) can be used to make the matrix. U.S. Patent Application No. 5,231,169, incorporated herein by reference, describes the production of mineralized collagen by forming a calcium phosphate mineral under gentle in situ mixing in the presence of dispersed collagen fibrils. Such a preparation can be used in the context of delivering a nucleic acid fragment to a bone tissue site. Mineralized collagen can be used, e.g., as part of a therapy kit for a gene activated matrix to repair fractures.

At least 20 different forms of collagen have been identified and each of these collagens can be used in the practice of the invention. For example, collagen can be purified from vitreous cartilage that has been isolated from two-joint junctions or growth plates. Type II collagen purified from cartilage s ^^ I ^^ this: in trade and can be purchased, e.g. in Sigma

Chemical Company, St. Louis. Type I collagen from rat tail tendons can be purchased, e.g., from Collagen Corporation. Any form of recombinant collagen that can be obtained from recombinant collagen expressing host cells, including bacterial, yeast, mammalian and insect cells can be used. When collagen is used as the matrix material, it may be beneficial to remove what is termed a "telopeptide" that lies at the end of the collagen molecule and is known to induce an inflammatory response.

The collagen used in the invention may, if necessary, be supplemented with additional mineral components such as calcium, e.g. in the form of calcium phosphate. In this way, it is possible to supplement both native and recombinant types of collagen, by admixture, absorption or, on the other hand, binding of additional minerals.

5.1.2 DNA

The present methods and compositions can utilize many different types of DNA molecules. DNA molecules can include genomic, cDNA, single-stranded DNA, double-stranded DNA, triple-stranded DNA, oligonucleotides and Z-DNA.

DNA molecules can encode a variety of factors that promote wound healing, including cellular, surface-cellular and intracellular RNAs and proteins. Examples of extracellular proteins are growth factors, cytokines, therapeutic proteins, hormones and hormone peptide fragments, cytokine inhibitors, peptide growth and differentiation factors, interleukins, chemokines, interferons, colony stimulating factors and angiogenic factors. Examples of such proteins include, but are not limited to, nadeodrine of TGF-β molecules, including five TGF-β isoforms and bone morphogenesis (BMP) proteins, late TGF-β binding proteins, LTBP; keratinocyte growth factor (KGF); hepatocyte growth factor (HGF); platelet derived growth factor (PDGF); insulin-like growth factor (IGF); basal fibroblast growth factors (FGF-1, FGF-2 etc.); vascular endothelial growth factor (VEGF); factor VIII and factor IX; erythropoietin (EPO); tissue plasminogen activator (TPA); activins and inhibins. Hormones that can be used in the practice of the invention include growth hormone (GH) and parathyroid hormone (PTH). Examples of extracellular proteins are also extracellular matrix proteins such as collagen, laminin and fibronectin. Examples of surface cell proteins include a family of cell adhesion molecules (e.g. integrins, selectins, members of the Ig family such as N-CAM and LI and cadherins); cytokine signaling receptors such as type I and type II TGF-β receptors and the FGF receptor; and non-signaling co-receptors such as betaglican and syndecan. Examples of RNA and intracellular proteins include the signal transducing kinase family, cell skeletal proteins such as talin and vinculin, cytokine binding proteins such as the family of late TGF-β binding proteins and nuclear proteins that act as trans, such as transcription factors and enhancers.

DNA molecules can also encode proteins that block pathological processes, thereby allowing them not to interfere with the natural wound healing process.

Examples of blocking agents include ribozymes that destroy RNA and DNA functions, which, e.g., encode tissue inhibitors of enzymes that destroy tissue integrity, e.g., inhibitors of arthritis associated with metalloproteinases.

189 174

A DNA fragment encoding the protein of interest can be obtained using various molecular biology techniques generally known to those skilled in the art. For example, cDNA or genomic libraries can be screened using primers or probes with sequences based on known nucleotide sequences. You can also use polymerase chain reaction (PCR) to form a DNA fragment encoding the protein of interest. Alternatively, the DNA fragment can be obtained from a commercial source.

Genes having sequences that differ from those described in the literature are also within the scope of the invention, as long as the changed or modified gene still encodes a protein that acts to stimulate wound healing in any direct or indirect manner. These sequences include those that caused point mutations, those that resulted from the degeneracy of the genetic code, or naturally occurring allelic variants, as well as modifications introduced by genetic constructs, i.e., the hand of man.

Techniques for introducing changes to nucleotide sequences intended to alter the functional properties of encoded proteins or polypeptides are well known in the art. Such modifications include base deletion, insertion or substitution that results in a change in the amino acid sequence. Changes can be made to increase the activity of the encoded protein, to increase its biological stability or half-life, to change its glycosylation pattern, make it sensitive to temperature, or change the pattern of protein expression and the like. All such modifications to the nucleotide sequence are within the scope of this invention.

DNA encoding interesting translation or transcription products can be recombined into various vector systems that provide large scale DNA replication to generate gene activated arrays. These vectors may contain the necessary elements to direct the transcription and / or translation of the DNA sequence absorbed by the repair cells in the wound in vivo.

Vectors that can be used include, but are not limited to, derived from recombinant phage DNA, plasmid DNA or cosmid DNA. For example, plasmid vectors such as pBR322, pUC 19/18, pUC 118, 119 and series of Ml 3 mp vectors can be used. Bacteriophage vectors may include ygt10, ygtłi, ygt18-23, yZAP / R, and a series of EMBL bacteriophage vectors. Cosmid vectors that can be used include, but are not limited to, pJB8, pCV 103, pCV 107, pCV 108, pTM, UMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16, and a series of charomid 9 vectors. Vectors that allow RNA transcription in in vitro, such as SP6 vectors, can also be used to produce large amounts of RNA introduced into the matrix. Alternatively, recombinant viral vectors may be constructed, including but not limited to, viruses such as herpes virus, vaccinia virus, adenoviruses, adeno-associated viruses or bovine papilloma virus. Although integrating vectors can be used, non-integrating systems that do not transfer the gene product to daughter cells for many generations are recommended for wound healing. In this way, the gene product is expressed during the wound healing process and when the gene is thinned in posterity, the expression level of the gene product is reduced.

Methods well known to those of skill in the art can be used to build expression vectors containing the protein coding sequence operably associated with the appropriate transcription / translation control signals. These methods include in vitro recombinant DNA techniques and synthetic techniques. See, e.g., the techniques described in Sambrook et al., 1992, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, NY and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates & Wiley Interscience, NY,

Genes encoding the proteins of interest can be operably linked to various promoter / enhancer elements. The elements of expression of these vectors can change their strength and specificity. Depending on the host / vector system used, any of the numerous transcription and translation elements may be used. The promoter may be in the form of a promoter naturally associated with the gene of interest. Alternatively, the DNA can be set up under the control of a recombinant or heterologous promoter, i.e. a promoter that is not normally associated with this gene. For example, it can be used

189 174 tissue-specific promoter / enhancer elements for regulating the expression of transferred DNA in specific cell types.

Examples of transcriptional control regions that exhibit tissue specificity that are described and could be used include, but are not limited to: the elastase I gene control region that is active in acinar cells of the pancreas (Swift et al., 1984, Cell 38: 639-646 ; Omitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50: 399-409; MacDonald, 1987, Hepatology 7: 42S-52S); insulin gene control region that is active in pancreatic beta cells (Hanahan 1985, Nature 315: 115-122); immunoglobulin gene control region active in lymphoid cells (Grosschedl et al., 1984, Cell: 38: 647-658; Adams et al., 1985, Nature 318: 533-538; Alexander et al., 1987, Mol. Cell. Biol . 7: 1436-1444); albumin gene control region active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5: 1639-1648; Hammer et al., 1987, Science 235: 53-58); the alpha-1-antitrypsin gene control region active in the liver (Kelsey et al., 1987, Genes and Devel 1: 161-171); beta-globin gene control region active in bone marrow cells (Magram et al., 1985, Nature 315: 338-340; Kollias et al., 1986, Cell 46: 89-94); myelin base protein gene control region active in brain oligodendrocyte cells (Readhead et al., 1987, Cell 48: 703-712); myosin light chain-2 gene control region active in skeletal muscle (Shani 1985, Nature 314: 283-286); and the gonadotropic hormone releasing hormone releasing gene control region (Mason et al., 1986, Science 234: 1372-1378). Promoters isolated from the genome of viruses that have grown in mammalian cells (e.g., RSV promoters, vaccinia virus 7.5K, SV40, HSV, MLP MMTC LtR and CMV adenoviruses) as well as promoters produced by recombinant DNA techniques or synthetically may be used.

In some cases, the promoter elements may be constitutive or inducible promoters and may be used under appropriate conditions to direct high-level or regulated expression of the gene of interest. Gene expression under the control of constitutive promoters does not require the presence of a specific substrate to induce gene expression and will occur under all cell growth conditions. In contrast, the expression of genes controlled by inducible promoters responds to the presence or absence of an inducing agent.

Specific initiation signals are also required for sufficient translation of the inserted protein coding sequence. These signals include the ATG initiation codon and adjacent sequences. In cases where the entire coding sequence has been inserted into the respective expression vectors, including the initiation codon and adjacent sequences, no additional translation control signals are needed. However, in cases where only part of the coding sequence has been inserted, exogenous translation control signals must be provided, including the ATG initiation codon. In addition, the initiation codon must be in phase with the reading frame of the protein coding sequences to ensure translation of the entire insert. These exogenous translation control signals and initiation codons can be of various origins, both natural and synthetic. Efficacy and expression control can be enhanced by introducing translation weakening sequences, enhancer elements etc.

In addition to DNA sequences encoding therapeutic proteins of interest, the use of ribozymes or DNA nasal molecules that can be transferred to mammalian repair cells is within the scope of the present invention. Such ribozymes and nonsense molecules can be used to inhibit the translation of RNA, encoding proteins, in genes that inhibit the disease process or wound healing process, thereby allowing tissue repair.

Expression of nonsense RNA molecules will work to directly block mRNA translation by binding to targeted mRNA and preventing protein translation. Expression of ribozymes, which are enzymatic RNA molecules capable of catalyzing specific RNA cleavage, can also be used to block protein translation. The mechanism of ribozyme action involves hybridization specific to the sequence of the ribozyme molecule with complementary target RNA followed by endonucleolytic cleavage. The scope of the invention includes a constructed hammerhead motif of a ribozyme molecule that specifically and effectively catalyzes endonucleolytic cleavage of RNA sequences. RNA molecules can be created by transcription of a DNA sequence coding for an RNA molecule.

189 174

Multiple genes may also be used, linked on a single genetic construct under the control of one or more promoters, or generated as separate constructs of the same or different type. Thus, almost endless combinations of different genes and genetic constructs can be used. Certain gene combinations may be intended, or their use may, on the other hand, result in synergistic effects of cellular stimulation and regeneration, each and all such combinations are intended to be within the scope of the present invention. Indeed, many synergistic effects have been described in the scientific literature so that one skilled in the art will easily identify likely synergistic combinations of genes or even gene-protein combinations.

5.1.3 Production of gene activated matrices

In preferred embodiments, the matrix or implant material contacts the DNA encoding the therapeutic product of interest by saturating the matrix material in the recombinant DNA mother liquor. The amount of dNa and the amount of contact time required to introduce DNA into the matrix will depend on the type of matrix used and can easily be determined by a skilled person without unnecessary experimentation. Alternatively, DNA can be encapsulated within a matrix of synthetic polymers, such as e.g. polylactic polyglycolic acid block copolymers (see Langer and Folkman, 1976, Nature 263: 797-800, which is incorporated herein by reference). Again, these parameters can easily be determined by a skilled person without unnecessary experimentation. For example, the amount of DNA construction used for an array can be determined taking into account various biological and medical factors. The individual gene, matrix, wound site, mammalian host age, sex and diet should be considered, as well as all additional clinical factors that may affect wound healing, such as serum levels of various factors and hormones.

In additional embodiments, both biological and synthetic matrix and DNA compositions can be lyophilized together to form a dry pharmaceutical powder. The gene activated matrix can be rehydrated prior to implantation into the body, or alternatively the gene activated matrix can naturally hydrate when placed in the body.

In some cases, medical devices such as implants, sutures, wound dressings etc. may be coated with the nucleic acid compositions using conventional coating techniques well known in the art. Such methods are, for example and without limitation, immersing the device in a nucleic acid composition, applying the nucleic acid composition with a brush, and / or spraying the device with an aerosol composition of said nucleic acids. The device is then dried, either at room temperature or by means of a drying oven, optionally under reduced pressure. The recommended method of coating seams is given in the examples.

For sutures coated with a polymer matrix containing plasmid DNA. Applicant has found that applying a coating composition containing a total amount of about .01 to 10 mg plasmid DNA, and preferably about 1-5 mg plasmid DNA, on a 70 cm long seam using about 5-100, preferably about 5-50, and more preferably about 15-30 coating applications, gave a therapeutically effective and homogeneous coating.

In this way, coated sutures, in particular sutures coated with a polymer matrix containing nucleic acids, encoding therapeutic proteins that stimulate wound healing in vivo can be formed.

Seams that can be coated according to the method described above include any seam of natural or synthetic origin. Typical seam material is, for example and without limitation, silk; Cotton; flax; polyolefins such as polyethylene and polyesters such as polyethylene terephthalate; homopolymers and copolymers of hydroxycarboxylic acid esters; collagen (ordinary or chromed); catgut (plain or chromed); and seam substitutes such as cyanoacrylates. Seams can take any convenient form, such as weaves or twists, and can have a wide range of sizes, as is commonly used in the art.

The benefits of coated sutures, especially sutures coated with a polymer matrix containing nucleic acids, encoding therapeutic proteins that stimulate wound healing, actually cover all areas of surgical applications in humans and animals.

189 174

5.2 Applications of gene activated matrix

Biologically compatible matrix can be used in many different wound healing situations in human medicine. These include, without limitation, bone repair, tendon repair, joining, repair, repair of blood vessels, skeletal muscle repair and skin repair. For example, using gene-activated matrix technology, cytokine growth factors produced by transfected repair cells will affect other cells in the wound by binding to cell surface signaling receptors, thereby stimulating and enhancing the cascade of physiological events normally associated with wound healing. The end result is increased tissue repair and regeneration.

The clinical goal may also be to block the disease process, thereby allowing natural healing of the tissue, or when the goal is to replace a genetically defective protein function.

Wounds may arise as a result of traumatic injury, or alternatively damage to tissue induced or resulting from a surgical procedure. The gene activated matrix can be transferred to a patient using various techniques. For example, matrices can be transferred directly to the site of injury with the hands of a physician, either in the form of a therapeutic implant or in the form of a coated tool (e.g. suture, tube used for skin transplants, coated tool, etc.). Arrays can be administered topically, also surgically placed in place of normal tissue to treat diseased tissue located at some distance.

The wound healing process is a coordinated sequence of events that include bleeding, clot formation, clot dissolution by competitive removal of damaged tissue, and the deposition of zaminam tissue as initial repair material. Iamin tissue is a mixture of fibroblasts and capillary blood vessels. The wound healing process involves various cell populations, including endothelial cells, stem cells, macrophages and fibroblasts. Regulatory factors associated with wound healing are known as systemic hormones, cytokines, growth factors, extracellular matrix proteins and other proteins that regulate growth and differentiation.

The methods of DNA transfer and matrix compositions of the invention will have a number of uses as a method of providing a drug to stimulate tissue repair and regeneration in various types of tissues. These include, but are not limited to bone repair, skin repair, connective tissue repair or regulation of angiogenesis and / or angiogenesis. The use of gene activated matrices may also take place in the treatment of patients with impaired healing ability, resulting e.g. from the effect of age or diabetes. Matrices can also be used to treat wounds that heal slowly for natural reasons, e.g. in the elderly and those who do not respond to existing therapies, i.e. those with chronic skin wounds.

An important feature of the present invention is that scar tissue formation can be regulated by controlling the expression level of therapeutic proteins. In cases such as the treatment of burns or connective tissue damage, it is particularly desirable to inhibit the formation of scar tissue.

Therapeutic methods may also include matrix transplants containing the DNA of interest to the host. Matrix transplant procedures may include surgical placement or injection of the matrix into the host. In cases where the matrices are injected, they are drawn into the syringe and injected into the patient at the site of injury. Multiple injections can be made in the area of ​​injury. Alternatively, arrays can be surgically placed at the wound site. The amount of matrix needed to achieve the purpose of the present invention, i.e. stimulation of wound healing and regeneration, varies depending on the size, age and weight of the host.

An essential feature of the invention is that regardless of when the gene activated matrix is ​​transferred to the host, whether by injection or surgery, local tissue destruction is sufficient to induce wound healing. This is a necessary condition for the induction of migration and proliferation of targeted mammalian repair cells to the site of the gene activated matrix.

189 174

Specific implementation forms are described in the following sections.

5.3 Bone regeneration

Bone has significant regenerative ability after a fracture. A comprehensive but serial sequence of fracture repair includes stopping bleeding, dissolving the clot, ingrowth of granulation tissue, scar formation and sculpting again for optimal structure (AW Ham., 1930, J. Bone Joint Surg. 12, 827-844). The cells involved in this process are platelets, inflammatory cells, fibroblasts, endothelial cells, pericytes, osteoclasts and ancestors of osteogenesis. Recently, several peptide growth and differentiation factors have been identified that have proven to control cellular events associated with bone formation and repair (A.Erlebacher, et al., 1995 Cell 80, 371-378). For example, bone morphogenesis proteins (BMPs) are soluble extracellular factors that control the destination of osteogenesis cells: BMP genes normally expressed by cultured fetal osteoblasts (SEs) Harris et al., 1994, J.Bone Min.Res 9, 389-394) and by osteoblasts during the formation of the mouse embryo skeleton (KM Lyons et al., 1989, Genes Dev. 3, 1657-1668; KM Lyons et al. ., 1990, Development 190, 833-844; MC Jones et al., 1991, Development 111, 531-542), recombinant BMP proteins initiate differentiation of cartilage and bone progenitor cells (A. Yamaguchi et al., 1991, J. Cell Biol. 113, 681-687; M. Ahrens et al., 1993, J. Bone Min. Res. 12, 871-880; SE Gitelman et al., 1994, J. Cell. Biol. 126, 1595-1609; V. Rosen et al., 1994, J.Cell.Biol.i27, 1755-1766), the delivery of recombinant BMPs induces a formation sequence similar to intra-cartilage bone formation (JMWozney 1992, Mol. Reprod. Dev. 32, 160-167; AH Reddi, 1994 Curr. Opin. Genet. Dev. 4, 737-744), and BMP-4 gene expression is not regulated early in the fracture repair process (T. Nakase et al., 1994, J. Bone Min. Res. 9 651-659). Osteogenic protein-1, a member of the family of BMP-related molecules (E. Ozkaynak et al., 1990 EMBO J. 9, 2085-2093) is capable of similar effects in vitro and in vivo (TKSampath et al. (1994) J. Bone Joint Surg. 76-A, 827-838). TGF-β has also been shown to stimulate cartilage and bone formation in vivo (M. Centrella et al., 1994, Endocrine Rev. 15, 27-38; DR Sumner et al., 1995, J. Bone Joint Surg. 77A, 1135 -1147). Finally, parathyroid hormone (PTH) is a 84 amino acid hormone that raises Ca levels¹ in plasma and extracellular fluid. In skeletal tissues, sporadic administration of a PTH fragment having structural requirements for biological activity (amino acids 1-34) gives a real anabolic effect: numerous in vivo and in vitro studies provide strong evidence that administration of PTH 1-34 to animals (including rats) gives the result of unconjugated, high-quality bone formation due to the combined inhibitory effect on osteoclasts and the stimulating effect on osteogenesis cells (DW) Dempster et al., 1993, Endocrine Rev. 14, 690-709). PTH1 peptide is known to interact synergistically with BMP-4 which upregulates the expression of functional PTH receptors on the cell surface in differentiating osteoblasts in vitro (M. Ahrens et al., 1993, J. Bone Min. Res. 12, 871- 880).

Like recombinant proteins, peptide growth and differentiation factors such as BMP and TGF-β are promising therapeutic alternatives for fracture repair (JM Wozney, 1992, Mol. Reprod. Dev. 32, 160-167; AH Reddi, 1994, curr. Opin Genet. Dev. 4, 737-744; M. Centrella et al., 1994, Endocrine Rev. 15, 27-38; DR Sumner et al., 1995, J. Bone Joint Surg. 77A, 1135-1147). However, relatively large doses (micrograms) are required to stimulate significant bone formation in animals, raising the question that future human therapies may be expensive and may have an increased risk of toxicity.

Gene activated matrices are surgically implanted into a 5 mm bone incision site in a rat, a comprehensive, non-healing fracture model in man. The present inventors have found that gene transfer to repair cells in a bone incision gap can easily be achieved.

Defects in the process of bone repair and regeneration are associated with significant complications in clinical orthopedic practice, e.g. fibrous failure to connect after fracture, inability to connect the implant and failure of large allogeneic transplants. Many complex fractures are currently treated with autogenous transplants but this technique is not effective and involves complications.

Naturally, any new technique designed to stimulate bone repair would be a valuable tool in treating bone fractures. Much of the broken bones are still healed by immobilization, allowing the injury to be repaired by the influence of natural mechanisms. Although fracture treatment has advanced in recent years, including improvement of tools, development of new processes, stimulating or complementing injury repair mechanisms, it would be a significant advance in this field.

A biocompatible matrix can be used to transfer the bone growth gene to promote fracture repair. Other important aspects of this technology include the use of gene transfer to treat patients with "weak bones" as in diseases such as osteoporosis; to improve poor healing, which can occur for various reasons, e.g. fibrous non-fusing; to promote implant integration and the operation of artificial connectors; for stimulating healing of other skeletal tissues such as Achilles tendons; and as an adjuvant to repair large cavities.

It is known that bone tissue has the ability to repair and regenerate, and the cellular and molecular basis of these processes are understood. The initiation of new bone formation includes stimulation, clonal expansion, and repair cell differentiation. Once initiated, bone formation is stimulated by various polypeptide growth factors. The newly formed bone is then maintained by a series of local and systemic growth and differentiation factors.

Recently, several genes of bone morphogenetic proteins have been cloned (Wozney et al., 1988; Rosen et al., 1989, Connect. Tissue Res., 20: 313: 319; discussed in Alper, 1994) and thanks to this work it was established that BMP are members transforming growth factor-β (TGF-β) superfamily based on DNA sequence homology. Cloning of different BMP genes led to the determination of individual BMP genes and proteins such as BMP-1 to at least BMP-8. BMP 2-8 is generally considered osteogenic, although BMP-1 may be a more general morphogen; Shimell et al., 1991, Cell, 67: 469-481). BMP-3 is also called osteogen (Luyten et al., 1989, J. Biol. Chem. 264: 13377-13380), and BMP-7 is also called OP-1 (Ozkaynak et al., 1990, EMBO J. 9 : 2085-2093). Each of tGf and BMP acts on cells through comprehensive tissue-specific interactions with surface-cell receptor families (Roberts and Sporn, 1989, MB Sporn and AB Roberts, Ed., Springer-Verlag, Heidelberg, 95 (part 1); Aralkar et al., 1991).

Transforming growth factors (TGFs) have also been shown to play a central role in regulating tissue healing by affecting cell proliferation, gene expression and matrix protein synthesis (Roberts and Sporn, 1989, MB Sporn and AB Roberts, Ed., Springer-Verlag. Heidelberg, 95 (part 1)). For example, TGF ^ i TGF ^ 2 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).

Other growth factors / hormones in addition to TGF and BMP can be used in the practice of the invention to affect new bone formation after fracture. For example, fibroblast growth factor injected into the fracture site of a rat (Jingushi et al., 1990) at many high doses (1.0 mg / ml) gives a significant increase in cartilage tissue in the fracture gap, while low doses have no effect.

Calcium regulating hormones such as parathyroid hormone (PTH) may also be used. PTH is a calcium-regulated hormone with 84 amino acids, whose main task is to increase the concentration of Ca '*' in plasma and extracellular fluid. Undamaged PTH has been shown to stimulate reabsorption in organ culture over 30 years ago and the hormone is known to increase the number and activity of osteoclasts. Studies on the native hormone and synthetic peptides have revealed that the amino terminus of the molecule (amino acids 1-34) contains structural requirements for biological activity (Tregear et al., 1973; Hermann-Erlee et al., 1976, Endocrine Research Commnications. 3: 21 -35; Riond, 1993, Clin. Sei. 85: 223-228).

Gene activated arrays are surgically implanted into the bone fracture site. Such surgical procedures may include direct injection of GAM to the fracture site, surgical repair of a complex fracture or arthroscopic surgery. In cases where activated matrix is ​​used to repair broken bone

189 In a gene, mammalian repair cells will naturally migrate and proliferate to the bone damage site.

The present inventors have surprisingly found that it is easy to obtain gene transfer to repair cells in regenerative tissue in a bone incision gap. Currently, recommended methods for obtaining gene transfer generally involve the use of fibrous collagen implant material saturated in DNA solution shortly before placing it in a place where bone growth is desired, or the use of a plasmid DNA preparation encapsulated in a synthetic matrix, such as a PLGA block copolymer. As the research presented here shows, the implant material facilitates the intentional uptake of exogenous plasmid constructions by cells in the bone incision gap that clearly participate in bone regeneration / repair. Transgenes, after uptake by cells, direct the expression of recombinant polypeptides, as demonstrated by the in vivo expression of functional marker gene products.

Further research is presented here showing that osteotropic gene transfer results in cellular expression of a recombinant osteotropic molecule whose expression is directly associated with stimulation of new bone formation. Specifically, a gene transfer vector encoding BMP-4 and a gene transfer vector encoding a human PTH1-34 fragment, alone or in combination, will stimulate new bone formation. Given the relatively large number of candidate genes, the gene transfer vector encoding the human parathyroid hormone fragment, hPTH1-34, will stimulate new bone formation in Sprague-Dawley rats, indicating that the human peptide can bind efficiently to the PTH / PTHrP receptor on cell surface of rat osteoblast.

5.4 Soft tissues

A gene activated matrix can also be used to stimulate the growth and regeneration of soft tissues, such as connective, tendon, baptism and skin. Destruction of skeletal connective tissue due to traumatic injury can be treated using matrices containing genes encoding various growth factors.

Connective tissue normally consists of cells and an extracellular matrix organized in characteristic tissue architecture. Tissue injury can disrupt this architecture and stimulate the wound healing response. The methods of the present invention are particularly suitable for stimulating the growth and regeneration of connective tissue as it is important that damaged connective tissue regenerates without scar tissue formation, since scar tissue can interfere with the normal mechanical functions of connective tissue.

Various growth factors can be used to promote connective tissue repair. They are, but are not limited to, members of the TGF-β superfamily (e.g., TGF-β alone) who stimulate the expression of genes encoding extracellular matrix proteins, and other cytokines such as EGF and PDGF. Examples of other genes that can be used are (a) cytokines such as peptide growth and differentiation factors; (b) angiogenic factors such as FGF and VEGF; (c) extracellular matrix proteins such as collagen, laminin and fibronectin; (d) a family of cellular adhesion molecules (e.g. integrins, selectins, members of the Ig family (such as N-CAM and LI, and cadherins); (e) surface-cell cytokine signaling receptors such as type I and type II TGF-β receptors; (f) non-signaling co-receptors such as betaglycan and syndecan; (g) a family of signal transducing kinases; (h) cellular backbone proteins such as talin and vinculin (i) cytokine binding proteins such as the family of late TGF-β binding proteins; and (j) nuclear proteins that act as trans, such as transcription factors.

Once formed, such matrices can then be placed in the mammalian host in the wounded area of ​​the connective tissue. Gene activated arrays can be injected directly into the connective tissue damage area.

Alternatively, surgical techniques such as arthroscopy can be used to deliver the matrix to the connective tissue wound area.

5.5 Regeneration of the organ

Gene activated arrays can also be used to stimulate the repair and regeneration of organ tissue. You can also treat organ damage caused by traumatic injury or surgery. In the case of the liver, it can be damaged by excessive consumption

189 174 alcohol or by various types of infections with various types of infectious agents, such as the hepatitis virus family. Normal kidney function may be similarly affected by damage caused by kidney disease. The mucous membranes of the esophagus, stomach and duodenum may contain ulcers caused by acid and pepsin in gastric juices. Ulcers may also arise as a result of colonization of gastric mucosa with Helicobacter pylori. These organs and diseases only serve as examples, in fact the methods of the invention can be used to treat diseases or to stimulate organ regeneration in any organ in the body.

Arrays containing DNA encoding cytokines that stimulate cell proliferation and differentiation and / or regulate tissue morphogenesis can be transplanted to the appropriate site of the organ. Such factors may include, but are not limited to, the transforming growth factor protein family of platelet-derived growth factors (PDGF), insulin-like growth factor (IGF) and fibroblast growth factor (FGF). In some cases, it may be useful to express growth factors and / or cytokines that stimulate the proliferation of organ-specific cell types, i.e. hepatocytes, kidney or heart cells, etc. For example, expression of hepatocyte growth factor may stimulate healing of the wound in the liver. For the treatment of ulcers resulting from Helicobacter infection, gene-activated arrays may contain DNA encoding antimicrobial proteins.

Gene-activated arrays can be surgically implanted in an organ that is undergoing treatment. Alternatively, surgical laparoscopy procedures can be used to transfer gene activated matrices into the body. In cases of treatment in response to tissue damage, the natural healing process will stimulate the migration and proliferation of repair cells into transplanted matrices. Alternatively, where gene activated matrices are transferred to organs that have not been damaged, e.g., when the matrices are implanted to express therapeutic proteins not related to wound healing, the wound healing process can be stimulated by inducing tissue damage.

5.6 Regulation of angiogenesis

The present invention is used to regulate the formation and spread of blood vessels or, respectively, angiogenesis and angiogenesis. Both these physiological processes play an important role in wound healing and organ regeneration.

Initially, at the site of injury, the iamin tissue accumulates, which is a mixture of collagen, matrix and blood vessels, and provides wound strength during tissue repair. The formation of new blood vessels involves the proliferation, migration and penetration of vascular endothelial cells and is known to be regulated by various polypeptide growth factors. Several polypeptides have been identified that have endothelial cell growth promoting activity, including acid and basic fibroblast growth factors (FGF), vascular endothelial growth factor (VEGF), and platelet derived growth factor (PDGF).

In order to stimulate the formation and spread of blood vessels, DNA encoding such growth factors can be introduced into the matrix and these matrices implanted into the host. In some cases, it may be necessary to induce wound healing by tissue damage.

It may be desirable to inhibit the proliferation of blood vessel formation, such as angiogenesis associated with the growth of solid tumors, which involves vascularization for growth. Tumor angiogenesis can be inhibited by transferring DNA encoding negative angiogenesis inhibitors such as thrombospondin or angiostatin. In specific embodiments, the DNA encoding e.g. thrombospondin or angiostatin can be introduced into the matrix after the matrix has been implanted in the patient at the tumor site.

5.7. Skin repair

The gene activated matrix can also be used to stimulate the growth and repair of skin tissues. In wounds that involve damage to skin areas, and especially for severe burns, it is important that the skin grows very quickly to prevent infection, reduce fluid loss, and reduce the area of ​​potential scarring. Skin damage resulting from burns, punctures, cuts and / or abrasions can be treated using gene activated matrices. You can also treat skin conditions such as psoriasis,

189 174 atopic dermatitis or skin damage resulting from fungal, bacterial and viral infections, treatment of skin cancers such as melanoma.

Arrays containing DNA encoding cytokines that stimulate the proliferation and differentiation of skin cells, including central primary stem cells, keratinocytes, melanocytes, Langerhans cells and Merkel cells, can be used to treat skin damage and diseases. The gene-activated matrix serves two purposes, protecting the wound against infections and dehydration, and supplying DNA that captures repair cells. The gene activated arrays of the invention may include skin patches, corpse skin, bandages, swabs, collagen skeletons, such as those described in US Patent Application No. 4,505,266 or US 4,485,097, topical creams or gels. Before applying the matrix to the wound site, you can remove damaged skin or necrotic tissue. DNA introduced into the matrix can encode various growth factors, including keratinocyte growth factor (KGF) or epidermal growth factor (EGF). DNA encoding lL-1, which is known to be a strong inducer of epithelial cell migration and proliferation as part of the healing process, can also be introduced into the matrix of the invention.

6. Example: Implant material for use in gene transfer to bone

Many implant materials can be used to transfer genes to a bone repair and / or regeneration site in vivo. These materials are saturated in the solution containing the DNA or gene to be transferred to the secondary bone growth site. Alternatively. DNA can be inserted into the matrix, which is the recommended way to do it.

A particular example of a suitable material is fibrous collagen, which can be freeze-dried after extraction and partial cleansing from tissue and then sterilized. Another particularly recommended collagen is type II collagen, with the most preferred collagen being either recombinant type II collagen or mineralized type II collagen. Before placing in places of damage, implant materials are saturated in DNA (or virus) solutions under sterile conditions. Saturation can last for any suitable and convenient period, e.g. from 6 minutes to the whole night. The DNA solution (e.g. plasmid) will be a sterile aqueous solution, such as sterile water or an acceptable buffer, at a concentration of generally 0.5-1.0 mg / ml. Currently recommended plasmids are pGL2 (Promega), pSV4p-gal, pAd.CMVlazZ and pcDNA3.

7. Example: In vivo protein detection after transgene expression

7.1. Beta-galactosidase β-galactosidase transgene can be detected by immunohistochemistry. Bone incision tissue samples were fixed in Bouins fixative, demineralized and then divided in half along the longitudinal plane. One half of each sample was embedded in paraffin for subsequent identification of bacterial β-galactosidase immunohistochemical protein.

For immunohistochemistry, cross sections (2-3 mm thick) were transferred to poly-L-lysine-coated microscope slides and fixed in acetone at 0 ° C for at least 20 minutes. Cross-sections are hydrated in PBS. Endogenous peroxidase activity was neutralized by immersion of tissue sections in 0.1% hydrogen peroxide (in 95% methanol) at room temperature for 10 minutes and the neutralized sections washed 3x in PBS. In some cases, cranial vault sections were demineralized by immersion in 4% EDTA, 5% polyvinyl pyridone and 7% sucrose, pH 7.4, for 24 hours at 4 ° C. Demineralized sections were washed 3x before applying to the antibodies. Primary antibodies without dilution were used as hybridoma supernatant. Purified antibodies were applied to tissue sections at a concentration of 5 mg / ml. Primary antibodies were detected with biotinylated anti-mouse rabbit IgG and peroxidase conjugated streptavidin (Zymed Histostain-SPkit). After peroxidase staining, the sections were counterstained with hematoxylin.

Bacterial β-gal was also detected by substrate consumption assays using commercially available kits (e.g. Promega) according to the manufacturer's instructions.

7.2. Luciferase transgene

Luciferase was detected by substrate consumption assays using commercially available kits (e.g. Promega) according to the manufacturer's instructions.

189 174

7.3. PTH transgene

Recombinant PTH, such as hPTHl-34 peptide, was assayed in bone lesion tissue homogenates, e.g., using two commercially available radioimmunoassay kits, according to manufacturers' protocols (Nichols Institute Diagnostics, San Juan Capistrano, CA).

One set is the Intact PTH-Parathyroid Hormone 100T Kit. This radioimmunoassay utilizes the carboxy-terminal antibody of the intact hormone and is used to measure endogenous hormone levels in bone tissue gap fracture. This assay can be used to establish a baseline value for PTH expression in a rat bone incision model.

The second set is a double-sided immunoradiometric kit for measuring rat PTH. This kit uses the affinity of purified antibodies specific for the amino terminus of the unnatural rat hormone (PTH 1-34) and will thus measure the production of endogenous PTH as well as recombinant protein. Previous studies have shown that these antibodies cross-react with human PTH and that recombinant molecules can be recognized in vivo.

The values ​​obtained for kit # 1 (carboxy-terminal antibody) were subtracted from the values ​​obtained for kit # 2 (amino-terminal antibody) to obtain accurate and sensitive measurements.

The level of recombinant peptide was thus correlated with the degree of new bone formation.

7.4 BMP transgen

BMP proteins, such as the murine BMP-4 peptide product, were detected by immunohistochemistry using a specific antibody that recognizes the HA epitope (Majmudar et al., 1991, J. Bone and Min. Res. 6: 869-881), such as an antibody monoclonal available at Boehringer-Mannheim. Antibodies to BMP proteins alone can also be used. Such antibodies, together with various immunoassay methods, are described in US Patent Application No. 4,857,456, which is incorporated herein by reference.

Bone incision tissue samples were fixed by Bouins fixation, demineralized and then split in half along the longitudinal plane. One half of each sample was embedded in paraffin for subsequent immunohistochemical identification of the recombinant mouse BMP-4 molecule.

8. Example: Transfer of the osteotropic gene stimulates bone regeneration / repair in vivo.

The following experiment was to investigate whether gene transfer could be used to form 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. During the dissection, the bone incision site was carefully stratified for histomorphometric analysis. The AP and ML dimensions of scar tissue were measured using a calipers.

The samples were then fixed by immersion in Bouins fixative, washed in ethanol and demineralized in buffered formic acid. Deposition of the decalcified material in plastic was used because of the dimensional stability of methacrylate during sample preparation and cutting.

Tissue blocks were dehydrated in increasing alcohol concentration and deposited. Sections 5 mm thick were cut in the coronary plane using a Reichert Polycot microtome. Sections were taken from the inside through the width of the medullary canal to protect sample randomness. Sections for light microscopy were stained using Goldner's three-color staining to differentiate bone, osteoid, cartilaginous and fibrous tissue. Sections were covered with Eukitta embedding medium (Calibrated Instruments, Ardsley, NY). Histomorphometric analyzes in the bright field were carried out using a Nikon Optiphot Research microscope and standard stereological techniques for point counting using a 10 mm x 10 mm eyepiece with a grille.

The total area of ​​the scar tissue at 125x magnification was measured as an index of the total intensity of the healing reaction. Fractions of the bone, cartilage and fibrous tissue area were measured at 250x magnification to examine the relative contribution of each tissue to scar formation. Because the dimensions of the bone incision gap reflect the baseline

189 174 (time 0), bone area measurement was used at successive intervals to determine the rate of bone filling. Statistical significance was estimated using analysis of variance, comparing between post-hoc groups using the Tukey interval test based on Student's t-distribution.

In the 5 mm wide bone incision model described above, it was found that expression of the PTH transgene can stimulate bone regeneration / repair in a live animal. This is a particularly important finding as it is known that hPTHl-34 is a more potent anabolic agent when administered intermittently as opposed to continuous, and is a continuous type delivery that results from the gene transfer methods used herein.

9. Example: Direct gene transfer to regenerated bone in vivo

Gene activated matrices containing mammalian expression plasmid DNA were implanted into large segmental gaps formed in the females of an adult male. Implantation of gene activated matrices containing p-galactosidase or luciferase plasmids led to DNA uptake and functional expression of the enzyme by repair cells growing into the gap. In addition, the implantation of a gene activated matrix containing either a bone morphogenetic protein plasmid or a plasmid encoding a parathyroid hormone fragment (amino acids 1-34) results in a biological response filling the fracture with new bone. Finally, implantation of a dual-plasmid gene-activated matrix encoding bone morphogenetic protein 4 and a parathyroid hormone fragment, which have been found to act synergistically in vitro, resulted in faster new bone formation than with both factors alone. These studies show for the first time that repair cells in bone can be genetically manipulated in vivo. Although it serves as a useful tool for studying fibroblast repair biology and wound healing responses, the gene activated arrays of the present invention also have broad therapeutic applications.

9.1 Materials and methods

9.1.1. Mammal host model

To create a 5 mm wide incision, four 1.2 mm diameter pins were screwed into the femoral shaft of adult Sprague-Dawley rats in general dormant and constant flushing. A parallel placement of the pins was performed using the surgical matrix, which was confirmed by fluorography (the pins were located 3.5 mm from the edge of the fixation site and 2.5 mm apart). Then the external fixation site (30 x 10 x 5 mm) was secured on pins. External fixation plates were made of aluminum alloy on a CNC mill to ensure high tolerance. The prefabricated fasteners with washers and threaded pins are made of stainless steel. All parts of the immobilization were sterilized prior to the operation with ethylene oxide gas. 5 mm wide segmental defects were created in the center of the bone shaft using a Hall Micro 100 oscillating saw (Zimmer Inc., Warsaw, IN). Collagen sponges were placed and held in the incision gap until they were surrounded by clotted blood; preliminary research showed that this maneuver attached the sponge to the incision site. The skin incision was closed with staples. The immobilization provided the necessary stability so that the walking of the mammal host was unlimited for a period of several weeks.

9.1.2. immunohistochemistry

Tissues for light microscopy were prepared and immunohistochemistry was performed as described (Wong et al., 1992, J. Biol. Chem. 267: 5592-5598). Histological cross-sections were incubated with commercially available anti-p-gal antibody (1: 200 dilution, 5 prim-3 primer) and with commercially available anti-HAH polyclonal antibody (1: 500 dilution, BAbCO).

9.1.3. Luciferase and β-gal enzyme assays

Luciferase activity was determined using Luciferase Assay System (Promega) and Enzyme Assay System (Promega) for β-galactosidase according to protocols provided by 15 manufacturers.

9.1.4. PGAMl expression plasmid

To assemble pGAMl, mouse embryo mRNA was generated from 13.5 days after fertilization using kit reagents and protocols (Poly AT Tract mRNA Isolation System I,

189 174

Promega). Equal amounts of mRNA were used to create cDNA using commercial reagents (Reverse Transcriptase System, Promega). The full-length coding sequence for BMP-4 mouse cDNA was generated by polymerase chain reaction (PCR) under the following conditions: 94 ° C, 4 minutes, 1 cycle; 94 ° C, 1 minute, 65 ° C, 1 minute, 72 ° C, 1 minute, 30 cycles; 72 ° C. 8 minutes, 1 cycle. The sequence of PCR primers was based on the known sequence of mouse bMP-4 (GenBank): 5'-CCATGATTCCTGGTAACCGAATGCTG-3 'up primer; down primer 5'-CTCAGCGGCATCCGCACCCCTC-3 '. A single PCR product of the expected size (1.3 kb) was purified by agarose gel electrophoresis and cloned into TA Invitrogen cloning vector). The 5 'end of the BMP-4 insert was modified by additions (PCR) by adding a 27-nucleotide sequence that encodes the HA epitope and the BMP-4 insert was cloned into the expression vector pcDNA3 (InVitrogen). Plasmid DNA was generated and sequenced (both strands) to ensure orientation and integrity of the BMP-4 insert.

Expression of the plasmid pGAM1 was caused using an in vitro transcription and translation kit (TNT T7 Coupled Reticulocyte Lysate System, Promega) according to the protocols provided by the manufacturers. Radiolabelling, immunoprecipitation, sample preparation and SDS-PAGE, autoradiography, transient transfection and Western blot analysis were performed as described (Yin et al., 1995, J. Biol. Chem. 270: 10147-10160).

9.1.5. PGAM2 expression plasmid

Human parathyroid hormone cDNA fragments encoding preprol-34 amino acids were generated by pCR. The sequence of PCR primers was based on the known sequence of human PTH (GenBank): 5'-GCGGATCCGCGATGATACCTGCAAAAGACATG-3 'up primer; down primer 5'-GCGGATCCGCGTCAAAAATTGTGCACATCC-3 '. This pair of primers formed BamHI sites at both ends of the PCR fragment. The fragment was digested with BamHI and ligated to the BamHI cloning site in the PLJ retroviral vector (Wilson et al., 1992, Endocrinol. 130: 2947-2954). A clone with an insert in coding orientation (pGAM2) was optionally isolated and characterized by DNA sequence analysis.

To create retroviral strains, the φ CRIP packed cell line (JMWilson et al., 1992, Endocrinology 130: 2947-2954) was transfected with 10 pg of recombinant vector DNA using the calcium phosphate method. After overnight incubation, culture medium (Eagle's modified Dulbecco'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 were collected and applied to cultured Rat-1 cells. Independent transduced clones were successfully obtained by standard infection and selection procedures. Briefly, cultured Rat-1 cells were grown to confluence, divided 1:10 and selected in G418 (1 mg / ml, Gibco-BRL Life Technologies, Inc.). In some cases, antibiotic resistant colonies were pooled into a single culture. In other cases, single colonies of resistant cells were retained. Similar methods were used to create clones of Rat-1 cells transduced with BAGT retrovirus that encode the bacterial β-gal enzyme.

HPTH1-34 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 pGAM2 encoded peptide was evaluated as described (McCauley et al., 1994, Mol. Cell. Endocrinol. 101: 331-336).

9.1.6. Production of gene activated collagen sponges

For each bone incision slot, lyophilized bovine tracheal collagen (10 mg, Sigma) was completely soaked in sterile 0.5-1.0 mg plasmid DNA solution and allowed to incubate for 1-16 hours at 4 ° C before implantation.

9.1.7. Radiography

Weekly ordinary radiographs (posterior-anterior view) were obtained while the mammalian host was awake, using a portable X-ray device (GE, model 100). The exposure was 1/10 of a second, at 57 kV and 15 ma.

9.2. Results

9.2.1. Osteotomy model

In our model, we used a 5 mm osteotomy in the middle of the femoral shaft of an adult rat. The osteotomy site was stabilized with a clamping apparatus equipped with four

189 174 pins. Bone reconstruction after osteotomy occurs in the rat 9 weeks after surgery; the method of reconstruction depends on the size of the break; a 2 mm gap heals by callus formation, but a 5 mm gap heals by creating a fibrous junction (Rouleau, JP et al., Trans. Ortho. Res. Soc. 20 :). Under controlled conditions in mammals observed for 13 weeks after surgery, it was confirmed that 5 mm long gaps typically heal by forming a fibrous junction. Weekly normal x-rays and histological examinations showed that no callus was produced in mammals with only 5 mm osteotomy (n = 3), 5 mm osteotomy with collagen sponge (n = 10) or 5 mm osteotomy with assuming a collagen sponge containing the exposed marker plasmid DNA gene (n = 23). In all 36 control cases, the healing of gaps was achieved by the accumulation of fibrous tissue. In femoral control bones, local periosteal new bone formation (complication after pin placement) was found. A local transient inflammatory response in break tissue was also observed after surgery.

9.2.2. Marker gene research

Initial studies successfully transferred lacZ DNA expression plasmids (--gal in vivo. The aim was to standardize the method of producing a gene activated matrix and postoperative course. GAM-encoding luciferase was placed in the interval after osteotomy of one rat, and (-gal coding matrix was placed in break after osteotomy in another animal. Three weeks later, a gap homogenizer (containing the zamin tissue) was prepared after thoroughly removing the surrounding bone, cartilage and skeletal muscles. Samples from each homogenate were evaluated for enzyme expression in a substrate utilization assay. The expected enzyme activity was detected in each sample of homogenate. In other experiments, positive results were obtained under changed conditions (e.g. DNA dose, time to expression of the test protein).

9.2.3. Transferring the BMP-4 gene

After proving that the break cells express functional enzymes after capturing the DNA plasmid from the matrix, we asked if gene transfer could be used to modulate bone renewal. For overexpression, we selected BMP-4, a factor that stimulates bone renewal, undergoing normal expression in stem cells during bone union after fracture. Full-length BMP-4 mouse cDNA cDNA was generated by PGR and subcloned into the pcDNAS (Imritrogen) eukaryotic expression vector (Figure 2). For specific detection of recombinant proteins, the 3 'end of the BMP-4 coding sequence was modified by the addition of a hemagglutinin (HA) epitope. Recombinant BMP-4 was expressed from this structure (pGAMI) using in vitro transcription and translation methods. Immunoprecipitation studies confirmed the ability of the HA epitope to recognize anti-HA polyclonal antibodies. The biosynthesis of recombinant BMP-4 was assessed after transient transfection of cultured 293T cells with pGAMI DNA plasmid. BMP-4 molecules, as demonstrated by immunoprecipitation, were combined into homodimers, isolation and further processing as expected. Together, these results confirm the recognition of the HA epitope by anti-HA polyclonal antibodies.

Collagen sponges containing pGAMI GNA were placed in gaps in nine adult rats kept alive for 4-24 weeks. In one mammal, killed 4 weeks after surgery, immunohistochemical studies using anti-HA antibodies demonstrated pGAMI expression by restorative fibroblasts within the gap. This was important, given that we did not observe false positive staining in observation of the gap tissue from thirteen control mammals. Microscopic visceral foci of callus originating from both edges of the surgical wound were also observed in samples obtained 4 weeks after surgery. As previously described for bone formation through auto-induction (Urist, 1965, Science 150: 893-899), these foci were formed from bone plates coated with large cube-shaped osteoblasts and supported by cellular connective tissue formed from spindle-shaped fibroblasts and capillaries. In seven mammals killed 5-12 weeks after surgery, the amount of radiographic callus grew steadily (Fig. 3A), although transgene encoded BMP-4 was not detected by immunohistochemistry. Sternum production, defined as callus spread from the edges of the surgical wound by a break after osteotomy, was typically seen after 9 weeks. The ninth mammal survived without complications 24 weeks after surgery. After 18 weeks, the amount of callus accumulated was sufficient to remove the fastening apparatus; the mammal was able to walk well for the next 6 weeks (Fig. 3A). After killing, it was found that the gap was filled with active callus remodeling, except for a thin band partially permeable to X-ray tissue in the distal (distal) edge of the gap. As the mammal was able to walk without the fastening apparatus, we believe that this band has partially mineralized. The result of biomechanical tests is consistent with this hypothesis (Frankenburg et al., 1994, Trans. Ortho. Res. Soc. 19: 513), which proved that the mechanical strength of the healed gap is essentially the same as the unoperated femur of the same mammal (6.3% difference in the maximum torsional load test). The radiological appearance of the contralateral (inoperable) femur remained unchanged in all nine cases, indicating that the effect of gene transfer and overexpression of BMP-4 was limited to the post-osteotomy pause.

9.2.4. Transfer and expression of plasmid cocktail (BMP-4 + PTH1-34)

Bone regeneration in normal conditions depends on many factors operating in a specific order; we were therefore wondering if the expression of several anabolic factors would stimulate bone formation more than a single factor. To test this hypothesis, we decided to use two-plasmid GAM encoding BMP-4 and a parathyroid hormone (PTH) peptide fragment. PTH is an 84-amino acid hormone that increases Ca^2L in plasma and extracellular fluid. In skeletal tissues, periodic administration of a PTH fragment meeting the structural criteria of biological activity (aa 1-34) gives a real anabolic effect: numerous in vivo and in vitro studies provide evidence that administered PTH 1-34 in mammals (including rats) causes formation of high quality bone-free bone tissue due to the combined osteoclast inhibitory and osteogenic cell stimulating effects (Dempster et al., 1993, Endocrin Rev. 14: 690-709). The PTH 1-34 peptide is known to act synergistically with BMP-4, which causes upregulation of expression of functional cell surface PTH receptors in differentiating osteoblasts (Ahrens et al., 1993, J. Bone Min. Res. 12: 871-880 ).

The cDNA fragment encoding human PTH1-34 was generated by PGR. To determine its biological activity, the fragment was subcloned into the PLJ retroviral vector (Wilson et al., 1992, Endocrin, 130: 2947-2954), producing the expression plasmid pGAM2 (Figure 4A). A stock of replication-defective recombinant retrovirus was generated and fed to Rat-I cell culture. Independent cells of transduced Rat-I cells were produced and stable integration and expression of retrovirus DNA was demonstrated by Southern and Northern analyzes. Radioimmunoassay was used to determine the concentration of human PTH 1-34 in the media of individual clones. ROS 17 / 2.8 cells have PTH receptors on the cell surface; these receptors belong to the superfamily of G protein-related receptors (Dempster et al., 1993, Endocrin. Rev. 14: 690-709). Incubation of ROS 17 / 2.8 cells with media samples from a stably transduced cell line (secreting> 2 pg / ml by radioimmunoassay) resulted in a 2.7-fold increase in CAMP response compared to control; this result confirms the biological activity of secreted PTH 1-34 peptide.

Gam containing only pGAM2 DNA plasmid and stimulated GAM containing both BMP-4 and PTH1-34 DNA expression plasmids were then implanted into the gap after osteotomy of a further three mammals. Sternal production after 4 weeks was observed in all three mammals (at this point one mammal was sacrificed for histological examination), and 12 weeks after surgery the amount of new bone tissue produced in the remaining mammals enabled removal of the external fixation apparatus (Fig. 5). Both mammals walked well at the time of publication, i.e. 15 and 26 weeks after implantation, respectively. Based on normal x-rays, it can be concluded that the effect of gene transfer and overexpression seemed limited to the post-osteotomy pause.

Following studies using a collagen sponge, it was also proved that plasmid DNA can be delivered to cells in a slow manner, allowing the co-polymerization of block copolymers of polylactic-polyglycol particles. Results

189 174 indicates that the cell culture can be transfected with plasmid DNA released from polylactate-polyglycol particles. The results also show that the repair fibroblasts (rat osteotomy model) in vivo capture the DNA of the pGAM2 plasmid, which is expressed in them, after the release of DNA from polylactic-polyglycol particles. As shown in Figure 7, expression encoded by plasmid PTH1-34 is accompanied by the formation of a significant amount of callus during the post-osteotomy interval.

All in all, these studies show that the gene-activated matrix technique does not require the use of collagen matrix. So this technique is wide enough to connect to both biological and synthetic arrays.

10: Example: Gene transfer to regenerative tendon and to regenerative cruciate ligament in vivo

There is a clinical need to stimulate scar formation during renewal of the Achilles tendon and ligaments (shoulder and knee) to increase the mechanical resistance of tissue after injury. A model system has been developed in which segmental Achilles tendon defects are created and a new biological material, submucosal layer of the small intestine or SIS is used as a tendon implant / particle delivery agent. In this example, the ability to deliver and express structures with a marker gene in regenerative tendon tissue using SIS transplantation was demonstrated.

10.1. Material and methods

Staple Achilles tendon defects were made and SIS was used as the tendon implant / particle delivery agent. Stock solutions of plasmids (psVogal, Promega) were prepared according to routine methods (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press). SIS transplant material was prepared from the jejunum of adult pigs (Badylak et al., 1989, J. Surg. Res. 47: 74-80). After collection, mesenteric tissues were removed, the segment was inverted and the mucosa and surface layer of submucosal tissue removed by mechanical abrasion. After the inversion of the section again, the serous and muscular layers were rinsed, sterilized by treatment with diluted paraacetic acid and stored at 4 ° C until use.

Dogs (mongrels) (in all studies) were anesthetized, intubated, placed on their right side on a heated pad and kept under anesthesia by inhalation. To visualize the Achilles tendon, a lateral incision was made from the muscular-tendon joint to the plantar fascia. A double SIS layer was wrapped around the central part of the tendon, both ends were sutured, a 1.5 cm section of the tendon was removed from the lateral opening in the graft material and the wound closed. The limb was immobilized for 6 weeks and then allowed to use the animal freely for 6 weeks. The graft tissue was collected at the following time points, fixed in Bouins solution and embedded in paraffin. Sections (tissue sections) (8 pm) were made and used for immunohistochemistry.

10.2. Results

In the first study, the implantation of SIS material alone ("SIS only" transplant) increased the renewal of the Achilles tendon after the formation of a staple defect in mongrel dogs for up to 6 months after surgery. As a result of the remodeling process, the iamamin tissue was quickly formed and eventually the graft disintegrated. No scar tissue was formed and no signs of immune rejection were observed.

In the second study, SIS was soaked in plasmid DNA solution ("SIS + plasmid" transplant) and then implanted as an Achilles tendon transplant (n = 2 dogs) or a cruciate ligament transplant (n = 2 dogs) in healthy mongrel dogs. The plasmid pSVPgal, using the monkey virus regulatory sequences for β-galactosidase (β-gal) activity, was detectable by immunohistochemistry using a specific antibody in 4/4 mammals. In the negative control, β-gal activity was not detected in the inoperable Achilles tendon and cruciate ligament of these mammals. It therefore appears that SIS facilitates reuptake and subsequent expression of plasmid DNA by new tendon tissue cells, both in the tendon and in the ligament.

A third study looked at the expression time of the P-gal transgene. SIS + plasmid grafts were implanted for 3, 6, 9 and 12 weeks (n = 2 dogs for each time point) and transgene expression was examined by immunohistochemistry. Sections were made from a cross section (8 μιη) fixed in Bouins solution and embedded in paraffin tissue and mounted on slides Probeon Plus (Fisher). According to the protocol, immunohistochemistry was performed using the Histostain-SP kit (Zymed). Briefly, slides were incubated with anti-β-galactosidase antibodies with known characteristics (dilution 12:00, Prime-33 Prime), washed with PBS, incubated with biotinylated second antibody, washed, stained with enzyme conjugate with substrateatchromogen, and then counterstained hematoxylin and eosin.

Activity (bacterial gallium was detected in tendons with SIS + plasmid implants (8/8 mammals). Although no strict quantitative relationship was found, transgene expression appeared to reach a maximum after 9-12 weeks. In 35 mammals that received the "only SIS, , no bacterial P-gal gene expression was detected.

11 Example: Adenoviral gene transfer to regenerative bone in vivo

An alternative way to achieve gene transfer in vivo to regenerative tissue is to use a transfer mediated by adenovirus. Effective adenoviral gene transfer - marker gene structure - into bone regeneration cells was achieved in a rat osteotomy model.

11.1. Materials and methods

The adenoviral pAd vector, CMVlacZ, is an example of a replication-deficient adenoviral vector capable of replication in permissive cells (Stratford-Perricaudet et al., 1992, J. Clin. Invest. 90: 626-630). In this lacZ transcription vector with the SV40 polyadenylation sequence cloned towards the 3 'end of the reporter gene, an early cytomegalovirus (CMV) enhancer / promoter (Davidson et al., 1993, Nature Genetics 3: 219-223) is used.

pAd.RSV4 has essentially the same core as pAdCMVlacZ, however the CMV promoter and the Bglll cloning site have been cassette replaced with the Bglll fragment containing the RSV promoter, multiple cloning site and poly (A⁺). The greater flexibility of this vector is beneficial in subcloning osteotropic genes, such as the hPTHl-34 cDNA fragment, for use in further studies.

The Ultra Fiber ™ implant was soaked for 6 minutes in the AdCMVlacZ virus solution (10^1ο-10¹¹plaque formation units (i.e. PFU / ml) and then implanted into the osteotomy site. The defect was allowed to heal for 3 weeks; at this time, wound healing was monitored by weekly X-ray examination. After three weeks, it was estimated that 4% of the defect was filled with callus tissue. The mammal was sacrificed and the tissues fixed in Bouins solution and then demineralized for 7 days in standard formic acid solutions.

11.2 Results

The results demonstrated expression of the marker gene product in chondrocyte-like break cells after osteotomy (Fig. 6). Nuclear-targeted signal has also been observed in preosteoblasts.

12. Example: Gene transfer to skeletal muscle

There is a clinical need to stimulate scar formation during the renewal of soft tissues other than the Achilles tendon and ligament (shoulder and knee) to increase the mechanical resistance of the tissue after injury. A model system was developed in which incisions are formed in the skeletal muscle of an adult rat and a suture coated with a slow release PLGA particle preparation and plasmid DNA is used as a skeletal muscle implant / gene delivery agent. To prove the applicability of the coating compositions and methods of the invention, the surgical suture was covered with marker DNA (encoding human placental alkaline phosphatase) and used to suture muscle rat tissue. In this example, the ability to transfer and express DNA in sutured tissue covered with sutures has been demonstrated.

12.1. Materials and methods

12.1.1. Preparation of the DNA-PLGA coating composition

189 174

To 1.5 ml of PLGA / chloroform solution (3% (by weight) 50/50 PLGA polylactate-polyglycolate polymer with an average molecular weight of 90,000 and a natural viscosity of 1.07), 0.2 ml of a solution containing marker DNA encoding human placental alkaline phosphatase (1 mg DNA, 0.5 mM Tris-EDTA, 0.5 mM EDTA, pH 7.3). The solution was emulsified by vortexing for 2 minutes and then sonication for 30 seconds at a temperature of about 0 ° C, using a micro-terminated probe sonicator and an output power of 55 watts. As a result of this process, an emulsion with a very milky appearance was obtained.

12.1.2. Covering the surgical suture

In a piece of Teflon-coated film (Norton Performance Plastic Corp., Akron. OH) a hole was pierced with a 22-gauge needle. A drop (about 60 β) of DNA-PLGA emulsion was placed on the hole. A 70 cm chrome 3-0 (Ethicon) seam was drawn through the hole to cover the seam. While the seam passed through the hole, it was covered with a thin (thickness of about 30 βΐη) uniform layer of the covering composition. The seam was allowed to air dry for approximately 3 minutes, and the coating process was repeated 15 times, leaving each coat layer to dry. The coated seam was examined by electron microscopy (150X) and the seam was found to be covered with a uniform DNA-PLGA layer. In addition, the coating remained intact even after repeated threading of the suture through the tissue.

12.1.3. Skeletal muscle renewal using a coated suture

The suture prepared as described above sutured the skeletal muscle tissue of healthy adult rats, obtaining a satisfactory surgical effect. The quality of the stitching was good. One week later, the muscle and suture were separated, frozen quickly in liquid nitrogen, and ground into powder. The powder was incubated in 200 p1 lysis buffer, exposed to three freezing and thawing cycles and clarified. The clear liquid was tested for alkaline phosphatase activity by standard methods after incubation at 65 ° C.

12.2. Results

The results indicate that rat skeletal muscle sutured with suture and removed a week later showed alkaline phosphatase activity, which means that the marker alkaline phosphatase gene was expressed in muscle tissue. No significant alkaline phosphatase activity was found in control samples. These data indicate that emulsions can be used to efficiently cover sutures and deliver genes to proliferating renewal cells in vivo.

13. Example: Gene transfer to blood vessels

There is a clinical need to prevent excessive fibrosis (restenosis), which can occur, for example, during blood vessel renewal after angioplasty. This can be achieved, for example, by administering genes encoding lysyl oxidase inhibitors, or by transferring genes encoding some TGF-β. There is also a clinical need to regulate angiogenesis, such as in vascular failure disorders, where the goal would be to stimulate the formation of new vessels to prevent hypoxia and cell death. A model system was developed in which renewal cells in large blood vessels in a rabbit are transfected with a preparation of PLGA particles with delayed release and plasmid DNA. Renewal cells are present because foam cells similar to clinical atherosclerosis in humans are present in such rabbit blood vessels. This example demonstrates the ability to deliver and express marker gene structures to large blood vessel renewal cells.

13.1. Materials and methods

White New Zealand rabbits of both sexes, weighing 3.1-3.5 kg, were used for this study. The rabbits were anesthetized with ketamine (35 mg / kg) and xylazine (5 mg / kg), administered intramuscularly, followed by maintenance anesthesia - intravenous ketamine (8 mg / kg) into the angular vein. Sections of about 2 cm in length of both iliac arteries were isolated between the descending aortic division and the inguinal ligament, they were sutured proximal and all small branches of these arteries were bound. The formation of local thrombi was prevented by administering heparin (100 mg) to the auricular angular vein. An angioplasty balloon catheter (2.0 mm balloon) was inserted into the iliac arteries by iliac arteriotomy and the balloon was inflated for 1 minute at 8 atm.

After inflating the balloon, the catheter was removed and 20 mg heparin was injected intra-arterially to prevent distal thrombosis. Both ends of the iliac artery were sutured with 10.0 silk suture and 5 mg / ml of DNA-nanoparticle suspension was administered to each iliac artery for 3 minutes at 0.5 atm. The wound was sewn. The rabbits were sacrificed 2 weeks after balloon angioplasty and administration of nanoparticles. Both iliac arteries were isolated by a vertical incision of the lower abdomen. A 2 cm segment of the iliac artery was dissected on both sides. Carotid arteries of rabbits were taken as controls. The tissue was fixed in liquid nitrogen for alkaline phosphatase testing.

13.2. Results

The results of the phosphatase expression study indicate that the nanoparticle preparation from DNA was able to deliver nucleic acids for the renewal of hip rabbit arteries cells after balloon catheter injury. Both the right and left iliac arteries were positive for phosphatase activity after exposure to the preparation of nanoparticles with DNA. There was no phosphatase · activity present in the control artery. These positive results indicate that when exposed to a gene-activated matrix, the repair cells of large blood vessels can capture nucleic acid molecules that can be expressed in them.

The scope of the present invention is not limited by the embodiments shown, which are illustrative of some aspects of the invention; all DNA and amino acid clones and sequences, functionally equivalent, are within the scope of the present invention. The skilled person can make various other modifications of the invention based on the above description and drawings. Such modifications are within the scope of the present invention as defined in the appended claims. It should also be understood that all base pair sizes given for nucleotides are approximate and used for illustration.

189 174

STRUCTURE pGA / Wi

<img file="PL189174B1_D0001.tif" />

Fig. 2

<img file="PL189174B1_D0002.tif" />

Fig. 3A

189 174

<img file="PL189174B1_D0003.tif" />

weeks after implantation (before killing)

Fig. 3B

<img file="PL189174B1_D0004.tif" />

Fig. 4 AB

189 174

<img file="PL189174B1_D0005.tif" />

Fig. 5

189 174

<img file="PL189174B1_D0006.tif" />

Fig. 6

<img file="PL189174B1_D0007.tif" />

Fig. 7

189 174

<img file="PL189174B1_D0008.tif" />

Fig. 1

UP Department of Publications. Circulation of 50 copies Price PLN 6.00.