Methods and compositions for stimulating bone cells

IS 2 139 889 T5

DESCRIPTION

Methods and compositions for stimulating bone cells.

The present application is a continuation - in part - of US application Serial Number 08 / 316,650, filed September 30, 1994; which is a continuation - in part - of US application Serial Number 08 / 199,780, filed February 18, 1994; the full text and figures of which are specifically incorporated herein by reference without rectification. The United States Government has certain rights in the present invention pursuant to grant HL-41926 from the National Institutes of Health.

1. Field of the invention

The present invention relates generally to the field of bone cells and tissues. More particularly, some embodiments relate to the transfer of genetic material to bone and other embodiments relate to type II collagen. In some examples, the invention relates to the use of type II collagen and nucleic acids to stimulate bone growth, repair and regeneration. Methods, compositions, kits, and devices are provided for transferring an osteotropic gene into progenitor bone cells, which has been shown to stimulate progenitor cells and promote increased bone formation in vivo.

two. Description of the prior art

Defects in the bone repair and regeneration procedure are related to the development of various human diseases and disorders, for example, osteoporosis and osteogenesis imperfecta. Failure of the bone repair mechanism is, of course, also associated with significant complications in clinical orthopedic practice, for example, fibrous nonunion after bone fracture, interphase graft failures, and large allograft failures. The lives of many people could be improved by developing new therapies designed to stimulate and enhance fracture repair procedures.

Naturally, any new technique to stimulate bone repair would be a valuable tool in treating bone fractures. A significant part of the fractured bones are still treated with plaster, leaving the natural mechanisms to repair the wound. Although there have been advances in the treatment of fractures in recent years, including improved devices, the development of new procedures to stimulate or complement wound repair mechanisms would represent significant advances in this field.

A very significant population of patients that would benefit from new therapies designed to promote fracture repair, or even prevent or reduce them, are those with osteoporosis. The term osteoporosis refers to a heterogeneous group of disorders characterized by decreased bone mass and fractures. Clinically, osteoporosis is divided into type I and type II. Type I occurs predominantly in middle-aged women and is associated with estrogen loss at menopause, while type II osteoporosis is associated with advanced age.

An estimated 20-25 million people are at increased risk of fractures, due to bone loss in specific locations. The cost of treating osteoporosis in the United States is routinely estimated to be in the order of $ 10 billion per year. Demographic trends, for example the gradually increasing age of the US population, suggest that these costs may increase 2 to 3 times in 2020 if a safe and effective treatment is not found.

The major focus of routine osteoporosis therapies is fracture prevention, not fracture repair. This is an important consideration, as significant morbidity and mortality are known to be associated with prolonged bed stay in older people, especially those with hip fractures. New methods are clearly needed to stimulate fracture repair, thus restoring mobility in these patients before complications arise.

Osteogenesis imperfecta (OI) refers to a group of inherited connective tissue diseases characterized by brittle bones and soft connective tissue (Byers and Steiner, 1912; Prockop, 1990). Men and women are equally affected, and the total incidence is usually estimated to be 1 in 5,000-14,000 live births. Hearing loss, imperfect tooth formation, respiratory failure, severe scoliosis, and emphysema are just some of the conditions associated with one or more types of OI. While precise estimates of health care costs are not available, the morbidity and mortality associated with OI certainly stem from the extreme propensity for fractures (OI types I-IV) and abnormal bone deformation after repair of the OI. fractures (OI types II-IV) (Bonadio and Goldstein, 1993). The most important consequence of the treatment of OI is to develop new methods by which to improve fracture repair and therefore improve the quality of life of these patients.

Bone reconstruction techniques, such as those used to reconstruct defects that occur as a result of trauma, cancer surgery, or developmental errors, would also be enhanced by new methods to promote bone repair. Commonly used reconstruction methods, such as using autologous bone grafts, or bone grafts with attached soft tissues and blood vessels, are associated with significant drawbacks.

ES 2 139 889 T5 cost and difficulty. For example, harvesting a useful amount of autologous bone is not easily achieved, and even autologous grafts are often infected or resorbed.

The bone repair and regeneration procedure is similar to that of wound healing in other tissues. A typical sequence of events includes: bleeding; clot formation; dissolution of this with concomitant renewal of damaged tissues; ingrowth of granulation tissue; cartilage formation; capillary internal growth and cartilage turnover; rapid bone formation (callus tissue); and, finally, remodeling of the callus in cortical and trabecular bone. Therefore, bone repair is a complicated procedure that involves many cell types and regulatory molecules. The various cell populations involved in fracture repair include supporting cells, macrophages, fibroblasts, vascular cells, osteoblasts, chondroblasts, and osteoclasts.

Regulatory factors involved in bone repair are known to include systemic hormones, cytokines, growth factors, and other molecules that regulate growth and differentiation. Various bone inducing agents have been purified, showing that they are polypeptide molecules such as growth factors. These stimulating factors refer to bone morphogenetic proteins or morphogenic proteins (BMPs) and have also been called bone osteogenic inducing proteins or osteogenic proteins (OPs). Several BMP (or OP) genes have now been cloned, the usual designations being BMP-1 through BMP-8. New BMPs are being discovered. Although the BMP terminology is widely used, it may be the case that an OP replica term exists for each individual BMP (Alper, 1994).

BMPs 2-8 are thought to be generally osteogenic, although BMP-1 is a more generalized morphogen (Shimell et al., 1991). BMP-3 is also called osteogenin (Luyten et al., 1989) and BMP-7 is also called OP-1 (Ozkaynak et al., 1990). BMPs are related to, or part of, the transforming growth factor β (TGF-β) superfamily and both TGF-βι and TGF- / t_; they also regulate osteoblastic function (Seitz et al., 1992). Various BMP (or OP) nucleotide sequences and polypeptides have been described in US Patent documents eg at 4,795,804; 4,877,864; 4,968,590; 5,108,753; which specifically include, BMP-1 which is disclosed in US Patent 5,108,922; BMP-2A (commonly referred to as BMP-2) in US Patent documents 5,166,058 and 5,013,649; BMP-2B (commonly referred to as BMP-4) which is disclosed in US Patent 5,013,649; BMP-3 in 5,116,738; BMP-5 in 5,106,748; BMP-6 in 5,187,076; BMP-7 in 5,108,753 and 5,141,905; and OP-1, COP-5 and COP-7 in document 5,011,691.

Other growth factors or hormones, which have been reported to have the ability to stimulate new bone tissue formation, include fibroblast acidic growth factor (Jingushi et al., 1990); estrogens (Boden et al., 1989); macrophage colony stimulating factor (Horowitz et al., 1989); and calcium regulatory agents such as parathyroid hormone (PTH) (Raisz and Kream, 1983).

Several groups have investigated the possibility of using bone stimulating proteins and polypeptides, particularly recombinant BMPs, to influence bone repair in vivo. For example, recombinant BMP-2 has been used to repair surgically created defects in the jaw of adult dogs (Touriumi et al., 1991) and high doses of this molecule have been shown to functionally repair segmental defects in the femurs of female dogs. rats (Yasko et al., 1992). Chen and colleagues demonstrated that a single application of 25-100 mg of recombinant TGF-βι adjacent to cartilage induced endochondral bone formation in full-thickness dermal wounds of the rabbit ear (Chen et al., 1991). It has also been reported that an application of TGF-βι in a 3% methylcellulose gel was able to repair large, surgically induced cranial defects that otherwise heal by fibrous connective tissue and never form bone (Beck et al., 1991) . WO 88/00205 discloses the use of a recombinant protein for bone repair.

The prior art (WO 94/01139) describes a method for transfecting a cell into a hinge structure, in which a DNA vector containing a nucleic acid cassette encoding a desired protein, for example an ablating agent cell or therapeutic agent, is injected directly into the joint. Cells that are transfected with the gene are, for example, synovial cells. EP No. 0 248 531 discloses a microcapsule for the controlled release of nucleic acid.

However, there are many drawbacks associated with these types of treatment protocols, especially the expensive and time-consuming purification of recombinant proteins from their host cells. Also, polypeptides, once administered to an animal, are more unstable than is generally desirable for a therapeutic agent, and are susceptible to proteolytic attack. Furthermore, the administration of recombinant proteins can initiate various inhibitory or otherwise damaging immune responses. It is clear, therefore, that a new procedure capable of promoting bone repair and regeneration in vivo would represent a significant medical and scientific advance with immediate benefits for a large number of patients. An easily adaptable method for use with various matrices and bone stimulator genes would be particularly advantageous.

Summary of the invention

The present invention overcomes one or more of these and other drawbacks inherent in the prior art, providing new procedures, compositions and devices for use in the transfer of nucleic acids to

ES 2 139 889 T5 bone cells and tissues, and to promote bone repair and regeneration. Some embodiments of the present invention are generally based on the inventors' surprising finding that nucleic acids can be effectively transferred to bone progenitor cells in vivo and that, in some of those embodiments, the transfer of a Osteotropic gene stimulates bone repair in an animal.

As used herein, "isolated nucleic acid segment refers to a nucleic acid" as defined in claim 1.

As used herein, the term "matrix" refers to a matrix according to claim 1.

The present invention, in general terms, therefore relates to methods, compositions and devices for transferring a nucleic acid segment to progenitor cells or bone tissues. The methods of the present invention generally comprise effectively contacting bone progenitor cells with a composition comprising a nucleic acid segment, to transfer the nucleic acid to the cells. The cells can be cultured cells or recombinant cells that are maintained in vitro, when all that is needed is to add the nucleic acid composition to the cells, for example by adding it to the culture medium.

Alternatively, the progenitor cells may be located within a parent tissue site in bone of an animal, when the nucleic acid composition is applied to that site in order to affect, or promote in vivo transfer of the nucleic acid to the cells. of the bony parent. In the transfer of nucleic acids to bone cells within an animal, a preferred procedure involves first adding the genetic material to a compatible bone matrix and then using the resulting matrix to contact an appropriate tissue site within the animal. The "resulting" matrix can, in some embodiments, be referred to as a matrix impregnated with genetic material, or it can take the form of a nucleic acid-matrix mixture, or else a conjugate.

To bone or tissue progenitor cells, using the compositions and methods of the present invention, an extremely wide variety of genetic material can be transferred as claimed in claim 1. For example, the nucleic acid segment can be DNA (mono or double stranded) or RNA. The nucleic acid segments can therefore be genomic sequences, including exons or introns only, or exons and introns, or coding cDNA regions, or indeed any construct that it is desired to transfer into a progenitor cell or bone tissue. Appropriate nucleic acid segments can be in virtually any form, such as naked DNA or RNA, including linear nucleic acid molecules and plasmids; functional insertions into the genomes of various recombinant viruses, including viruses with DNA genomes and retroviruses; and any form of nucleic acid segment, plasmid or virus associated with a liposome or a gold particle, the latter being able to be used in connection with gene gun technology.

The invention can be used to promote the expression of a desired gene in bone cells or tissues and to impart a particular desired phenotype to them. This expression could constitute the increased expression of a gene that is normally expressed, or it could (ie, "over-expression") be used to express a gene that does not normally associate with bone progenitor cells in their natural environment. Alternatively, the invention can be used to suppress the expression of a gene that is naturally expressed in such cells and tissues, and again, to change or alter the phenotype. Gene suppression may be a way of expressing a gene that encodes a protein that exerts a down-regulatory function.

1. Bone progenitor cells and tissues

In some embodiments, the present invention provides advantageous methods of using genes that stimulate bone progenitor cells. As used herein, the term "bone progenitor cells" refers to any or all of those cells that have the ability to ultimately form, or contribute to the formation of new bone tissue. This includes various cells in different stages of differentiation, such as, for example, supporting cells, macrophages, fibroblasts, vascular cells, osteoblasts, chondroblasts, osteoclasts, and the like. Bone progenitor cells also include cells that have been isolated and manipulated in vitro, eg, subjected to stimulation with agents such as cytokines or growth factors or even genetically engineered cells. The particular type or types of bone progenitor cells that are stimulated using the methods and compositions of the invention are not important, as long as the cells are stimulated in such a way that they activate and, in the context of the in vivo embodiments, produce finally new bone tissue.

The term "bone progenitor cell" is used to refer particularly to those cells that are located within, are in contact with, or migrate to (ie, "home") the bone progenitor tissue and whose cells directly or indirectly stimulate the formation of mature bone. As such, the progenitor cells can be cells that eventually differentiate themselves into mature bone cells, that is, cells that "directly" form new bone tissue. Cells that, upon stimulation, further attract progenitor cells or promote neighboring cells to differentiate into bone-forming cells (e.g. osteoblasts, osteocytes and / or osteoclasts) are also considered progenitor cells in the context of this discussion - already that its stimulation leads "indirectly" to bone repair or regeneration. Cells that affect bone formation can do this indirectly through the manufacture of various growth factors or cytokines, or through their physical interaction with other cell types. Although of scientific interest, the direct or indirect mechanisms through which

ES 2 139 889 T5 progenitor cells stimulate bone or wound repair is not a consideration for the practice of this invention.

Bone progenitor cells and bone progenitor tissues can be cells and tissues that, in their natural environment, reach an area of ​​active bone growth, repair, or regeneration (also referred to as a wound repair site). In terms of bone progenitor cells, these can also be cells that are attracted or recruited to that area. These can be cells that are present within an artificially created osteotomic site in an animal model, such as those discussed herein. Bone progenitor cells can also be isolated from animal or human tissues and preserved in an in vitro environment. Appropriate areas of the body from which to obtain bone progenitor cells are areas such as the bone tissue and fluid surrounding a fracture or other skeletal defect (this is an artificially created site anyway) or, indeed, from bone marrow. Isolated cells can be stimulated using the methods and compositions disclosed herein, and, if desired, returned to an appropriate site in an animal where bone repair is to be stimulated. In such cases, the cells containing the nucleic acid would themselves be a form of therapeutic agent. Such ex vivo protocols are well known to those of skill in the art.

In important embodiments of the invention, the bone progenitor cells and tissues will be those cells and tissues that reach the area of ​​bone fracture or damage to be treated. Accordingly, in embodiments of the treatment, there is no difficulty associated with identifying appropriate target progenitor cells to which the present therapeutic compositions are to be applied. All that is needed in such cases is to obtain an appropriate stimulatory composition, as set forth, and contact the site of the bone fracture or defect with the composition. The nature of this biological environment is such that appropriate cells will be activated in the absence of any further targeting or cell identification by the physician.

Some methods of the present invention generally involve contacting bone progenitor cells with a composition comprising one or more osteotropic genes (with or without additional genes, proteins or other biomolecules), so as to promote the expression of said gene. in these cells. As stated above, cells can be contacted in vitro or in vivo. This is achieved, in the most direct way, by simply obtaining an osteotropic gene functional construct and applying it to the cells. The inventors surprisingly found that there are no particular molecular biological modifications that need to be carried out in order to promote effective expression of the gene in progenitor cells. Contacting cells with DNA, for example, a linear DNA molecule, or DNA in the form of a plasmid or other recombinant vector, that contains the gene of interest under the control of a promoter along with the appropriate termination signals, is sufficient for the uptake and expression of DNA to be obtained, with no further steps being necessary.

In preferred embodiments, the method of contacting the progenitor cells with the osteotropic gene composition is carried out in vivo. Again, a direct consequence of this procedure is that the cells take up and express the gene and that, without additional steps, they function to stimulate the growth, repair or regeneration of bone tissue.

An assay for an osteoinductive gene can be carried out using the bone induction assay of Sampath and Reddi (1981; incorporated herein by reference). This is a rat bone formation assay that is used routinely to assess the osteogenic activity of bone inducing factors. However, to analyze the effects of osteotropic genes on bone growth, it is generally directed to the use of the new osteotomy model disclosed here.

two. Osteotropic genes

As used herein, the terms "osteotropic" and "osteogenic gene" are used to refer to a gene or coding region of DNA that encodes a protein, polypeptide, or peptide that is capable of promoting, or assisting in promoting. of bone formation, or one that increases the rate of primary bone growth or healing of skeletal connective tissue (or also a gene that increases the rate of growth or healing of skeletal connective tissue). The terms "promote", "induce", and "stimulate" are used interchangeably throughout this text to refer directly or indirectly to procedures that ultimately result in the formation of new bone tissue or an increased rate of bone repair. Thus, an osteotropic gene is a gene that, when expressed, causes the phenotype of a cell to change so that the cell differentiates, stimulates other cells to differentiate, attracts bone-forming cells, or functions in another way. so that it eventually gives rise to new bone tissue.

When using the new osteotomy model of the present invention, an osteotropic gene is a gene characterized as one that is capable of stimulating adequate bone growth in the osteotomy space to any degree greater than that observed in control studies, for example, parallel studies employing an irrelevant marker gene such as jd-galactosidase. This stimulation of "adequate bone growth" includes both the type of tissue growth and the rate of bone formation. When using the model with an osteotomic gap of 5 mm, an osteotropic gene is generally characterized as a gene that is capable of promoting or inducing new bone formation, rather than the abnormal repair of a bone fracture, that is, non-union fibrous. Using the 2mm osteotomic space, osteotropic genes can be characterized as genes that increase the rate of primary bone healing

ES 2 139 889 T5 when compared to controls, and more preferably, genes capable of stimulating repair of the osteotomic defect in less than 9 weeks.

In general terms, an osteotropic gene can also be characterized as a gene capable of stimulating the growth or regeneration of the connective tissues of the skeleton such as, for example, tendons, cartilage and ligaments. Thus, in some embodiments, the methods and compositions of the invention can be used to stimulate the growth or repair of both the bone tissue itself and the connective tissues of the skeleton.

Various osteotropic genes are now known, all of which are suitable for use in connection with the present invention. Osteotropic genes and the proteins they encode include, for example, systemic hormones, such as parathyroid hormone (PTH) and estrogens; many different growth factors and cytokines; chemotactic or adhesive peptides or polypeptides; molecules such as activin (US Patent 5,208,219, incorporated herein by reference); specific bone morphogenetic proteins (BMPs); and also growth factor receptor genes.

Examples of appropriate osteotropic growth factors include the gene family of transforming growth factors (TGFs) which include TGFs 1-3 and particularly TGF-Si, TGF- / T and TGF- / 7._; (US Patent 4,886,747 and 4,742,003, which are incorporated herein by reference) with TGF-α (US Patent 5,168,051, which is incorporated herein by reference) also being of possible use; and also fibroblast growth factors (FGF), previously referred to as acidic and basic FGFs and now referred to as FGF1-9; granulocyte / macrophage colonial stimulating factor (GMCSF); epidermal growth factor (EGF); platelet derived growth factor (PDGF); insulin-like growth factors (IGF), including IGF-I and IGF-II; and leukemic inhibitory factor (LIF), also known as HILDA and DIA. Any of the above or other related genes, or DNA segments encoding the active portions of said proteins, can be used in the new methods and compositions of the invention.

Some preferred osteotropic genes and DNA segments are those of the TGF superfamily, such as TGF / ή, TGF- / T, TGF-6F, and members of the BMP family of genes. For example, several BMP genes have been cloned that are ideal candidates for use in nucleic acid transfer or delivery protocols of the invention. Appropriate BMP people are called BMP-2 to BMP-12. BMP-1 is not considered particularly useful at this stage.

There is considerable variation in the terminology commonly used in the literature when referring to these genes and polypeptides. It will be understood by those of skill in the art that all BMP genes encoding an active osteogenic protein are considered useful in the present invention, despite different terminology that may be employed. For example, BMP-3 is also called osteogenin and BMP-7 is also called OP-1 (osteogenic protein-1). It is likely that the family of factors called OP (s) is as broad as the one called BMP (s), and that these terms, in fact, describe the same set of molecules (Alper, 1994).

DNA sequences for various BMP (or OP) genes have been described both in scientific articles and in US Patent documents such as 4,877,864; 4,968,590; 5,108,753. Specifically, the BMP-1 sequences are set forth in US Pat. 5,108,922; BMP-2A (commonly referred to as BMP-2), in Patent Nos. 5,166,058 and 5,013,649; BMP-2B (commonly referred to as BMP-4) is disclosed in US Patent 5,013,649; BMP-3 in 5,116,738; BMP-5 at 5,106,748; BMP-6 at 5,187,076; and BMP-7 at 5,108,753 and 5,141,905; all are incorporated herein by reference). The article by Wozney et al., (1988; incorporated herein by reference) is considered particularly useful for the description of molecular BMP clones and their activities. The sequences encoding the osteogenic proteins designated OP-1, COP-5 and COP-7 are also disclosed in US Patent 5,011,691.

All previously published US Patent documents are incorporated herein by reference and are intended to be used in order to supplement the present teachings regarding the preparation of the BMP and OP genes and the DNA segments expressing the osteotropic polypeptides. . As disclosed in the above patents, and as is known to those skilled in the art, the original source of a recombinant gene or DNA segment to be used in a therapeutic regimen need not be of the same species as the animal to be treated. In this regard, it is contemplated that any recombinant PTH, TGF or BMP gene can be used to promote bone repair or regeneration in a human individual or animal; for example, a horse. Particularly preferred genes are those of human, murine, and bovine origin, because such genes and DNA segments are readily available, with murine and human forms of the gene being the most preferred for use in human treatment regimens. Recombinant proteins and polypeptides encoded by isolated DNA and gene segments are often referred to by the prefix "r" for recombinant, and "rh" for recombinant human. " As such, DNA segments encoding rBMPs, such as rhBMP-2 or rhBMP-4, are viewed as particularly useful in connection with the present invention.

The definition of a "BMP gene" as used herein is a gene that hybridizes, under relatively stringent hybridization conditions (see, eg, Maniatis et al., 1982) to currently known DNA sequences. include BMP gene sequences.

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To prepare an osteotropic gene segment or cDNA, the teachings disclosed herein and also those of any of the patents or scientific documents specifically referenced may be followed. Various nucleotide sequences encoding active BMPs are disclosed in US Pat. Nos. 5,166,058, 5,013,649, 5,116,738. 5,106,748, 5,187,076, 5,108,753 and 5,011,691, each of which is incorporated herein by reference. As an example only, US Patent 5,166,058 teaches that hBMP-2 is encoded by a nucleotide sequence ranging from nucleotide # 356 to nucleotide # 1543 of the sequence shown in Table II of the patent. An hBMP-2 DNA segment can be obtained in this way using molecular biology techniques, such as polymerase chain reaction (PCR ™) or by searching a cDNA or genomic library, using primers or probes with sequences based on the sequence. anterior nucleotide. The practice of such techniques is a routine matter for those skilled in the art, as taught in various scientific articles such as Sambrook et al., (1989), which is incorporated herein by reference. Certain documents particularly further describe suitable mammalian expression vectors, for example, US Patent 5,168,050, which is incorporated herein by reference.

Osteotropic genes and DNA segments that are particularly preferred for use in certain aspects of the present compositions and methods are the TGF, PTH and BMP genes. TFG genes are described in US Patent Documents: 5,618,051; 4,886,747 and 4,742,003, each of which will be incorporated herein by reference. TGF-α may not be as widely applicable as TGF / t, but it is proposed for use particularly in applications involving skeletal soft tissues. The PTH gene or a DNA segment that encodes its active fragment, such as a DNA segment that encodes a polypeptide that includes amino acids 1-34 (hPTH 1-34; Hendy et al., 1981; incorporated herein. memory for reference) is another preferred gene; as are the BMP genes designated BMP-4 and BMP-2, such as the gene or cDNA encoding BMP-4 disclosed herein.

It is also contemplated that genes or cDNAs encoding an osteotropic protein or polypeptide may be subsequently cloned. Techniques for cloning DNA molecules, that is, obtaining a specific coding sequence from a genomic library that is distinct from other portions of DNA, are well known in the art. This can be obtained from, for example, searching for an appropriate DNA library as disclosed herein in Example XV, which relates to the cloning of a wound healing gene. The search procedure may be based on the hybridization of oligonucleotide probes, designed from the consideration of parts of the amino acid sequence of known DNA sequences that encode related osteogenic proteins. The performance of such search protocols is well known to those of skill in the art and is described in detail in the scientific literature, for example, in Sambrook et al., (1989), which is incorporated herein by reference.

Osteotropic genes with sequences varying from those described in the literature are also encompassed by the invention, provided that the altered or modified gene still encodes a protein that acts to stimulate bone progenitor cells in any direct or indirect way. These sequences include those caused by point mutations, those due to degenerations of the genetic code or its allelic variants that occur naturally, or subsequent modifications that have been introduced by genetic engineering, that is, by man.

Techniques for introducing changes in nucleotide sequences that are designed to alter the functional properties of encoded proteins or polypeptides are well known in the art, for example, US Patent 4,518,584 which is incorporated herein as reference, which techniques are described herein in more detail. Such modifications include the deletion, insertion or substitution of bases, and therefore changes in the amino acid sequence. Changes can be made to increase a protein's osteogenic activity, to increase its biological stability or half-life, to change its glycosylation pattern, and the like. All such modifications to nucleotide sequences are encompassed by the present invention.

It will be understood, however, of course, that one or more than one osteotropic gene may be used in the methods and compositions of the invention. Nucleic acid delivery methods can thereby result in the delivery of one, two, three, or more osteotropic genes. The maximum number of genes that can be applied is limited only by practical considerations, such as the effort involved in simultaneously preparing a large number of gene constructs or even the possibility of causing a significant adverse cytotoxic effect. The particular combination of genes can be of two or more different BMP genes; or it can be such that a growth factor gene is combined with a hormone gene, for example a BMP gene and a PTH gene; a hormone or growth factor gene can even be combined with a gene encoding a cell surface receptor capable of interacting with the polypeptide product of the first gene.

By using multiple genes, they can be combined into a single gene construct under the control of one or more promoters, or they can be prepared as separate constructs of the same or different type. Thus, an almost endless combination of different genes and genetic constructs can be used. Certain gene combinations may be designed for, or their use may otherwise lead to, achieving synergistic effects on cellular stimulation and bone development, with any and all such combinations intended to be included 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 could identify probable synergistic gene combinations, or even gene-protein combinations.

IS 2 139 889 T5

It will also be understood that, if desired, the nucleic acid segment or gene could be administered in combination with other agents, such as, for example, proteins or polypeptides or various pharmaceutically active agents. As long as the genetic material is part of the composition, there is virtually no limit to other components that can also be included, since the additional agents do not cause a significant adverse effect on contact with the target cells or tissues. Nucleic acids can thus be administered in conjunction with various other agents, for example, in certain embodiments it may be desired to administer an angiogenic factor and / or an inhibitor of bone resorption, as disclosed in patent documents. US 5,270,300 and 5,118,667, respectively, each of which is incorporated herein by reference.

3. Gene constructs and DNA segments

As used herein, the terms "gene" and "DNA segment" are both used to refer to a DNA molecule that has been isolated free from total genomic DNA of a particular species. Thus, a gene or DNA segment encoding an osteotropic gene refers to a DNA segment that contains sequences encoding an osteotropic protein, but is isolated out or purified free from the total genomic DNA of the species from which it is derived. the DNA. Included in the term "DNA segment" are smaller DNA segments and fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phages, retroviruses, adenoviruses, and the like.

The term "gene" is used for simplicity to refer to a peptide coding unit or a functional protein. As will be understood by those of skill in the art, this functional term includes both genomic sequences and cDNA sequences. "Isolated substantially out of other coding sequences" means that the gene of interest, in this case an osteotropic gene, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large pieces of coding DNA that occurs naturally, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions, such as sequences encoding leader peptides or target sequences, later added to the segment by man.

The present invention provides new ways of utilizing various known osteotropic segments of recombinant DNA and vectors. As described above, many such vectors are readily available, and a particular detailed example of a vector suitable for expression in cells is described in US Pat. No. 5,168,050, which is incorporated herein by reference. of a mammal. However, it is not necessary that a highly purified vector be used, as long as the coding segment that is used encodes an osteotropic protein and does not include coding or regulatory sequences that have a significant adverse effect on bone progenitor cells. Thus, it will also be understood that useful nucleic acid sequences may include additional residues, such as additional noncoding sequences flanking the 5 'or 3' portions of the coding region or may include various internal sequences, i.e., introns, that they are known to be found inside genes.

After identifying an appropriate osteotropic gene or DNA molecule, it can be inserted into any of the many vectors that are commonly known in the art, in a manner that directs the expression and production of the osteotropic protein when incorporated into a bone progenitor cell. In a recombinant expression vector, the coding portion of the DNA segment is under the control of a promoter. This can be in the form of the promoter that is naturally associated with an osteotropic gene, as can be obtained by isolating non-coding sequences from the 5 'end that are located above the coding segment or exon, for example, using recombinant cloning and / or PCR technology. ™, in connection with the compositions disclosed herein.

In other embodiments, it is contemplated that certain advantages will be obtained by placing the coding segment of DNA under the control of a recombinant or heterologous promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with an osteotropic gene in its natural environment. Such promoters can include those normally associated with other osteotropic genes, and / or promoters isolated from other bacterial, viral, eukaryotic or mammalian cells. Naturally, it will be important to use a promoter that effectively drives the expression of the DNA segment in bone progenitor cells.

The use of recombinant promoters to obtain protein expression is generally known to those skilled in molecular biology, see for example Sambrook et al., (1989). The promoters used can be constitutive or inducible, and can be used under the appropriate conditions to drive high level expression, or regulated expression of the introduced DNA segment. Commonly employed promoters are those such as CMV, RSV LTR, the SV40 promoter alone, and the SV40 promoter in combination with various enhancement elements.

Osteotropic genes and DNA segments can also be in the form of a DNA insert that is located within the genome of a recombinant virus, such as for example a recombinant adenovirus, adeno-associated virus (AAV) or retrovirus. In said embodiments, to put the gene in contact with a bone progenitor cell, the recombinant viral particles, whose genome includes the insertion of the osteotropic gene, will be prepared, simply by putting the progenitor cells or tissues in contact with the virus, therefore the virus infects cells and transfers genetic material.

IS 2 139 889 T5

In certain preferred embodiments, a matrix or implant material would be impregnated with virus by immersing the material in a recombinant virus stock solution, for example, for 1 to 2 hours, and then contacting the bone progenitor cells or tissues. with the resulting impregnated matrix. The cells then penetrate, or enter, the matrix, thereby coming into contact with the virus and allowing viral infection, which results in the cells taking up the desired gene or cDNA and expressing the encoded protein.

In other preferred embodiments, a matrix-nucleic acid mixture would be formed, using naked DNA or a plasmid or viral vector, and the bone progenitor cells or tissues would be contacted with the resulting mixed matrix. The matrix can then introduce the nucleic acid into the cells after dissociation at the cell surface, or into the immediate cellular environment. Likewise, the matrix mixture itself, especially a particle or fiber-DNA mixture, can then be absorbed by cells to provide subsequent intracellular release of genetic material. The matrix can then be expelled from the cell, catabolized by it, or even stored within it. The molecular mechanism by which a bone-compatible matrix achieves transfer of DNA into a cell is indifferent to the practice of the present invention.

Four. Bone-compatible matrices

In certain preferred embodiments, the methods of the invention were applied to the preparation of a composition in which the osteotropic gene, genes, DNA segments or cells that already incorporate said genes or segments, is associated with, is impregnated in the interior of, or even coupled to, a compatible bone matrix according to claim 1, to form a "matrix-gene composition", then bringing this into contact with the bone progenitor cells or tissue. The matrix can then be impregnated with a gene DNA segment simply by immersing the matrix in a solution containing the DNA, such as a plasmid solution, for a short period of time from about 5 minutes, up to two weeks.

Matrix-gene compositions are all those in which genetic material is adsorbed, absorbed, impregnated, conjugated to, or generally otherwise kept in contact with the matrix. "Maintained in contact with the matrix" means that an effective amount of the nucleic acid composition must remain functionally associated with the matrix until its transfer to the bone progenitor cell or its release at the bone tissue site.

The type of matrix that can be used in the compositions, devices, and methods of the invention is a "bone-compatible matrix." This means that the matrix possesses all the characteristics commonly associated with being "biocompatible", in that it is in a form that does not produce a significant allergic or adverse reaction, or other undesirable reaction when administered to an animal, and that it is also suitable for putting it in contact with bone tissue. A "significant" adverse effect is one that outweighs the side effects that are normally accepted with any given therapy.

The term "bone-compatible", as used herein, means that the matrix (and gene) does not produce a significant adverse or inconvenient reaction when contacted with bone. In certain embodiments, when choosing to use a particular bone-compatible matrix, various other factors may optionally be taken into consideration, for example, the ability of the matrix to provide structure for the bone being developed, the ability for resorption in the body after the bone has been repaired, and such factors. However, these properties are not necessary to practice the invention and are only examples of factors that may be considered.

In other embodiments, the possibility of the matrix being transported into the cell, for example by active or passive membrane transport, may also be considered. When such transport and subsequent nucleic acid release are contemplated, other properties of the matrix and the gene can be evaluated to optimize the matrix-gene formulation. For example, adenovirus vectors can provide advantageous DNA delivery in such embodiments. In these, matrices that are readily metabolized in the cytoplasm would also probably be preferred. Matrices that are later released from the cell, and preferably also removed from the surrounding tissue area, would be another preferred form of matrix for use in such embodiments.

The selection of matrix material will differ depending on the particular circumstances and the bone site to be treated. Matrices such as those described in US Patent 5,270,300 (incorporated herein by reference) may be employed. Physical and chemical characteristics, such as, for example, biocompatibility, biodegradability, intensity, stiffness, interface properties, even cosmetic appearance, can be considered when choosing a matrix, as is well known to those skilled in the art. Appropriate matrices will release the gene composition and in certain circumstances, they can be incorporated into a cell, or they can provide a surface for new bone development, that is, they can act as an in situ scaffold through which progenitor cells can migrate. .

A particularly important aspect of the present invention is its use in connection with orthopedic grafts and artificial interfaces and joints, including the grafts themselves and the functional parts of a graft, such as, for example, surgical screws, nails, and the like. In preferred forms of organization, it is contemplated that the metal surface (s) of a graft or a part thereof, such as a titanium surface, will be upholstered with a material possessing affinity for nucleic acids, more preferably, with hydroxylapatite and then the

ES 2 139 889 T5 coated metal will be further upholstered with the gene or nucleic acid to be transferred. The available chemical groups of the absorption material, such as hydroxylapatite, can be easily manipulated to control its affinity for nucleic acids, as is known to those of skill in the art.

In certain embodiments, non-biodegradable matrices can be used, such as sintered hydroxylapatite, aluminates, other bioceramics, and metallic materials, such as titanium. A suitable ceramic system is that described in US Patent 4,596,574, which is incorporated herein by reference. Polymeric matrices can also be used, including acrylic ester polymers, lactic acid polymers, and polyglycolic polylactic acid (PLGA) block copolymers, which have been described (US Patent 4,526,909, 4,563,489, Simons et al. ., 1992, and Langer and Folkman, 1976, respectively, each of which is incorporated herein by reference).

In certain embodiments, it is contemplated that a biodegradable matrix will likely be the most useful. A biodegradable matrix is ​​generally defined as one that is capable of undergoing resorption in the body. Potential biodegradable matrices for use in connection with the compositions, devices, and methods of the present invention include, for example, chemically defined and biodegradable calcium sulfate, tricalcium phosphate, hydroxylapatite, PLGA block copolymers, polyanhydrides, purified protein matrices, and matrix compositions. semi-purified extracellular.

A preferred group of matrices are collagenous matrices, including those obtained from tendon or dermal collagen, for example type I collagen, which is generally prepared from the dermis; those obtained from cartilage, such as type II collagen; and various other types of collagen. Collagens can be obtained from various commercial sources; for example, Sigma which provides type II collagen obtained from bovine tracheas; and the Collagen Corporation. Collagen matrices can also be prepared as described in US Pat. Nos. 4,394,370 and 4,975,527, each of which is incorporated herein by reference.

The various collagen materials can also be in the form of mineralized collagen. A preferred mineralized collagen material is called UltraFiber ™, available from Norian Corp (Mountain View, CA). US Patent No. 5,231,169, which is incorporated herein by reference, describes the preparation of mineralized collagen through the formation of calcium phosphate mineral under gentle in situ agitation in the presence of dispersed collagen fibrils. Such a formulation can be used in the context of the release of a nucleic acid segment at a site in bone tissue.

Some other preferred collagen materials are those based on type II collagen. Type II collagen preparations have been found to have the surprising and advantageous property of stimulating bone progenitor cells in the absence of any osteotropic genes. Prior to the present invention, type II collagen was thought to have only a structural role in cartilaginous extracellular matrix and the present finding that type II collagen is currently an osteoconductive / osteoinductive material is unexpected. The present invention thus contemplates the use of a variety of type II collagen preparations as gene transfer matrices or bone cell stimulants, with or without DNA segments, including the original type II collagen, prepared from cartilage, and recombinant type II collagen.

PLGA block copolymers can also be used as gene transfer matrices. Such polymers have been shown to readily incorporate DNA, are commercially available, non-toxic, and hydrolyze in defined proportions (that is, they facilitate sustained release of pharmaceutical agents). PLGA block copolymers possess two particular advantageous properties: first, they exhibit reversible thermal gelation, and second, they can be combined with other agents to allow radiographic visualization.

5. Ways of carrying out nucleic acid transfer

Once an appropriate matrix-gene composition has been prepared or obtained, all that is required to deliver the osteotropic gene into bone progenitor cells within an animal is to bring the matrix-gene composition into contact with the site at the organism in which it is desired to promote bone growth. This can be achieved by physically placing the matrix-gene composition in contact with the organism site, or by applying an injectable dosage form of the matrix-gene composition to the appropriate area.

The matrix-gene composition can be applied to the site of a simple bone fracture to be repaired, an area of ​​weak bone, such as in a patient with osteoporosis, or to a bone cavity to be filled with new bone tissue. Bone cavities can occur as a result of an inherited disorder, birth defect, or they can come from dental or periodontal surgery or after removal of an osteosarcoma.

The use of PLGA compounds and the like as matrices allows the matrix-DNA composition to be syringed, which is generally achieved by mixing the matrix-gene composition with a pluronic agent. The resulting matrix-gene / pluronic agent composition can be stored within a heat-jacketed syringe, maintained at a temperature of about 4 ° C, immediately prior to administration to the body. At this temperature and with this environment, the composition will be liquid. After insertion into the body, the composition will equilibrate with respect to body temperature, and in doing so will form a gelatinous matrix.

IS 2 139 889 T5

The above phenomenon is called "reversible thermal gelation" and allows a controlled rate of gelation to be achieved. How to use pluronic agents in this context will be known to those skilled in the art in light of the present disclosure. The matrix-gene / pluronic agent compositions can also be mixed, or generally associated with an imaging agent, so that the present gene transfer technology can be used in various imaging modalities. In these cases, the attending physician or veterinarian must be able to control the release and positioning of the matrix-gene composition. Many safe and effective imaging agents are available, such as the radiographic compound calcium phosphate, which can be used in conjunction with fluoroscopy, or even with tomography, to image the body or tissue site while the composition is being applied. is releasing.

Where an image of the tissue site is to be provided, it will be desired to use a detectable imaging agent, such as a radiographic agent, or else, a paramagnetic or radioactive agent. Many radiographic diagnostic agents are known in the art to be useful for imaging, including, for example, calcium phosphate.

In the case of paramagnetic ions, examples include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III) , samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III), with gadolinium being generally preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially, bismuth (III).

Although not generally preferred, radioactive isotopes are not excluded and can be used for imaging, if desired. Suitable ions include iodine¹³¹, iodine¹²³, technetium^99m, indian¹¹¹, rhenium¹⁸⁸, rhenium¹⁸⁶, gallium⁶⁷, copper⁶⁷, yttrium⁹⁰, iodine¹²⁵ and astatus²¹¹.

The amount of gene construct that is applied to the matrix and the amount of matrix-gene material that is applied to the bone tissue will be determined by the attending physician or veterinarian considering various medical and biological factors. For example, one would want to consider the particular osteotropic gene and matrix, the amount of bone weight to be formed, the site of bone damage, the condition of the damaged bone, the age, sex and diet of the patient or animal, the severity of any infection, the time of administration, and any subsequent clinical factors that may affect bone growth, such as serum levels of various factors and hormones. The appropriate dosage regimen will therefore be readily determinable by any person skilled in the art in light of the present disclosure, taking into account individual circumstances.

In treating animals and man, progress can be monitored by periodic evaluation of bone growth and / or repair, for example using X-rays. The therapeutic methods and compositions of the invention are contemplated for use in medical and veterinary applications, due to to the lack of species specificity in bone inducing factors. In particular, it is contemplated that domestic, farm and zoo animals, as well as purebred horses, would be treatable using the nucleic acid transfer protocols disclosed herein.

Current methods and compositions may also have prophylactic uses in the reduction of open and closed fractures and also in the improved fixation of artificial joints. The invention is applied to stimulate bone repair in congenital craniofacial defects, induced by trauma or by oncological resection, and is also useful in the treatment of periodontal diseases and in other dental repair procedures and also in cosmetic plastic surgery. The matrix-gene compositions and devices of the present invention may also be used in wound healing and related tissue repair, including, but not limited to, healing of burns, incisions, and ulcers.

The present invention also encompasses DNA-based compositions for use in cell transfer to treat bone defects and disorders. The compositions of the present invention generally comprise an osteotropic gene associated with a bone-compatible matrix, such as type II collagen, wherein the composition is capable of stimulating bone growth, repair or regeneration after administration to, or grafting into, a site of the bone parent tissue of an animal. The osteotropic gene or genes can be any of those described above, the TGF-α genes (for soft skeletal tissues), TGF-6 being generally preferred.₁, TGF- / T, TGF-e₃, PTH, BMP-2 and BMP-4. Likewise, regardless of gene selection, the compatible bone matrix can be any of those described above, with biodegradable matrices such as collagen, and more particularly, type II collagen being preferred.

In still further embodiments, the present invention relates to osteotropic devices, which can generally be considered as gene-matrix-engineered or molded compositions. The devices of the present invention naturally comprise a bone-compatible matrix in which an osteotropic gene associates with the matrix. The combination of genes and matrix components is such that the device is capable of stimulating bone growth or healing when grafted into an animal. The devices can be of virtually any size or shape so that their dimensions are adapted to fit a bone fracture or bone cavity site in the animal to be treated, allowing the fracture joint and / or bone regrowth are more uniform. Other devices that are particularly contemplated are those that are designed to act as an artificial joint. Titanium and hydroxylapatite-coated titanium devices will be preferred in

ES 2 139 889 T5 certain embodiments. Device parts in combination with an osteotropic nucleic acid segment, such as a DNA-coated screw for an artificial joint, and the like, are also within the scope of the present invention.

Therapeutic kits comprising, in appropriate container means, a bone-compatible matrix, such as type II collagen or a PLGA block copolymer, and an osteotropic gene, constitute another aspect of the invention. Such kits will generally contain a pharmaceutically acceptable formulation of the matrix and a pharmaceutically acceptable formulation of an osteotropic gene, such as PTH, BMP, TGF-β, FGF, GMCSF, EGF, PDGF, IGF or a LIF gene. Commonly preferred genes include PTH, TGF-61, TGF-62, TGF- / 13, and BMP-4.

The kits may comprise a single container medium that contains both the biocompatible matrix and the osteotropic gene. The container means may, if desired, contain a sterile pharmaceutically acceptable injectable matrix, having associated therewith the composition of the osteotropic gene, and, optionally, a detectable marker or imaging agent. The injectable DNA-matrix formulation may be in the form of a gelatinous composition, for example a type II collagen-DNA composition, or it may be in a more fluid form which nevertheless gives rise to a gel-like composition after administration to the body. In these cases, the container means can itself be a syringe, pipette, or other similar apparatus, from which the DNA-matrix material can be applied to a site of the bone tissue or the wound area. However, the single container medium may contain a dry or lyophilized mix, and osteotropic gene and matrix composition, which may or may not require pre-wetting prior to use.

Alternatively, the kits of the invention may comprise different container means for each component. In such cases, one container will contain the osteotropic gene, either as a sterile DNA solution or in lyophilized form, and the other container will include the matrix, which may or may not in turn be pre-moistened with a sterile solution, or be in sterile form. gelatinous, liquid or other injectable type.

The kits may also comprise a second or third container means for containing a sterile pharmaceutically acceptable buffer, diluent or solvent. Such a solution may be required to formulate either the DNA component, the matrix component, both components separately, or a premixed combination of the components, into a form more suitable for application to the body, eg, a more gelatinous form. It should be noted, however, that all components of a kit must be supplied in a dry form (lyophilized), which will allow them to be “wetted” after contact with body fluids. Thus, the presence of any type of pharmaceutically acceptable buffer or solvent is not a necessity for the kits of the invention. The kits may also comprise a second or third container means for containing a pharmaceutically acceptable detectable imaging agent or composition.

The container means will generally be a container such as a vial, test tube, flask, bottle, syringe or other container means, in which the components of the kit can be arranged. The matrix and gene components can also be aliquoted into smaller containers, if desired. The kits of the present invention may also include a means for containing the individual containers in a closed packaging for commercial sale, such as injection or blow molded plastic containers in which the desired vials or syringes are retained.

Regardless of the number of containers, the kits of the invention may also comprise, or be packaged with, an instrument to aid in the application of the defined matrix-gene composition within the organism of an animal. Such an instrument can be a syringe, pipette, forceps, or any suitable medical delivery vehicle.

6. Type II collagen as an osteoconductive / inducer material

The present invention also provides methods for stimulating bone progenitor cells, as it can be applied, in certain circumstances, to promote new bone formation, or to stimulate wound healing. As such, the bone progenitor cells that are the targets of the invention may also be referred to as "wound healing bone progenitor cells". Although the function of wound healing itself may not always be required to practice all aspects of the invention, and although mechanical knowledge is not required to carry out the invention, it is generally thought that the healing procedure wound treatment operates during the performance of the present invention.

To stimulate a bone progenitor cell in accordance with these aspects of the invention, a bone progenitor cell will generally be contacted with a composition comprising a biologically effective amount of type II collagen. Although crushed bone and mineralized collagen preparations have been shown to be osteoconductive, this property had not previously been ascribed to type II collagen. The inventors have found that type II collagen alone is surprisingly effective in promoting new bone formation, being able to bridge a 5mm osteotomy space in just eight weeks in all animals tested (Fig. 5A, Fig. 5B , Fig. 6A, Fig. 6B, Fig. 6C, Fig. 6D, Fig. 7A, Fig. 7B, Fig. 8A, Fig. 8B, and Fig. 8C).

The forms of type II collagen that can be used in the present invention are virtually unlimited. For example, type II collagen can be purified from the hyaline cartilage of bovine trachea, or isolated from

ES 2 139 889 T5 of the diarthrosic joints or growth plates. Purified type II collagen is commercially available and can be obtained from, for example, Sigma Chemical Company, St. Louis, MO. Any form of recombinant type II collagen can also be used, as it can be obtained from a recombinant host cell expressing type II collagen, including bacterial, yeast, mammalian, and insect cells. A particular example of a recombinant type II collagen expression system is a yeast cell that includes an expression vector encoding type II collagen, as disclosed herein in Example VI.

The type II collagen used in the present invention can, if desired, be supplemented with additional minerals, such as calcium, for example in the form of calcium phosphate. Both the original and recombinant type II collagen can be supplemented by mixing, adsorbing , or associating with additional minerals in this way. Such type II collagen preparations are clearly distinguished from the types of "mineralized collagen" which have been previously described, for example, in US Patent: 5,231,169 which describes the preparation of mineralized total collagen fibrils.

An objective of this aspect of the present invention is to provide a source of matrix osteoconductive material that can be reproducibly prepared in a simple and cost-effective manner and that can be used, with an osteotropic gene segment, to stimulate bone progenitor cells. Recombinant type II collagen was surprisingly found to meet these criteria. The present invention also encompasses, although it is not clearly necessary for effective results, the combination of original or recombinant type II collagen with mineral supplements, such as calcium.

A biologically effective amount of type II collagen is an amount of type II collagen that functions to stimulate a bone progenitor cell, as described herein. As an example, a measurement of a biologically effective amount is an amount effective to stimulate bone progenitor cells until new bone formation is evidenced. In this regard, the inventors have shown that 10 mg of lyophilized collagen works effectively to close a 5 mm osteotomy space in three weeks. This information can be used by those skilled in the art to optimize the amount of type II collagen that is needed for any given situation.

Depending on individual cases, the skilled person will be able, in light of this discussion, to easily calculate an appropriate amount, or dose, of type II collagen to stimulate bone cells and promote bone growth. In terms of small animals or human individuals, appropriate effective amounts of collagen include between about 1 mg and about 500 mg, and preferably between about 1 mg and 100 mg, of lyophilized type II collagen per bone tissue site. Of course it is likely that variations will exist due to, for example, individual responses, particular tissue conditions, and the speed with which bone formation is required. While 10 mg proved useful in the illustrative example, the inventors contemplate that 1,5,10,15,20,30,40,50,75,100,125,150,200,300 mg and the like can be usefully used for patients. humans and small animals. Of course, any of the values ​​contemplated can be useful in any particular case.

Naturally, one of the main variables to consider is the amount of new bone that needs to be generated in a particular area or bone cavity. This can be largely a function of the size of the animal to be treated, for example a cat or a horse. Thus, there is usually no upper limit on the amount of type II collagen or indeed the amount of any matrix-gene composition that can be employed in the methods of the present invention, given careful supervision by the physician.

By contacting or applying type II collagen, with a DNA segment, to bone progenitor cells located within a bone progenitor tissue site of an animal, bone growth will be stimulated. Thus, bone socket sites and bone fractures can be filled and repaired.

The use of type II collagen in combination with a nucleic acid segment encoding a polypeptide or protein that stimulates bone progenitor cells when expressed in such cells, as described above, is preferred. Nucleic acid segments comprising an isolated PTH gene, a BMP gene, a growth factor gene, a growth factor receptor gene, a cytokine gene, or a chemotactic factor gene are preferred, the PTH genes being, TGF-β and BMP most preferred. The genes function subsequent to their transfer to, and expression in, the progenitor cells of the treated animal, thereby promoting bone growth.

Although type II collagen is effective alone, its use in combination with an osteotropic gene segment may prove to have synergistic and particularly advantageous effects. Type II collagen, native or recombinant, can thus also be formulated in a therapeutic kit with an osteotropic gene segment, according to the kits described hereinabove. This includes the use of single or multiple container means, and combination with any medically approved delivery vehicle, including, but not limited to, syringes, pipettes, forceps, additional diluents, and the like.

IS 2 139 889 T5

Brief description of the drawings

The drawings are part of the present specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by referring to one or more of these drawings in conjunction with the detailed description of specific embodiments presented herein.

Fig. 1. depicts a DNA therapy model for bone repair.

Fig. 2A. represents a schematic model of the cellular and molecular basis of the direct mechanism of DNA transfer to osteogenic cells in vivo. The procedure of creating an osteotomy and placing the genetically activated matrix in situ is depicted.

Fig. 2B. represents a schematic model of the cellular and molecular basis of the direct mechanism of DNA transfer to osteogenic cells in vivo. The repair cell fracture procedure is depicted where blood vessels grow in the gene activated matrix (Fig. 2A).

Fig. 2C. represents a schematic model of the cellular and molecular basis of the direct mechanism of DNA transfer to osteogenic cells in vivo. Fractured cells are shown incorporating DNA as an episomal element, ie direct gene transfer in vivo.

Fig. 2D. represents a schematic model of the cellular and molecular basis of the direct mechanism of DNA transfer to osteogenic cells in vivo. Fractured repair cells that synthesize and secrete recombinant proteins encoded by episomal DNA are depicted.

Fig. 2E. represents a schematic model of the cellular and molecular basis of the direct mechanism of DNA transfer to osteogenic cells in vivo. The resulting new bone formation is depicted.

Fig. 3A. represents Achilles tendon gene transfer is represented as an overview of the time course at 3 weeks postoperative.

Fig. 3B. represents Achilles tendon gene transfer is represented as an overview of the time course at 9 weeks postoperative.

Fig. 3C. represents Achilles tendon gene transfer is represented as an overview of the time course at 12 weeks postoperative.

Fig. 3D. represents Achilles tendon gene transfer is represented as an immunohistochemical study over time. The microscopy of the tendon tissue that received the SIS graft impregnated with the expression plasmid DNA is represented. Note positive cytoplasmic staining of fibroblast cells 9 weeks after surgery.

Fig. 3E. represents Achilles tendon gene transfer is represented as an immunohistochemical study over time. Microscopy of tendon tissue that received the SIS graft alone, without DNA, is depicted. Note the relative absence of cytoplasmic staining.

Fig. 4. Depicts the control of cruciate ligament gene transfer using a substrate utilization assay. Three weeks after implantation of the SIS soaked in a solution of the expression plasmid pSV40d-gal, the tendon tissue was harvested, briefly fixed in 0.5% glutaraldehyde, and then incubated with X-gal according to published methods. . The tissues were then embedded in paraffin and sectioned and stained with H and E. Note the positive staining (arrows) in the cytoplasm of the fibroblasts of the granulation tissues.

Fig. 5A. represents Direct transfer of DNA to regenerating bone: β-gal activity. The figure compares β-galactosidase activity in osteotomic cleft tissue homogenates from two Sprague-Dawley rats. In animal # 1, the UltraFiber ™ graft material was soaked in a solution of pSV4o / l-gal DNA (Promega) encoding bacterial β-galactosidase. In animal # 2, the graft material was soaked in a pure solution of Vector DNA-Promoter pGL2 (Promega) encoding insect luciferase. Enzyme activity was determined using substrate assay kits (β-galactosidase and Luciferase Assay Systems, Promega). Note that significant β-galactosidase activity was found only in homogenate prepared from animal # 1.

Fig. 5B. represents the direct transfer of DNA into regenerating bone: luciferase activity. The figure compares the luciferase activity in aliquots of the homogenates described in Fig. 5A. Luciferase activity was determined using the commercial reagents and protocols (Promega) described in Fig. 5A. Note that significant luciferase activity is found only in homogenate prepared from animal # 2.

Fig. 6A. represents osteotomic gene transfer controlled by PTH studies. In this study, an expression plasmid encoding a 34 amino acid functional peptide fragment of human parathyroid hormone

ES 2 139 889 T5 (PTH1-34) was transferred and expressed in vivo using GAM technology. The progress of new bone formation in the cleft was radiographically monitored for three weeks and the animals were sacrificed. A radiograph of the osteotomic cleft of the animal that received the hPTH1-34 GAM sense construct is depicted. Note the presence of radiodense tissue in the cleft (arrow).

Fig. 6B. represents osteotomic gene transfer (Fig. 6A) controlled by PTH studies. A radiograph of the osteotomic cleft of the control animal that received an hPTH1-34 GAM antisense construct is depicted. There was no evidence of radiodense tissue in the cleft.

Fig. 6C. represents osteotomic gene transfer (Fig. 6A) controlled by PTH studies. A histological section of the osteotomic repair tissue from the same control animal as in Fig. 6B is represented. The section is characterized by the presence of fibroblasts from granulation tissue and capillaries.

Fig. 6D. represents osteotomic gene transfer (Fig. 6A) controlled by PTH studies. A histological section of osteotomic repair tissue from the same animal that received the hPTH1-34 GAM sense construct (as in Fig. 6A) is depicted. The section is characterized by the presence of trabecular bone plates that extend into the cleft from the surgical margin.

Fig. 7A. represents osteotomic gene transfer studies with BMP-4. Immunohistochemical evidence of transgene expression of BMP-4 by fibroblasts in granulation tissue near the center of an osteotomy cleft three weeks after surgery is depicted. Note the positive staining (arrows) of the axial cells. The BMP-4 transgene included an epitope tag (HA epitope, Pharmacia) that facilitated the identification of the BMP-4 transgenic molecules. Tissue staining was performed using commercially available polyclonal anti-HA antibodies and standard procedures. The immunostaining was localized only to the tissues of the cleft. Control sections included serial sections stained with rabbit preimmune serum and tissue sections from 13 control osteotomy slits. In both cases, all controls were negative for peroxidase staining of granulating tissue fibroblasts.

Fig. 7B. represents osteotomic gene transfer studies with BMP-4. The histology of the newly formed bone is depicted as early as three weeks after gene transfer (Fig. 7A).

Fig. 8A. represents radiographic evidence of the formation of a new bone junction (arrows) as a consequence of the transfer of the BMP-4 gene and expression at six weeks after surgery. 9 and 16 weeks after surgery are presented in Fig. 8B and Fig. 8C, respectively, to demonstrate the anterior growth of new bone in situ over time. This animal, which has been kept for 23 weeks, has been moving normally without an external fixator for the past 7 weeks. Similar results have been obtained in a second long-term animal (out of two) that is now in the 17^to week after the operation.

Fig. 8B. represents radiographic evidence of the formation of a new bone splice (arrows) as a consequence of the transfer of the BMP-4 gene and its expression, nine weeks after surgery (see Fig. 8A).

Fig. 8C. represents radiographic evidence of the formation of a new bone splice (arrows) as a consequence of the transfer of the BMP-4 gene and its expression, at the 16th week after surgery (see Fig. 8A).

Fig. 9A. represents the animal shown represents the control group that received an osteotomy plus a collagen sponge without DNA of any kind. The animal was kept for 9 weeks after surgery and was then sacrificed. The progress of new bone formation in the cleft was monitored radiographically and histologically. A radiograph of the osteotomic cleft is depicted at 9 weeks. Note the absence of radiodense tissue in the cleft.

Fig. 9B. represents a histological section of the osteotomic cleft tissue of the control animal used in Fig. 9A. The cut is characterized by the presence of fibroblasts and capillaries from the granulation tissue.

Fig. 10. Depicts an expression construct PLJ-HPTH1-34. A cDNA fragment encoding a prepro-hPTH1-34 peptide was generated by PCR ™ (Hendy et al., 1981) then ligated into a BamHI cloning site (Wilson et al., 1992). Several independent clones with the insert in the coding orientation were isolated and characterized.

Fig. 11. represents the Southern analysis of retroviral integration in clone YZ-15. 10 mg of YZ-15 genomic DNA was digested with Kpnl (for which there is a unique site in the LTR vector) and analyzed by Southern blotting. A cDNA fragment encoding prepro-hPTH1-34 was used as a probe. The positive control for the Southern hybridization conditions was a KpnI digest of genomic DNA from Rat-1 cells infected and selected with the recombinant retrovirus PLJ-hPTH1-84 incomplete in replica (Wilson et al., 1992). KpnI DNA digests were also prepared from two negative controls: original Rat-1 cells and BAG-selected and infected Rat-1 cells ("BAG cells"), (Wilson et al., 1992), an incomplete recombinant retrovirus in replica encoding lajd-galactosidase, which is an irrelevant marker gene in these studies. The assignments

ES 2 139 889 T5 lanes were as follows: 1, PLJ-hPTH1-84 cells; 2, BAG cells; 3, YZ-15; 4, Rat-1 parent cells. DNA sizes (kb) are represented on the left of the figure. As expected, a fragment of the predicted size (eg, 4.3 kb) is seen only in lane 1 (positive control) and lane 3 (YZ-15 DNA).

Fig. 12. depicts Northern blot analysis of a transduced Rat-1 clone. Poly-A (+) RNA was prepared from clone YZ-15 and analyzed by Northern blotting as described (Chen et al., 1993). Fig. 12 contains two frames on a single sheet. Poly-A (+) RNA prepared from PLJ-hPTH1-84 cells, BAG cells, and parent Rat-1 cells were used as positive and negative controls. Four probes were applied to a single blot following the sequence: hPTH1-34, β-gal, Neo, and jd-actin. The lane assignments were as follows: PLJ-hPTH1-84 cells; 2, BAG cells; 3, YZ-15 cells; 4, Rat-1 parent cells. As expected, the hPTH1-34 transcript is seen only in lane 1 (positive control) and lanes 3-4; a Neo transcript is seen only in lanes 1-3; a d-gal transcript is seen only in lane 2; and jd-actin transcripts are seen in lanes 1-4.

Fig. 13. depicts Northern analysis of poly-A (+) RNA demonstrating PTH / PTHrP receptor expression in osteotomic repair tissue.

Fig. 14. Depicts the overlapping murine cDNA Clones representing the LTBP-like sequence (LTBP3). A partial representation of the restriction sites is shown. N, NcoI; P, PvuII; R, RsaII; B, BamHI; H, HindIII. The numbering system in the background assumes that the "A" of the Met initiation codon is nt # 1.

Fig. 15A. is a schematic depicting the structure of the murine fibrillin-1 gene product. The structural domains are represented below the diagram. Symbols designating various structural elements are defined in the legend of Fig. 15B.

Fig. 15B. is a scheme that represents the structure of the LTBP-like molecule (LTBP-3). Domains # 1-5 are indicated below the diagram. The symbols designate the following structural elements: EGF-CB repeats: open rectangles; TGF base pair repeats: open ovals; Fib motif: open circle; TGF base pair type repeats: oval pattern; cysteine-rich sequences: rectangle drawing; proline / glycine rich region: thick curved line, domain # 2; proline rich region, thick curved line, domain # 3. Note that the symbols designating the signal peptide have been deleted for the sake of simplicity. Additionally, the scheme assumes that the EGF and eGf-CB-like repeats can extend several amino acids behind the C position.₆.

Fig. 15C. is a schematic representing the structure of human LTBP-1. Domains # 1-5 are noted below the diagram. The symbols designating the structural elements are defined in the legend of Fig. 15B.

Fig. 16. is an overview of the expression of the new LTBP-like gene (LTBP-3) during murine development, determined by tissue in situ hybridization. Fig. 16 is composed of autoradiograms made by direct exposure of tissue sections to photographic film, after hybridization with radioactively labeled probes. On day 8.5-9.0 the sections contained embryos surrounded by intact membranes, uterine tissues, and the placental disc, sliced ​​into random planes. On days 13.5 and 16.5, the sections contained isolated whole embryos, sectioned in the sagittal plane close to or close to the median axis. Identical conditions were maintained during autoradiography and photography, thus allowing a comparison of the overall intensity of hybridization in all tissue sections. The transcript is expressed in connective tissue, mesenchyme, liver, heart, and CNS.

Fig. 17A. are selected microscopic views of mouse LTBP-3 gene expression in mouse developing tissues on day 8.5-9.0. All photographs in Figs. 17A-17D were taken from identical slides used to prepare the mounted whole sections (after dipping the slides in the radiographic emulsion). The neural tube is represented, brightfield image 1 cm = 20 mm.

Fig. 17B. are selected microscopic views of mouse LTBP-3 gene expression in mouse developing tissues on day 8.5-9.0. The neural tube is represented, dark field image. Note the expression by the neuroepithelial cells and by the surrounding mesenchyme. 1 cm = 20 mm.

Fig. 17C. are selected microscopic views of mouse LTBP-3 gene expression in mouse developing tissues on day 8.5-9.0. The heart is represented, image in bright field. The figure demonstrates expression by mine and endocardial cells (arrows). 1 cm = 20 mm.

Fig. 17D. are a selected microscopic view of mouse LTBP-3 gene expression in mouse developing tissues on day 8.5-9.0. Heart shown, darkfield image. The figure demonstrates expression by mine and endocardial cells (arrows). Dark field photomicrographs were taken after exposure of the tissues to the photographic emulsion for 2 weeks. In this image and the one represented in Fig. 17B, ​​red blood cells and other plasma membranes gave rise to a pale white signal that contributes to the background of the experiment. 1 cm = 20 mm.

Fig. 18A. is a microscopy of mouse LTBP-3 gene expression in mouse developing tissues on day 13.5 and day 16. All photographs in Figs. 18A-18P were taken from identical slides used to prepare the mounted whole sections (after dipping the slides in the radiographic emulsion).

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The cartilage model of the long bone that develops from the lower limb is represented, brightfield image. Expression by chondrocytes and perichondrial cells is seen in Fig 18B. 1 cm = 20 mm.

Fig. 18B. is a microscopy of the expression of the mouse LTBP-3 gene in the developing tissues of the mouse on day 13.5 and 16. The cartilage model of the long bone that develops from the lower limb is represented, darkfield image. Note the expression by chondrocytes and perichondrial cells. In all darkfield views of Fig. 18, the red blood cells and other plasma membranes gave rise to a pale white signal that contributes to the background of the experiment. Note the absence of a spurious hybridization signal in areas of the slide lacking cellular elements. 1cm = 20mm.

Fig. 18C. is a microscopy of mouse LTBP-3 gene expression in mouse developing tissues on day 13.5 and 16. Lungs are represented, brightfield image. 1cm = 20mm.

Fig. 18D. is a microscopy of mouse LTBP-3 gene expression in mouse developing tissues on day 13.5 and 16. Heart is represented, brightfield image. 1 cm = 20 mm.

Fig. 18E. is a microscopy of mouse LTBP-3 gene expression in mouse developing tissues on day 13.5 and day 16. Lungs are depicted, dark field image. Note expression by developing airway epithelial cells and surrounding parenchymal cells. 1 cm = 20 mm.

Fig. 18F. is a microscopy of mouse LTBP-3 gene expression in mouse developing tissues on day 13.5 and day 16. The heart is represented, dark field image. Note the continued expression by myocardial cells. 1 cm = 20 mm.

Fig. 18G. It is a microscopy of the expression of the mouse LTBP-3 gene in the developing tissues of the mouse on day 13.5 and day 16. The pancreas is represented, image in bright field. 1 cm = 20 mm.

Fig. 18H. It is a microscopy of the expression of the mouse LTBP-3 gene in the developing tissues of the mouse on day 13.5 and day 16. The intestine is represented, image in bright field. 1 cm = 20 mm.

Fig. 18I. It is a microscopy of the expression of the LTBP-3 gene in the mouse in its developing tissues on day 13.5 and 16. The pancreas is represented, dark field image. Note the expression by acinar epithelial cells, 1 cm = 20 mm.

Fig. 18J. It is a microscopy of the expression of the mouse LTBP-3 gene in the developing tissues of the mouse on day 13.5 and 16. The intestine is represented, dark field image. Note the expression in epithelial and subepithelial cells. 1 cm = 20 mm.

Fig. 18K. It is a microscopy of the expression of the mouse LTBP-3 gene in the developing tissues of the mouse on day 13.5 and 16. The kidney is represented, image in bright field. 1 cm = 20 mm.

Fig. 18L. It is a microscopy of the expression of the mouse LTBP-3 gene in the developing tissues of the mouse on day 13.5 and 16. The skin is represented, image in bright field. 1 cm = 20 mm.

Fig. 18M. It is a microscopy of the expression of the mouse LTBP-3 gene in the developing tissues of the mouse on day 13.5 and 16. The kidney is represented, dark field image. Note expression by blasthemic cells below the renal capsule, by developing epithelial cells of nephrons and tubules, and by interstitial mesenchyme. 1 cm = 20 mm.

Fig. 18N. It is a microscopy of the expression of the mouse LTBP-3 gene in the developing tissues of the mouse on day 13.5 and day 16. The skin is represented, dark field image. Note the expression by the epidermal, adnexal, and dermal cells of the developing skin. 1 cm = 20 mm.

Fig. 18O. It is a microscopy of the expression of the mouse LTBP-3 gene in the developing tissues of the mouse on day 13.5 and day 16. The retina is represented, image in bright field. 1 cm = 20 mm.

Fig. 18P. It is a microscopy of the expression of the LTBP-3 gene in the developing tissues on day 13.5 and 16. The retina is represented, dark field image. Note the expression by retinal epithelial cells and by adjacent connective tissue cells. 1 cm = 20 mm.

Fig. 19. Depicts the time-dependent expression of the LTBP-3 gene by MC3T3-E1 cells. The mRNA preparation and Northern blotting were carried out as described in Example XIV. Equal aliquots of total RNA determined by UV spectroscopy were loaded onto each lane of the Northern gel. As demonstrated by methylene blue staining (Sambrook et al., 1989), equal amounts of RNA were transferred to the nylon membrane. The results demonstrate a precise and intense peak in LTBP-3 gene expression after 14 days in culture. Weaker signals that revealed the expression of the LTBP-3 gene can also be observed after 5 and 28 days in culture.

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Fig. 20. Depicts antiserum # 274 that specifically binds LTBP-3 epitopes. Transfection of 293T cells with a full-length mouse LTBP-3 expression plasmid, followed by radioactive labeling, medium sample preparation, immunoprecipitation, and 4-18% gradient SDS-PAGE, were performed as is described in Example XIV. The figure represents an SDS-PAGE autoradiogram of samples of the medium following a 2-day exposure to photographic film. The lane assignments are as follows: lane 1, radioactively labeled 293 T medium (prior to transfection) immunoprecipitated with preimmune serum; Lane 2: radioactively labeled 293T medium (prior to transfection) immunoprecipitated with antibody # 274; lane 3, radiolabelled 393T medium (after transfection and preincubation with 10 pg of the synthetic peptide cocktail LTBP) immunoprecipitated with antibody # 274; and lane 4, radioactively labeled 293T medium (after transfection) immunoprecipitated with antibody # 274. As indicated by the bar, the full length LTBP-3 molecule migrated at 180-190 kDa.

Fig. 21. Represents the co-immunoprecipitation of LTBP-3 and TG-βι produced by MC3T3-E1 cells. Aliquots (~ 10⁶ of incorporated CMP) of radioactively labeled medium produced by MC3T3-E1 cells after 7 days in culture were immunoprecipitated as described in Example XIV. Bars indicate the position of cold molecular weight standards used to estimate molecular weight (Rainbow mix, Amersham). Immunoprecipitates were separated using SDS-PAGE with a 4-18% gradient and reducing conditions. The figure shows a negative control lane 1 consisting of MC3T3-E1 medium immunoprecipitated with anti-LTBP-3 antibody # 274. Western blotting was carried out using the bottom of the gradient gel and a commercially available TGF-β antibody (Santa Cruz Biotechnology, Inc.). Antibody staining was detected using commercially available reagents and protocols (ECL Western Blotting Reagent, Amersham). MC3T3E1 medium was immunoprecipitated with anti-LTBP-3 antibody # 274.

Fig. 22A. Represents radiographic analysis of type II collagen osteotomic cleft three weeks after surgery.

Fig. 22B. Represents radiographic analysis of type I collagen osteotomic cleft three weeks after surgery.

Fig. 22C. Represents the histological analysis of the type II collagen osteotomy shown in Fig. 22A.

Fig. 23A. Represents adenovirus-mediated gene transfer into bone repair / regeneration cells in vivo. Positive β'-gal cytoplasmic staining (arrows) is observed in fracture repair cells.

Fig. 23B. Represents adenovirus-mediated gene transfer into bone repair / regeneration cells in vivo. Negative control of serial sections stained with the β-gal antibody vehicle plus a cocktail of non-specific rabbit IgG antibodies.

Fig. 23C. Represents adenovirus-mediated gene transfer into repair / regeneration cells in vivo. The osteotomy site was filled with a fibrous collagen graft material soaked in a solution of the incomplete recombinant adenovirus in the AdRSVe-gal replica (-10¹¹ plaque-forming units / ml). Note the positive β-gal nuclear staining (arrow) of the chondrocytes within the osteotomic site, as demonstrated by immunohistochemistry using a specific anti-e-gal antibody.

Fig. 24 is the murine amino acid sequence BMP-4, SEQ ID NO: 1. The HA epitope is represented in bold type at the carboxy terminal end of the sequence.

Fig. 25 is the DNA sequence of the murine LTBP-3 gene (SEQ ID NO: 2).

Fig. 26 is the amino acid sequence of the murine LTBP-3 gene product (SEQ ID NO: 3).

Fig. 27. is the DNA sequence of the murine LTBP-2 gene (SEQ ID NO: 17)

Fig. 28 is the amino acid sequence of the murine LTBP-2 gene product (SEQ ID NO: 18).

Description of the preferred embodiment

1. Applications of bone repair technology to human treatment

What follows is a brief consideration of four human situations to exemplify the variety of diseases and conditions that will benefit from the development of new technology to improve bone repair and healing procedures. In addition to the following, various other conditions, for example, vitamin D deficiency; wound healing in general; soft skeletal tissue repair; and tendon repair and regeneration can also benefit from technology that relates to the stimulation of bone progenitor cells.

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The first example is the otherwise healthy individual who suffers a fracture. Often times, a clinical bone fracture is treated by casting to relieve pain and allow natural repair mechanisms to act on the wound. Although progress has recently been made in the treatment of fractures, and although the various complications that can arise when treating fractured bones are not considered, any new procedure that increases bone healing under normal circumstances would represent a breakthrough.

A second example that can benefit from newer treatment methods is osteogenesis imperfecta (OI). OI encompasses a variety of inherited connective tissue diseases involving soft connective tissue and bone fragility in man (Byers & Steiner, 1992; Prockop, 1990). OI affects approximately one child in every 5,000-20,000 children born and the disease is associated with significant morbidity throughout life. A number of deaths also occur, resulting in part from the high propensity for bone fractures and abnormal bone deformation after fracture repair (OI types II-IV; Bonadio & Goldstein, 1993). The important consequence here is the quality of life; clearly, the lives of affected individuals would be improved by the development of new therapies designed to stimulate and reinforce fracture repair processes.

Type I OI is a benign disorder characterized by nondeformed bone fractures, blue sclerae, normal or near-normal height, and autosomal dominant inheritance (Bonadio & Goldstein, 1993). Osteopenia is associated with an increased proportion of isolated bone fractures on ambulation (the frequency of fractures decreases significantly at puberty and during life in young adults, but increases again in older age). Hearing loss, which often begins in the second or third decade, is a feature of this disease in about half of families and can progress despite an overall decrease in the frequency of fractures. Imperfect dental genesis is observed in a subset of individuals.

In contrast, OI types II-VI represent a spectrum of more severe disorders associated with a shorter life span. Type II OI, the perinatal lethal form, is characterized by short stature, soft cranial vault, blue sclerae, fragile skin, small chest, lower limbs that appear pendulous (due to external rotation and abduction of the femurs), tendons and fragile ligaments, bone fractures with significant deformities, and death in the perinatal period due to respiratory failure. Radiographic signs of bone weakness include compression of the femurs, bowed tibiae, wide ribs composed of small portions, and thinning of the cranial vault.

Type III OI is characterized by short stature, triangular facies, severe scoliosis, and moderately deformed bone fractures. Scoliosis can lead to emphysema and a shortened life span due to respiratory failure. Type IV OI is characterized by normal sclerae, bone fractures with moderate to moderate deformities, dental defects, and a natural history that is essentially intermediate between type II and type I OI.

More than 200 OI mutations have been characterized since 1989 (reviewed in Byers and Steiner, 1992; Prockop, 1990). Most of it occurs in the COL1A1 and COL1A2 genes of collagen type I. Most cases of type I Oi appear to be the result of heterozygous mutations in the COL1A1 gene that decrease collagen production but do not alter the primary structure, that is, heterozygous null mutations affect COL1A1 expression. Most cases of OI types II-IV result from heterozygous mutations in the COL1A1 and COL1A2 genes that alter the structure of collagen.

A third important example is osteoporosis. The term "osteoporosis" refers to a heterogeneous group of disorders characterized by decreased bone mass and fractures. There is an estimated 20-25 million people who are at increased risk of fractures due to site-specific bone loss. Risk factors for osteoporosis include increasing age, gender (more women), low bone mass, early menopause, race (Caucasians), low calcium intake, reduced physical activity, genetic factors, environmental factors (including cigarette smoking and alcohol or caffeine abuse), and deficiencies in neuromuscular control that create a propensity to fall.

More than a million fractures in the USA each year can be attributed to osteoporosis, and in 1986 alone the cost of its treatment was estimated at 70,000-100,000 million dollars. Demographic trends (that is, the gradual increase in the age of the US population) suggest that these costs may increase 2-3 times in 2020 if no effective and safe treatment is found. Clearly, osteoporosis is a significant health care problem.

Clinically, osteoporosis is divided into type I and type II. Type I occurs predominantly in middle-aged women and is associated with loss of estrogen at menopause, while type II osteoporosis is associated with advanced age. A large part of the morbidity and mortality associated with osteoporosis comes from immobilization of elderly patients after fractures.

Routine therapies for osteoporosis patients emphasize fracture prevention, not fracture repair. This remains an important consideration, because of the literature, which clearly states that significant morbidity and mortality are associated with prolonged bed rest in older people, particularly those who have suffered hip fractures. Complications of bedtime include blood clots and pneumonia. These complications are recognized and measures are commonly taken to avoid them, but these measures hardly represent the best approach to therapy. Thus, the patient population

ES 2 139 889 T5 osteoporotics will benefit from new therapies designed to strengthen the bones and speed up the fracture repair processes, thus keeping these people on their feet before complications come.

A fourth example refers to bone reconstruction, and specifically the ability to reconstruct defects in bone tissue that come from traumatic damage; cancer or its surgery; birth defects; an error in development or an inherited alteration; or by age. There is a significant orthopedic need for more stable total joint grafts, and the cranial and facial bones are a particular target for this type of reconstructive need. The availability of new graft materials, for example titanium, has allowed the repair of relatively large defects. Titanium grafts provide excellent temporary stability to bone defects. However, experience has shown that a lack of viable bone union can cause the defect to lead to device exposure, infection, structural instability, and ultimately failure to repair the defect.

Autologous bone grafts are another possible reconstructive modality, but they have a number of proven disadvantages because they must be taken from a donor site such as the iliac crest or ribs, thus typically providing insufficient bone to completely fill the defect. and the bone it forms is sometimes prone to infection and resorption. Partially purified xenogeneic preparations are impractical for clinical use because microgram quantities are purified from kilograms of bovine bone, making large-scale commercial production costly and impractical. Allografts and demineralized bone preparations are therefore often used.

Microsurgical transfers of free bone grafts with hooked soft tissue and blood vessels can close bone defects with an immediate source of blood supply to the graft. However, these techniques take time, have been shown to cause great morbidity, and can only be used by specially trained individuals. Furthermore, the bone graft is often limited in quantity and not easily outlined. In the jaw, for example, most patients cannot wear dental devices using currently accepted techniques (even if continuity is established later), and thus little improvement in chewing ability. Toriumi et al., Have written that, “reconstructive surgeons will have at their disposal a bone substitute that will be safe, biocompatible, easy to use, and that will last a long time and will restore mandibular continuity with little associated morbidity.

In relation to bone reconstruction, the specific problem areas for improvement are those that refer to the treatment of large defects, such as those created by trauma, birth defects, or particularly after tumor resections. The success of orthopedic grafts, interfaces, and artificial joints would be imaginable if the graft surface, or a functional part of it, were coated with one or more appropriate materials, in order to promote a more effective interaction with the surrounding biological site. to the graft, and, ideally, to promote tissue repair.

two. Bone repair

It is known that bone tissue has the capacity for repair and regeneration and there is a certain knowledge of the molecular and cellular bases of these processes. The initiation of new bone formation involves the involvement, clonal expansion, and differentiation of progenitor cells. Once initiated, bone formation is promoted by various polypeptide growth factors. The newly formed bone is then maintained by a series of systemic and local growth and differentiation factors.

The concept of specific agents that promote bone growth is derived from the work of Huggins and Urist. Huggins et al., 1936. demonstrated that autologous grafting of canine incisor teeth to skeletal muscle resulted in local new bone formation (Huggins et al., 1936). Urist et al. Reported that freeze-dried and demineralized bone segments induced bone formation (Urist, 1965; Urist et al., 1983), a process that involved macrophage chemotaxis; the recruitment of progenitor cells; the formation of granulation tissue, cartilage, and bone; bone remodeling; and the differentiation of the marrow. The initiation of cartilage and bone formation at an extraskeletal site, a process referred to as osteinduction, has allowed the unequivocal identification of initiators of bone morphogenesis (Urist, 1965; Urist et al., 1983; Sampath et al. ., 1984; Wang et al., 1990; Cunningham et al., 1992).

Significant progress has been made in the characterization of biological agents made by active bone tissue during growth and natural bone healing. Demineralized bone matrix is ​​highly insoluble; Sampath and Reddi (1981) showed that only 3% of proteins can be extracted using strong combinations of denaturants and detergents. They also demonstrated that unfractionated demineralized bone extract will initiate bone morphogenesis, a critical observation that led to the purification of "osteoinductive" molecules. Families of proteinaceous osteoinductive factors have now been purified and characterized. In the literature, they have been referred to in various ways as morphogenic or morphogenetic proteins (BMPs), osteogenic bone inducing proteins or osteogenic proteins (OPs).

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3. Bone repair and bone morphogenetic proteins (BMPs)

After their initial purification, several bone morphogenetic protein genes have now been cloned using molecular techniques (Wozney et al., 1988; Rosen et al., 1989; summarized in Alper, 1994). This work has established BMPs as members of the transforming growth factor-β (TGF-β) superfamily based on DNA sequence homologies. Other TGF molecules have also been shown to participate in the formation of new bone, and TGF-β is considered a complex multifunctional regulator of osteoblastic function (Centrell et al., 1988; Carrington et al., 1988; Seitz et al. ., 1992). Indeed, the family of transforming growth factors (TGF- / 1, TGF- / 2, and TGF- / 3) has been proposed as potentially useful in the treatment of bone diseases (US Pat. 5,125,978, which is incorporated herein by reference).

Cloning of different BMP genes has led to the designation of individual BMP genes and proteins as BMP-1 through BMP-8. BMPs 2-8 are those thought to be generally osteogenic (BMP-1 may be a more generalized morphogen; Shimell et al., 1991). BMP-3 is also called osteogenin (Luyten et al., 1989)^Y

BMP-7 is also called OP-1 (Ozkaynak et al., 1990). Each of the TGFs and BMPs acts on cells through complex histospecific interactions with cell surface receptor families (Roberts and Sporn, 1989; Paralkar et al., 1991).

In the patent literature, various nucleotide sequences of BMP (or OP) and vectors, cultured host cells, and polypeptides have been described. For example, US Patent Documents 4,877,864, 4,968,590 and 5,108,753 all refer to osteogenic factors. More specifically, BMP-1 is disclosed in US Patent 5,108,922; Type BMP-2, which includes BMP-2A and BMP-2B, are disclosed in US Pat. 5,166,058, 5,013,649, and 5,013,649; BMP-3 at 5,116,738; BMP-5 at 5,106,748; BMP-6 at 5,187,076; and BMP-7 at 5,108,753 and 5,141,905; all of which are incorporated herein by reference. Various BMP clones and their activities have been described particularly by Wozney et al., 1988; (incorporated herein by reference). The DNA sequences encoding the osteogenic proteins designated OP-1, COP-5 and COP-7 are also disclosed in US Patent 5,011,691. Although the terminology of BMP is widely used, it may be the case that there is an OP “counterpart” term for each individual BMP (Alper, 1994).

Four. Bone repair and growth factors and cytokines

Transforming growth factors (TGFs) play a central role in the regulation of tissue healing due to their effect on cell proliferation, gene expression, and matrix protein synthesis (Roberts and Sporn, 1989). Although not necessarily exerting a direct effect, Bolander et al. Have provided evidence that TGF- / 1 and TGF- / 2 can initiate both chondrogenesis and osteogenesis (Joyce et al., 1990; Izumi et al., 1992; Jingushi et al., 1992; Jingushi et al. al., 1992). In these studies, bone and cartilage formation appeared to be dose dependent (ie dependent on the local concentration of growth factor). The data also suggested that TGF- / 1 and TGF- / 2 stimulated cell differentiation by a similar mechanism, even though they differed in terms of the final amount of new cartilage and bone that was formed.

Other growth factors / hormones in addition to TGF and BMP can influence the formation of new bone after the fracture. Bolander et al. Injected recombinant acidic fibroblast growth factor into a rat fracture site (Jingushi et al., 1990). The greatest effect of multiple high doses (1.0 mg / 50 ml) was a significant increase in cartilage tissue at the fracture cleft, while low doses had no effect. These investigators also used the reverse transcriptase polymerase chain reaction (PCR ™) technique to demonstrate the expression of estrogen receptor transcripts in callus tissue (Boden et al., 1989). These results suggested a role for estrogens in normal fracture repair.

Horowitz et al. Have shown that osteoblasts synthesize cytokines, macrophage colony-stimulating factors (Horowitz et al., 1989). Osteotropic agents used in this study included lipopolysaccharides, PTH1-84, PTH1-34, vitamin D, and al-trans retinoic acid. This observation has led to the suggestion that activation of osteoblasts after fracture may lead to the production of cytokines that regulate both hematopoiesis and new bone formation. Several other proteins and polypeptides that have been found to be expressed at high levels in osteogenic cells, such as, for example, the polypeptide designated Vgr-1 (Lyons et al., 1989), also have potential for use in connection with the present invention.

5. Bone repair and hormones that regulate calcium

Calcium regulatory hormones such as parathyroid hormone (PTH) participate in the formation of new bone and in its remodeling (Raisz and Kream, 1983). PTH is an 84 amino acid hormone that regulates calcium whose main function is to raise the concentration of Ca²⁺ in plasma and extracellular fluid. Studies with the original hormone and with synthetic peptides have shown that the amino terminal of the molecule (aa 1-34) contains the structural requirements for biological activity (Tregear et al., 1973; Hermann-Erlee et al., 1976; Riond , 1993). PTH functions by binding to a specific cell surface receptor that belongs to the superfamily of G protein-coupled receptors (Silve et al., 1982; Rizzoli et al., 1983; Juppner et al., 1991).

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Using a retroviral approach, a full-length human PTH gene construct has been introduced into cultured rat fibroblasts to create recombinant PTH-secreting cells. These cells were then transplanted into syngeneic murine recipients who were found to develop hypercalcemia mediated by increased serum concentrations of PTH (Wilson et al., 1992). The aim of these studies was to create an animal model of primary hyperparathyroidism.

PTH has a dual effect on new bone formation, a somewhat puzzling aspect of hormonal function, despite extensive research. PTH has been shown to be a potent direct inhibitor of type I collagen production by osteoblasts (Kream et al., 1993). 30 years ago, intact PTH was also shown to stimulate bone resorption in organ culture, and the hormone is known to increase osteoclast number and activity. Recent studies by Gay and collaborators have shown the union of (I¹²⁵) PTH (1-94) to osteoclasts in histological sections and that osteoclasts bind to intact PTH in a way that is both saturable and dependent on time and temperature (Agarwala and Gay, 1992). Although these properties are consistent with the presence of PTH / PTHrP receptors on the osteoclastic cell surface, this hypothesis is still considered controversial. A more accepted view, perhaps, is that osteoclast activation occurs through an osteoblast signaling mechanism.

On the other hand, osteosclerosis can occur in human patients with primary hyperparathyroidism (Seyle, 1932). It is well known that individuals with hyperparathyroidism do not inexorably lose bone mass, but eventually reach a new equilibrium state of new bone remodeling after an initial period of net bone loss. Chronic administration at low doses of the amino terminal fragment of PTH (aa 1-34) can also induce new bone formation according to a dose-time dependent scheme (Seyle, 1932; Parsons and Reit, 1974).

Human PTH 1-34 has recently been shown to stimulate DNA synthesis in cultured chicken osteoblasts and chondrocytes (Van der Plas, 1985; Schluter et al., 1989; Somjen et al., 1990); increases the number of bone cells in vivo (Malluche et al., 1986) enhances the in vitro growth of cartilage and bone of chick embryos (Kawashima, 1980; Burch and Lebovitz, 1983; Lewinson and Silbermann, 1986; Endo et al. , 1980; Kleind-Nulend et al., 1990); enhances superficial bone formation (both trabecular and cortical) in normal and osteogenic animals and in man with osteoporosis (Reeve et al., 1976; Reeve et al., 1980; Tam et al., 1982; Hefti et al., 1982; Podbesek et al., 1983; Stevenson and Parsons, 1983; Slovik et al., 1986; Gunness-Hey and Hock, 1984; Tada et al., 1988; Spencer et al., 1989; Hock and Fonseca, 1990; Liu and Kalu, 1990; Hock and Gera, 1992; Mitlak et al., 1992; Ejersted et al., 1993); and delays and reverses the catabolic effects of estrogen deprivation on bone mass (Hock et al., 1988; Hori et al., 1988; Gunness-Hey and Hock, 1989; Liu et al., 1991). Evidence of synergistic interactions between hPTH-1-34 and other anabolic molecules has been presented, including insulin-like growth factor, BMP-2, growth hormone, vitamin D, and TGF-β (Slovik et al., 1986; Spencer et al., 1989; Mitlak et al., 1992; Canalis et al., 1989; Linkhart and Mohan, 1989; Seitz et al., 1992; Vukicevic et al., 1989).

Anecdotal observation has shown that serum PTH levels may rise after bone fracture (Meller et al., 1984; Johnston et al., 1985; Compston et al., 1989; Hardy et al., 1993), but the meaning of this observation is not understood. There are apparently no reports in the literature regarding attempts to localize PTH or the PTH / PTHrP receptor in situ at human fracture sites or in experimental models. Furthermore, no attempt has been made to enhance repair by exogenous addition of PTH peptides. Although hPTH134 is known to function as an anabolic agent for bone prior to the present invention, much remains to be learned about the role (if any) of PTH during bone regeneration and repair.

6. Protein administration and bone repair

Several studies have been carried out in which protein growth factor preparations, including BMPs, were administered to animals in an effort to stimulate bone growth. The results of four such exemplary studies are described below.

Toriumi et al. Studied the effect of recombinant BMP-2 on the repair of surgically created defects in the jaw of adult dogs (Toriumi et al., 1991). 26 Adult dogs were arranged in three groups after the creation of a mandibular defect with a total thickness of 3 cm: 12 animals received test grafts composed of inactive dog bone matrix carriers and human BMP-2, 10 animals received control grafts. composed of transporters without BMP-2, and BMP-4 animals received no graft. Dogs were sacrificed at 2.5-6 months, and the reconstructed segments were analyzed by radiography, histology, histomorphometry, and biomechanical assays. Animals that received test grafts were euthanized after 2.5 months, due to the presence of well mineralized bones splicing the defect. The new bone allowed these animals to chew on a solid diet, and the average bending force of the reconstructed jaws was 27% of normal ("normal" in this case represents the unoperated and contralateral hemimandible). In contrast, the grafts in the other two groups were not functional even after 6 months and showed minimal bone formation.

Yasko et al. Published a related study examining the effect of BMP-2 on the repair of segmental defects in the rat femur (Yasko et al., 1992). The study design included a group receiving a 1.4 mg dose of BMP-2, another group receiving an 11.0 mg dose of BMP-2, and a control group receiving only the carrier matrix. Endochondral bone formation was observed in the two groups of animals that

ES 2 139 889 T5 received BMP-2. As demonstrated by radiography, histology, and whole bone mechanical integrity (torsion) testing, the largest dose that resulted in functional repair of 5mm defects beginning 4.5 weeks after surgery. The lower dose resulted in radiographic and histological evidence of new bone formation, but functional attachment was not observed until 9 weeks after surgery. There was also no evidence of bone formation in the control animals at this time.

Chen et al. Demonstrated that a single application of 25-100 mg of recombinant TGF-β 1 adjacent to cartilage induced endochondral bone formation in full-thickness dermal wounds of rabbit ears (Chen et al., 1991 ). Bone formation started 21 days after wound creation and reached a maximum on day 42, as demonstrated by morphological methods. Active bone remodeling was observed after this time.

In a related study, Beck et al. Demonstrated that a single application of TGF-β 1 in a 3% methylcellulose gel was able to repair large, surgically-induced cranial defects that are otherwise healed by fibrous connective tissue and never form bone. (Beck et al., 1991). Bone closure was achieved within 28 days of the application of 200 mg of TGF-61 and the rate of healing was shown to be dose dependent.

Studies such as those described above have thus established that exogenous growth factors can be used to stimulate new bone formation / repair / regeneration in vivo. Certain US Patent documents also relate to methods for treating bone defects or inducing bone formation. For example, US Patent 4,877,864 relates to the administration of a bone-inducing protein therapeutic composition to treat cartilage and / or bone defects; US Patent 5,108,753 refers to the use of a device containing a pure osteogenic protein to induce endochondral bone formation and for use in periodontal, dental or craniofacial reconstructive procedures.

However, nowhere in this extensive literature is there any suggestion that the same osteogenic genes can be applied to an animal in order to promote bone repair or regeneration. Indeed, even through the patent literature that refers to genes encoding various bone stimulating factors and their expression in vitro in host cells to produce recombinant proteins, the possibility of using nucleic acid transfer in a effort to express an osteogenic gene in bone progenitor cells in vivo or to promote new bone formation in an animal or human individual.

7. Biocompatible matrices for use in bone repair

There is a considerable amount of work that has been directed to the development of biocompatible matrices for use in medical grafts, including those specifically for bone implant work. In the context of the present invention, an array can be used in association with the gene or coding region of DNA that encodes the osteotropic polypeptide, in order to easily deliver the gene to the site of bone damage. Such matrices can be formed from various materials that are used today for grafted medical devices.

In certain cases, the matrix can also act as a "bio-filler" to provide structure for developing bone and cartilage. However, the formation of such a scaffold structure is not a primary need, because, rather, the main requirements of the matrix are to be biocompatible and to be able to deliver a nucleic acid segment to a bone cell or bone tissue site.

Matrices that can be used in certain embodiments include chemically defined, non-biodegradable matrices, such as sintered hydroxylapatite, bioglass, aluminates, and other ceramics. Bioceramics can be altered in their composition, such as in calcium-aluminate-phosphate; and they can be processed to modify particular physical and chemical characteristics, such as pore size, particle size, particle shape, and biodegradability. Certain polymeric matrices can also be used if desired, including acrylic ester polymers and lactic acid polymers, as disclosed in US Patent Nos. 4,526,909, and 4,563,489, respectively, each incorporated herein. herein for reference. Particular examples of useful polymers are those of orthoesters, anhydrides, propylene-cofumarates, or a polymer of one or more of the monomers of α-hydroxycarboxylic acid, for example, α-hydroxyacetic acid (glycolic acid) and / or acid α-hydroxypropionic (lactic acid).

Some of the preferred matrices for use for current purposes are those that are capable of being reabsorbed into the body. Potential biodegradable matrices for use in bone gene transfer include, for example, PLGA block copolymers, chemically defined and biodegradable calcium sulfate, tricalcium phosphate, hydroxyapatite, and polyanhydrides. In addition , biomatrices composed of pure proteins and / or extracellular matrix components can be used.

The inventors have shown the use of dermal or bone collagen materials as matrices, as they can be prepared from various commercially available lyophilized collagen preparations, such as those from rat or bovine skin, as well as from PLGA block copolymers. Collagen matrices can also be formulated as described in US Pat. 4,394,370, which is incorporated herein by reference, which refers to the use of collagen matrices as delivery vehicles for osteogenic protein.

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UltraFiber ™, available from Norian Corp (Mountain View, CA), is a preferred matrix. Preferred matrices are those formulated with type II collagen, and more preferably with recombinant type II collagen and mineralized type II collagen.

Appropriate matrices can subsequently be prepared from combinations of materials, such as PLGA block copolymers, which allow for sustained release; hydroxyapatite; or collagen and tricalcium phosphate. While sufficient retention and subsequent release of an osteotropic gene is by no means a limit to the present invention, it would be desirable if a porous matrix and gene pool could be delivered to the bone tissue site in combination with an autologous blood clot. The basis for this is that blood clots have previously been used to increase the sequestration of osteogenic proteins for use in bone treatment (US Patent 5,171,579, which is incorporated herein by reference), not excluding any way their use in connection with the present invention (they can even attract growth factors or cytokines).

8. Collagen

Although not previously proposed for use with a nucleic acid molecule, the use of collagen as a pharmaceutical delivery vehicle has been described. The biocompatibility of collagen matrices is well known in the art. US Patent Documents 5,206,028, 5,128,136. 5,081,106, 4,585,797. 4,390,519, and 5,197,977 (all incorporated herein by reference) describe the biocompatibility of collagen-containing matrices in the treatment of dermal lesions, use as a wound dressing, and as a means of controlling bleeding. In light of these documents, therefore, it does not offer the slightest doubt as to the appropriateness of applying a collagen preparation to a bone site of an animal.

US Patent 5,197,977 describes the preparation of a collagen impregnated vascular graft that includes medicinal materials complexed with the collagen to be slowly released from the graft, after implantation. US Patent 4,538,603 refers to an occlusive dressing useful for treating dermal lesions and to a granular material capable of interacting with wound exudate. US Patent Document 5,162,430 discloses a pharmaceutically acceptable non-immunogenic composition comprising a telopeptide collagen chemically conjugated to a synthetic hydrophilic polymer.

Subsequent documents, readily available to one of ordinary skill in the art, include US Patents 4,837,285,4,703,108,4,409,332 and 4,347,234, each of which is hereby incorporated by reference. These references describe the uses of collagen as a non-immunogenic, biodegradable, and bioresorbable binding agent.

The inventors believe that collagen from many sources will be useful in the present invention. Particularly useful are the amino acid sequences of type II collagen. Examples of type II collagen are well known in the art. For example, the amino acid sequences of man (Lee et al., 1989), rat (Michaelson et al., 1994), and mouse (Ortman et al., 1994) have been determined (SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14), respectively).

Although it was not previously known to be capable of stimulating bone progenitor cells by itself, it is surprisingly shown here that type II collagen possesses this property, thus opening up new possibilities for clinical uses.

9. Nucleic acid release

The transfer of nucleic acids into mammalian cells has provided a method for treating certain diseases or disorders. Nucleic acid transfer or release is often referred to as "gene therapy." Initial efforts toward postnatal (somatic) gene therapy depended on indirect means of introducing genes into tissues, for example, target cells were obtained from the body, infected with viral vectors carrying recombinant genes, and implanted there again. . These types of techniques are generally referred to as ex vivo treatment protocols. In vivo gene transfer has recently been achieved with liposome-entrapped DNA formulations (Ledley et al., 1987); or in proteoliposomes containing virus envelope receptor proteins (Nicolau et al., 1983); Calcium phosphate co-precipitated DNA (Benvenisty and Reshef, 1986); and DNA coupled to a glycoprotein-polylysine transporter complex (Wu and Wu, 1988). The use of incomplete replication viral recombinant vectors to infect target cells in vivo has also been described (eg, Seeger et al., 1984).

Recently, Wolff et al. Demonstrated that direct injection of purified DNA and RNA preparations into murine skeletal muscle resulted in significant expression of a reporter gene (Wolff et al., 1990). This was an unexpected finding, and the mechanism of gene transfer could not be defined. The authors speculated that muscle cells may be particularly gifted to take up and express polynucleotides in vivo or that damage associated with injection of DNA may allow transfection to take place.

Wolff et al. Suggested several potential applications of the direct injection method, including (a) the treatment of heritable muscle disorders. (b) modification of non-muscular alterations through muscle tissue expression of therapeutic transgenes, (c) vaccine development, and (d) a reversible type of transfer

ES 2 139 889 T5 genetics, in which DNA is administered as a conventional pharmaceutical treatment. In an elegant study, Liu et al. Recently demonstrated that the direct injection method can be successfully applied to the problem of developing an influenza vaccine (Ulmer et al., 1993).

The use of gene transfer to synoviocytes as a means of treating arthritis has also been considered (Bandara et al., 1992; Roessler et al., 1993). The protocols under consideration have included both the ex vivo treatment of isolated synoviocytes and their reintroduction into the animal and also direct gene transfer in which appropriate vectors are injected into the joint. Transfer of marker genes to synoviocytes has already been demonstrated using retroviral and adenoviral technology (Bandara et al., 1992 Roessler et al., 1993).

Despite the exclusive emphasis on protein treatment by those working in the field of bone regrowth, the inventors saw that there was great potential for using the same nucleic acids to promote bone regeneration / repair in vivo. This provides a more sophisticated type of pharmaceutical release. In addition to the ease and cost of preparing DNA, it was also reasoned that using transfer of DNA rather than peptides would provide many further advantages. For example, DNA transfer allows the expression or overexpression of integral membrane receptors on the surface of bone repair / regeneration cells, whereas this cannot be done using peptide transfer because the latter (a priori) it is an extracellular manipulation. Importantly, DNA transfer also allows the expression of modified polypeptides in a site-directed manner, with the minimum amount of additional work (i.e., clear molecular biological manipulation without protein purification), as well as release sustained use of injectable therapies.

The advantages of using DNA are also numerous considering the development of effective pharmaceuticals and means of delivery. Here, important advantages include the ability to prepare injectable formulations, especially those compositions that exhibit reversible thermal gelation, and the opportunity to combine such injectable with on-delivery imaging technologies. "Sustained release" is also an important advantage of using DNA, because exogenously added DNA continues to drive the production of a protein product after incorporation into a cell. The use of certain matrix-DNA compositions also allows for a more typical "sustained release" phenomenon because the operative release of DNA from the matrix mixture can also be manipulated.

The inventors envisioned that both naked and virally mediated DNA could be used in an effort to transfer genes to bone progenitor cells. When starting to study this, the most appropriate animal model had to be employed, that is, one in which the possibilities of using nucleic acids to promote bone repair could be adequately tested in controlled studies.

10. Osteotomy model

Prior to the present invention, three model systems were available for study in this area, including the Mov13 mouse, an animal model of OI. Unfortunately, each of the models suffers from significant drawbacks. With the Mov13 mouse, first, these mice typically die in early coming of age, due to retrovirus-induced leukemia (Schnieke et al., 1983); Second, gene transfer studies in the Mov13 mouse carried out between postnatal weeks 8-16 (that is, before the development of leukemia) may be complicated by a natural adaptation in which a significant amount of new bone it is deposited on the periosteal surface (Bonadio et al., 1993); and third, an osteotropic gene transferred to an osteotomy site can synergize with the active retrovirus and make it even more virulent.

Another system is the in vivo bone fracture model created by Einhorn and colleagues (Bonnarens and Einhorn, 1984). However, this model is a closed system that would not easily allow initial in vivo gene transfer studies. The organ culture model developed by Bolander et al. (Joyce et al., 1990) was also available, but again, this model is not appropriate for studying gene transfer in vivo. Because the above-cited models are not appropriate for studying the effects of gene transfer on bone repair and regeneration, the inventors used a murine osteotomic system, as described below.

Important features of the murine osteotomic model are as follows: Under general anesthesia, four 1.2 mm diameter nails are screwed into the femoral shaft of normal adult Sprague Dawley rats. A surgical template ensures parallel nail placement. A fixator is then secured over these, and a 2mm or 5mm segmental defect (incision) is created on the central shaft with a microoscillating Hall 100 saw. A biodegradable graft material, embedded in a plasmid DNA solution, or in another genetic construct or in a recombinant virus preparation, is then placed in the intramedullary canal and the defect is closed (Fig. 5A, Fig. 5B, Fig. 6A, Fig. 6B, Fig. 6C, Fig. 6D, Fig. 7A, Fig. 7B, Fig. 8A, Fig. 8B, Fig. 8C).

New bone formation can be detected as early as three weeks later at the 2mm incision, although new bone formation is generally allowed to take place up to 9 weeks. The fixative provided the necessary stability, and there were no limitations in the movement of the animals. To date, the surgical protocol has been performed successfully in 21/21 animals. None of them have died. After sacrifice, new bone formation tests are carried out, except for the complete radiograph, which is performed weekly between the date of surgery and the sacrifice.

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Previous studies in Sprague Dawley rats have shown that the 5mm osteotomic incision will heal as a fibrous nonunion, whereas a less than 3mm incision, (such as the 2mm incision used routinely in the studies described herein ) will heal through primary bone formation. Studies using the 5 mm incision thus allow a determination of whether transgene expression can stimulate the formation of new bone when healing by fibrous tissue is normally expected. On the other hand, studies with the 2 mm incision allow a determination of whether transgene expression can accelerate natural primary bone healing. Controls were also carried out in which the animals did not receive DNA (Fig. 9A and Fig. 9B).

eleven. Gene transfer promotes bone repair in vivo

The inventors surprisingly found that gene transfer to bone progenitor cells in vivo (that is, cells in the regenerating tissue of the osteotomic cleft) could be easily achieved. Usually, the preferred methods of achieving gene transfer involve the use of a fibrous collagen graft material embedded in a DNA solution shortly before being placed at the site where it is desired to promote bone growth. As the studies presented show, the grafted material facilitates the uptake of exogenous plasmid constructs by cells (in the osteotomic cleft) that are clearly involved in bone regeneration / repair. Transgenes, after cell uptake, direct the expression of recombinant polypeptides, as evidenced by the in vivo expression of functional marker gene products.

Further studies are presented here demonstrating that the transfer of an osteotropic gene results in the cellular expression of a recombinant osteotropic molecule, the expression of which is directly associated with the stimulation of new bone formation. After considering a relatively large number of candidate genes, a transfer gene vector encoding a fragment of human parathyroid hormone (hPTH1-34) was selected for our initial studies. Several factors were considered in making this selection: (a), the recombinant peptides hPTH1-34 can be discriminated from any endogenous rat hormones present in osteotomic tissues; (b), hPTH1-34 peptides will stimulate new bone formation in Sprague Dawley rats, indicating that human peptide can efficiently bind to the PTH / PTHrP receptor on the rat osteoblastic cell surface; and (c), there is only one PTH / PTHrP receptor, the gene of which has been cloned, and cDNA probes are available for the receptor.

Thus, in terms of understanding the mechanism of action of the transgene on new bone formation in vivo, the inventors resolved that the clearest thing was to correlate the expression of the recombinant peptide hPTH1-34 and its receptor with the formation of new bone in the osteotomic model. of the rat. Of course, following these initial studies, it is considered that any of a wide variety of genes can be used in connection with the bone gene transfer embodiments of the present invention.

Previous studies have indicated that hPTH1-34 is a more potent anabolic agent when given intermittently than when given continuously. Despite the fact that an anabolic effect is still expected to occur with continuous dosing, as documented by studies by Parsons et al. (Tam et al., 1982; Spencer et al., 1989), there was concern. that the PLJ-hPTH1-34 transgene may not function very effectively as transfected cells are expected to express recombinant hPTH134 molecules constitutively. The finding that transfection and expression of the LPH-hPTH1-34 transgene did indeed stimulate bone formation in the rat osteotomic model was therefore an important result.

As the osteotomic site in this model is highly vascularized, a possible complication of studies with the PLJ-hPTH1-34 transgene is the secretion of recombinant human PTH from the osteotomic site with consequent hypercalcemia and (potentially) death of the animal. When using this transgene, serum calcium levels should therefore be determined weekly. The fact that no evidence of altered serum calcium levels has been found in this study is therefore a further encouraging finding.

These studies complement others carried out by the inventors in which direct gene transfer was used to introduce genes into the Achilles tendon and the cruciate ligament, as described in Example XI.

After these surprising findings, several immediate applications to utilize nucleic acid release in connection with bone abnormalities became clear to the inventors. The direct transfer of an osteotropic gene to promote fracture repair in clinical orthopedic practice is just one use. Other important aspects of this technology include the use of gene transfer to treat patients with "brittle bones", such as in diseases such as osteoporosis; to improve poor scarring that may occur for unknown reasons, for example fibrous nonunion; to promote graft integration and artificial joint function; to stimulate the healing of other skeletal tissues, such as the Achilles tendon; and as an adjunct to repair large defects. In all of these embodiments DNA is used as a direct pharmaceutical agent.

12. Biological functional equivalents

As mentioned above, modifications and changes can be made in the structure of an osteotropic gene and still obtain a functional molecule that encodes a protein or polypeptide with characteristics

ES 2 139 889 T5 desirable. The following is a consideration based on changing the amino acids of a protein to create an equivalent, or even an improved second generation molecule. Changes in amino acids can be achieved by changing the codons of the DNA sequence, according to the following codon table. :

TABLE 1

Amino acids

Codons

<td>To the girl</td><td>To</td><td>TO</td><td>GCA</td>

<td>Cysteine</td><td>Cys</td><td>C</td><td>UGC</td>

<td>Aspartic acid</td><td>Asp</td><td>D</td><td>GAC</td>

<td>Glutamic acid</td><td>Glu</td><td>AND</td><td>GAA</td>

<td>Phenylalanine</td><td>Phe</td><td>F</td><td>UUC</td>

<td>Wisteria</td><td>Gly</td><td>G</td><td>GGA</td>

<td>Histidine</td><td>His</td><td>H</td><td>CAC</td>

<td>Isoleucine</td><td>Ile</td><td>I</td><td>AUA</td>

<td>Lysine</td><td>Lys</td><td>K</td><td>AAA</td>

<td>Leucine</td><td>Leu</td><td>L</td><td>UUA</td>

<td>Methionine</td><td>Met</td><td>M</td><td>AUG</td>

<td>Asparagine</td><td>Asn</td><td>N</td><td>AAC</td>

<td>Proline</td><td>Pro</td><td>P</td><td>CCA</td>

<td>Glutamine</td><td>Gln</td><td>Q</td><td>CAA</td>

<td>Arginine</td><td>Arg</td><td>R</td><td>AGA</td>

<td>Serine</td><td>To be</td><td>S</td><td>AGC</td>

<td>Threonine</td><td>Thr</td><td>T</td><td>HERE</td>

<td>Valine</td><td>Val</td><td>V</td><td>GUA</td>

<td>Tryptophan</td><td>Trp</td><td>W</td><td>UGG</td>

<td>Tyrosine</td><td>Tyr</td><td>Y</td><td>UAC</td>

<td>GCC</td><td>GCG</td><td colspan="3">GCU</td>

<td>UGU</td><td></td><td></td><td></td><td></td>

<td>GAU</td><td></td><td></td><td></td><td></td>

<td>GAG</td><td></td><td></td><td></td><td></td>

<td>UUU</td><td></td><td></td><td></td><td></td>

<td>GGC</td><td>GGG</td><td>GGU</td><td></td><td></td>

<td>CAU</td><td></td><td></td><td></td><td></td>

<td>AUC</td><td>AUU</td><td></td><td></td><td></td>

<td>AAG</td><td></td><td></td><td></td><td></td>

<td>UUG</td><td>CUA</td><td>CUC</td><td>CUG</td><td>CUU</td>

<td>AAU</td><td></td><td></td><td></td><td></td>

<td>CCC</td><td>CCG</td><td>CCU</td><td></td><td></td>

<td>CAG</td><td></td><td></td><td></td><td></td>

<td>AGG</td><td>CGA</td><td>CGC</td><td>CGG</td><td>CGU</td>

<td>AGU</td><td>UCA</td><td>UCC</td><td>UCG</td><td>UCU</td>

<td>ACC</td><td>ACG</td><td>ACU</td><td></td><td></td>

<td>GUC</td><td>GUG</td><td>GUU</td><td></td><td></td>

UAU

For example, certain amino acids can be substituted for others in a protein structure without seeming loss of interactive binding ability with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since the interactive capacity and nature of a protein define its biological functional activity, certain sequential amino acid substitutions can be made in a protein sequence, and, of course, in its underlying coding DNA sequence, and nevertheless obtain a protein with properties similar. It is thus then considered by the inventors that various changes can be made to the osteotropic gene sequences without appreciable loss of their utility or biological activity.

In making such changes, the hydropathic amino acid index can be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resulting protein, which in turn defines its interaction with other molecules, eg, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), which are: Isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine ​​/ cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is known in the art that certain amino acids can be substituted for others having a similar hydropathic index or rating and still result in a protein with similar biological activity, ie, still obtain a functionally biological equivalent protein. In making such changes, substitution of amino acids whose hydropathic indices are within ± 2 is preferable, substitution of those within ± 1 is particularly preferred, and even more preferred is substitution of those within ± 0.5.

It is also understood in the art that substitution of like amino acids can be effectively accomplished on the basis of hydrophilicity. US Patent Document: 4,554,101, which is incorporated herein by reference, discloses that the higher local average hydrophilicity of a protein, governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. .

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As detailed in US Patent document 4,554,101, the following hydrophilicity values ​​have been assigned to amino acid residues: arginine, (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent one, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values ​​are within ± 2 is preferred, those within ± 1 are particularly preferred, and those with values ​​within ± 0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are therefore generally based on the relative similarity of the amino acid side chain substituents, eg, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that consider several of the above characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

13. Site-specific mutagenesis

Site-specific mutagenesis is a useful technique in the preparation of individual peptides, or biologically functional proteins or equivalent peptides, by specific mutagenesis of the underlying DNA. The technique further provides an ability to prepare and test sequence variants, for example, incorporating one or more of the above-mentioned considerations, introducing one or more nucleotide sequence changes in DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer 17 to 25 nucleotides in length is preferred, with approximately 5 to 10 residues on either side of the splice of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications. As will be appreciated, the technique typically utilizes a phage vector that exists in both single and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as phage M13. These phages are readily available commercially and their use is generally well known to those of skill in the art. Double-stranded plasmids are also routinely used in site-specific mutagenesis that eliminates the step of transferring the gene of interest from a plasmid to phage.

In general, site-directed mutagenesis according to the accompanying considerations is carried out first by obtaining a single-stranded vector or by fusing separately two strands of a double-stranded vector that includes within its sequence a DNA encoding the desired osteotropic protein. An oligonucleotide primer carrying the desired mutated sequence is prepared, generally synthetically. This primer is then hybridized with the single-stranded vector, and subjected to DNA polymerizing enzymes such as the Klenow fragment of E. coli polymerase I, in order to complete the synthesis of the filament carrying the mutation. In this way, a heteroduplex is formed in which one strand encodes the original, non-mutated sequence and the second strand carries the desired mutation. The heteroduplex vector is then used to transform appropriate cells, such as E. coli, and clones are selected that include recombinant vectors that carry the mutated sequence arrangement.

The preparation of sequence variants of the selected osteotropic gene using site-directed mutagenesis is provided as a means of producing potentially useful types and is not meant to be limiting, as are other ways by which sequence variants of osteotropic genes can be obtained. For example, recombinant vectors encoding the desired osteotropic gene can be treated with mutagenic agents such as hydroxylamine to obtain sequence variants.

14. Generation of monoclonal antibodies

Means for preparing and characterizing antibodies are well known in the art (See, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).

Methods for generating monoclonal antibodies (MAbs) generally start according to the same guidelines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition according to the present invention and recovering the antisera from that immunized animal. A wide variety of animal species can be used for the production of antisera. Typically the animal used for the production of the antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is the preferred selection for the production of polyclonal antibodies.

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As is well known in the art, a given composition can vary in its immunogenicity. It is often necessary, therefore, to revaccinate the host's immune system, as can be accomplished by linking a peptide or polypeptide immunogen to a vehicle. Exemplary and preferred vehicles are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins, such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as vehicles. Means for attaching a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide, and bis-binitrogenated benzidine.

As is also well known in the art, the immunogenicity of a particular immunogenic composition can be stimulated through the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants, and aluminum hydroxide adjuvant.

The amount of immunogenic composition used in the production of polyclonal antibodies varies according to the nature of the immunogen as well as according to the animal used for immunization. Different routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies can be monitored by sampling blood from the immunized animal at various points after immunization. A second booster injection may also be given. The revaccination and titration procedure is repeated until an appropriate titer is reached. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and / or the animal can be used to generate MAbs.

MAbs can be readily prepared using well known techniques, such as those exemplified in Patent 4,196,265 which is incorporated herein by reference. Typically, this technique involves immunizing an appropriate animal with a selected immunogenic composition, eg, a purified or partially purified LTBP-3 protein, polypeptide, or peptide. The immunizing composition is administered effectively to stimulate antibody-producing cells. Rodents such as mice and rats are preferred animals, however the use of rabbit, sheep and frog cells is also possible. The use of rats can provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with BALB / c mice being the most preferred, as they are the most routinely used and result in higher percentages of stable fusions.

After immunization, somatic cells with the potential to produce antibodies, specifically B lymphocytes (B cells), were selected for use in the MAb generation protocol. These cells can be obtained from biopsied spleens, tonsils, or lymph nodes, or from a peripheral blood sample. Spleen cells and blood cells are preferred, the former because they constitute a rich source of antibody-producing cells which are in the hemocytoblastic phase of division, and the latter because peripheral blood is easily accessible. Often times, a pool of animals will be immunized and the spleen of the animal with the highest antibody titer will be removed and lymphocytes from this organ will be obtained by homogenizing them with a syringe. Typically, a spleen from an immunized mouse contains approximately 5 x 10⁷ to 2 x 10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are then fused with cells from an immortal myeloma cell line, generally one of the same type as the animal that was immunized. Myeloma cell lines that are suitable for use in hybridoma-producing fusion procedures do not preferentially produce antibodies, possess high fusion efficiency, and enzyme deficiencies that render them unable to grow on certain selective growth-supporting media. of only the fused cells that are desired (hybridomas).

Any of a number of myeloma cells can be used as is known to those of skill in the art (Goding, pp. 6566, 1986; Campbell, pp. 75-83, 1984). for example, when the immunized animal is a mouse, P3X63 / Ag8, X63-Ag8.653, NS1 / 1.Ag 4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45- can be used. GTG 1.7 and S194 / 5XX0 Bul; for rats, R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210 can be used; and U-266, GM1500-GRG2, LICRLON-HMy2 and UC729-6 all being useful in connection with human cell fusions.

A preferred murine myeloma cell is the myeloma cell line NS-1 (also referred to as P3-NS-1Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting the cell line repository number GM3573. Another mouse myeloma cell line that can be used is the non-8-azaguanine resistance-producing murine myeloma cell line SP2 / 0.

Methods for generating antibody-producing lymph node or spleen cell hybrids and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2: 1 ratio, although this can range from about 20: 1 to 1: 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976) and those using polyethylene glycol (PEG), such as 37% (v / v) PEG, by Gefter et al., (1977 ). The use of electrically induced fusion methods is also appropriate (Goding, pp. 71-74, 1986).

IS 2 139 889 T5

Fusion procedures typically produce viable hybrids at low frequencies, around 1 x 10 ⁶to 1 x 10^-8. However, this is not a problem, as viable fused hybrids are differentiated from parental unfused cells (particularly unfused myeloma cells which will normally continue to divide indefinitely) by culturing them in selective medium. The selective medium is generally one that contains an agent that blocks de novo nucleotide synthesis in the tissue culture medium. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. When aminopterin or methotrexate are used, the medium is supplemented with hypoxanthine and thymidine as a nucleotide source (HAT medium). When azaserine is used, the medium is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of nucleotide salvage pathways are capable of surviving in the HAT medium. Myeloma cells lack key salvage pathway enzymes, eg, hypoxanthine phosphoribosyl transferase (HPRT), and cannot survive. B cells can perform this pathway, but have a limited life in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective medium are the hybrids formed from myeloma and B cells.

This culture provides a population of hybridomas from which specific hybridomas are selected. Typically, hybridoma selection is carried out by culturing cells by dilution of single clones in microtiter plates, followed by testing individual clonal supernatants (after two to three weeks) for the desired reactivity. The assay should be sensitive, simple, and rapid, such as radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assays, immunoblotting assays, and the like.

The selected hybridomas will then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide MAbs. Cell lines can be exploited for MAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors that secrete the specific monoclonal antibody produced by the fused cell hybrid. The animal's body fluids, such as serum or ascites fluid, can then be drained to provide MAbs at high concentrations. Individual cell lines could also be cultured in vitro, where MAbs are naturally secreted into the culture medium from which they can be easily obtained at high concentrations. MAbs produced by either means can be further purified, if desired, using filtration, centrifugation, and various chromatographic methods such as HPLC or affinity chromatography.

fifteen. LTBP-3

Other aspects of the present invention relate to isolated segments of DNA and to recombinant vectors that encode LTBP-3, and to the creation and use of recombinant host cells through the application of DNA technology, which express products of the LTBP-3 gene. . As such, the present invention relates to a DNA segment comprising an isolated gene encoding a protein or peptide that includes an amino acid sequence as essentially set forth by a contiguous sequence of SEQ ID NO: 3. These DNA segments are represented by those that include a nucleic acid sequence as set forth essentially by a contiguous sequence of SEQ ID NO: 2 (Fig. 25). The invention also encompasses compositions that include a purified protein possessing an amino acid sequence, as essentially set forth by the amino acid sequence of SEQ ID NO: 3 (Fig. 26).

TGF-ds represent a family of structurally related molecules with diverse effects on mammalian cell shape, growth, and differentiation (Roberts and Sporn, 1990). Initially synthesized as a precursor consisting of an amino-terminal propeptide followed by mature TGF-β, two nascent pro-TGF-β chains associate in most tissues to form an inactive disulfide-linked dimer of a Mr of approximately 106,000. Homodimers are more common, but heterodimers have also been described (Cheifetz et al., 1987; Ogava et al., 1992). During biosynthesis the mature TGF-β dimer is cleaved from the propeptide dimer. The latent state of TGF-β comes in part from the non-covalent association of propeptide and mature TGF-β dimers (Pircher et al., 1984, 1986; Wakefield et al., 1987; Millan et al., 1992; Miyazono and Heldin, 1989). Consequently, the propeptide dimer is often referred to as the Latency Associated Protein (LAP), and LAP plus the disulfide-linked TGF-β dimer is also known as the Latency Small Complex. In the extracellular space, small latent complexes must dissociate to activate mature TGF-βs. The mechanism of activation of the latency complex is believed to be one of the most important steps governing the effects of TGF-β (Lyons et al., 1988; Antonelli-Orlidge et al., 1989; Twardzik et al., 1990 ; Sato et al., 1993).

In certain cultured cell lines the small dormant complexes of growth factors may contain additional high molecular weight proteins. The best characterized of these high molecular weight proteins is the latent TGF-β or LTPB binding protein (Miyazono et al., 1988; Kanzaki et al., 1990; Tsuji et al., 1990; Olofsson et al., 1992; Taketazu et al., 1994). LTBP produced by different cell types is heterogeneous in size, perhaps due to alternative splicing or due to processing, specific tissue proteolytic (Miyazono et al., 1988; Wakefield et al., 1988; Kanzaki et al., 1990; Tsuji et al. ., 1990). Latency TGF-β complexes containing

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LTBP are known as large latency complexes. LTPB has no covalent bond to mature TGF-β, but is disulfide bonded to LAP.

Regarding the new LTBP-3 protein, the present invention relates to DNA segments, which can be isolated from virtually any mammalian origin, which are free from total genomic DNA and which encode proteins having LTBP-3-like activity . DNA segments encoding LTBP-3-like types can be tested to encode proteins, polypeptides, subunits, functional domains, and the like.

As used herein, the term "DNA segment" refers to a DNA molecule that has been isolated free from the total genomic DNA of a particular species. Thus, a DNA segment encoding LTBP-3 refers to a DNA segment containing sequences encoding LTBP-3 that is isolated away from, or free purified from, total genomic DNA of the species from which the segment of DNA is obtained. Included within the term "DNA segment" are DNA segments and fragments of such smaller segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phages, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified LTBP-3 gene refers to a DNA segment that includes LTBP-3 coding sequences and, in certain aspects, regulatory sequences, isolated substantially away from other genes found naturally or from protein-coding sequences. In this regard, the term "gene" is used for simplicity to refer to a unit that encodes a functional protein, polypeptide, or peptide. As will be understood by those of skill in the art, this functional term includes both genomic sequences, cDNA sequences, and smaller genetically engineered gene segments that express, or can be adapted to express, proteins, polypeptides, or peptides.

"Isolated substantially away from other coding sequences" means that the gene of interest, in this case, a gene encoding LTBP-3, constitutes the significant part of the coding region of the DNA segment, and that the DNA segment contains no portions large naturally occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the originally isolated DNA segment, and does not exclude genes or coding regions that were later added to the segment by man.

In particular embodiments, the invention relates to isolated DNA segments and to recombinant vectors that incorporate DNA sequences that encode a type of LTBP-3 that includes within its amino acid sequence an amino acid sequence, as essentially set forth in SEQ ID NO: 3. In other particular embodiments, the invention relates to isolated DNA segments and recombinant vectors that incorporate DNA sequences that include within them a nucleotide sequence as essentially set forth in SEQ ID NO: 2.

The term "a sequence as set forth essentially in SEQ ID NO: 3" means that the sequence corresponds substantially to a part of SEQ ID NO: 3 and possesses relatively few amino acids that are not identical to, or biologically equivalent. functional amino acids of SEQ ID NO: 3. The term "biologically functional equivalent" is well understood in the art and is defined in detail hereinafter (eg, see section 7, preferred embodiments). In agreement with this, the sequences that have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of the amino acids that are identical or functionally equivalent to the amino acids of SEQ ID NO: 3, will be sequences that are "as essentially set forth in SEQ ID NO: 3".

In some other embodiments, the present invention relates to isolated DNA segments and recombinant vectors that include in their sequences a nucleic acid sequence as essentially set forth in SEQ ID NO: 2. The term "as set forth essentially in SEQ ID NO: 2 is used in the same sense as that described above and means that the nucleic acid sequence corresponds substantially to a part of SEQ ID NO: 2 and has relatively few codons that are not identical, or functionally equivalent, to the codons of SEQ ID NO: 2. Again, DNA segments encoding proteins exhibiting LTBP-3-like activity will be most preferred.

It will also be understood that the amino acid and nucleic acid sequences may include additional residues, such as N or C terminal amino acids or 5 'or 3' end sequences, and will still be as essentially set forth in one of the sequences set forth herein. disclose, provided that the sequence meets the criteria set forth above, including the maintenance of the biological activity of the protein as regards protein expression. The addition of terminal sequences particularly affects nucleic acid sequences which may, for example, include several non-coding sequences flanking some of the 5 'or 3' end portions of the coding region or may include several internal sequences, i.e. , introns, which are known to be inside genes.

Naturally, the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO: 2. Nucleic acid sequences that are "complementary" are those that are capable of pairing according to the standard WatsonCrick complementarity rules. As used herein, the term "complementary sequences" means sequences of

ES 2 139 889 T5 nucleic acids that are substantially complementary, as can be verified by the same nucleotide comparison as set forth above, or that is defined as being capable of hybridizing with the nucleic acid segment of SEQ ID NO: 2, under relatively stringent conditions such as those described here.

The nucleic acid segments of the present invention, despite the length of the same coding sequence, can be combined with other DNA sequences, such as promoters, polyadenylation signals, additional enzymatic restriction sites, multiple cloning sites, other segments. encoders, and the like, so that their total length can vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be used, the entire length being preferably limited by ease of preparation and use in the recombinant DNA protocol it is intended to carry out. For example, nucleic acid fragments can be prepared that include a continuous short piece identical to or complementary to SEQ ID NO: 2, such as about 14 nucleotides, and that are up to about 10,000 base pairs in length or about 5,000 base pairs, with segments of about 3,000 being preferred in some cases. DNA segments with total lengths of about 1,000, 500, 200, 100, and 50 base pairs in length are also contemplated as useful.

It will be readily understood that "intermediate lengths" in these contexts, means any length between the quoted intervals, such as 14, 15, 16, 17, 18, 19, 20, etc; 21,22,23, etc; 30, 31, 32, etc; 50, 51, 52, 53, etc; 100, 101, 102, 103, etc; 150, 151, 152, 153, etc; including all integers within the 200-500 range; 5001,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; 5,000-10,000, up to and including sequences of about 12,001, 12,002, 13,001, 13,002, and the like.

It will also be understood that the present invention is not limited to the particular nucleic acid and amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3. Recombinant vectors and isolated DNA segments may thus variously include the LTBP-3 coding regions themselves, coding regions showing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that, however, include the LTBP-3 coding regions or may encode biologically functional equivalent proteins or peptides possessing variable amino acid sequences.

The DNA segments of the present invention encompass LTBP-3 proteins and biologically functional equivalent peptides. Such sequences can occur as a consequence of codon redundancy and functional equivalence known to occur naturally within nucleic acid sequences and proteins thus encoded. Alternatively, functionally equivalent proteins or peptides can be created through the application of recombinant DNA technology, in which changes in protein structure can be technologically brought about, based on considerations of the properties of the amino acids being changed. Man-made changes can be introduced by applying single-site mutagenesis techniques, for example, to introduce improvements in protein antigenicity or to test mutants to examine activity at the molecular level.

If desired, fusion proteins and peptides can also be prepared, eg, where the LTBP-3 coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, for purification purposes. or immunodetection (eg, proteins that can be purified by affinity chromatography and regions encoding enzyme markers, respectively).

Recombinant vectors constitute further aspects of the present invention. Particularly useful vectors are considered to be those in which the coding portion of the DNA segment, whether it encodes an entire protein or a smaller peptide, is placed under the control of a promoter. The promoter can be in the form of that which is naturally associated with a LTBP-3 gene, as it can be obtained by isolating the non-coding sequences of the 5 'end located above the coding segment or exon, for example, using recombinant cloning and / or PCR ™ technology, in relation to the compositions disclosed herein.

In other embodiments, it is believed that certain advantages will be obtained by placing the coding DNA segment under the control of a recombinant or heterologous promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a LTBP-3 gene in its natural environment. Such promoters can include LTBP-3 promoters that are normally associated with other genes, and / or promoters isolated from any bacterial, viral, eukaryotic, or mammalian cell. Naturally, it will be important to use a promoter that effectively drives the expression of the DNA segment in the cell type, organism, or even animal, that is selected for expression. The use of promoter and cell type combinations for protein expression is known to those skilled in the art of molecular biology, for example, see Sambrook et al., 1989. The promoters used can be constitutive, or inducible, and can be used under conditions appropriate to direct high-level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides. Appropriate promoter systems that are considered for use in high-level expression include, but are not limited to, the Pichia expression vector system (Pharmacia LKB Biotechnology) (see Example XVI here).

In relation to the expression embodiments to prepare the recombinant LTBP-3 proteins and peptides, it is considered that the longer DNA segments will be used more often, with the DNA segments encoding the LTBP protein being the most preferred. -3 integer or its functional domains, subunits, etc. Without

ES 2 139 889 T5 however, it will be appreciated that the use of shorter DNA segments to direct the expression of LTBP-3 peptides or epitopic core regions, as can be used to generate anti-LTBP-3 antibodies, is also within the scope of the present invention. DNA segments encoding peptide antigens from about 15 to about 50 amino acids long, or more preferably, from about 15 to about 30 amino acids long, are considered to be particularly useful.

The LTBP-3 gene and DNA segments can also be used in connection with somatic expression in an animal or in the creation of a transgenic animal. Again, in such embodiments, the use of a recombinant vector that directs the expression of the entire or active LTBP-3 protein is particularly considered.

In addition to their use to direct expression of the LTBP-3 protein, the nucleic acid sequences disclosed herein have various other uses as well. For example, they are also useful as probes or primers in nucleic acid hybridization embodiments. As such, nucleic acid segments comprising a sequential region that is made up of at least one continuous sequence 14 nucleotides in length are considered to have identical sequence that is, or is complementary to, a continuous sequence of length 14 nucleotides of SEQ ID NO: 2 will find particular utility. Longer contiguous complementary or identical sequences for example, are those around 20, 30, 40, 50, 100, 200, 500, 1000 (including all lengths in between) even up to full sequences, they will be useful in certain ways of realization.

The ability of such nucleic acid probes to specifically hybridize to LTBP-3 encoding sequences will allow them to be useful in detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including using the sequence information for the preparation of mutant-type primers, or primers for use in the preparation of other genetic constructs.

Nucleic acid molecules that possess sequential regions formed by contiguous nucleotide pieces of 10-14, 15-20, 30, 50 or even 100-200 nucleotides or so, identical or complementary to SEQ ID N °: 2, are considered particularly as hybridization probes for use in eg Northern or Southern blotting. This would allow the structural and regulatory genes of LTBP-3 to be analyzed, both in various cell types, as well as in various mammalian cells. The overall size of the fragment, as well as the size of the complementary piece (s), will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally have utility in hybridization embodiments, where the length of the contiguous complementary region can vary, such as between about 1014 and about 100 nucleotides, but larger contiguous pieces of complementarity can be used, depending on the length of the complementary sequences to be detected.

The use of a hybridization probe of about 10-14 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules possessing contiguous complementary sequences on pieces greater than 10 bases in length are generally preferred, though, in order to increase the stability and selectivity of the hybrid, and thus improve the quality and degree of specific hybrid molecules obtained. It will generally be preferred to design nucleic acid molecules that possess complementary pieces of 15 to 20 contiguous nucleotides, or even longer, if desired.

Hybridization probes can be selected from any part of any of the sequences disclosed herein. All that is needed is to review the sequence set forth in SEQ ID NO: 2 and select any continuous portion of the sequence, from about 10-14 nucleotides in length up to and including the entire sequence, that you wish to use as a probe or initiator. The selection of probe and primer sequences can be governed by a number of factors, such as, for example only, that one may wish to use primers towards the end of the overall sequence.

The process of selecting and preparing a nucleic acid segment that includes a contiguous sequence within SEQ ID NO: 2 can alternatively be described as preparing a nucleic acid fragment. Of course, fragments can also be obtained by other techniques such as eg mechanical shear or by restriction enzymatic digestion. Small nucleic acid segments or fragments can be readily prepared, for example, by directly synthesizing the fragment by chemical means, as is commonly done using an automated oligonucleotide synthesizer. Fragments can also be obtained by applying nucleic acid reproduction technology, such as the PCR ™ of Patent 4,603,102 (incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques that are generally known to those of skill in molecular biology.

Accordingly, the nucleotide sequences of the present invention can be used for their ability to selectively form duplex molecules with complementary pieces of LTBP-3 or cDNA gene fragments. Depending on the intended application, it will be desirable to use varying hybridization conditions to achieve varying degrees of selectivity of the probe over the target sequence. For applications requiring high selectivity, it will typically be desired to use relatively stringent conditions to form the hybrids, e.g., low salt and / or high temperature solution conditions will be selected, such as provided by about 0.02 M at 0 15 M NaCl approximately at temperatures of 50 ° C to 70 ° C. Such conditions

Selective ES 2 139 889 T5 tolerate minimal, if any, pairing between probe and target filament or mold, and will be particularly suitable for isolating LTBP-3 genes.

Of course, for some applications, for example when it is desired to prepare mutants using a mutant starter strand hybridized to an underlying template or if it is desired to isolate the LTBP3 coding sequences from related species, functional equivalents or analogs, conditions will typically be required. less stringent hybridization in order to allow heteroduplex formation. Under these circumstances, it may be desired to use conditions such as salts of about 0.15 M to about 0.9 M, at temperatures ranging from 20 ° C to 55 ° C. Species that cross-hybridize can be quickly identified as positive hybridization signals relative to control hybridizations. In any event, it is generally appreciated that conditions can be made more stringent by adding increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same way as increased temperature. Thus, the hybridization conditions can be easily manipulated, and thus will generally constitute a selection method depending on the desired results.

In some embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with appropriate means, such as a marker, to determine hybridization. A wide variety of appropriate indicator media are known in the art, including fluorescent, radioactive, enzymatic, or other ligands, such as avidin / biotin, that are capable of eliciting a detectable signal. In preferred embodiments, it will probably be desired to employ a fluorescent or enzymatic label, such as urease, alkaline phosphatase, or peroxidase, rather than radioactive or other environmentally undesirable reagents. In the case of enzyme markers, colorimetric reporter substrates, which are known to provide a means visible to the human eye or spectrophotometrically, can be used to identify specific hybridization with samples containing complementary nucleic acids.

In general it is anticipated that the hybridization probes described herein will be useful both as reagents in the hybridization solution and in embodiments using a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise attached to a selected matrix or surface. This fixed single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The conditions selected will depend on the particular circumstances that are based on the particular criteria that are required (depending for example, on the content of G + C, type of target nucleic acid, source of the nucleic acid, size of the hybridization probe, etc.) . After washing the hybridized surface so as to remove molecules that are not specifically bound, specific hybridization is detected, even quantified by the marker.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors in order to enable the practice of the invention to be well carried out, and thus may be considered to constitute modes. preferred for your practice. However, those skilled in the art should, in light of the present discussion, appreciate that many changes can be made to the specific embodiments set forth and still obtain a similar or analogous result without departing from the spirit and scope of the invention. . Example I

Animal model to evaluate new bone formation

Since various animal models were not suitable for studying the effects of nucleic acid transfer on bone formation, the inventors employed the following model system. The important characteristics of the rat osteotomic model are as described in the following protocol (which is generally completed in 25-35 minutes).

The osteotomy was performed on one femur per animal. There were no significant differences between the right or left extremities, but these differences were controlled in these studies, as the extremity receiving the osteotomy is random.

After pre-operative preparation (i.e., shaving and rubbing with Betadine®), adult male SprageDawley rats (~ 500 g, removed breeding males) were anesthetized using a mixture of 3% halothane and 97% oxygen (700 ml / min flow rate). On one of the extremities, a lateral approach to the femur was carried out. Using specially designed surgical guides, four 1.2 mm diameter nails were driven into the shaft after pre-drilling with a high-speed precision drill. A surgical template ensured the precise and parallel arrangement of the nails. The order of nail arrangement was always the same: first proximal external and then distal external, proximal internal and distal internal (with “external” and “internal” referring to the distance from the hip joint). Nail placement in the center of the femur was ensured by fluoroscopic imaging during nail placement. The external fixator was secured over the nails and a 1 or 2 mm segmental defect was created in the central shaft by incision using a Hall Micro 100 oscillating saw (Hall surgical blades # 5053-60) under constant irrigation. Apart from the size of the segmental defect, there is no difference between the 5 mm and 2 mm of the osteotomy protocols (Fig. 5A, Fig. 5B, Fig. 6A, Fig. 6B, Fig. 6C, Fig. 6D, Fig. 7A, Fig. 7B, Fig. 8A, Fig. 8B, Fig. 8C).

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The contents of the osteotomy site were flushed with sterile saline and fibrous collagen graft material, previously soaked in a plasmid DNA solution or other DNA construct if appropriate, was placed in situ. The wound was then closed in layers. Since the fixator provided the necessary stability, there were no limitations for ambulation of the animals, and no other supports were needed. The surgical protocol has been successfully performed in 53 animals to date, including 35 controls (Table 2 and Fig. 24). None of these animals died and no significant side effects have been observed, other than complications that could be associated with surgical repair of the fracture. Minor complications that were experienced were one animal that developed postoperative osteomyelitis and one animal in which 2/4 nails became loose as a consequence of the postoperative bone fracture.

Example II

Graft material for use in bone gene transfer

Various graft materials can be used to transfer genes to the site of bone repair and / or regeneration in vivo. These materials are soaked in a solution containing the DNA or gene to be transferred to the bone regrowth site. Alternatively, DNA can be incorporated into the matrix as a preferred method of performance.

A particular example of a suitable material is fibrous collagen, which can be lyophilized after extraction and partial purification of the tissues and then sterilized. A particularly preferred collagen is the fibrous collagen graft material called UltraFiber ™, as available from Norian Corp., (Mountain View, CA). Detailed descriptions of the composition and use of Ultra Fiber ™ are provided in Gunasekaran et al., (1993a, 1993b; each incorporated herein by reference).

A more particularly preferred collagen is type II collagen, the most particularly preferred collagen being recombinant type II collagen or mineralized type II collagen. Before placement in the osteotomy sites, the graft materials are soaked in DNA (or virus) solutions under sterile conditions. Soaking can be done for any appropriate and convenient period, for example, from 6 minutes to a full night. The DNA solution (eg plasmid) will be a sterile aqueous solution, such as a sterile aqueous buffer or an acceptable one, the concentration generally being between 0.5 and 1.0 mg / ml. Commonly preferred plasmids are those such as pGL2 (Promega), pSV40yd-gal, pAd.CMVlacZ, and pLJ.

Example III

Gene Constructs of Parathyroid Hormone

The active fragment of the human parathyroid hormone gene (hPTH1-34) was selected as the first of the osteotropic genes to be incorporated into an expression vector for its use in gene transfer, in order to promote the formation of new bone. in the osteotomic model of the rat.

The inventors chose to construct the hPTH1-34 transgene in the expression vector pLJ (Fig. 10), since this vector was appropriate for studies of transgenic function, both in vitro and in vivo. A schematic of the PLJ-hPTH1-34 transgene is shown in Fig. 10. The DNA and amino acid sequences of hPTH1-34 are well known, for example, see Hendy et al., 1981 (incorporated herein by reference). To insert the transgene into the expression vector pJL, PCR ™ of a full-length recombinant PTH clone was used, followed by standard molecular biological manipulation.

A retroviral pool was then generated after CaPO-mediated transfection.₄ of O cells with the hPTH1-34 construct, all according to standard protocols (Sambrook et al., 1989). Independent transduced Rat-1 clones were obtained by standard infection and selection procedures (Sambrook et al., 1989).

One clone (YZ-15) was analyzed by Southern analysis, demonstrating that the PLJ-hPTH1-34 transgene had stably integrated into the Rat-1 genome (Fig. 11). The next analysis that was performed, a Northern analysis, was to show that clone YZ-15 expressed the PLJ-hPTH1-34 transgene, as evidenced by the presence of specific PLJ-hPTH1-34 transcripts (Fig. 12).

Example IV

Expression and activity of polypeptide parathyroid hormone

A specific and sensitive radioimmunoassay was carried out to demonstrate that YZ-15 cells expressed and secreted a recombinant hPTH1-34 molecule (Table 2). The radioimmunoassay was carried out in the medium of transduced Rat-1 clones. To quantify the secretion of the recombinant peptide hPTH1-34 produced by the YZ15 cells, the culture medium from a confluent 100 mm plate was collected over a period of 24 hours and assayed with the NH kit.₂-terminal hPTH RIA (Nichols Institute Diagnostics) according to the manufacturer's protocol. PLJhPTH1-84 cells and BAG cells served as positive and negative controls, respectively.

IS 2 139 889 T5

The protein concentrations in Table 2 are expressed as the average of three tests plus the standard deviation (in parentheses). The concentration of peptides 1-34 and full-length (1-84) was determined relative to a standard curve generated with commercially available reagents (Nichols Institute Diagnostics).

TABLE 2

<td>Cell lines</td><td>PTH (pg / ml)</td>

<td>YZ-15</td><td> 247 (± 38)</td>

<td>PLJ-hPTH1-84</td><td> 2616(± 372)</td>

<td>BAG</td><td> 13 (± 3)</td>

As shown in Table 2, PTH expression was detected in both cells, YZ-15 and PLJ-hPTH184. The BAG cells did not produce detectable PTH and served as a baseline for the RIA. These results demonstrate that YZ-15 cells expressed the recombinant protein hPTH1-34.

The recombinant molecule hPTH1-34 was added to rat osteosarcoma cells and a cAMP response assay was carried out in order to determine whether the secreted molecule had biological activity. Unconcentrated medium was collected from YZ-15 cells, PLJ-hPTH1-84 cells, and BAG cells and used to treat ROS17 / 2.8 cells for 10 minutes, as described (Majmudar et al., 1991). The cAMP was then extracted from the treated cells and quantified by RIA (Table 3). The amount of cAMP shown is the average of three tests. The standard deviation of the mean is shown in parentheses.

TABLE 3

<td>Cell lines</td><td>cAMP (pmol)</td>

<td>YZ-15</td><td> 20,3 (± 0,25)</td>

<td>PLJ-hPTH184</td><td> 88,5 (± 4,50)</td>

<td>BAG</td><td> 7,6 (± 0,30)</td>

A cAMP response was induced by recombinant PTH secreted by YZ-15 cells and by PLJhPTH1-84 cells. The BAG cells did not produce PTH and served as a baseline for the cAMP assay. These results provide direct in vitro evidence that the PLJ-hPTH1-34 transgene directs the expression and secretion of a functional osteotropic agent.

Example V

Gene constructs of bone morphogenetic protein (BMP)

Murine bone morphogenetic protein-4 (BMP-4) was selected as the next of the osteotropic genes to be incorporated into the expression vector for use in promoting bone repair and regeneration.

A full length murine BMP-4 cDNA was generated by searching a murine 3T3 cell cDNA library (Stratagene). The human sequence for BMP-4 is well known to those of skill in the art and has been deposited with Genbank. Degenerate oligonucleotide primers were prepared and used in a standard PCR ™ to obtain a murine cDNA sequence.

The ends of the cDNA clone were further modified using the polymerase chain reaction so that the full-length cDNA (5 '3' direction) encodes the natural murine initiation Met codon, the full-length murine coding sequence, a marker of 9 amino acids (known as the HA epitope) and the natural murine stop codon. The amino acid sequence encoded by the murine BMP-4 transgene is shown in Fig. 24; this entire sequence, including the marker, is represented by SEQ ID NO: 1.

The location of the HA epitope at the carboxyterminal end will not interfere with the sequence function of the recombinant molecule in vitro or in vivo. The advantage of the epitope is that it can be used in immunohistochemical methods to specifically identify the recombinant murine BMP-4 molecule in osteotomic tissues in vivo, e.g., the epitope can be identified using a monoclonal antibody that is commercially available (Boehringer-Mannheim ), as described here.

Studies to demonstrate that the murine BMP-4 transgene encodes a functional osteotropic agent, include, for example, (a) transfection of COS cells and immunoprecipitation of a protein band of the correct size using an anti-HA monoclonal antibody (Boehringer-Mannheim) ; and (b) a quantitative in vivo bone induction assay.

ES 2 139 889 T5 (Sampath and Reddi, 1981) which involves implanting proteins from the medium of COS transfected cells under the skin of male rats and assessing new bone formation at the ectopic site.

Example VI

Detection of mRNA by tissue in situ hybridization

The following technique describes the detection of mRNA in tissue obtained from the site of bone regeneration. This can be useful for detecting the expression of the mRNA transgene itself, and also for detecting the expression of the hormone or growth factor receptors or other molecules. This method can be used in place of, or in addition to, Northern analyzes, such as those described in Fig. 13.

The DNA of a plasmid containing the gene for which the mRNA is to be detected is linearized, extracted and precipitated with ethanol. Sense and antisense transcripts are generated from 1 mg of the matrix with T3 and T7 polymerases, e.g., in the presence of (S)³⁵ UTP at> 6 mCi / ml (Amersham Corp.,> 1200 Ci / mmol) and 1.6 U / ml RNsin (Promega), with the remaining in vitro transcription reagents provided in a kit (SureSite, Novagen Inc). After transcription at 37 ° C for 1 hour, the DNA matrices are removed by a 15-minute digestion at 37 ° C with 0.5 U / ml RNase-free DNase I, extracted, and ethanol precipitated. . Riboprobes are hydrolyzed to an average final length of 150 base pairs by incubation in 40 mM NaHCO₃, 60 mMdeNa₂ CO₃, 80 mM DTT at 60 ° C, according to the previously determined formula. The hydrolysis is terminated by adding sodium acetate, pH 6.0, and glacial acetic acid at 0.09 M and 0.005% (v / v), respectively, and the probes are then ethanol precipitated, dissolved in 0.1 M DTT, are counted, and stored at -20 ° C until use.

RNase precautions are taken at all stages of slide preparation. The histological sections fixed in Bouin's fluid and embedded in paraffin are heated to 65 ° C for 10 minutes, then deparaffinized in 3 xylene changes for 5 minutes, and rehydrated in a descending series of ethanol, ending in a buffered solution of phosphate (PBS). Slides will be soaked in 0.2N HCl for 5 minutes, rinsed in PBS, digested with 0.0002% proteinase K in PBS for 30 minutes at 37 ° C, and briefly rinsed with DEPC-treated water. After equilibration for 3 minutes in 0.1 M triethanolamine-HCl (TEA-HCl), pH 8.0, sections are acetylated in 0.25% (vol / vol) acetic anhydride in 0.1 M TEA-HCl for 10 minutes at room temperature, rinse in PBS, and dehydrate in an ascending series of ethanol. Each section receives 100-200 ml of prehybridization solution (0.5 mg / ml RNase-free, denatured tRNA (Boehringer-Mannheim), 10 mM DTT, 5 mg / ml denatured sulfurylated salmon sperm DNA, 50% formamide, 10% dextran sulfate, 300 mM NaCl, 1X RNase-free Denhardt's solution (prepared with RNase-free bovine serum albumin, Sigma), 10 mM Tris-HCl, pH 7.4, 1 mM EDTA) and incubate on a slide warmer at 50 ° C in a humidified room for 2 hours. Sulfurylated Salmon Sperm DNA Blocking Reagent is used in both the prehybridization and hybridization solutions to help reduce non-specific binding to tissues by SH groups.³⁵ on the probe. It is prepared by labeling RNase-free salmon sperm DNA (Sigma) with α-thiodCTP and non-radioactive α-thio-dATP (Amersham) in a standard oligonucleotide-initiated random DNA labeling reaction. Excess prehybridization solution is removed with a short rinse in 4X SSC before probe is applied.

Riboprobes, fresh tRNA and sulfurylated salmon sperm DNA will be denatured for 10 minutes at 70 ° C, and chilled on ice. The hybridization solution is applied, identical to the prehybridization solution except that a denatured probe will be added to it at 5 x 10⁶ CPM / ml, the slides being incubated at 50 ° C overnight in a slide warmer located in a sealed humidified chamber. Antisense and sense probes are applied to serial histological sections. The slides are rinsed 3 times in 4X SSC, washed with 2X SSC, 1 mM DTT for 30 minutes at 50 ° C, digested with RNase A (20 mg / ml RNase A, 0.5 M NaCl, 10 mM Tris , pH 8.0, 1 mM EDTA, pH 8.0) for 30 minutes, at 37 ° C, and briefly rinsed with 2X SSC, 1 mM DTT. Three additional washes are carried out, each at 50 ° C for 30 minutes: once with 2X SSC, 50% formamide, 1 mM DTT, and twice with 1X SSC, 0.13% sodium pyrophosphate (wt / vol), 1 mM DTT.

The slides are dehydrated in an ascending ethanolic series (supplementing the diluted ethanols (50% and 70%) with SSC and DTT at 0.1X and 1 mM, respectively). The slides are exposed to X-ray film for 20-60 hours to visualize the total hybridization patterns, immersed in an autoradiographic emulsion (Kodak NTB-2, diluted up to 50% with 0.3 M ammonium acetate), dried slowly over 2 hours, and exposed (4 ° C) for periods ranging from 8 days to 8 weeks. After developing the emulsion, sections were secondarily stained with hematoxylin and eosin, dehydrated, and mounted with xylene-based medium. The hybridization signal is visualized under dark field microscopy.

The previously discussed in situ hybridization protocol can be used, for example, to detect the spatial and temporal pattern of PTH / PTHrP receptor expression. An appropriate rat PTH / PTHrP receptor cDNA probe (R15B) is one that consists of a 1810 base pair region encoding the rat full-length bone PTH / PTHrP receptor (Abou-Samra et al., 1992). The cDNA fragment is subcloned into pcDNA (Invitrogen Corp., San Diego, CA) and fragmented using XbaI and BamHI. This probe has provided positive signals for Northern blot analysis of rat, murine and human osteoblastic cell lines, rat primary cranial cells, and murine bone tissue. The plasmid pcDNA I contains a T7 and SP6 promoter that facilitates the generation of cRNA probes for in situ hybridization. The full-length transcript has been used to

ES 2 139 889 T5 detect the PTH / PTHrP receptor in bone sections (Lee et al., 1994). The PTHrP cDNA probe (Yasuda et al., 1989) is a 400 base pair fragment subcloned into pBluescript (Stratagene). This probe has been used for in situ hybridization, generating an antisense cRNA probe using a BamHI fragmentation and the T3 primer and a sense cRNA probe using an EcoRI fragmentation and the T7 primer.

Example VII

In vivo protein detection after transgene expression

1. Transgene β-galactosidase

Bacterial jd-galactosidase can be detected immunohistochemically. Osteotomic tissue preparations are fixed in Bouin's fluid, demineralized, and then divided in half along the longitudinal plane. Half of each preparation is embedded in paraffin for subsequent immunohistochemical identification of the bacterial protein jd-galactosidase.

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

Bacterial β-gal can also be detected by substrate utilization assays. This is done using commercially available kits (eg Promega), according to the manufacturer's instructions.

two. Luciferase transgene

Luciferase can be detected by substrate utilization assays. This is done using commercially available kits (eg Promega) according to the manufacturer's instructions.

3. PTH transgenes

Recombinant PTH, such as the hPTH1-34 peptide, is assayed in homogenates of osteotomic cleft tissue, for example, using two commercially available radioimmunoassay kits according to manufacturer's protocols (Nichols Institute Diagnostics, San Juan Capistrano, CA).

One kit is the "Intact Parathyroid Hormone-PTH Kit 100T". This radioimmunoassay uses an antibody to the carboxyl terminus of the intact hormone, and is thus used to measure endogenous hormone levels in osteotomic cleft tissue. This assay can be used to establish a baseline PTH expression in the rat osteotomy model.

The second kit is a two-site immunoradiometric kit for the measurement of rat PTH. This kit uses affinity purified antibodies that are specific for the amino terminus of intact rat hormone (PTH134) and thus will measure endogenous PTH production as well as recombinant protein. Previous studies have shown that these antibodies cross-react with human PTH and are therefore capable of recognizing recombinant molecules in vivo.

Values ​​obtained with kit # 1 (antibodies to the carboxyl end) are subtracted from the values ​​obtained with kit # 2 (antibodies to the amino end) to obtain sensitive and accurate measurements. The level of recombinant peptide thus correlates with the degree of new bone formation.

Four. Transgen BMP

Preferably, BMP proteins, such as the murine BMP-4 transgenic peptide product, are immunohistochemically detected using a specific antibody that recognizes the HA epitope (Majmudar et al., 1991), such as the monoclonal antibody available from Boehringer-Mannheim. Antibodies to the same BMP proteins can also be used. Such antibodies, along with various immunoassay methods, are described in US Pat. 4,857,456, which is incorporated herein by reference.

Osteotomic tissue preparations are fixed in Bouin's fluid, demineralized, and then divided in half along the longitudinal plane. Half of each preparation is embedded in paraffin for subsequent immunohistochemical identification of the recombinant murine BMP-4 molecule.

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Example VIII

Direct gene transfer to bone that regenerates in vivo

To assess the feasibility of direct gene transfer to bone regenerating in vivo, transfer of a marker gene to cells was used in the rat osteotomic model. These studies involved two marker genes: bacterial β-galactosidase and insect luciferase.

Aliquots of a fibrous collagen graft material were embedded in solutions of pure marker gene DNA. The graft materials were then applied to the osteotomy site, and their expression was determined as described above.

Both marker genes were found to be successfully transferred and expressed, without failure, as demonstrated by substrate utilization assays (Figs. 5A, 5B, 6A, 6B, 6C and 6D). Since mammalian cells do not normally synthesize marker gene products either, this provides direct evidence that osteotomic repair cells were transfected in vivo and then expressed β-galactosidase and luciferase transgenes as functional enzymes.

Example IX

Adenoviral gene transfer to regenerating bone in vivo

One of the alternative methods of obtaining in vivo gene transfer to regenerating bone is to use adenovirus-mediated transfer. Adenoviral gene transfer of a marker gene construct into repair bone cells has been successfully achieved in the rat osteotomic model (Figs. 23A, 23B and 23C).

The inventors used the adenoviral vector pAd.CMV.lacZ, which is an example of an incompletely replicated adenoviral vector that can replicate in facultative cells (Stratford-Perricaudet et al., 1992). In pAd.CMVlacZ, the cytomegalovirus (CMV) early enhancer / promoter is used to govern lacZ transcription with an SV40 polyadenylation sequence cloned downstream of this reporter (Davidson et al., 1993).

The vector pAd.RSV4 is also used by the inventors. This vector has essentially the same basic structure as pAd.CMVlacZ, but nevertheless the CMV promoter and the unique Bg1II cloning site have been replaced in the form of a cassette with the Bg1II fragment which is formed by an RSV promoter, a multiple cloning site, and a poly (A *) site. The greater flexibility of this vector is considered to be useful for subcloning osteotropic genes, such as the hPTH1-34 cDNA fragment, for use in further studies.

To generate recombinant adenovirus PTH, a 100 mm plate of 293 cells is transfected using calcium phosphate with 20 mg of a plasmid construct, e.g., the plasmid containing the NheI linearized hPTH1-34 insert, plus 2 mg of the DNA wild-type adenovirus digested with XbaI and ClaI. Adenoviral DNA is derived from adenovirus type 5, which contains only a single XbaI and ClaI site and has a partial deletion of the E3 region. Approximately 7 days after transfection, cells and media were recovered and a lysate was prepared by repeated freeze-thaw cycles. This lysate was diluted and used to infect 60 mm plates of 293 confluent cells for 1 hour. The cells are then covered with 0.8% agar / 1X MEM / 2% calf serum / MgCl₂ 12.5 mM. Ten days after infection, individual plaques were selected and used to infect 60 mm plates of 293 cells to spread the amount of virus. Positive plaques were selected for further purification and generation of adenovirus stocks.

To purify recombinant adenoviruses, 150 mm plaques of 293 cells showing 75-90% confluence were infected with 2-5 PFU / cell, a titer that avoids the potential cytotoxic effects of adenoviruses. 30 hours after infection, cells were rinsed, removed from plates, pelleted, and resuspended in 10 mM Tris-HCl, pH 8.1. A viral lysate is generated by three freeze-thaw cycles, cell debris being removed by centrifugation for 10 minutes at 2,000 rpm, and the adenovirus being purified by density gradient centrifugation. The adenoviral band is stored at -20 ° C in sterile glycerol / BSA until needed.

The virus particle solution was sterilized and incubated with the graft material (from 6 min to overnight), and the virus impregnated material was grafted into the osteotomic cleft, where the cellular viral infection clearly took place. The obtained results clearly demonstrated the exquisite specificity of the anti-d-gal antibody (Sambrook et al., 1989), and conclusively demonstrated the expression of the marker gene product in chondrocytic cells of the osteotomic cleft. The signal directed to the nucleus has also been observed in preosteoblasts.

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Example X

Osteotropic gene transfer stimulates bone regeneration / repair in vivo

In order for a parathyroid hormone (PTH) transgene to function as an osteotropic agent, it is likely that the PTH / PTHrP receptor is required to be expressed in the same bone repair tissue. Therefore, the inventors investigated the expression of the PTH / PTHrP receptor in the rat osteotomic model.

A Northern analysis of poly-A (+) RNA was carried out which demonstrated that the PTH / PTHrP receptor was expressed in osteotomy repair tissue (Fig. 13).

The inventors then investigated whether gene transfer could be used to create transfected cells that constitutively express hPTH1-34 in vivo, and whether this transgene can stimulate bone formation. The rate of new bone formation is analyzed as follows. At necropsy, the osteotomy site is carefully dissected for histomorphometric analysis. The AP and ML dimensions of the callus tissue are measured using calipers. The samples are then fixed by immersion in Bouin's liquid, washed in ethanol, and demineralized in buffered formic acid. Plastic soaking of decalcified materials is used because of the superior dimensional stability of methacrylate during sample preparation and cutting.

Tissue blocks dehydrate in increasing alcohol concentrations and become embedded. 5 mm thick cuts are made in the coronal plane using a Reichert Polycut microtome. Sections are prepared from the center across the width of the medullary canal to guard against sampling bias. The sections for light microscopy are stained using modified Goldner's trichrome stain, to differentiate bone, osteoid, cartilage and fibrous tissue. Sections are covered using Eukitt's mounting medium (Calibrated Instruments, Ardsley, NY). Histomorphometric analyzes are carried out under bright field using a Nikon Optiphot Research microscope. Standard point counting stereology techniques are used using a 10mm x 10mm lattice eye grid.

The total area of ​​the callus is measured at a magnification of 125X as an index of the total intensity of the healing reaction. At 250X magnification the fractions of the bone, cartilage and fibrous tissue areas are measured to examine the relative contribution of each tissue to callus formation. Since the dimensions of the osteotomic cleft reflect the baseline (time 0), a measurement of the bone area at subsequent time intervals is used to indicate the rate of bone filling. Statistical significance is assessed using analysis of variance, with subsequent appropriate comparisons between groups using Tukey's t student test.

In the 5 mm rat osteotomic model described above, it was found that the expression of the PTH transgene can stimulate regeneration / repair in living animals (Figs. 6A, 6B, 6C and 6D). This is a particularly important finding as hPTH1-34 is known to be a more potent anabolic agent when administered intermittently than when compared to continuous administration, and it is the continuous-type release that originates from gene transfer methods here. used.

Although the inventors have already demonstrated the success of direct gene transfer to in vivo regenerating bone, the use of ex vivo treatment protocols is also considered. In such embodiments, the bone progenitor cells will be isolated from a particular animal or man, and maintained in an in vitro environment. Appropriate areas of the body from which bone progenitor cells can be obtained are areas such as bone tissue and fluid that surround a fracture or other skeletal defects (whether this is an artificially created site or not) and from the marrow. that is. The isolated cells will then be contacted with the DNA (or recombinant viral) composition, with, or preferably without, a matrix, when the cells take up the DNA (or are infected with the recombinant virus). The stimulated cells will then be returned to the site in the animal or patient where bone repair is to be stimulated.

Example XI

Gene transfer to the Achilles tendon and cruciate ligament in vivo

The studies on regenerating bone described above complement others of the inventors in which gene transfer was used successfully to introduce genes into the Achilles tendon (Figs. 3A, 3B, 3C, 3D and 3E) and in the cruciate ligament (Fig. 4).

The Achilles tendon is made up of organized cells and extracellular matrix with a characteristic tissue architecture. Wound tissues can disrupt this architecture and stimulate a wound healing response. The injured tendon will regenerate, opposing the scar, if the elements of its connective tissue remain approximately intact. Regeneration is advantageous because scar tissue is not optimally designed to support normal mechanical function. Segmental defects in the tendon due to traumatic damage can be treated with biological or synthetic grafts that encourage new tendon formation. This strategy is limited, however, by the availability of effective (autologous) biological grafts, the long-term stability and compatibility of synthetic prostheses, and the slow rate of incorporation often seen with both types of grafts.

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The inventors hypothesized that the effectiveness of biological grafts can be enhanced by the overexpression of molecules that regulate the regenerative response of tissue. To this end, they developed a model system in which segmental defects are created in the Achilles tendon and a new biomaterial is used as a tendon / molecular graft delivery agent. In the present example, the ability to deliver and express marker gene constructs in regenerating tendon tissue is demonstrated.

Plasmid storage solutions (pSV / gal, Promega) were prepared according to standard protocols (Sambrook et al., 1989). SIS graft material was prepared from a jejunum segment of adult pigs (Badylak et al., 1989). At harvest, the mesenteric tissues were removed, the segment was inverted, and the mucosa and superficial submucosa were removed using a mechanical abrasion technique. After returning the segment to its original orientation, the serosa and muscle layers were rinsed, sterilized by treatment with dilute peracetic acid, and stored at 4 ° C until use.

Crossbreed dogs (all studies) were anesthetized, intubated, placed in a reclining position on a heating pad, and maintained under inhalant anesthesia. A lateral incision from the musculotendinous junction to the plantar aponeurosis was used to expose the Achilles tendon. A thick double sheet of SIS was wrapped around a central portion of the tendon, both ends were sutured, a 1.5 cm segment of the tendon was removed through a lateral opening in the graft material, and the graft and surgical site they closed. The leg was immobilized for 6 weeks and then used freely for another 6 weeks. The graft tissues were harvested at specified times indicated below, fixed in Bouin solution, and embedded in paraffin. Histological sections (8 pm) were cut and used for immunohistochemistry.

In an initial study, SIS material alone (SIS-only graft) was grafted and promoted Achilles tendon regeneration after the creation of a segmental defect in mongrel dogs within a time period of 6 months after surgery. The remodeling process involved the rapid formation of granulation tissue and eventual graft degradation. No scar tissue formed, and no evidence of immune-mediated rejection was observed.

In a second study, SIS was embedded in a plasmid DNA solution (SIS + plasmid graft) and then grafted as an Achilles tendon graft (n = 2 dogs) or a cruciate ligament graft (n = 2 dogs ) in normal mongrel dogs. A plasmid pSV / gal utilizing simian virus 40 regulatory sequences to govern // -galaclosidase (/ -gal) activity was detectable by immunohistochemistry using a specific antibody in 4/4 of the animals. As a negative control, //-gal activity was not detected in the unoperated Achilles tendon and in the cruciate ligament of these animals. It therefore appeared that SIS facilitated the uptake and subsequent expression of plasmid DNA by wound healing cells in both the tendon and the ligament.

A third study was designed to evaluate the course of transgene expression of // - gal. SIS + plasmid grafts were implanted for 3, 6, 9, and 12 weeks (n = 2 dogs, pre-time point) and transgene expression was assayed by immunohistochemistry and in situ hybridization. Cross sections (8 pm) of Bouin-fixed, paraffin-embedded tissue were cut and mounted on Probeon Plus slides (Fisher). Immunohistochemistry was carried out according to the protocol provided with the Histostain-SP kit (Zymed). Briefly, slides were incubated with a well-characterized anii - // - galaclosidase antibody (1: 200 dilution, 5 '3'), washed in

PBS, incubated with a second biotinylated antibody, washed, stained with the enzyme conjugate plus a chromogen-substrate mixture, and stained a second time with hematoxylin and eosin.

Bacterial // - gal activity was detected in tendons that received the SIS + plasmid graft (8/8 animals). Although not rigorously quantitative, the expression of the transgene appeared at its peak at 9-12 weeks. Expression of the //-gal gene was not detected in animals that received only SIS grafts (n = 2, 3 weeks and 12 weeks). As before, no scar tissue formed and no evidence of immune-mediated rejection was observed.

This study demonstrated that the mucosal SIS biomaterial can function as an autologous graft that promotes the regeneration of tissues such as the Achilles tendon and the anterior cruciate ligament. SIS can also be used to release a marker gene construct to regenerate tissue.

Example XIII

Mechanical properties of new bone formation

The mechanical properties of the new bone formed during gene transfer can be measured using, for example, total bone torsion tests that create a state of tension in which the maximum extension stresses will occur in planes that lie obliquely to the longitudinal axis of the bone. bone. Such tests can provide inferences regarding the mechanical anisotropy of the callus tissue and the degree of bone integration of the new bone tissue. These tests are particularly advantageous in evaluating fracture specimens, for example, the irregular shape of the callus tissue typically precludes the use of 4-point joint testing of the whole bone, because it is impossible to align properly. reproducible points from sample to sample.

The femurs are tested on an MTS Servohydraulic Testing machine while there is humidity and at room temperature. A torque sensor and variable rotary displacement transducers provide data

ES 2 139 889 T5 for the torque-angular displacement curves. Specially designed devices support each bone near the metaphyseal-shaft joints, and apply a 2-point load to the shaft. The tests are carried out at a constant speed of travel equal to 20 degrees / sec. A 250-inch-ounce load cell measures the total applied force. All bones are tested while moist and at room temperature. Torque and angular displacement data are obtained using an analog-digital converter and a Macintosh computer and software. From these data, the following variables are calculated: a) maximum torque, b) torsional stiffness, the slope of the pre-performance portion of the curve determined from a linear regression of the data, c) energy for failure, the area under the angular torque displacement curve to the point of failure, and d) the angular displacement ratio, the ratio of displacement at failure to displacement in performance. Statistical significance is determined by analysis of variance followed by multiple comparisons with appropriate corrections (eg Bonferroni).

The present invention also provides a means of utilizing osteotropic gene transfer in connection with reconstructive surgery and various bone remodeling procedures. The techniques described here can therefore be used in conjunction with the technology described by Yasko et al., 1992; Chen et al., 1991; and Beck et al., 1991, each of which is incorporated herein by reference.

Example XIV

Type II collagen promotes new bone growth

Certain matrix materials are capable by themselves of stimulating at least some new growth, that is, they are "osteoconductive materials". Potential examples of such materials are well known in the field of orthopedic research and include hydroxyapatite preparations; crushed bone and mineralized collagen preparations; PLGA and polyanhydride block copolymers. The ability of these materials to stimulate new bone formation distinguishes them from inert graft materials, such as methylcellulose, which has been used in the past to release BMPs at fracture repair sites.

This Example refers to a study using the rat osteotomic model with grafts made from type I collagen (Sigma), type II collagen (Sigma), and UltraFiber ™ (Norian Corp). These materials have been placed in situ without DNA of any kind. Five animals received an osteotomy with 10 mg of a type II collagen only graft (10 mg refers to the original amount of lyophilized collagen). Five out of five control animals received an osteotomy with 10 mg of a type I collagen only graft. Animals were housed for three weeks after surgery and then sacrificed.

The results of these studies were that SIS appeared to retard new bone formation; type I collagen resulted in a moderately strong inflammatory response; and UltraFiber ™ acted as an osteoconductive agent. Type II collagen graft studies produced surprising results because 10 mg of this collagen was found to promote new bone formation in the 5 mm osteotomic model (Figs. 22A, 22B, 22C). New bone splicing the osteotomic cleft was identified three weeks after surgery in 5/5 animals that received a type II collagen only graft (ie, less DNA of any type). In contrast, fibrous granulation tissue was obtained but there was no evidence of new bone formation in 5/5 animals that received only one type I collagen graft.

Radiographic analysis conclusively demonstrated that all animals that received an osteotomy with a type II collagen graft without exception showed radiodense material in the osteotomic cleft (Fig. 22A). In acute contrast, radiographic analysis of all animals that received a type I collagen graft did not reveal formation of radiodense material in the osteotomic cleft (Fig. 22B). The arrow in Fig 22A points towards the growth of new bone formed in the osteotomic cleft of the type II collagen grafted animals. Such new bone growth was not observed in animals that received type I collagen grafts (Fig. 22B).

Fig. 22C demonstrates the results of osteotomy with a type II collagen graft. The arrow points to the area of ​​the new bone formed in the osteotomic cleft. In contrast, fibrous granulation tissue was only identified in the type I collagen cleft.

Previous studies suggested that type II collagen plays only a structural role in the extracellular matrix. The results of type II collagen graft studies are interesting because they demonstrate a new and osteoconductive role for type II collagen during endochondral bone repair. To further optimize the osteoconductive potential of type II collagen, a yeast expression vector encoding type II collagen (full-length collagen α1 (II)) will be used to produce recombinant collagen α1 (II) protein.

Example XV

Identification of subsequent osteotropic genes: isolation of a new gene (LTBP-3) of the latent TGF-β binding protein type

TGF-ds represent a family of structurally related molecules with diverse effects on mammalian cell shape, growth, and differentiation (Roberts and Sporn, 1990). Initially synthesized

ES 2 139 889 T5 as a precursor consisting of an amino-terminal propeptide followed by mature TGF-β, two nascent pro-TGF-β chains, associate in most tissues to form an inactive disulfide-linked dimer of a Mr of approximately 106,000. Homodimers are more common, but heterodimers have also been described (Cheifetz et al., 1987; Ogava et al., 1992). During biosynthesis the mature TGF-β dimer is cleaved from the propeptide dimer. The latent state of TGF-β comes in part from the non-covalent association of propeptide and mature TGF-β dimers (Pircher et al., 1984, 1986; Wakefield et al., 1987; Millan et al., 1992; Miyazono and Heldin, 1989). Consequently, the propeptide dimer is often referred to as the Latency Associated Protein (LAP), and LAP plus the disulfide-linked TGF- / 1 dimer is also known as the Latency Small Complex. In the extracellular space, small latent complexes must dissociate to activate mature TGF-βs. The mechanism of activation of the latency complex is believed to be one of the most important steps governing the effects of TGF-β (Lyons et al., 1988; Antonelli-Orlidge et al., 1989; Twardzik et al., 1990 ; Sato et al., 1993).

In certain cultured cell lines the small dormant complexes of growth factors may contain additional high molecular weight proteins. The best characterized of these high molecular weight proteins is the latent TGF-β or LTPB binding protein (Miyazono et al., 1988; Kanzaki et al., 1990; Tsuji et al., 1990; Olofsson et al., 1992; Taketazu et al., 1994). LTBP produced by different cell types is heterogeneous in size, perhaps due to alternative splicing or due to specific tissue proteolytic processing (Miyazono et al., 1988; Wakefield et al., 1988; Kanzaki et al., 1990; Tsuji et al. ., 1990). TGFβ latent complexes containing LTBP are known as large latent complexes. LTPB has no known covalent bond to mature TGF-β, but is disulfide bonded to LAP.

To date, two LTBPs have been isolated. The deduced amino acid sequence human LTBP-1 comprises a signal peptide, 16 epidermal growth factor-like repeats with the potential to bind calcium (EGF-CB repeats), 2 copies of a single motif containing 8 cysteine ​​residues, a motif of binding to RGD cells, and an 8 amino acid motif identical to the cellular binding domain of laminin B2 chain (Kanzaki et al., 1990). There is evidence that LTBP-1 binds calcium, which, in turn, induces a structural change that protects LTBP from proteolytic attack (Colosetti et al., 1993). LTBP-2 show a sequence identity of 41% with LTBP1 and its structural domains show a similar overall organization (Moren et al., 1994).

While the functions of LTBP-1 and LTBP-2 are currently unknown, several ideas have been proposed in the literature. First, LTBP can regulate the intracellular biosynthesis of latent TGF-β precursors. Cultured erythroleukemic cells efficiently pool and secrete large latent TGF-β complexes, while they slowly secrete small latent TGF-β complexes containing abnormal disulfide bonds (Miyazono et al., 1991; Miyazono et al., 1992). Therefore, LTBP can facilitate the normal assembly and secretion of latent TGFβ complexes. Second, LTBP can target latent TGF-βs to specific types of connective tissue. Recent evidence suggests that the large latent TGF-β complex is covalently bound to the extracellular matrix by LTBP (Taipale et al., 1994). Based on these observations, LTBP has been referred to as a "matrix receptor", that is, a secreted protein that targets and stores latent growth factors such as TGF-β for the extracellular matrix. Third, LTBP can modulate the activation of latent complexes. This idea is based in part on recent evidence suggesting that mature TGF-β is released from extracellular storage sites by proteases such as plasmin and thrombin and that LTBP can protect small latent complexes from proteolytic attack (Falcone et al., 1993; Benezra et al., 1993; Taipale et al., 1994), that is, protease activity can govern the effect of TGF-β in tissues, but LTBP can modulate this activity. Fourth, LTBP may play an important role in directing the TGF-β complex to the cell surface, allowing latent TGF-β to be efficiently activated (Flaumenhaft et al., 1993).

A. Materials and procedures

1. CDNA cloning

Aliquots (typically 40-50,000 PFU) of phage particles from a cDNA library in the eZAPII® vector made from mRNA from NIH 3T3 cells (Stratagene) and fresh XL1-Blue ™ cells (grown in Luria culture broth supplemented with maltose al 0.4% in 10 mM MgSO₄) were mixed, incubated for 15 min at 37 ° C, mixed again with 9 ml of a liquid surface layer (50 ° C) of agarose (NZY culture medium plus 0.75% agarose), and spread evenly on 150 mm NZY-agar plates that had been freshly prepared. Standard procedures were used for the preparation of "raised" plaques and filters for hybridizations (42 ° C, in buffer containing 50% formamide, 5X SSPE, 1X Denhardt's, 0.1% SDS, 100 mg / ml DNA of salmon sperm, 100 mg / ml heparin). Filters were washed progressively to very severe conditions (0.1X SSC / 0.1% SDS, 65 ° C). The cDNA probes were radioactively labeled by the nick displacement method using commercially available reagents and protocols (Nick Translation Kit, Boehringer Mannheim). The purified phage clones were converted to pBluescript® plasmid clones, which were sequenced using Sequenase (v2.0) as described (Chen et al., 1993; Ying et al., 1995). Sequence alignment and identity was determined using Genetics Computer Group (MacVector) sequential analysis programs.

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two. Tissue in situ hybridization

To prepare normal sense and antisense probes, a single 342 base pair fragment from the 3 'end untranslated region (+3973 to +4314, counting the "A" of the Met start codon as +1; see " ish ”, Figure 1) was subcloned into plasmid pBSKS + (Stratagene, Inc). The parent DNA was linearized with either EcoRI or BamHI, extracted, and ethanol precipitated. Sense and antisense transcripts were generated from 1 mg of the matrix with T3 and T7 polymerases in the presence of (S³⁵) UTP at> 6 mCi / ml (Amersham,> 1200 Ci / mmol) and 1.6 U / ml RNasin (Promega), with the remaining in vitro transcription reagents provided in a kit (SureSite, Novagen Inc) . After transcription at 37 ° C for 1 hour, the DNA matrices were removed by a 15 minute digestion at 37 ° C with 0.5 U / ml RNase-free DNase I, extracted, and ethanol precipitated. The riboprobes were hydrolyzed to an average final length of 150 base pairs by incubation in 40 mM NaHCO₃, 60 mM Na₂CO₃, 80 mM DTT for approximately 40 minutes at 60 ° C. The hydrolysis was terminated by adding sodium acetate, pH 6.0, and glacial acetic acid to 0.09 M and 0.56% (v / v), respectively, and the probes were then ethanol precipitated, dissolved in DTT 0 , 1M, were counted, and stored at -20 ° C until use. On days 8.5-9.0, 13.5 and 16.5 the mouse embryo tissue sections (Novagen) and the in situ hybridization protocol were exactly as described (Chen et al., 1993 ; Yin et al., 1995).

3. Northern analysis

Poly A (+) RNA from MC3T3-E1 cells (2-10 mg aliquots) was electrophoresed on a 1.25% agarose / 2.2 M formaldehyde gel and then transferred to a nylon membrane. (Hybond-N, Amersham). RNA was cross-linked to the membrane by exposure to a UV light source (1.2 x 10⁶ mJ / cm² , UV Stratalinker 2400, Stratagene) and then prehybridized for> 15 min at 65 ° C in Rapid-Hyb buffer (Amersham, Inc). A specific cDNA probe that consisted only of the untranslated sequence of the 3 'end of the transcript was labeled with P³² by random priming and was used for hybridization (2 h at 65 ° C). The blots were progressively washed to very severe conditions (0.1X SSC / 0.1% SDS, 65 ° C) and then placed against X-ray film with intensifying screens (XAR, Kodak) at -86 ° C .

Four. Antibody preparation

LTBP-3 antibodies were raised against a unique peptide sequence found in domain # 2 (amino acids 155-167). Peptide # 274 (GESVASKHAIYAVC) (SEQ ID NO: 16) was synthesized using an ABI model 431A synthesizer using FastMoc chemistry. The sequence was confirmed using an ABI473 protein sequencer. A cysteine ​​residue was added to the carboxyl terminus to facilitate cross-linking with carrier proteins. For antibody production, the synthetic peptide was coupled with rabbit serum albumin (RSA) using MBS (m-maleimidobenzoic ester -N-hydroxysuccinimide) at a substitution of 7.5 mg of peptide per mg of RSA. One mg of the peptide-RSA conjugate in 1 ml of Freund's complete adjuvant was injected subcutaneously at 10 different sites along the backs of the rabbits. Starting at 3 weeks after the initial immunization, rabbits were given biweekly booster injections of 1 mg of peptide-RSA in 100 µl of incomplete Freund's adjuvant. IgG was prepared by mixing immune serum with caprylic acid (0.7 ml caprylic acid per ml serum), shaking for 30 minutes, and centrifuging at 5,000 xg for 10 minutes. The supernatant was decanted and dialyzed against two changes of buffered saline (PBS) overnight at 4 ° C. The antibody solution was then affinity purified by passing it through a column containing the immunizing peptide coupled to an Affi-gel 10 affinity support. Bound antibodies were eluted with 0.2M glycine (pH 2.3), immediately dialyzed against PBS, and concentrated to 1mg / ml before being stored at -70 ° C.

5. Transfection

Transient transfection was carried out using standard protocols (Sambrook et al., 1989). Briefly, subconfluent cells (covering less than 20% of a 100mm plastic tissue culture plate) were washed 2X in DMEM tissue culture medium (GIBCO) and then incubated for 3 hours at 37 ° C in a sterile mixture. DEAE-dextran (0.25 mg / ml), chloroquine (55 mg / ml) and 15 mg of plasmid DNA (Courey and Tjian, 1988). The cells were then shocked by incubating them with 10% DMSO in sterile PBS for 2 minutes, at 37 ° C, washed 2X with DMEM (Sambrook et al., 1989), and incubated in DMEM plus fetal calf serum at 10% and antibiotics for 72 hours, at 37 ° C.

6. Immunoprecipitation

For immunoprecipitation, 1 ml of antibodies (1: 400 final concentration, in PBS-TDS buffer: 0.38 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 1% Triton X-100, 0.5% Deoxycholic Acid, and 0.1% SDS) was added to 1 ml of radioactively labeled proteins in the medium. The mixture was incubated with shaking at 4 ° C for 1 hour, adding protein A-Sepharose CL-4B beads (200 ml suspension, 10%) and this mixture was incubated with shaking for an additional hour at 4 ° C. Immunoprecipitated proteins were pelleted by brief centrifugation, the pellet was washed 6x with PBS-TDS buffer, 2x loading protein dye was added, and the samples were boiled for 5 minutes and then fractionated on a 4- gradient SDS-PAGE. 18% (Bonadio et al., 1985). Cold molecular weight markers (200 kDa-14.3 kDa, Rainbow mix, Amersham) were used to estimate molecular weight. The gel was dried and exposed to the film for the indicated time at room temperature.

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7. Western analysis

Fractionated proteins inside SDS-polyacrylamide gels were transferred to a nitrocellulose filter for 2 hours using Tris-glycine-methanol buffer, pH 8.3 at 0.5 mA / cm². The filter was blocked, incubated with nonfat milk plus antibodies (1: 1000 dilution) for 2 hours, and washed. Antibody staining was visualized using ECL Western blot reagent (Amersham) according to manufacturer's protocols.

B. Results

In this study, the inventors isolated and characterized a novel murine fibrillin-like cDNA encoding LTBP-3. To clone the murine LTBP-3 gene, cDNA from a 3T3 cell cDNA library was amplified using human fibrillin-1 PCR primers under mild conditions (i.e., hybridization initially at 37 ° C for 10 cycles, followed by hybridization at 60 ° C for 30 cycles). The results indicated that a murine DNA fragment of unexpectedly low homology (approx. 50%) was obtained to human fibrillin-1. Molecular cloning of the authentic murine fibrillin-1 transcript was also carried out, confirming that the sequences encoding murine and human fibrillin-1 share> 95% sequence identity. The murine fibrillin-1 and PCR ™ sequences were different, suggesting that the PCR ™ product may have been derived from a related fibrillin-like cDNA. The 3T3 cell cDNA library was severely screened using the murine PCR ™ product as a probe in order to test this hypothesis. A cDNA screening strategy eventually resulted in seven overlapping cDNA clones (Fig. 14). It provides a single mRNA of 4,314 nucleotides, with an open reading frame of 3,753 nucleotides (SEQ ID NO: 2). The deduced molecule is a unique 1,251 amino acid polypeptide (SEQ ID NO: 3). Excluding the signal peptide (21 amino acids), the new fibrillin-like molecule is made up of five structurally distinct regions (Region 1-Region 5) and although similar to murine fibrillin1 (Fig. 15A), its domain structure is unique, as evidenced by the schematic representation of LTBP-3 shown in Fig. 15B.

Domain # 1 is a 28 amino acid segment with a net base charge (st. PI, 12.36) that can allow the binding of acidic molecules in the extracellular matrix (eg, acidic proteoglycans). Sequences rich in basic amino acids can also function as endoproteolytic processing signals (Barr, 1991; Steiner et al., 1992), suggesting that the NH terminus₂ it can be proteolytically processed. Domain # 2 spans 390 amino acids, consisting of an EGF-like repeat, a 135 amino acid segment that was rich in proline (20.7%) and rich in glycine (11.8%) but not cysteine. a Fib motif (Pereira et al., 1993), an EGF-CB repeat, and a TGF-bp-like repeat. Domain # 3 is a segment of 113 amino acids characterized by its high proline content (21%). Domain # 4 spans 678 amino acids and is made up of 14 consecutive cysteine-rich repeats. Based on structural homologies, 12/14 repeats were epidermal growth factor (EGF-CB) calcium-binding motifs (Hanford et al., 1991), while 2/14 were transforming growth factor-bound protein motifs - β (TGF-bp) (Kanzaki et al., 1990). Finally, domain # 5 is a 22 amino acid segment at the carboxyl terminus. The conceptual 45 amino acid sequence encoded by the open reading frame consisted of 1,251 amino acids (Fig. 15B) with an estimated pI of 5.92, a predicted molecular mass of 134,710 Da, and five potential N-linked glycosylation sites. The RGD sequence was not present.

Northern blot analysis of murine embryonic RNA using a 3 'end untranslated region probe identified a transcription band of approximately 4.6 kb. In this regard, 4,310 nt have been isolated by cDNA cloning, including a 3 'end untranslated region of 401 nt and a sequence upstream of the 5' end of 156 nt. The apparent discrepancy between the Northern analysis result and the cDNA sequence analysis suggested that the sequence upstream of the 5 'end may include approximately 300 nt of additional upstream sequence. This estimate was in agreement with preliminary primer extension mapping studies indicating that the sequence upstream of the 5 'end is 400-500 nt in length.

A total of 19 cysteine-rich repeats were found in domains # 2 and # 4 of the murine LTBP-like polypeptide (LTBP-3). Thirteen were of the EGF type and 11/13 contained the calcium-binding consensus sequence. This consensus was derived from an analysis of 154 EGF-CB repeats in 23 different proteins and from structural analyzes of the EGF-CB repeat, both bound and unbound to the calcium ion (Selander-Sunnerhagen et al., 1992). Variations over consensus have been noted previously and one of these, DLN / DE-Ci, was identified in the third EGF-like repeat of domain # 4. In addition, a potential calcium-binding sequence was identified that has not been previously reported (ETN / DEC₁), in the first EGF-like repeat of domain # 4. Ten of the 13 EGFCB repeats also contained a second consensus sequence representing a recognition sequence for an Asp / Asn hydroxylase that co- and post-translationally modifies the D / N residues (Stenflo et al., 1987; Gronke et al., 1989).

Although about half the size, the deduced polypeptide was organized like fibrillin-1 in that it consisted of a signal peptide followed by 5 structurally distinct domains, that is, two domains with numerous EGF, EGF-CB, and Fib and a third with a sequence rich in proline (Pereira et al., 1993). However, the comparison of each of these domains using the GAP and BESTFIT (Genetics Computer Group) programs has revealed a low level of amino acid homology of only 27% over the five structural domains shared by the deduced murine polypeptide and fibrillin- 2 human. These values ​​are low for a putative member of the fibrillin family because fibrillin-1 and fibrillin-2 share approximately 50% identity (Zhang et al., 1994).

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An investigation of available databases revealed that the deduced murine polypeptide was most similar to the latent TGF-β binding proteins of rat and man (Kanzaki et al., 1990; Tsuji et al., 1990). In this regard, LTBP was found to be similar to fibrillin in that it could also be divided into five structurally distinct domains (Figs. 15A, 15B, 15C). These include a relatively short domain downstream of the signal peptide with a net base charge (amino acids 21-33, est. pI, 11.14); a domain consisting of EGF, EGF-CB, TGF-bp and Fib-like motifs plus a sequence rich in proline and glycine (amino acids 34-407); a proline rich domain (amino acids 408-545); a large domain consisting of EGF-CB, TGF-bp, and TGF-bp-like repeat motifs (amino acids 546-1379); and a relatively short domain at the carboxyl terminus (amino acids 1380-1394). Comparison of the amino acid sequence of the deduced human and murine polypeptides shows 60% identity for domain # 1, 52% for domain # 2, 30% for domain # 3, 43% for domain # 4 and 7 % for domain # 5. The average identity with respect to the five domains shared by murine polypeptide and human LTBP was 38.4%. Significantly, the cysteine ​​residues in both polypeptide sequences were highly conserved.

Fibrillins are expressed exclusively by connective cells in developing tissues (Zhang et al., 1994), while LTBP will be co-expressed with TGF-β by both conjunctival and epithelial cells (Tsuji et al., 1990). The structural homology data therefore predict that the murine LTBP-3 gene shown in Fig. 15B will be expressed by both connective tissue and epithelial cells. Tissue in situ hybridization was used to test this hypothesis.

An overview of the expression pattern as determined by tissue in in situ hybridization is presented in Figs. 17A, 17B, 17C and 17D. Approximate midsagittal sections of normal murine embryos on days 8.5-9.0, 13.5 and 16.5 pc of development were hybridized with a single-stranded normal sense riboprobe labeled with S³⁵ of the same cDNA construct that was used. On days 8.5-9.0 of development, intense gene expression was observed in mesometrial and anti-mesometrial uterine tissues, ectoplacental cone, placenta, placental membranes. The transcript appeared to be widely expressed in murine embryomesenchymal / conjunctival tissue compartments, including facial mesenchyme, on days 8.5-9.0, 13.5, and 16.5 of development. A particularly strong expression of the transcript was noted in the liver.

Microscopy on day 8.5-9.0 of the embryos confirmed the extended expression of the murine gene by mesenchymal cells. Significant expression of the transcript was also observed by cells of the developing central nervous system, somites, and cardiovascular tissue (myocardium plus endocardium).

Microscopy on day 13.5 and day 16.5 of the embryos demonstrated the expression of the murine gene by skeletal muscle cells and by cells involved in intramenbranous and endochondral bone formation. The transcript was expressed by osteoblasts and periosteal cells of the cranial vault, mandible and maxilla. The transcript was also identified in cartilage and lower limb bone. A positive signal was detected in the perichondrial cells and in the chondrocytes (proliferating> mature> hypertrophic) of the articular cartilage, in the presumed growth plate, and in the cartilage model within the central canal. The positive signal was also expressed by the endothelial cells of the vessels inside the mid-diaphysis, and in the muscle cells that surrounded it (Figs. 18A, 18B, 18C, 18D, 18E, 18F, 19G, 18H, 18I, 18J, 18K, 18L, 18M, 18N, 18O and 18P).

Respiratory epithelial cells that line the small developing airways and connective tissue cells in the lung interstitium expressed the murine transcript, as did myocardial cells (atrium and ventricles) and endocardial pad tissue. Cells within the walls of the great arteries also expressed the transcript. The expression of the murine gene was identified in various organs of the alimentary system, including the tongue, esophagus, stomach, small and large intestine, pancreas and liver. The mucous epithelial cells that line the upper and lower digestive tracts, plus the smooth muscle cells and connective tissue cells found in the submucosa, expressed the transcript, as did the acinars of the exocrine pancreas. Despite the high level of expression of the transcript in the liver, these results suggest that both cell populations express the LTBP-3 transcript.

In the kidney, an expression higher than the basal level was observed in the cells of the developing nephrons, in the ureteric primordium, renal blastema and in the renal interstitium. In the skin, the murine transcript was expressed by epidermal and adnexal keratinocytes, dermal connective tissue cells, and brown fat cells within the dorsal subcutis. In the central and peripheral nervous systems, ganglion cells within the brain, brainstem, spinal cord, and peripheral nerves, expressed the murine transcript. The transcript was also strongly expressed by developing murine retinal cells.

Thus, the murine gene is widely expressed by both types of cells, epithelial and connective tissue, a pattern that was expected for a latent TGF-β binding protein. Three final observations reason that the LTBP-like sequence (LTBP-3) depicted in Fig. 25 is not simply the murine homologue of human LTBP. First, domain # 4 of the murine LTBP-like sequence (LTBP-3) has a smaller number of EFG-like repetitive motifs than rat and human LTBP (8 vs. 11). Second, portions of the rat and human LTBP-like coding sequence were characterized and found to share approximately 90% identity with murine and human LTBP, but only 65% ​​identity with the murine type gene. LTBP. Third, the human LTBP and LTBP-like genes are located on separate chromosomes. Human LTBP was assigned to human chromosome 2 based on analysis of hybrid somatic cell progeny46

ES 2 139 889 T5 rodent x human cas (Stenman et al., 1994). The present invention represents the realization of the first map of a LTBP gene in the mouse. The human LTBP-like gene was recently localized to band q12 of chromosome 11, while the murine gene was localized to band B of chromosome 19 (a region of conserved genome collinearity), using several independent approaches including fluorescent hybridization. in situ.

The first indication of alternative splicing came from molecular cloning studies in the murine, in which independent cDNA clones were isolated with a 51 base pair deletion from the coding sequence. PCR ™ / Southern blot analysis provided additional evidence that the 51 base pair homologous sequence was alternatively spliced ​​in normal murine embryonic tissues.

Northern blot analysis also showed that the new fibrillin gene was also expressed in rat callus three weeks after osteotomy, after mineralization had begun. Expression of the gene in normal adult rat bone tissue was negligible, suggesting that microfibrils are an important part of the bone fracture healing response. The new fibrillin-like gene was expressed in the callus as a pair of alternately splicing transcripts. This result has been reproduced independently on three occasions. Molecular cloning of the novel fibrillin gene in both murines and rats has identified potential splice sites for this alternative event.

MC3T3 murine preosteoblasts were used to demonstrate that the murine gene product was capable of binding TGF-β. MC3T3-E1 cells were used because they synthesize and secrete TGF-β, which can act as an autocrine regulator of osteoblastic proliferation (Amarnani et al., 1933); Van Vlasselaer et al., 1994; López-Casillas et al., 1994).

To determine whether or not MC3T3-E1 cells co-expressed the murine TGF-β gene product, they were seeded in 100 mm plates under differentiation conditions (Quarles et al., 1992) and the medium was replaced twice weekly. Parallel discs were seeded and tested for cell number and alkaline phosphatase activity, confirming that osteoblastic differentiation was indeed taking place. Equal aliquots of total cellular RNA were prepared from these MC3T3-E1 cells after 5, 14 and 28 days of culture, for Northern blot analysis. As shown in Fig. 19, the expression of the new murine gene reached its maximum concentration on day 14 of culture. Since MC3T3-E1 cells also show a maximum concentration in alkaline phosphatase activity on day 14 of culture (Quarles et al., 1992), the results suggest for the first time that the expression of the LTBP-2 gene constitutes a early marker of osteoblast differentiation.

C. Discussion

This study reports the molecular cloning of a new LTBP-like gene that contains numerous EGF-like repeats. Northern analysis indicates that the gene encodes a single transcript of approximately 4.6 kb in murine embryonic tissues. The deduced amino acid sequence of the murine gene product appears to be a 1,251 amino acid secreted polypeptide. Although similar to fibrillin, the overall structural organization and expression pattern of this gene product closely resembles LTBP, a latent TGF-β binding protein that was originally isolated and characterized by Heldin et al. , 1990). Several observations strongly suggest that LTBP and the murine LTBP-like gene product are therefore derived from distinct but related genetic loci. First, LTBP and the LTBP-like coding sequence share approximately 40% identity and there are differences in the number of EGF-CB repeats in the deduced polypeptide sequence of the two molecules. Second, a portion of the murine LTBP gene has been cloned and shown to share approximately 90% identity with human and rat LTBP. Conversely, portions of the rat and human LTBP-like genes have been cloned and are shown to share approximately 90% identity with the murine LTBP-like gene. Third, the LTBP and LTBP-like genes reside on different human chromosomes (Stenman et al., 1994). When these data are taken together, they suggest that there is a family of at least two LTBP genes.

Similarities in the structural organization of LTBP-1 and fibrillin-1 and fibrillin-2 polypeptides have previously been emphasized (Pereira et al., 1933; Zhang et al., 1994; Taipale et al., 1994). For example, LTBP-1 and fibrillins are all secreted constituents of the extracellular matrix. Furthermore, each polypeptide can be organized into five domains, two of which are predominantly made up of EGF-CB and TGFbp repetitive motifs. LTBP-1 and fibrillin-1 also share a domain that is rich in proline, and LTBP possesses an 8 cysteine ​​repeat previously referred to as the “Fib motif” because it was assumed to be unique in fibrillin (Pereira et al., 1993). These similarities probably explain the initial isolation and cloning of the LTBP2 PCR ™ product, especially since the human oligonucleotide primers used to initially amplify murine cDNA were designed to direct the synthesis of an EGF-CB repeat in domain # 4.

Another point of distinction between LTBP-2 and fibrillin concerns the spacing of conserved C4 and C5 cysteines in EGF-like repeats. Fibrillin-1 and fibrillin-2 each contain> 50 of these repeats, and in each the spacing is C₄-C₅. While this pattern is repeated in most EGF-like repeats in LTBP-1 and LTBP-2, both genes also contain C-spaced repeats.₄-XXC₅. Although the significance of this observation is unclear, the variation in the number of amino acids between C₄ and C₅ it is not expected to alter the function of the EGF-like repeat. Mature EGF is a 48 amino acid secreted polypeptide that is made up of two subdomains that have few interdomain contacts (Engel, 1989; Davis, 1990). The largest NH subdomain₂-terminal is formed by residues 1-32 and is stabilized by a pair of disulfide bonds (C₁-C₃

ES 2 139 889 T5 and C₂-C₄), while the smaller COOH-terminal subdomain (amino acids 33-48) is stabilized by a single disulfide bond (C₅-C₆). The COOH-terminal subdomain has a highly conserved conformation that is only possible if certain residues and the distances between them are well conserved, whereas the conformationsequence requirements for the NH subdomain₂-terminal are relatively relaxed. Variation in spacing C₄-C₅ it is not expected to alter the conformation because these residues do not normally form a disulfide bond and the variation in spacing occurs at the interface of the subdomains that is not predicted to interact. Cloning of additional genes will decide whether the variation in C spacing₄-C₅ it is a reliable discriminator between members of the fibrillin and LTBP gene families.

The LTBP-2 gene is more widely expressed during development than fibrillin-1 or fibrillin-2. Studies in developing murine tissues have shown that the Fbn-1 gene is expressed by the mesenchymal cells of developing connective tissue, while the murine LTBP-like gene is strongly expressed by epithelial, parenchymal and stromal cells. Previous reports have suggested that TGF-β plays a role in differentiation and morphogenesis during murine development (Lyons and Moses, 1990), when TGF-β is produced by epithelial, parenchymal, and stromal cells. Tsuji et al., (1990) and others have suggested that the expression of TGF-β binding proteins should mirror that of TGF-β itself; the expression pattern of the LTBP-2 gene over the course of murine development agrees with this expectation. However, the LTBP-2 gene may not be fully co-regulated with TGF-β. The TGF-β gene and protein expression during murine development has been extensively surveyed (Heine et al., 1987; Lehnert and Akhurst, 1988; Pelton et al., 1989; Pelton et al., 1990 a, b; Millan et al. al., 1991); these studies have not identified expression by skeletal muscle cells, chondrocytes, hepatocytes, ganglion cells, mucosal cells lining the digestive tract, and epithelial cells of developing nephrons. It is conceivable that the LTBP-2 molecule has an additional function in certain connective tissues in addition to targeting TGF-β.

The binding properties of the LTBP-2 gene product are under investigation. Formally, the LTBP-2 polypeptide can bind to a specific TGF-β isoform, another member of the TGF-β superfamily (eg, a bone morphogenetic protein, inhibin, activin, or Mullerian inhibiting factor), or a factor growth rate not related to TGF-β. Anti-peptide antibodies have been generated to the murine LTBP-2 polypeptide and osteoblastic cell lines have been identified that express the molecule at relatively high levels. Studies with these reagents suggest that LTBP-2 assembles intracellularly into large latent complexes with a growth factor that is characterized by immunological procedures.

The presence of dibasic amino acids in the LTBP-2 sequence suggests that it may undergo specific cell and tissue proteolysis. TGF-β regulates the production of the extracellular matrix by suppressing its degradation (through a decrease in the expression of proteases such as collagenase, plasminogen activator, and stromelysin plus an increase in the expression of proteinase inhibitors such as the plasminogen activator1 and tissue inhibitor of metalloproteinase-1) and by stimulating the synthesis of matrix macromolecules (for recent review, see Lyons and Moses, 1990; Massague, 1990; Laiho and Keski-Oja, 1992; Miyazono et al., 1992). Conversely, extracellular matrix production has been shown to downregulate TGFβ gene expression (Streuli et al., 1993). TGF-β can therefore regulate the production of the extracellular matrix through a sophisticated feedback loop that influences the expression of a relatively large number of genes. LTBP-1 and LTBP2 can contribute to this regulation by facilitating the assembly and secretion of latent growth factor complexes and then targeting the complex to specific connective tissues (Taipale et al., 1994).

If LTBP-3 is like LTBP-1, it has the potential to function as a secreted extracellular structural protein. As shown here, domain # 1 of LTBP-3 appears to be a unique sequence that probably has a globular conformation. Domain # 1 is also very basic and can facilitate the binding of LTBP-2 to acidic molecules (eg, acidic proteoglycans) within the extracellular space. Sequences rich in basic amino acids have also been shown to function as endoproteolytic processing signals for various peptide hormones (Barr, 1991; Steiner et al., 1992). It is therefore possible that the NH end₂ LTBP-3 is proteolytically processed into a specific tissue form. Domains # 2 and # 4 are made up of consecutive cysteine-rich repeats, most of which are of the EGF-CB type. In addition to binding calcium (Corson et al., 1993), these repeats can provide LTBP-3 with a conformation of the regions capable of interacting with other macromolecules of the matrix (Engel, 1989). Domain # 3 is rich in proline and may be able to bind (or function as a hinge) in three-dimensional space (MacArthur and Thornton, 1991). (In this regard, domain # 2 is of interest because it has a similar 135 amino acid chunk that is rich in proline and glycine. Since glycine-rich sequences are also believed to be capable of binding or hinging in three-dimensional space, this amino acid sequence can disrupt the extended conformation of domain # 2, thereby providing a degree of flexibility in space three-dimensional). Domain # 5 also appears to have a unique sequence that possesses a globular conformation. The absence of a known cellular binding motif may indicate that, in contrast to LTBP-1, the LTBP-3 molecule may have a more limited role in the extracellular matrix (i.e., that of a structural protein) in addition to its ability to to target latent TGF-β complexes to specific connective tissues.

MC3T3 preosteoblasts co-express LTBP-3 and TGF-β 1 and these proteins form a complex in the culture medium. These results are particularly interesting because bone represents one of the largest known pools of latent TGF-βs (200 jug / kg bone; Seyedin et al., 1986 and 1987), and because this growth factor plays a critical role in the determination of bone structure and function. For example, it is thought that

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TGF- / (i) provides a powerful stimulus for bone formation in developing tissues, (ii) functions as a potential “coupling factor” during bone remodeling (a process that coordinates bone resorption and formation), and (iii) exerts a powerful osteo-inducing stimulus after fracture. Activation of the latent complex can be an important step governing the effects of TGF-β, and LTBP can modulate the activation process (eg, it can "protect" small latent complexes from proteolytic attack).

The expression of large TGF- / latent complexes bearing LTBP may be physiologically important, ie, it may be part of the preosteoblast osteoblast differentiation cascade mechanism. This is based on the evidence that MC3T3-E1 cells express large TGF- / latent complexes that carry PTBP-2 precisely when the transition from the preosteoblast to the osteoblast phenotype occurs (approximately day 14 in culture, or, at beginning of alkaline phosphatase expression; see Quarles et al., 1992). The organ culture model, for example, is probably composed of differentiated osteoblasts but few conjoined parents, making the best of it a difficult model for studying the differentiation cascade (Dallas et al., 1984). MG63, ROS17 / 2.8 and UMR 106 cells are also known to divide rapidly and express the osteoblast phenotype. Thus, these osteoblast-like cell lines do not show the uncoupling of cell proliferation and differentiation that characterizes the normal pre-osteoblast osteoblast transition (physiologically important) (Gerstenfeld et al., 1984; Stein and Lian, 1993). Therefore, the production of small relative to large TGF- / latent complexes may be related to specific phases in bone cell maturation.

LTBP-3 can bind calcium, as EGF-CB repeats have been shown to mediate high-affinity calcium binding in LTBP-1 and other proteins (Colosetti et al., 1993). Binding to calcium, in turn, can contribute to molecular conformation and the regulation of its interactions with other molecules. The presence of dibasic amino acids suggests that it may undergo specific cell and tissue proteolysis. TGF- / regulates the production of the extracellular matrix by suppressing its degradation (through a decrease in the expression of proteases such as collagenase, plasminogen activator, and stromelysin plus an increase in the expression of proteinase inhibitors such as the plasminogen activator-1 and tissue inhibitor of metalloproteinase-1) and by stimulating the synthesis of matrix macromolecules (for recent review, see Lyons and Moses, 1990; Massague, 1990; Laiho and Keski-Oja, 1992; Miyazono et al., 1992). Conversely, extracellular matrix production has been shown to down-regulate TGF- / gene expression (Streuli et al., 1993). TGF- / can therefore regulate the production of the extracellular matrix by means of a sophisticated feedback loop that influences the expression of a relatively large number of genes. LTBP-1, LTBP-2 and LTBP-3 can contribute to this regulation by facilitating the assembly and secretion of large complexes of latent growth factors and then directing the complex to specific connective tissues (Taipale et al., 1994).

Example XVI

Preparation of antibodies against the LTBP-3 gene product

An affinity purified antibody (# 274) capable of immunoprecipitation was prepared against the murine LTBP-3 gene product. A full-length murine cDNA was pooled into a mammalian pcDNA3 expression vector (Invitrogen) and expressed after transient transfection of 293T cells. Nascent polypeptides, radioactively labeled by addition of Cys S³⁵ The transfected cells were immunoprecipitated using affinity purified # 274 antibodies. As shown in Fig. 20, the new murine polypeptide was estimated to be about 180-190 kDa. To ensure specificity of binding to # 274, preincubation with 10 pg of synthetic peptide is shown to block immunoprecipitation of the 180-190 kDa band.

Finally, MC3T3 cells were cultured for 7 days under differentiation conditions and double-labeled with 30 pCi / ml of S³⁵cysteine ​​and S³⁵methionine in deficient media. Radiolabelled medium was dialyzed in cold PBS with protease inhibitors. Aliquots of the dialyzed medium sample (10⁶ Incorporated CPMs) were analyzed by a combined immunoprecipitation / Western analysis protocol. The murine polypeptide was clearly and reproducibly secreted by MC3T3 cells, migrating under reduced conditions as a single 180-190 kDa band (Fig. 21). Consistent with the results of previous studies (eg, Miyazono et al., 1988; Dallas et al., 1994; Moren et al., 1994), 70 and 50 kDa bands corresponding to the TGF-β precursor were co-immunoprecipitated. with the 180 kDa LTBP-3 protein. Weak bands of 40 and 12 kDa were also identified in experiments in which only immunoprecipitation was carried out. The latter were not included in Fig. twenty-one because they migrated into the portion of the gel included in the Western analysis. The 70-12.5 kDa protein bands are not variant forms of LTBP-3; Fig. 20 demonstrates that LTBP-3 migrates as a single 180-190 kDa band after transient transfection of 293T cells, which TGF- / does not. By immunoprecipitation, a unique band was found in the LTBP-2 immunoprecipitate according to the mature TGF- / monomeric. Antibody # 274 is unable to bind TGF / as determined by radioimmunoassay using commercially available reagents (R&amp;D Systems) and manufacturer's suggested protocols. These results have been reproduced in 6 independent experiments using 3 separate batches of MC3T3-E1 medium. Thus, the novel murine LTBP-3 polypeptide binds TGF- / in vitro.

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Example XVII

Isolation of a gene encoding murine LTBP-2

In addition to determining the DNA and corresponding polypeptide sequence of the murine LTBP-3 gene, the murine LTBP2 gene was also cloned and sequenced.

The complete cDNA nucleotide sequence for murine LTBP-2 is shown in Fig. 27 (SEQ ID NO: 17). The deduced amino acid sequence is shown in Fig. 28 (SEQ ID NO: 18).

Example XVIII

Expression of recombinant type II collagen

The Pichia Expression Kit (Invitrogen, Inc) can be used to prepare recombinant type II collagen. This kit, based on the methylotrophic yeast, Pichia pastoris, allows a high level of expression of recombinant proteins in an easy-to-use and relatively inexpensive system. In the absence of the preferred carbon source, glucose, P.pastoris uses methanol as the carbon source. The AOX1 promoter controls the gene that encodes the expression of the enzyme alcohol oxidase, which catalyzes the first step in methanol metabolism. This promoter, which is induced by methanol, has been characterized and incorporated into a series of Pichia expression vectors. This characteristic of Pichia has been exploited to express high levels of recombinant proteins often in the range of grams per liter. Because it is eukaryotic, Pichia pastoris uses post-translational modification pathways that are similar to those used by mammalian cells. This implies that the recombinant type II collagen will be glycosylated and contain disulfide bonds.

The inventors consider the following particular elements as useful in the expression of recombinant type II collagen: the DNA sequence of human type II collagen (SEQ ID NO: 11) (Lee et al., 1989); type II rat collagen (SEQ ID NO: 13) (Michaelson et al., 1994); and / or mouse type II collagen (SEQ ID NO: 15) (Ortman et al., 1994). As other sources of DNA sequences encoding type II collagen are available, these three are examples of many sequential elements that may be useful in the present invention.

For the preparation of a recombinant type II collagen, the original type II collagen cDNA is modified by adding a commercially available marker epitope (HA epitope, Pharmacia, LKB Biotechnology, Inc). Said fragments can be easily prepared, for example, by directly synthesizing the fragment by chemical means, by applying nucleic acid reproduction technology, such as that of PCR ™ in US Patent Document 4,603,102 (incorporated herein by reference) or by introducing selected sequences into recombinant vectors for recombinant production. (PCR ™ is a registered trademark of Hoffmann-LaRoche, Inc). This is followed by cloning into the Pichia expression vector. The resulting plasmid is characterized by DNA sequence analysis, linearized by digestion with NotI, and spheroplasts with the linearized construct will be prepared and transformed according to the manufacturer's recommendations.

Transformation facilitates an in vivo recombination event between the 5 'and 3' AOX1 sequences in the Pichia vector and the sequences in the Pichia genome. The result is the replacement of AOX1 with the gene of interest.

Transformants are then seeded in histidine-deficient media, which will be selected to become successfully transformed cells. Transformants will be further selected for slow growth in growth media containing methanol. Positive transformants are grown for 2 days in liquid culture and then for 2-6 days in medium using methanol as the sole carbon source. Protein expression is assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western hybridization, using a commercially available polyclonal antiserum for the HA epitope (Pharmacia).

Recombinant type II collagen protein can be purified according to the manufacturer's recommendations, dialyzed against double distilled and deionized water, and lyophilized in 10 mg aliquots. Aliquots are sterilized and used as graft material for bone conductive matrices.

All of the compositions and procedures disclosed and claimed herein can be carried out without experimentation, in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it is clear to those skilled in the art that variations can be applied to the composition, to the procedures, and to the steps or sequence of the steps. procedure described herein, without departing from the concept, spirit and scope of the invention. More specifically, it is clear that certain agents that are chemically and physiologically related can substitute for the agents described herein as long as the same or similar results are achieved. Such similar substitutes and modifications clear to those skilled in the art, are considered to belong to the spirit, scope and concept of the invention, as defined in the appended claims.

IS 2 139 889 T5

References

The following literature citations as well as those cited above are incorporated herein, in the corresponding part, by reference for the reasons cited in the preceding text.

Abou-Samra et al., “Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone related peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol triphosphates and increases intracellular free Calcium”, Proc. Natl. Acad. Sci. USA, 89: 2732-2736, 1992.

Agarwala and Gay, J. Bone Min. Res., 7: 531, 1992.

Alper, "Boning up: newly isolated proteins heal bad breaks", Science, 263: 324-325, 1994.

Amarnani et al., J. Bone Min. Res., 8: 157-164, 1993.

Antonelli-Olridge et al., Proc. Natl. Acad. Sci. USA, 86: 4544-4548, 1989.

Badylak et al., J. Surg. Res., 47: 74-80, 1989.

Bandara et al., "Gene Transfer to Synoviocytes: Prospects for Gene Treatment of Arthritis", DNA Cell Biol., 11: (3): 227-231, 1992.

Barr, Cell, 66: 1-3, 1991.

Beck et al., "TGF-beta 1 induces bone closure of skull defects", J. Bone Miner. Res., 11: 1257-65, 1991.

Benezra et al., Blood, 81: 3324-3331, 1993.

Benvenisty and Reshef, Proc. Natl. Acad. Sci. USA, 83: 9551, 1986.

Boden et al., "Estrogen receptor mRNA expression in callus during fracture healing in the rat", Calcif. Tissue Int., 45: 34-325, 1989.

Bonadio et al., “An adaptive response by murine skeletal tissues that significantly increases the mechanical properties of cortical bone: implications for the treatment of skeletal fragility”, J. Clin. Invest., 92: 1697-1705, 1993.

Bonadio et al., J. Biol. Chem., 260: 1734-1742, 1985.

Bondaio and Goldstein, “Our understanding of inherited skeletal fragility and what this has taught us about bone structure and function, In: Molecular and Cellular Biology of Bone”, Noda, M., ed., Academic Press, Inc., San Diego, CA . pp. 169-189, 1993.

Bonewald et al., Mol. Endocrinol., 5: 741-751, 1991.

Bonnarens and Einhorn, "Production of a standard closed fracture in laboratory animal bone", J. Orthop. Res., 2: 97-101, 1984.

Burch and Lebovitz, "Parathyroid hormone stimulates growth of embryonic chick pelvic cartilage in vitro," Calcif. Tissue Int., 35: 526-532, 1983.

Byers and Steiner, "Osteogenesis imperfecta", Annu. Rev. Med., 43: 269-289, 1992.

Campbell, in Monoclonal Antibody Technology, Laboratory Techniques in Biochemistry and Molecular Biology Vol. 13, Burden and Von Knippenberg, Eds. P. 75-83, Amsterdam, Elseview, 1984.

Canalis et al., “Insulin-like growth factor-1 mediates selective anabolic effects of parathyroid hormone in bone culture”, J. Clin. Invest., 83: 60-65, 1989.

Carrington et al., "Accumulation, localization, and compartmentation of transforming growth factor b during endochondral bone development", J. Cell Biol., 107: 1969-1975, 1988.

Centrella et al., "Skeletal tissue and transforming growth factor-b", FASEB J., 22: 23066-3073, 1988.

Cheifetz et al., Cell, 48: 409-415, 1987.

Chen et al., "Bone morphogenetic protein-2b stimulation of growth and osteogenic phenotypes in rat osteoblastlike cells: comparison with TGF-beta 1", J. Bone Miner. Res., 6: 1387-93, 1991.

IS 2 139 889 T5

Chen et al., "Structure, chromosomal localization, and expression pattern of the murineMagp gene", J. Biol. Chem., 268: 27381-27389, 1993.

Colosetti et al., FEBS Letters, 320: 140-144, 1993.

Compston et al., “Elevated serum intact parathyroid hormone levels in elderly patients with hip fracture”, Clin. Endo., 31: 667-672, 1989.

Courey and Tjian, Cell, 55: 887-898, 1988.

Cunningham et al., "Osteogenic and recombinant bone morphogenetic protein 2B are chemotactic for human monocytes and stimulate transforming growth factor bl mRNA expression", Proc. Natl. Acad. Sci. USA, 89: 1174011744, 1992.

Dallas et al., J. Biol. Chem., 269: 6815-6822, 1994.

Davidson et al., "A model system for in vivo gene transfer into the central nervous system using an adenoviral vector", Nature Genetics, 3: 219-223, 1993.

Davis, New Biologist, 2: 410-419, 1990.

Ejersted et al., "Human parathyroid hormone (1-34) and (1-84) increase the mechanical strength and thickness of cortical bone in rats", J. Bone Min. Res., 8: 1097-1101, 1993.

Endo et al., "Vitamin D3 metabolites and PTH synergistically stimulate bone formation of chick embryonic femur in vitro", Nature, 286: 262-264, 1980.

Engel, FEBS Letters, 251: 1-7, 1989.

Falcone et al., J. Biol. Chem., 268: 11951-11958, 1993.

Flaumenhaft et al., J. Cell Biol., 120: 995-1002, 1993.

Franseschi and Iyer, J. Bone Min. Res., 7: 235-246, 1992.

Gefter et al., Somatic Cell Genet. 3: 231-236 (1977)

Gerstenfeld et al., Dev. Biol., 122: 49-60, 1987.

Goding, 1986, in Monoclonal Antibodies: Principles and Practice, 2^to ed., Orlando, Fla., Academic Press, 1986, pp. 60-61.65-66.71-74.

Gronke et al., Proc. Natl. Acad. Sci. USA, 86: 3609-3613, 1989.

Gunasekaran et al., “Mineralized Collagen As A Substitute for Autograft Bone That Can Deliver Bone Morphogenic Protein”, The 19th Annual Meeting of the SOCIETY FOR BIOMATERIAL, April 28, p. 253, 1993a.

Gunasekaran et al., Norian Corporation, Mountain View, CA. Abstract V7.5, p. 426, 1993b. The 19th Annual Meeting of the SOCIETY FOR BIOMATERIALS, April 28.

Gunness-Hey and Hock, “Increased trabecular bone mass in rats treated with human synthetic parathyroid hormone”, Metabl. Bone Dis., 5: 177-180, 1984.

Gunness-Hey and Hock, "Loss of the anabolic effect of parathyroid hormone on bone after discontinuation of hormone in rats", Bone, 10: 447-452, 1989.

Handford et al., EMBO J., 9: 475-480, 1990.

Hardy et al., "Serum ionized calcium and its relationship to parathyroid hormone after tibial fracture", J. Bone Jt. Surg., 75: 645-649, 1993.

Hefti et al., Clin. Sci., 62: 389-396, 1982.

Heine et al., J. Cell Biol., 105: 2861-2876, 1987.

Hendy et al., "Nucleotide sequence of cloned cDNAs encoding human preproparathyroid hormone", Proc. Natl. Acad. Sci. USA, 78: 7365-7369, 1981.

IS 2 139 889 T5

Herrmann-Erlee et al., "Effects on bone in vitro of bovine parathyroid hormone and synthetic fragments representing residues 1-34, 2-34 and 3-34", Endocrine Research Communications, 3: 21-35, 1976.

Hock and Fonseca, "Anabolic effect of human synthetic parathyroid hormone (1-34) depends on growth hormone", Endocrinology, 127: 1804, 1990.

Hock and Gera, "Effects of continuous and intermittent administration and inhibition of resorption on the anabolic response of bone to parathyroid hormone", J. Bone and Min. Res., 7: 65-72, 1992.

Hock et al., "Human parathyroid hormone- (1-34) increases bone mass in ovariectomized and orchidectomized rats", Endocrinology, 122: 2899-2904, 1988.

Hori et al., "Effect of human parathyroid hormone [PTH (1-34)] on experimental osteopenia of rats induced by ovariectomy", J. Bone Min. Res., 3: 193-199, 1988.

Horowitz et al., "Functional and molecular changes in colony stimulating factor secretion by osteoblasts", Connective Tissue Res., 20: 159-168, 1989.

Huggins et al., “Experiments on the theory of osteogenesis. The influence of local calcium deposits on ossification; the osteogenic stimulus of epithelium ”, Arch. Surg., 32: 915, 1936.

Izumi et al., "Transforming growth factor bl stimulates type II collagen expression in cultured periosteal-derived cells", J. Bone Min. Res., 7: 115-11, 1992.

Jingushi et al., “Acidic fibroblast growth factor injection stimulates cartilage enlargement and inhibits cartilage gene expression in rat fracture healing”, J. Orthop. Res., 8: 364-371, 1990.

Jingushi et al., "Genetic expression of extracellular matrix proteins correlates with histologic changes during fracture repair", J. Bone Min. Res., 7: 1045-1055, 1992.

Johnston et al., "Heterogeneity of fracture syndromes in postmenopausal women", J. Clin. Endo. Metab., 61: 551556, 1985.

Joyce et al., "Transforming growth factor-b and the initiation of chondrogenesis and osteogenesis in the rat femur", J. Cell Biol., 110: 195-2007, 1990.

Juppner et al., "A G-protein-linked receptor for parathyroid hormone and parathyroid hormone-related peptide", Science, 254: 1024-1026, 1991.

Kanzaki et al., Cell, 61: 1051-1061, 1990.

Kawashima, "Growth stimulative effect of parathyroid hormone, calcitonin and N6, 02'-dibutyryl adenosine 3 ', 5'-cyclic monophosphoric acid on chick embryonic cartilage cultivated in a chemically defined medium", Endocrinol. Jpn., 27: 349-356, 1980.

Klein-Nulend et al., "Comparison of the effects of synthetic human parathyroid hormone-related peptide of malignancy and bovine PTH- (1-34) on bone formation and resorption in organ culture", Endocrinology, 126: 223-227, 1990 .

Kohler and Milstein, Nature 256: 495-497 (1975)

Kohler and Milstein, Eur. J. Immunol. 6: 511-519 (1976)

Kovacina et al., Biochem. Biophys. Res. Commun., 160: 393-403, 1989.

Kozak, J. Biol. Chem., 266: 19867-19870, 1991.

Kream et al., "Parathyroid hormone represses al (I) collagen promoter activity in cultured calvariae from neonatal transgenic mice", Mol. Endocrinology, 7: 399-408, 1993.

Kyte and Doolittle, J. Mol. Biol., 157: 105-132, 1982.

Laiho and Keski-Oja, 1992.

Langer and Folkman, "Polymers for the sustained release of proteins and other macromolecules." Nature, 263: 797-800, 1976.

Ledley, J. Pediatrics, 110: 1, 1987.

IS 2 139 889 T5

Lee et al., "Identification of the molecular defect in a family with spondyloepiphyseal dysplasia", Science, 244: 978-980, 1989.

Lee et al., “Parathyroid hormone induces sequential c-fos expression in bone cells in vivo: in situ localization of its receptor and c-fos mRNAs”, Endocrinology,

Lehnert and Akhurst, Development, 104: 263-273, 1988.

Lewinson and Silbermann, "Parathyroid hormone stimulates proliferation of chondroprogenitor cells in vitro", Calcf Tissue Int. 38: 155-162, 1986.

Li et al., Mammalian Genome, in press, 1994.

Linkhart and Mohan, "Parathyroid hormone stimulates release of insulin-like growth factor-1 (IGF-1) and IGFII from neonatal mouse calvaria in organ culture", Endocrinology, 125: 1484-1491, 1989.

Liu and Kalu, "Human parathyroid hormone (1-34) prevents bone loss and augments bone formation in sexually mature ovariectomized rats", J. Bone Min. Res., 5: 973-982, 1990.

Liu et al., “Preexisting bone loss associated with ovariectomy in rats is reversed by parathyroid hormone”, J. Bone and Mins. Res., 6: 1071-1080, 1991.

Lopez-Casillas et al., J. Cell Biol., 124: 557-568, 1994.

Luyten et al., "Purification and partial amino acid sequence of osteogenic, a protein initiating bone differentiation", J. Biol. Chem., 264: 13377-13380, 1989.

Lyons and Moses, Eur J. Biochem., 187: 467-473, 1990.

Lyons et al., J. Cell Biol., 106: 1549-1665, 1988.

Lyons et al., Proc. Natl. Acad. Sci. USA, 86: 4554-4558, 1989.

MacArthur and Thornton, J. Mol. Biol., 218: 397-412, 1991.

Majmudar et al., "Bone cell culture in a three-dimensional polymer bead stabilizes the differentiated phenotype and provides evidence that osteoblastic cells synthesize type III collagen and fibronectin", J. Bone and Min. Res., 6: 869-881, 1991 .

Malluche et al., "1,25-dihydroxyvitamin D maintains bone cell activity, and parathyroid hormone modulates bone cell number in dogs", Endocrinology, 119: 1298-1304, 1986.

Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982.

Massague, Annu. Rev. Cell Biol., 6: 597-641, 1990.

Meller et al., “Mineral and endocrine metabolism during fracture healing in dogs”, Clin. Orthop. Rel. Res., 187: 289-295, 1984.

Michaelson et al., "T-cell recognition of carbohydrates on type II collagen" J. Exp. Med., 180: 745-749, 1994.

Millan et al., Development, 111: 131-144, 1991.

Mitlak et al., "Intermittent administration of bovine PTH- (1-34) increases serum 1,25-dihydroxyvitamin D concentrations and spinal bone density in senile (23 month) rats", J. Bone Min. Res., 7: 479 -484, 1992.

Miyazono and Heldin, Nature, 338: 158-160, 1989.

Miyazono et al., EMBO J., 10: 1091-1101, 1991.

Miyazono et al., J. Biol. Chem., 267: 5668-5675, 1992.

Miyazono et al., J. Biol. Chem., 263: 6407-6415, 1988.

Nicolau et al., Proc Natl. Acad. Sci. USA 80: 1068, 1983.

Ogawa et al., J. Biol. Chem., 267: 2325-2328, 1992.

IS 2 139 889 T5

Ohlin et al., J. Biol. Chem., 263: 7411-7414, 1988.

Olofsson et al., J. Biol. Chem., 267: 19482-19488, 1992.

Ortman et al., "BUB / BnJ (H-2q) is a TCR deletion mutant mouse strain (TCR V beta to KJ16-) that is susceptible to type II collagen-induced arthritis", J. Immunol., 152: 4175- 4182, 1994.

Ozkaynak et al., "OP-1 cDNA encodes an osteogenic protein in the TGF-b famil". EMBO J., 9: 2085-2093, 1990.

Paralkar et al., "Identification and characterization of cellular binding proteins (receptors) for recombinant human bone morphogenetic protein 2B, an initiator of bone differentiation cascade", Proc. Natl. Acad. Sci. USA, 88: 33973401, 1991.

Parsons and Reit, "Chronic response of dogs to parathyroid hormone infusion", Nature, 250: 254-257, 1974.

Pelton et al., Development, 106: 759-767, 1989.

Pelton et al., Dev. Biol., 141: 456-460, 1990a.

Pelton et al., Development, 110: 609-620, 1990b.

Pereira et al., Human Mol. Genet., 2: 961-968, 1993.

Persson et al., J. Biol. Chem., 264: 16897-16904, 1989.

Pircher et al., Biochem. Biophys. Res. Commun., 136: 30-37, 1986.

Pircher et al., Cancer Res., 44: 5538-5543, 1984.

Podbesek et al., "Effects of two treatment regimes with synthetic human parathyroid hormone fragment on bone formation and the tissue balance of trabecular bone in greyhounds", Endocrinology, 112: 1000-1006, 1983.

Prockop, “Mutations that alter the primary structure of type I collagen. The perils of a system for generating large structures by the principle of nucleated growth ”, J. Biol. Chem., 265: 15349-15352, 1990.

Quarles et al., J. Bone Min. Res., 7: 683-692, 1992.

Raisz and Kream, "Regulation of bone formation", N. Engl. J. Med., 309: 29-35, 1983.

Reeve et al., "Anabolic effect of human parathyroid hormone fragment on trabecular bone in involutional osteoporosis: a multicenter trial", Br. Med. J., 1340-1344, June 7, 1980.

Reeve et al., "Anabolic effect of low doses of a fragment of human parathyroid hormone ont he skeleton in postmenopausal osteoporosis", Lancet, 1035-1038, May 15, 1976.

Riond, “Modulation of the anabolic effect of synthetic human parathyroid hormone fragment (1-34) in the bone of growing rats by variations in dosage regimen”, Clin. Sci., 85: 223-228, 1993.

Rizzoli et al., "Binding of radioiodinated parathyroid hormone to cloned bone cells", Endocrinology, 113: 1832, 1983.

Roberts and Sporn, “The transforming growth factor-betas. In: Handbook of Experimental Pharmacology: Peptide Growth Factors and Their Receptors ”, MB Sporn and AB Roberts, Eds., Springer-Verlag, Heidelberg, 95 (Part 1): 419-472, 1990.

Roessler et al., "Adenoviral-mediated Gene Transfer to Rabbit Synovium In Vivo.", J. Clin. Invest., 92: 1085-1092, 1993.

Rosen et al., “Purification and molecular cloning of a novel group of BMPs and localization of BMP mRNA in developing bone”, Connect. Tissue Res., 20: 313-319, 1989.

Sambrook et al., Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory Press, pp. 18.60, 1989.

Sampath and Reddi, “Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation”, Proc. Natl. Acad. Sci. USA, 78: 7599-7603, 1981.

IS 2 139 889 T5

Sampath et al., “In vitro transformation of mesenchymal cells derived from embryonic muscle into cartilage in response to extracellular matrix components of bone”, Proc. Natl. Acad. Sci. USA 81: 3419-3423, 1984.

Sato et al., J. Cell Biol., 123: 1249-1254, 1993.

Schluter et al., "The central part of parathyroid hormone stimulates thymidine incorporation of chondrocytes", J. Biol. Chem., 264: 11087-11092, 1989.

Schnieke et al., "Embryonic lethal mutation in mice induced by retrovirus insertion into the al (I) collagen gene", Nature, 304: 315-320, 1983.

Seeger et al., Proc. Natl. Acad. Sci. USA, 81: 5849, 1984.

Seitz et al., "Effect of transforming growth factor b on parathyroid hormone receptor binding and cAMP formation in rat osteosarcoma cells", J. Bone Min. Res., 7: 541-546, 1992.

Selander-Sunnerhagen et al., J. Biol. Chem., 267: 19642-19649, 1992.

Selye, Endocrinology, 16: 547, 1932.

Seyedin et al., J. Biol. Chem., 261: 5693-5695, 1986.

Seyedin et al., J. Biol. Chem., 262: 1946-1949, 1987.

Shimell et al., "The Drosophila dorsal-ventral patterning gene tolloid is related to human bone morphogenetic protein 1", Cell, 67: 469-481, 1991.

Silve et al., "Parathyroid hormone receptor in intact embryonic chicken bone: characterization and cellular localization", J. Cell. Biol., 94: 379, 1982.

Simons et al., "Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo", Nature, 359: 67-70, 1992.

Slovik et al., "Restoration of spinal bone in osteoporotic men by treatment with human parathyroid hormone (134) and 1,25-dihydroxyvitamin D", J. Bone Min. Res., 1: 377-381, 1986.

Somjen et al., "Stimulation of cell proliferation in skeletal tissues of the rat by defined parathyroid hormone fragmenus", Biochem J., 272: 781-785, 1990.

Spencer et al., "Parathyroid hormone potentiates the effect of insulin-like growth factor-I on bone formation", Acta Endocrinological (Copenh), 121: 435-442, 1989.

Stenflo et al., 1987.

Stein and Lian, Enddocr. Rev., 14: 424-442, 1993.

Steiner et al., J. Biol. Chem., 267: 23435-23438, 1992.

Stenflo et al., Proc. Natl. Acad. Sci. USA, 84: 368-372, 1987.

Stenman et al., Cytogenet. Cell Genet., 66: 117-119, 1994.

Stevenson and Parsons, "Effects of parathyroid hormone and the synthetic 1-34 amino-terminal fragment in rats and dogs", J. Endocr., 97: 21-30, 1983.

Stratford-Perricaudet et al., "Widespread long-term gene transfer to mouse skeletal muscles and heart", J. Clin. Invest., 90: 626-630, 1992.

Streuli et al., J. Cell Biol., 120: 253-260, 1993.

Tada et al., Bone, 11: 163-169, 1988.

Taipale et al., J. Cell Biol., 124: 171-181, 1994.

Taketazu et al., Lab. Invest., 70: 620-630, 1994.

Tam et al., "Parathyroid hormone stimulates the bone apposition rate independently of its resorptive action: differential effects of intermittent and continuous administration", Endocrinology, 110: 506-512, 1982.

IS 2 139 889 T5

Toriumi et al., "Mandibular reconstruction with a recombinant bone-inducing factor", Arch. Otolaryngol. Head NeckSurg., 117: 1101-1112, 1991.

Tregear et al., "Bovine parathyroid hormone: minimal chain length ol", Endocrinology, 93: 1349-1353, 1973. Tsuji et al., Proc. Natl. Acad. Sci. USA, 87: 8835-8839, 1990.

Twardzik et al., Ann. NY Acad. Sci., 593: 276-284, 1990.

Ulmer et al., "Heterologous protection against influenza by injection of DNA encoding a viral protein", Science, 259: 1745-1749, 1993.

Urist, "Bone formation by autoinduction", Science, 150: 893-899, 1965.

Urist et al., "Bone cell differentiation and growth factors", Science, 220: 680-686, 1983.

Van der Plas, “Direct effect of parathyroid hormone on the proliferation of osteoblast-like cells; a possible involvement of cyclic AMP ”, Biochem. Biophys. Res. Comm., 129: 918-925, 1985.

Van Vlasselaer et al., J. Cell Biol., 124: 579-577, 1994.

Vukicevic et al., "Stimulation of the expression of osteogenic and chondrogenic phenotypes in vitro by osteogenic", Proc. Natl. Acad. Sci. USA, 86: 8793-8797, 1989.

Wakefield et al., J. Cell Biol., 105: 965-975, 1987.

Wakefield et al., J. Biol. Chem., 263: 7646-7654, 1988.

Wang et al., "Recombinant human bone morphogenetic protein induces bone formation", Proc. Natl. Acad. Sci. USA, 87: 2220-2224, 1990.

Wilson et al., "Somatic gene transfer in the development of an animal model for primary hyperparathyroidism", Endocrinology, 130: 2947-2954, 1992.

Wolff et al., "Direct gene transfer into mouse muscle in vivo", Science, 247: 1465-1468, 1990.

Wozney et al., "Novel regulators of bone formation: molecular clones and activities", Science, 242: 1528-1534, 1988.

Yasko et al., “The healing of segmental bone defects, induced by recombinant human bone morphogenetic protein (rhBMP-2). A radiographic, histological, and biomechanical study in rats ”, J. Bone Joint Surg., 5: 659-70, 1992.

Yasuda et al., "Rat parathyroid hormonelike peptide: Comparison with the human homologue and expression malignant and normal tissue", Mol. Endocrinol., 3: 518-525, 1989.

Zhang et al., J. Cell Biol., 124: 855-863, 1994.