WO1999047177A1 - Vecteur de therapie genique avec promoteur d'osteocalcine et genes de proteines morphogenetiques osseuses ou de facteurs de croissance osseuse - Google Patents

Vecteur de therapie genique avec promoteur d'osteocalcine et genes de proteines morphogenetiques osseuses ou de facteurs de croissance osseuse Download PDF

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WO1999047177A1
WO1999047177A1 PCT/US1999/005867 US9905867W WO9947177A1 WO 1999047177 A1 WO1999047177 A1 WO 1999047177A1 US 9905867 W US9905867 W US 9905867W WO 9947177 A1 WO9947177 A1 WO 9947177A1
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bone
factor
gene
polynucleotide
growth factor
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PCT/US1999/005867
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English (en)
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Gregory A. Helm
Gerald R. Hankins
Tord D. Alden
Leland W. K. Chung
Song-Chu Ko
Chinghai Kao
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The University Of Virginia Patent Foundation
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Priority to AU31898/99A priority Critical patent/AU3189899A/en
Publication of WO1999047177A1 publication Critical patent/WO1999047177A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • the present invention relates generally to the field of gene therapy. More particularly, the present invention relates to methods and compositions related to novel, viral vectors which can be used as therapeutic agents for curing a wide variety of bone and spinal disorders.
  • the viral vectors described in the present invention are used to deliver bone growth factor genes, which induce endochondral bone formation, to numerous tissues.
  • Promoters utilized with the viral vectors of the present invention are capable of selectively driving expression of a bone growth factor gene in a tissue specific manner. More particularly, the promoters are capable of selectively increasing endochondral bone formation in pre-existing bone.
  • the viral vectors of the present invention are effective therapeutic agents not only when administered via direct application, such as by injection, but also when administered systemically to the body via intravenous administration, oral administration or the like, because gene expression will be limited and localized to specific cell types.
  • Somatic cell gene therapy is a strategy in which a nucleic acid, typically in the form of DNA, is administered to alter the genetic repertoire of target cells for therapeutic purposes.
  • a nucleic acid typically in the form of DNA
  • somatic cell gene therapy to treat human diseases has caught the imagination of numerous scientists, mainly because of two recent technologic advancements.
  • increasing support for the human genome project will allow for the identity and sequence of the estimated 80,000 genes comprising the human genome in the very near future.
  • Gene therapy was originally conceived of as a specific gene replacement therapy for correction of heritable defects to deliver functionally active therapeutic genes into targeted cells.
  • Initial efforts toward somatic gene therapy relied on indirect means of introducing genes into tissues, called ex vivo gene therapy, e.g., target cells are removed from the body, transfected or infected with vectors carrying recombinant genes and re-implanted into the body ("autologous cell transfer").
  • ex vivo gene therapy e.g., target cells are removed from the body, transfected or infected with vectors carrying recombinant genes and re-implanted into the body (“autologous cell transfer").
  • a variety of transfection techniques are currently available and used to transfer DNA in vitro into cells; including calcium phosphate-DNA precipitation, DEAE-Dextran transfection, electroporation, liposome mediated DNA transfer or transduction with recombinant viral vectors.
  • ex vivo treatment protocols have been proposed to transfer DNA into a variety of different cell
  • fibroblasts Palmer et al, 1987, Proc. Natl. Acad. Sci. USA 84:1055-1059; Anson et al, 1987, Mol. Biol. Med. 4:11-20; Rosenberg et al, 1988, Science 242:1575-1578; Naughton & Naughton, U.S. Patent 4,963,489), lymphocytes (Anderson et al, U.S. Patent No. 5,399,346; Blaese, R.M. et al, 1995, Science 270:475-480) and hematopoietic stem cells (Lim, B. et al. 1989, Proc. Natl. Acad. Sci. USA 86:8892-8896; Anderson et al, U.S. Patent No. 5,399,346).
  • Viral vectors are regarded as the most efficient system, and recombinant replication-defective viral vectors have been used to transduce (i.e., infect) cells both ex vivo and in vivo.
  • Such vectors have included retroviral, adenoviral, adeno-associated viral and herpes viral vectors.
  • the major disadvantages associated with the use of viral vectors include the inability of many viral vectors to infect non-dividing cells, problems associated with insertional mutagenesis, inflammatory reactions to the virus and potential helper virus production and/or production and transmission of harmful virus to other human patients.
  • many targeted cell populations are found in such low numbers in the body that the efficiency of presentation of DNA to the specific targeted cell types is diminished even further.
  • Retro viruses represent one class of viruses that have been studied extensively for use in gene therapy (Miller, A.D., 1990, Human Gene Ther. 1 :5-14). Unfortunately, there are a number of disadvantages associated with retroviral use, including the random integration of retro viruses into the host genome, which often leads to insertional mutagenesis or the inadvertent activation of proto-oncogene expression due to the promoter activity associated with retroviral LTRs (long terminal repeats). Adeno-associated viruses (“AAV”) also have been studied as an alternative system for delivery of stable genetic information into a cell. These viruses have the desirable feature of potentially integrating in specific regions of the host genome.
  • AAV Adeno-associated viruses
  • retroviral and AAV vectors are limited by their inability to accept heterologous DNA fragments greater than 3-5 Kb, their inability to produce larger quantities of viral stocks and, in the case of retro viruses, their instability and inability to infect non-dividing cells.
  • Adeno virus is a large, non-enveloped virus consisting of a dense protein capsid and a large linear (36 kb) double stranded DNA genome.
  • Adenovirus infects a variety of both dividing and non-dividing cells, gaining entry by receptor-mediated uptake into endosomes, followed by internalization. After uncoating, the adenovirus genome expresses a large number of different gene products that are involved in viral replication, modification of host cell metabolism and packaging of progeny viral particles.
  • Three adenovirus gene products are essential for replication of viral genomes: (1) the terminal binding protein which primes DNA replication, (2) the viral DNA polymerase and (3) the DNA binding protein (reviewed in Tamanoi and Stillman, 1983, Immunol.
  • adenovirus 23kDa L3 protease processing of the terminal binding protein by the adenovirus 23kDa L3 protease is required to permit subsequent rounds of reinfection (Stillman et al, 1981, Cell, 23:497-508) as well as to process adenovirus structural proteins, permitting completion of self-assembly of capsids (Bhatti and Weber, 1979, Virology, 96:478-485).
  • Packaging of nascent adenovirus particles takes place in the nucleus, requiring both cis-acting DNA elements and trans-acting viral factors, the latter generally construed to be a number of viral structural polypeptides.
  • Packaging of adenoviral DNA sequences into adenovirus capsids requires the viral genomes to possess functional adenovirus encapsidation signals, which are located in the left and right termini of the linear viral genome (Hearing et al, 1987, J. Virol. 61:2555-2558). Additionally, the packaging sequence must reside near the ends of the viral genome to function (Hearing et al, 1987, J. Virol. 61 :2555-2558; Grable and Hearing, 1992, J. Virol., 66:723-731).
  • the El A enhancer, the viral replication origin and the encapsidation signal compose the duplicated inverted terminal repeat (ITR) sequences located at the two ends of adenovirus genomic DNA.
  • the replication origin is defined loosely by a series of conserved nucleotide sequences in the ITR which must be positioned close to the end of the genome to act as a replication-priming element (reviewed in Challberg and Kelly, 1989, Biochem, 58:671-717; Tamanoi and Stillman, 1983, Immunol. 109:75-87). As shown by several groups, the ITRs are sufficient to confer replication to a heterologous DNA in the presence of complementing adenovirus functions. Adenovirus "mini-chromosomes" consisting of the terminal ITRs flanking short linear DNA fragments (in some cases non- viral
  • DNAs were found to replicate in vivo at low levels in the presence of infecting wild-type adenovirus, or in vitro at low levels in extracts prepared from infected cells (e.g., Hay et al,
  • replication-defective viruses are produced by replacing part, or all, of essential region El with a heterologous gene of interest, either by direct ligation to viral genomes in vitro, or by homologous recombination within cells in vivo (procedures reviewed in Berkner, 1992, Curr. Topics Micro. Immunol., 158:39-66). These procedures all produce adenovirus vectors that replicate in complementing cell lines such as 293 cells which provide the El gene products in trans.
  • Replication competent adenovirus vectors also have been described that have the heterologous gene of interest inserted in place of non-essential region E3 (e.g., Haj-Ahmad and Graham, 1986, J. Virol. 57:267-274), or between the right ITR and region E4 (Saito et al, 1985, J. Virol., 54:711-719).
  • the heterologous gene of interest is incorporated into viral particles by packaging of the recombinant adenovirus genome.
  • retroviral or adeno-associated viral vectors which are
  • transgenes encode, for example, bone growth factors
  • transient expression may be advantageous, since prolonged gene expression could lead to serious side-effects.
  • a non-integrating viral vector such as adenovirus, would be preferred.
  • a typical sequence of events includes; hemorrhage, clot formation, dissolution of the clot with concurrent removal of damaged tissues, ingrowth of granulation tissue, formation of cartilage, capillary ingrowth and cartilage turnover, rapid bone formation (callus tissue) and, finally, remodeling of the callus into cortical and trabecular bone. Therefore, bone repair and regeneration are complex processes that involve many cell types and regulatory molecules. The diverse cell populations involved in bone repair and regeneration include stem cells, macrophages, fibroblasts, vascular cells, osteoblasts, chondroblasts and osteoclasts.
  • bone repair and regeneration therapies involve the administration of therapeutic proteins.
  • therapeutic proteins may include regulatory factors such as systemic hormones, cytokines, growth factors and other proteins that regulate proliferation and differentiation of cells.
  • Various osteoinductive agents have been purified and shown to be polypeptide growth-factor-like molecules. These stimulatory factors are referred to as bone morphogenetic or morphogenic proteins (BMPs) and also have been termed osteogenic bone inductive proteins or osteogenic proteins (OPs).
  • BMPs bone morphogenetic or morphogenic proteins
  • OPs osteogenic bone inductive proteins
  • BMPs are polypeptides which induce ectopic bone formation in standard rat in vivo assay systems. The initial evidence suggesting that such proteins exist was first
  • Urist demonstrated that bone formation could be induced in extra- skeletal sites using decalcified bone matrix and non-collagenous fractions of dentin, bone and osteosarcoma tissue. Urist's research group demonstrated that these implants stimulated pluripotent cells in the host to initiate endochondral bone formation. In 1979, Urist was the first to isolate and purify an osteoinduction factor from demineralized rat tibia matrix, which would likewise induce ectopic bone formation in rats and mice. This protein was a low molecular weight glycoprotein and confirmed the idea that individual proteins could have osteoinductive properties. The BMP field exploded in 1989, when John Wozney was successful in cloning four BMPs. Using molecular biology techniques, more than twelve such proteins now have been identified.
  • the BMPs are a family of proteins which belong to the TGF- ⁇ superfamily, based on amino acid similarities (Seitz, PA, et al, (1992), J. Bone Min. Res., 7, 541-546). All members of the superfamily contain seven conserved cystine residues.
  • the proteins in the TGF- ⁇ superfamily are signaling molecules which are involved with specific morphogenetic events during tissue and organ development. TGF- ⁇ is capable of transforming fibroblast cells in a monolayer culture and stimulating colony formation. It is thought that TGF- ⁇ is involved also in the control of mesenchymal cell division during development of the musculoskeletal system. BMP-2, however, seems to be involved in the differentiation of osteoblasts from pluripotent progenitor cells resident in bone marrow and extraskeletal sites.
  • BMPs are initially synthesized as large precursors that are approximately three times larger than the mature protein.
  • the recombinant protein which has been studied the most heavily to date has been BMP -2, which is a 32-KD homodimeric glycoprotein.
  • Implantation of purified recombinant BMP -2 leads to the formation of fully functional new bone, as assessed by alkaline phosphatase activity, calcium content and histology.
  • the recombinant protein recruits nearby mesenchymal pluripotent stem cells and triggers their proliferation and differentiation into chondrocytes in less than a week. This cartilaginous tissue is then invaded by capillaries and the chondrocytes become calcified, hypertrophy .and are replaced by newly formed bone in 9-12 days.
  • days 14-21 the mineralized bone is remodeled and is converted into a mature ossicle, filled with functional bone marrow elements.
  • -7- therapeutic uses in numerous disease processes, ranging from fracture healing to osteoporosis.
  • BMP-1 As discussed, several BMP (or OP) genes now have been cloned and the common designations are BMP-1 through BMP-8.
  • BMPs 2-8 generally are thought to be 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).
  • BMP (or OP) nucleotide sequences and polypeptides have been described in U.S. patents, e.g., U.S. Pat. Nos.
  • recombinant BMP-2 has been employed to repair surgically created defects in the mandible of adult dogs (Toriumi et al, 1991) and high doses of this molecule have been shown to functionally repair segmental defects in rat femurs (Yasko et al, 1992). Chen and colleagues showed that a single application of 25-100 ng of recombinant TGF- ⁇ l adjacent to cartilage induced endochondral bone formation in rabbit ear full thickness skin wounds (Chen et al, 1991). It also has been reported that an application of TGF- ⁇ l in a 3% methylcellulose gel was able to repair surgically induced large skull defects that otherwise heal by fibrous connective tissue and never form bone (Beck et al, 1991).
  • systemic delivery of therapeutic proteins can be associated with serious unwanted side effects. Due to inefficient delivery to specific cells and tissues in the body, administration of high doses of protein are required to ensure that sufficient amounts of the protein reach the appropriate tissue target. Further, because of the short half life in the body due to proteolytic degradation, the proteins also must be administered repeatedly which may give rise to an immune reaction to the therapeutic proteins. Moreover, the circulation of high doses of therapeutic proteins often is toxic due to pleiotropic effects of the administered protein and may, thus, give rise to serious side effects.
  • the present invention relates to novel compositions containing nucleotide sequences which can be used as therapeutic agents for curing a wide variety of bone and spinal disorders.
  • the present invention further relates to novel methods for using the therapeutic compositions. More specifically, the present invention relates to novel viral vectors which are used to deliver bone growth factor genes which induce endochondral bone formation to numerous tissues.
  • the methods of the invention further involve administering a bone growth factor gene, either directly or systemically, to a site where bone repair and/or regeneration is necessary.
  • the invention is based, in part, on the discovery that nucleotide sequences encoding bone growth factor genes contained within viral vectors can be administered in a cell and tissue specific manner, with the use of promoters which allow for tissue specific expression of the nucleotide sequence.
  • An example of such a promoter is the osteocalcin promoter which is activated only within cells of osteoblastic lineage.
  • a bone growth factor gene contained within a viral vector and controlled by an osteocalcin promoter can be expressed effectively and specifically in targeted bone cells and tissues, thereby reducing side effects of expression of the bone growth factor gene in non-osteoblastic cells.
  • the viral vectors of the present invention are effective therapeutic agents not only when administered via direct application, such as by injection, but also when administered systemically to the body via intravenous administration, oral administration or the like, because gene expression will be limited and localized to specific, osteoblastic cell and tissue types.
  • the present invention encompasses vectors using inducible promoters. Inducible promoters have the advantage that they can be switched on and off, depending on the clinical state of the patient. Therefore, if a cell is
  • DNA sequences While any number of DNA sequences can be used in the methods and compositions of the present invention, preferred DNA sequences are those that encode translational products (i.e. proteins) or transcriptional products (i.e. antisense or ribozymes) that (a) promote bone repair or regeneration; or (b) are capable of disrupting a disease process (thereby allowing normal bone morphogenesis to take place).
  • translational products i.e. proteins
  • transcriptional products i.e. antisense or ribozymes
  • the present invention overcomes the shortcomings of procedures currently used for bone repair or regeneration involving the administration of therapeutic proteins.
  • DNA which is both stable and non-toxic, can be administered safely in high doses in vivo.
  • repeated administration while possible, is not required.
  • the cells which take up and express the DNA provide a supply of gene product at the target site.
  • the invention could be practiced in a way that addresses the temporal requirements of dosing.
  • the DNA can be presented in vectors that integrate into the genome of the targeted cell. In this case, all daughter cells within the targeted cell will contain and express the transferred DNA, thereby acting as a continuous source for the therapeutic agent.
  • non-integrating systems may be utilized wherein the DNA does not integrate into the genome and the gene is not passed on to daughter cells. In such an instance, when the bone repair or regeneration process is completed and the gene product is no longer needed, the gene product will not be expressed.
  • the invention is demonstrated by way of examples, which show that genes can be transferred reproducibly and expressed in a variety of bone tissues in vivo. Specifically, it is shown that the method of the invention overcomes the problems associated with currently available gene therapy protocols.
  • the method of the invention provides gene transfer to a suitable number of bone cells to achieve functional effects, i.e., in the absence of any further targeting or cellular identification by the practitioner.
  • In vivo methods of gene therapy require some form of targeting which very often does not work.
  • targeting is not a problem since a tissue specific promoter will be utilized.
  • expression of a nucleotide sequence such as a bone growth factor, will occur only in osteotropic cells where the promoter is active.
  • the method of the invention may be used as a drug delivery system through transfer of DNA into mammalian cells for the purpose of stimulating
  • the cells will be those which, when stimulated with the DNA of the invention, will express bone growth factors and thus, induce bone formation.
  • the DNA to be used in the practice of the invention may include any DNA encoding translational products (i.e. proteins) or transcriptional products (i.e. antisense or ribozymes) that promote bone repair or regeneration or are capable of disrupting a disease process.
  • the DNA may comprise genes encoding therapeutically useful proteins such as growth factors, cytokines, hormones, etc.
  • the DNA may encode antisense or ribozyme molecules that may inhibit the translation of mRNAs encoding proteins that inhibit bone repair or regeneration or which induce excessive bone morhogenesis.
  • the invention is illustrated by way of working examples, wherein efficient in vivo transfer and tissue specific expression of genes into bone tissue undergoing repair and regeneration is demonstrated.
  • FIG. 1 Osteocalcin Promoter Activity. This graph compares the Vitamin D augmentation of mouse OC (mOC) promoter activity in human colon (LOVO) cancer cell line with human OC (AOC) activity. To the right, the /zOC promoter activity in luciferase constructs is compared to the level seen with vitamin D stimulation, which is greatly enhanced (5-100x). Thus, the human OC promoter is specific for cells of osteoblastic lineage and is induced by vitamin D.
  • mOC mouse OC
  • AOC human OC
  • the present invention relates to methods and compositions for the in vivo presentation and transfer of DNA into mammalian cells for the purpose of expressing therapeutic agents.
  • the methods of the invention involve viral vectors containing genes encoding bone growth factors controlled by tissue specific and/or inducible promoters.
  • Bone regeneration and repair is usually a coordinated, stereotyped sequence of events that includes (a) hemorrhage; (b) clot formation; (c) dissolution of the clot with
  • bone repair and regeneration are complex processes that involve many cell types and regulatory molecules.
  • the diverse cell populations involved in bone repair and regeneration include stem cells, macrophages, fibroblasts, vascular cells, osteoblasts, chondroblasts and osteoclasts.
  • the invention is based on the discovery that nucleotide sequences encoding bone growth factors contained within viral vectors can be administered in a cell and tissue specific manner, with the use of promoters which allow for tissue specific expression of the nucleotide sequences. Further, because the viral vectors of the invention utilize these promoters to control the expression of bone growth factor genes, the viral vectors of the invention are effective therapeutic agents not only when administered via direct application, but also when administered systemically to the body, because the bone growth factor genes will be expressed only in specifically targeted cells, i.e., within cells of osteoblast lineage.
  • the methods of the present invention are designed to efficiently transfer one or more DNA molecules encoding therapeutic agents to a site where bone repair and/or regeneration is necessary.
  • the methods involve the administration of a viral vector containing DNA encoding translational products (i.e. therapeutic proteins) or transcriptional products (i.e. antisense or ribozymes) within a mammalian host to a site where bone regeneration or repair is necessary.
  • the viral vector infects cells where bone repair and/or regeneration is necessary, the DNA of interest, i.e., bone growth factor genes, is expressed, thereby amplifying the amount of the therapeutic agent, protein or RNA.
  • the cells may take up and express DNA encoding proteins that inhibit the activity of antagonists of the bone repair process.
  • the DNA may encode also antisense or ribozyme RNA molecules that may be used to inhibit translation of mRNAs encoding inflammatory proteins or other factors that inhibit the bone healing process.
  • Stimulation of the bone repair process via direct DNA transfer from a viral vector to a mammalian cell offers a number of advantages.
  • the present invention relates also to pharmaceutical compositions comprising viral vectors containing DNA for use in bone repair.
  • the compositions of the invention generally are comprised of a bio-compatible, or bone-compatible, material containing the viral vector containing DNA encoding a therapeutic protein of interest, i.e., a bone growth factor.
  • a bio-compatible composition is one that is in a form that does not produce an allergic, adverse or other untoward reaction when administered to a mammalian host.
  • the invention overcomes shortcomings specifically associated with current recombinant protein therapies for bone repair applications.
  • transfected cells to (a) make physiological amounts of therapeutic protein, modified in a tissue- and context-specific manner, and (b) deliver this protein to the appropriate cell surface signaling receptor under the appropriate circumstances.
  • exogenous delivery of such molecules is expected to be associated with significant dosing and delivery problems.
  • repeated administration while possible, is not required with the methods of the invention because various promoters, including inducible promoters, can be used to control the level of expression of the therapeutic protein of interest.
  • integration of transfected DNA can be associated with long term recombinant protein expression.
  • osteotropic and osteogenic gene are used to refer to a gene or DNA coding region that encodes a protein, polypeptide or peptide that is capable of promoting, or assisting in the promotion of, bone formation, or one that increases the rate of primary bone growth or healing.
  • the terms promoting, inducing and stimulating are used interchangeably throughout this text to refer to direct or indirect processes that ultimately result in the formation of new bone tissue or in an increased rate of bone repair.
  • an osteotropic gene is a gene that, when expressed, causes the phenotype of a cell to change so that the cell either differentiates, stimulates other cells to differentiate, attracts bone-forming cells or otherwise functions in a manner that ultimately gives rise to new bone tissue.
  • the present methods and compositions may employ a variety of different types of DNA molecules encoding osteotropic genes.
  • the DNA molecules may include genomic, cDNAs, single stranded DNA, double stranded DNA, triple stranded DNA, oligonucleotides and Z-DNA.
  • the DNA molecules may code for a variety of factors that promote bone repair including extracellular, cell surface and intracellular RNAs and proteins. Examples of extracellular proteins include growth factors, cytokines, therapeutic proteins, hormones and peptide fragments of hormones, inhibitors of cytokines, peptide growth and differentiation factors, interleukins, chemokines, interferons, colony stimulating factors and angiogenic factors.
  • TGF- ⁇ proteins include, but are not limited to, the superfamily of TGF- ⁇ molecules, including the five TGF- ⁇ isoforms and bone morphogenetic proteins (BMP), latent TGF- ⁇ binding proteins, LTBP; keratinocyte growth factor (KGF); hepatocyte growth factor (HGF); platelet derived growth factor (PDGF); insulin-like growth factor (IGF); the basic fibroblast growth factors (FGF-1, FGF-2, etc.), vascular endothelial growth factor (VEGF); Factor VIII and Factor IX; erythropoietin (EPO); tissue plasminogen activator
  • BMP bone morphogenetic proteins
  • KGF keratinocyte growth factor
  • HGF hepatocyte growth factor
  • PDGF platelet derived growth factor
  • IGF insulin-like growth factor
  • FGF-1, FGF-2, etc. the basic fibroblast growth factors
  • VEGF vascular endothelial growth factor
  • EPO tissue plasminogen activator
  • TPA activins and inhibins.
  • Hormones which may be used in the practice of the invention include, for example, growth hormone (GH) and parathyroid hormone (PTH).
  • extracellular proteins also include the extracellular matrix proteins such as collagen, laminin and fibronectin.
  • cell surface proteins include the family of cell adhesion molecules (e.g., the integrins, selectins, Ig family members such as N-CAM and
  • cytokine signaling receptors such as the type I and type II TGF- ⁇ receptors and the FGF receptor and non-signaling co-receptors such as betaglycan and
  • RNAs and proteins include the family of signal transducing kinases, cytoskeletal proteins such as talin and vinculin, cytokine binding proteins such as the family of latent TGF- ⁇ binding proteins and nuclear trans acting proteins such as transcription factors and enhancing factors.
  • the DNA molecules may code also for proteins that block pathological processes, thereby allowing the natural bone repair process to occur unimpeded.
  • blocking factors include ribozymes that destroy RNA function and DNAs that, for example, code for tissue inhibitors of enzymes that destroy tissue integrity, e.g., inhibitors of metalloproteinases associated with arthritis.
  • DNA segment encoding the protein of interest may be obtained using a variety of molecular biological techniques, generally known to those skilled in the art. For example, cDNA or genomic libraries may be screened using primers or probes with sequences based on the known nucleotide sequences. Polymerase chain reaction (PCR) also may be used to generate the DNA fragment encoding the protein of interest. Alternatively, the DNA fragment may be obtained from a commercial source.
  • PCR Polymerase chain reaction
  • Genes with sequences that vary from those described in the literature also are encompassed by the invention, so long as the altered or modified gene still encodes a protein that functions to stimulate bone repair in any direct or indirect manner.
  • sequences include those caused by point mutations, those due to the degeneracies of the genetic code or naturally occurring allelic variants and further modifications that have been introduced by genetic engineering, i.e., by the hand of man.
  • -16- can be designed to contain the necessary elements for directing the transcription and/or translation of the DNA sequence taken up by the bone cells at the repair site in vivo.
  • Vectors that may be used include, but are not limited to, those derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA.
  • plasmid vectors such as pBR322, pUC 19/18, pUC 118, 119 and the Ml 3 mp series of vectors may be used.
  • Bacteriophage vectors may include ⁇ gtlO, ⁇ gtl 1, ⁇ gt 18-23, ⁇ ZAP/R and the EMBL series of bacteriophage vectors.
  • Cosmid vectors that may be utilized include, but are not limited to, pJB8, pCV 103, pCV 107, pCV 108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16 and the charomid 9 series of vectors.
  • Vectors that allow for the in vitro transcription of RNA such as SP6 vectors, also may be used to produce large quantities of RNA that may be incorporated into viral vectors.
  • recombinant virus vectors including, but not limited to, those derived from viruses such as herpes virus, retroviruses, vaccinia viruses, adenoviruses, adeno- associated viruses or bovine papilloma virus may be engineered. While integrating vectors may be used, non-integrating systems, which do not transmit the gene product to daughter cells for many generations, are preferred for non-disease related bone repair and regeneration. In this way, the gene product is expressed during the bone repair process, and as the gene is diluted out in progeny generations, the amount of expressed gene product is diminished.
  • the genes encoding the proteins of interest may be associated operatively with a variety of different promoter/enhancer elements.
  • the expression elements of these vectors may vary in their strength and specificities. Depending on the host/vector system utilized, any one of a number of suitable transcription and translation elements may be used.
  • the promoter may be in the form of the promoter which is associated naturally with the gene of interest.
  • the DNA may be positioned under the control of a recombinant or heterologous promoter, i.e., a promoter that is not associated normally with that gene.
  • tissue specific promoter/enhancer elements may be used to regulate the expression of the transferred DNA in specific cell types.
  • transcriptional control regions that exhibit tissue specificity include, but are not limited to: elastase I gene control region which is active in pancreatic acinar cells (Swift et al, 1984, Cell 38:639-646; Ornitz et al, 1986, Cold Spring Harbor Symp. Quant. Biol.
  • beta-globin gene control region which is active in myeloid cells
  • myelin basic protein gene control region which is active in oligodendrocyte cells in the brain
  • myosin light chain-2 gene control region which is active in skeletal muscle
  • gonadotropic releasing hormone gene control region which is active in the hypothalamus
  • Promoters isolated from the genome of viruses that grow in mammalian cells may be used, as well as promoters produced by recombinant DNA or synthetic techniques.
  • promoters specifically activated within bone i.e., the osteocalcin promoter, which is specifically activated within cells of osteoblastic lineage, may be used to target expression of nucleotide sequences within bone cells.
  • bone growth factor genes are expressed in host animals in a tissue specific manner.
  • the present invention utilizes a tissue specific promoter specific for osteoblasts, the osteocalcin promoter, which can be utilized to increase bone deposition specifically in, and around, bone.
  • Osteocalcin a noncollagenous Gla protein produced specifically in osteoblasts, is synthesized, secreted and deposited at the time of bone mineralization (Price,
  • the OC promoter specifically expresses OC only in cells of osteoblastic lineage.
  • use of viral vectors containing bone growth factor genes controlled by an OC promoter would be very effective in obtaining expression of the bone growth factor genes only in cells of osteoblastic lineage, thereby, reducing or eliminating harmful side effects caused by expression of the growth factor genes in non-osteoblastic cells.
  • tissue specific promoters to drive therapeutic gene expression would decrease further a toxic effect of the therapeutic gene on neighboring normal cells when virus-mediated gene delivery results in the infection of the normal cells. This would be important especially in diseases where systemic administration could be utilized to deliver a therapeutic vector throughout the body, while maintaining transgene expression to a limited and specific number of cell types. Moreover, since many bone growth factors, such as TGF- ⁇ , have pleiotropic effects, numerous, harmful side effects likely would be exhibited if the growth factor genes are expressed in all cells.
  • the promoter elements may be constitutive or inducible promoters and can be used under the appropriate conditions to direct high level or regulated expression of the gene of interest. Expression of genes under the control of constitutive promoters does not require the presence of a specific substrate to induce gene expression and will occur under all conditions of cell growth. In contrast, expression of genes controlled by inducible promoters is responsive to the presence or absence of an inducing agent. For example, if a cell is stably transfected with a therapeutic, inducible transgene, its expression could be controlled over the life-time of the individual. In fact, the OC promoter, itself, is induced by vitamin D. See, Fig. 1. In the case of a disease such as osteoporosis, for example, bone growth factor production by bone cells could be increased whenever bone resorption becomes significant. Levels of bone resorption can be assessed easily using quantitative techniques well known to those of skill in the art.
  • Specific initiation signals also are required for sufficient translation of inserted protein coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where the entire coding sequence, including the initiation codon and adjacent sequences, are inserted into the appropriate expression vectors, no additional translational control signals may be needed. However, in cases where only a portion of the
  • exogenous translational control signals including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the protein coding sequences to ensure translation of the entire insert.
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency and control of expression may be enhanced by the inclusion of transcription attenuation sequences, enhancer elements, etc.
  • ribozymes or antisense DNA molecules that may be transferred into mammalian cells.
  • Such ribozymes and antisense molecules may be used to inhibit the translation of RNA encoding proteins of genes that inhibit a disease process or the bone repair process thereby allowing tissue repair to take place.
  • Such molecules also may be used to inhibit a disease wherein over-expression of a bone growth factor gene occurs.
  • RNA molecules will act directly to block the translation of mRNA by binding to targeted mRNA and preventing protein translation.
  • ribozymes which are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA, also may be used to block protein translation.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences.
  • RNA molecules may be generated by transcription of DNA sequences encoding the RNA molecule.
  • genes combined on a single genetic construct under control of one or more promoters, or prepared as separate constructs of the same or different types, may be used.
  • an almost endless combination of different genes and genetic constructs may be employed.
  • Certain gene combinations may be designed to, or their use may otherwise result in, achieving synergistic effects on bone stimulation and regeneration, any and all such combinations are intended to fall within the scope of the present invention. Indeed, many synergistic effects have been described in the scientific literature, so that one of ordinary skill in the art readily would be able to identify likely synergistic gene combinations, or even gene-protein combinations.
  • Bone has a substantial capacity to regenerate following fracture.
  • the complex, but ordered, fracture repair sequence includes hemostasis, clot dissolution, granulation tissue ingrowth, formation of a callus and remodeling of the callus to an optimized structure (A.W. Ham., 1930, J. Bone Joint Surg. 12, 827-844).
  • Cells participating in this process include platelets, inflammatory cells, fibroblasts, endothelial cells, pericytes, osteoclasts and osteogenic progenitors.
  • Recently, several peptide growth and differentiation factors have been identified that appear to control cellular events associated with bone formation and repair (Erlebacher, A., et al, 1995, Cell 80, 371-378).
  • Bone morphogenetic proteins are soluble extracellular factors that control osteogenic cell fate.
  • BMP genes are normally expressed by cultured fetal osteoblasts (Harris, S.E., et al, 1994, J. Bone Min. Res. 9, 389-394) and by osteoblasts during mouse embryo skeletogenesis (Lyons, KM., et al, 1989, Genes Dev. 3, 1657-1668; Lyons, K.M., et al, 1990, Development 190, 833-844; Jones, M.C., et al, 1991, Development 111, 531-542), recombinant BMP proteins initiate cartilage and bone progenitor cell differentiation
  • BMP-4 gene expression is unregulated early in the process of fracture repair (Nakase, T., et al, 1994, J. Bone Min. Res. 9, 651-659).
  • Osteogenic protein-1 a member of a family of molecules related to the BMPs (Ozkaynak, E., et al, 1990, EMBO J. 9, 2085-2093), is capable of similar effects in vitro and in vivo (Sampath, T.K., et al, 1992, J. Biol. Chem. 267, 20352-20362; Cook, S.D., et al, (1994) J. Bone Joint Surg. 76-A, 827-838).
  • TGF- ⁇ also has been shown to stimulate cartilage and bone formation in vivo (Centrella, M., et al, 1994, Endocrine Rev. 15, 27-38; Sumner, D.R., et al, 1995, J. Bone Joint Surg. 77 A, 1135-1147).
  • parathyroid hormone (PTH) is an 84 amino acid hormone that raises the plasma and extracellular fluid Ca +2 concentration.
  • PTH parathyroid hormone
  • the PTH 1-34 peptide is known to interact synergistically with BMP-4, which up-regulates the expression of functional cell surface PTH receptors in differentiating osteoblasts in vitro (Ahrens, M., et al, 1993, J. Bone Min. Res. 12, 871-880).
  • peptide growth and differentiation factors such as BMP and TGF- ⁇ l
  • BMP and TGF- ⁇ l represent promising therapeutic alternatives for fracture repair (Wozney, J.M., 1992, Mol. Reprod. Dev. 32, 160-167; Reddi, A.H., 1994, Curr. Opin. Genet. Dev. 4, 737-744; Centrella, M., et al, 1994, Endocrine Rev. 15, 27-38; Sumner, D.R., et al, 1995 J. Bone Joint Surg. 77-A, 1135-1147).
  • relatively large doses are required to stimulate significant new bone formation in animals, raising the concern that future human therapies may be expensive and may possess an increased risk of toxicity.
  • any new technique designed to stimulate bone repair would be a valuable tool in treating bone fractures.
  • a significant portion of fractured bones still are treated by casting, allowing natural mechanisms to effect wound repair.
  • the development of new processes to stimulate, or complement, the wound repair mechanisms would represent significant progress in this area.
  • the present invention may be used to transfer a bone growth factor gene to promote fracture repair.
  • Other important aspects of this technology include the use of gene transfer to treat patents with "weak bones", such as in diseases like osteoporosis, to improve poor healing which may arise for unknown reasons, e.g. , fibrous non-union and to promote implant integration and the function of artificial joints.
  • Retroviral and adeno-associated viral vectors are ideal vectors for obtaining this type of transgene integration.
  • retroviral and adeno-associated viral vectors are ideal vectors for obtaining this type of transgene integration.
  • Adenovirus is a vector which is ideal for obtaining this type of non-integrated transgene expression.
  • Bone tissue is known to have the capacity for repair and regeneration and there is a certain understanding of the cellular and molecular basis of these processes. The initiation of new bone formation involves the commitment, clonal expansion and differentiation of repair cells. Once initiated, bone formation is promoted by a variety of polypeptide growth factors. Newly formed bone then is maintained by a series of local and systemic growth and differentiation factors.
  • BMPs as members of the transforming growth factor- ⁇ (TGF- ⁇ ) superfamily based on DNA sequence homologies.
  • TGF- ⁇ transforming growth factor- ⁇
  • BMPs 2-8 generally are thought to be osteogenic while BMP-1 may be a more generalized morphogen; Shimell et al, 1991, Cell, 67:469-481).
  • BMP-3 is also called osteogen (Luyten et al, 1989, J. Biol. Chem., 264:13377-13380) and BMP-7 is also called OP-1 (Ozkaynak et al, 1990, EMBO J., 9:2085-2093).
  • TGFs and BMPs each act on cells via complex, tissue-specific interactions with families of cell surface receptors (Roberts & Sporn,
  • TGFs Transforming growth factors
  • TGF- ⁇ l and TGF- ⁇ 2 can initiate both chondrogenesis and osteogenesis (Joyce et al, 1990, J. Cell Biol., 110:195-2007; Izumi et al, 1992, J. Bone Min. Res., 7:115-11; Jingushi et al, 1992, J. Orthop. Res., 8:364-371).
  • TGF and BMP growth factors/hormones besides TGF and BMP can be used in the practice of the invention to influence new bone formation following fracture.
  • fibroblast growth factor injected into a rat fracture site (Jingushi et al, 1990) at multiple high
  • PTH parathyroid hormone
  • PTH is an 84 amino acid calcium-regulated hormone whose principal function is to raise Ca +2 concentration in plasma and extracellular fluid.
  • amino acids 1-34 contains the structural requirements for biological activity (Tregear et al, 1973; Hermann-Erlee et al, 1976, Endocrine Research Communications, 3:21-35; Riond, 1993, Clin. Sci., 85:223-228).
  • a human full-length PTH gene construct has been introduced into cultured rat fibroblasts to create recombinant PTH-secreting cells. These cells then were transplanted into syngeneic rat recipients that were observed to develop hypercalcemia mediated by the increased serum concentrations of PTH (Wilson et al, 1992).
  • PTH has a dual effect on new bone formation, a somewhat confusing aspect of hormone function despite intensive investigation.
  • PTH has been shown to be a potent direct inhibitor of type I collagen production by osteoblasts (Kream et al, 1993). Intact PTH also was shown to stimulate bone resorption in organ culture over 30 years ago, and the hormone is known to increase the number and activity of osteoclasts. It currently is accepted that osteoclast activation occurs via an osteoblast signaling mechanism.
  • osteosclerosis may 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 achieve a new bone remodeling steady state after an initial period of net bone loss. Chronic, low dose administration of the amino-terminal fragment of PTH (amino acids 1-34) also can induce new bone formation according to a time- and dose-dependent schedule (Seyle, 1932; Parsons & Reit, 1974).
  • Human PTH1-34 recently has been shown to: (a) stimulate DNA synthesis in chick osteoblasts and chondrocytes in culture (van der Plas, 1985; Schluter et al, 1989; Somjen et al, 1990); (b) increase bone cell number in vivo (Malluche et al, 1986);
  • PTH/PTHrP receptor in situ in human fracture sites or in experimental models. Furthermore, no attempt has been made to augment bone repair by the exogenous addition of PTH peptides. Although hPTHl-34 was known to function as an anabolic agent for bone, prior to the present invention, much remained to be learned about the role (if any) of PTH during bone regeneration and repair.
  • the present inventors surprisingly have found that direct gene transfer into areas where bone repair and/or regeneration is necessary can be achieved readily with the use of viral vectors containing a bone growth factor gene of interest under the control of a tissue specific promoter, such as the osteocalcin promoter.
  • a tissue specific promoter such as the osteocalcin promoter.
  • the preferred methods for achieving gene transfer generally involve using a fibrous collagen implant material soaked in a solution of DNA shortly before being placed in the site in which one desires to promote bone growth or using a preparation of plasmid DNA encapsulated in a synthetic matrix such as a block copolymer of PLGA.
  • no implant material is necessary to facilitates the targeted uptake of exogenous viral constructs by cells which participate in bone regeneration/repair.
  • the need for long term spinal stability is a frequent requirement in neurosurgical patients.
  • anterior or posterior subluxation especially at the C1-C2 level, is frequently encountered and often produces myelopathic symptoms, including a spastic gait and weakness.
  • Progressive chronic instability may require surgical reduction with fusion, such as occiput to C2 or C3 fusion, posterior C1-C2 fusion or anterior cervical corpectomy with bone graft fusion.
  • fusion such as occiput to C2 or C3 fusion, posterior C1-C2 fusion or anterior cervical corpectomy with bone graft fusion.
  • degenerative changes including ligamentum hypertrophy, facet hypertrophy, disk bulging and subluxation can lead to lumbar stenosis causing neurogenic claudication.
  • the symptomatology of lumbar stenosis includes weakness, numbness and pain, especially while walking and standing.
  • Osteoporosis is characterized histologically by the parallel loss of bone mineral and bone matrix. Its cause may be physiologic, such as post-menopausal estrogen deficiency, or may be a primary age related decline in bone mass. The current treatment of osteoporosis is far from satisfactory. Although prophylaxis is the most effective form of management, there are no therapeutic options currently available to reverse the loss of bone mass in advanced osteoporosis. Diphosphonates, which are slowly hydrolyzable diphosphate analogs which inhibit osteoclastic bone resorption, and fluoride therapy, which stimulates bone formation and can substantially increase vertebral bone mass, are both being evaluated for clinical efficacy. Since bone growth factors, i.e. bone morphogenetic proteins, are characterized by their ability to induce endochondral bone formation de novo, they are
  • compositions and methods of the present invention would be ideally suited for the treatment of compression fractures that typically arise in a patient suffering from osteoporosis.
  • Bone growth factors certainly are ideal candidates to increase the amount of bone apposition and/or ingrowth to the implant, allowing for early and long term prosthesis stabilization.
  • Cook et al demonstrated that BMPs can enhance osseointegration of porous metal implants and significantly can increase the amount of bone deposition at the implant-bone interface (Cook SD, et al, 1996, Clinical Orthopedics and Related Research 324:29-38).
  • BMP coated dental implants increase new bone formation adjacent to the implant surface.
  • bone morphogenetic proteins delivered either locally or via gene therapy could increase the efficacy of these treatment modalities and decrease post-operative complication rates, such as implant extrusion, osteonecrosis, bone erosion and fractures.
  • osteotropic gene results in cellular expression of a recombinant osteotropic molecule, which expression is directly associated with stimulation of new bone formation.
  • a gene transfer vector coding for BMP-2 will stimulate new bone formation.
  • EXAMPLE 1 Construction of Recombinant Ad-OC-BMP-2 Vector
  • an adenoviral vector containing the OC promoter to drive the BMP -2 transgene was constructed. Briefly, to make recombinant adenovirus with BMP-2 under control of the human OC promoter, the cytosine
  • Primers (Integrated DNA Technologies, Coralville, IA) designed to give a 529 bp product were: forward: 5'-CCAGGTTGGTGAATCAGAATGC and reverse: 5'-GAGATAGCACTGAGTTCTGTCG.
  • PCR was carried out under the following conditions: 10 min. at 95 °C followed by 30 cycles of: 30 sec. denaturing at 95 °C, 1 min. annealing at 56°C and 2 min. extension at 72°C; the reaction was finished by a final extension step of 7 min. at 60°C.
  • a reaction with no template was run as a negative control and reactions with pBMP2-125 and p ⁇ Elspl-OC-BMP2 were run as positive controls.
  • Amplification products were visualized on a 2% agarose gel.
  • Animals A total of twelve rats are used in this study. Because host immune responses may limit transgene expression in adenoviral vectors, athymic nude rats (Harlan Sprague Dawley, Inc.) are utilized in this study (Festin et al, 1978). These rats do not respond to T-cell dependent mitogens present when adenoviruses infect normal cells.
  • Adenoviral constructs The recombinant Ad-OC-BMP-2 vector described in Example 1 , above is used to monitor bone formation.
  • Another recombinant adenovirus type 5 with the ⁇ -galactosidase ( ⁇ -gal) gene under the control of the CMV promoter (Ad-CMV- ⁇ -gal) is used as a control. Both of these adenoviruses were made replication defective through complete deletion of the Ela and Elb and partial deletion of the
  • the viruses were propagated on 293 cells and purified by two rounds of cesium chloride centrifugation. The purified virus was dialyzed in PBS. The
  • -28- virus are stored until use at -80°C in PBS, 10% glycerol at a concentration of 5 x 10 8 particles/ ⁇ l.
  • Radiology The animals are sedated and CT scans through the thigh are performed at weeks 3 and 5 post-injection. Scanning is performed using a Picker PQ- 2000 (version 4.2; Picker International, Cleveland, OH). Axial images with a 1 mm colhmation and 1 mm table increment are obtained using the standard algorithm with 130 kV, 100mA, a 2 second scan time and a 40 mm image size. Three dimensional reconstruction is performed using a Voxel Q workstation (Picker International).
  • Histology At day 3 post-injection, 4 of the animals are sacrificed and the thigh muscles are harvested, fixed with 0.05% glutaraldehyde solution and stained with Xgal reagent made with 35 mM K 3 Fe(CN) 6 , 35 mM K 4 Fe(CN) 6 and 2mM MgCl 2 (Sigma, St.
  • HU Hounsfield Units
  • An increase in the bone density will be seen at 5 weeks as compared to that at 3 weeks.
  • CT scanning through the abdomen, with special attention to the liver, will show no evidence of systemic ectopic calcification.
  • Histologic sections through the Ad-OC-BMP-2 injection site at about six weeks post-injection will reveal evidence of endochondral bone formation, including primitive mesenchymal cells, cartilage and bone. Further, bone induction within the thigh muscles likely will be limited to the injection site.
  • BMPs have been dramatically demonstrated in several species (Takagi and Urist, 1982a Yasko et al, 1992). Through improving the osseointegration of prosthetic arthroplasty devices, BMPs have improved the stability and increased the longevity of artificial joints.
  • Ad-BMP-2 has demonstrated the use of Ad-BMP-2 in ex vivo models. These have involved ex vivo transfection of cells in tissue culture by Ad-BMP-2 and then placement of these transfected cells into rat thighs with subsequent bone formation.
  • the present invention is based upon the surprising discovery that direct injection of Ad- BMP-2 also results in endochondral bone formation.
  • the limitations of direct in vivo application of the rh BMPs include poor control of bone formation and the inability to deliver the protein systemically to treat diffuse bone diseases.
  • the utilization of gene therapy techniques to express BMP-2 has several theoretical and practical advantages. Firstly, sustained BMP-2 gene expression at the treatment site would be possible using viral vectors such as Ad-OC-BMP-2.
  • viral vectors such as Ad-OC-BMP-2.
  • adenoviral vectors typically provide transgene expression for several weeks, improved bone formation may be possible compared to a single rh BMP treatment.
  • a lenti viral delivery of the BMP -2 gene may produce stably transfected cells, which could be useful for the treatment of chronic, progressive osteopenic states, such as osteoporosis.
  • modifications of the viral construct would make it possible to target BMP-2 gene expression to specific cells or tissues.
  • These techniques could include genetically altering the viral coating to establish tissue specific transduction or utilizing tissue specific promoters, such as the OC promoter, to drive transgene expression.
  • tissue specific promoters such as the OC promoter
  • These approaches also make it possible to deliver the BMP-2 vector systemically, while maintaining tissue specific BMP-2 gene expression.
  • inducible promoters such as a tetracycline responsive promoter, may make it possible to up-regulate or down-regulate BMP-2 production in vivo after successful gene delivery. Gossen, M., et al, 1992, Proc. Natl. Acad.
  • Ad-OC-BMP-2 induces bone formation only at the site of vector injection, without evidence of diffuse toxicity, the present technique has several clear clinical applications.
  • the adenoviral vector could be applied to fracture sites to stimulate
  • Ad-OC- BMP-2 vector additive to autologous bone grafts during spine stabilization procedures may improve their clinical efficacy by improving osteointegration of the graft and also may decrease the amount of bone graft required.
  • direct injection of the adenoviral construct alone adjacent to the spine may lead to long-term spine arthrodesis, eliminating the need for an open spine procedure.
  • an inducible promoter to the construct could be used to control the rate and amount of bone deposition at sites of bone healing, which certainly would be of practical benefit to ensure adequate bone formation and to prevent excessive bony overgrowth.
  • Bone morphogen gene therapy also may be useful in the treatment of several genetic diseases which probably involve BMP overexpression, including autosomal dominant fibrodysplasia ossificans progressiva and myositis ossificans.
  • Gannon et al. clearly demonstrated in an immunohistochemical study that BMP-2 and BMP-4 are present at increased levels in early fibromatous lesions in fibrodysplasia ossificans progressiva (Gannon et al, 1997).
  • Shafritz et al. demonstrated using Northern blot analysis and ribonuclease protection assays that BMP-4 mRNA levels are increased in lymphoblastoid cell lines from patients with this progressive disease (Shafritz et al, 1996).
  • anti-sense gene therapy for example, in these patients could be utilized to inactivate the over-expressed mRNA, leading to a successful treatment for this devastating condition.
  • the twelve athymic nude rats are sedated with a mixture of ketamine and xylazine, and the lumbosacral area is prepared in a sterile fashion.
  • the rats divided into three groups of four animals each, undergo paraspinal, percutaneous injection at the lumbosacral junction with 7.5 ⁇ l of virus.
  • the three groups are as follows: 1) bilateral injection of Ad-OC-BMP-2, 2) bilateral injection of Ad-CMV- ⁇ -gal, and 3) injection of Ad-
  • the lumbosacral spines are removed and decalcified with decalcifying solution composed of 10%) HC1 and 0.1 % ethylenediaminetetraacetic acid.
  • the specimens are then dehydrated through a series of graded ethanols, xylol and, finally, xylene after which the spines are infiltrated and embedded in paraffin.
  • the Ad-OC-BMP-2 vector could be added to autologous bone grafts to enhance bone deposition at the fusion site. Therefore, BMP-gene therapy could have the potential to improve long-term stability significantly after spine instrumentation and limit the need for extensive harvesting of autograft from the iliac crest or fibula.
  • percutaneous injection of the vector into the paraspinal musculature, facet joints, or the annulus fibrosis could fuse the spine in a minimally invasive fashion.
  • percutaneous injection of the vector into spinal fractures could increase the rate of bone repair and decrease the risk of nonunion and spinal instability.
  • this example demonstrates the feasibility of utilizing direct, percutaneous injections of adenoviral OC-BMP-2 to induce spinal arthrodesis.

Abstract

La présente invention concerne de nouvelles compositions contenant des séquences nucléotidiques pouvant s'utiliser comme agents thérapeutiques pour le traitement d'une grande variété d'affections osseuses ou spinales. Plus spécifiquement, la présente invention concerne de nouveaux vecteurs viraux utilisés pour l'administration de gènes de facteur de croissance osseuse et entraînant une formation endochondrale de l'os dans de nombreux tissus. L'expression des gènes de facteur de croissance osseuse est régulée par des promoteurs spécifiques et/ou inductibles de tissus. La présente invention concerne en outre de nouvelles méthodes d'utilisation des compositions thérapeutiques. Les méthodes selon la présente invention concernent spécifiquement l'administration d'un gène de facteur de croissance osseuse, directement ou de manière systémique, dans un site où une réparation et/ou une régénération osseuse est nécessaire.
PCT/US1999/005867 1998-03-18 1999-03-18 Vecteur de therapie genique avec promoteur d'osteocalcine et genes de proteines morphogenetiques osseuses ou de facteurs de croissance osseuse WO1999047177A1 (fr)

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WO2001082973A2 (fr) * 2000-04-28 2001-11-08 University Of Pittsburgh Of The Commonwealth System Of Higher Education Vecteurs viraux et non viraux en tant que vehicules d'administration de transgenes pour le traitement de pathologies osseuses
WO2001082973A3 (fr) * 2000-04-28 2003-02-06 Univ Pittsburgh Vecteurs viraux et non viraux en tant que vehicules d'administration de transgenes pour le traitement de pathologies osseuses
US7226587B2 (en) * 2001-06-01 2007-06-05 Wyeth Compositions and methods for systemic administration of sequences encoding bone morphogenetic proteins
WO2003006621A2 (fr) * 2001-07-13 2003-01-23 University Of Virginia Patent Foundation Super agent promoteur de l'osteocalcine pour traiter les tumeurs calcifiees et les tissus calcifies
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US7704272B2 (en) 2002-01-31 2010-04-27 University Of Rochester Method for introducing an ultraviolet light activated viral vector into the spinal column

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