JP2008536826A - Methods for reducing levels of tumor necrosis factor (TNF) in TNF-related disorders - Google Patents

Abstract

The present invention relates to recombinant adeno-associated virus (rAAV) vectors encoding tumor necrosis factor (TNF) antagonists for the treatment of TNF-related disorders or conditions, such as bone loss, wound healing or bone healing disorders, and oral buccal diseases. Provide a method to use. The present invention is directed to methods of treating or preventing bone loss, wound healing and bone healing disorders, and TNF-related disorders or conditions such as periodontitis in mammals. These methods generally use rAAV vectors to deliver polynucleotides encoding TNF antagonists to mammals, resulting in lower levels of TNF in the affected area and alleviating some TNF-related cascade events .

Description

This application claims the benefit of priority of US Provisional Patent Application No. 60 / 667,388, filed March 31, 2005. This provisional patent application is incorporated by reference in its entirety.

The present invention relates to methods of treating TNF-related diseases using adeno-associated virus (AAV) vectors encoding tumor necrosis factor (TNF) antagonists to reduce the level of TNF in an individual.

BACKGROUND OF THE INVENTION Tumor necrosis factor-α (TNFα) and tumor necrosis factor-β (TNFβ) are homologous multifunctional cytokines; tumor necrosis factor, or “TNF”, due to the remarkable similarity in their structural and functional characteristics. Is collectively called. Activities commonly attributed to TNF include the release of other cytokines such as IL-1, IL-6, GM-CSF and IL-10, induction of chemokines, increased adhesion molecules, blood vessel growth, tissue destruction enzymes Release and T cell activation. For example, Feldmann, et al. , 1997, Adv. Immunol. 64: 283-350, Nawroth, et al. , 1986, J. Am. Exp. Med. 163: 1363-1375; Moser, et al. , 1989, J. et al. Clin. Invest. 83: 444-455; Shingu, et al. 1993, Clin. Exp. Immunol. 94: 145-149; MacNaul, et al. 1992, Matrix Suppl. , 1: 198-199; and Ahmadzadeh, et al. , 1990, Clin. Exp. Rheumatol. 8: 387-391. All of these activities can act to enhance the inflammatory response.

  TNF induces biological effects through interaction with specific cell surface receptors on TNF-responsive cells. There are two different forms of cell surface tumor necrosis factor receptor (TNFR), labeled p75 (or type II) and p55 (or type I) (Smith, et al., 1990, Science 248). : 1019-1023; Loetscher, et al., 1990, Cell 61: 351-359). Both TNFR type I and TNFR type II bind to both TNFα and TNFβ. Soluble truncation forms of TNFR with a ligand binding domain are present in body fluids and joints (Engelmann, et al., 1989, J. Biol. Chem. 264: 11974-11980; Roux-Lombard, et al., 1993, Arthritis). Rheum. 36: 485-489).

  Some disorders are associated with elevated TNF levels, many of which have considerable medical significance. Such TNF-related disorders include congestive heart failure, inflammatory bowel disease (including Crohn's disease), arthritis, and asthma. US Pat. No. 6,537,540 and PCT WO 00/65038 administer a recombinant AAV vector comprising a polynucleotide encoding a TNF antagonist to reduce the level of TNF and treat a TNF-related disease A method is disclosed.

  Other examples of TNF-related disorders or conditions include impaired wound healing and fracture repair, and impaired bone allograft or autograft healing. This class of TNF-related disorders can be induced by infection with microorganisms, bacteria, molds or viral bodies, or by host immune-mediated defects that trigger cascades of inflammatory cytokines including TNF. .

  Studies with mouse models of allogeneic or autologous bone grafts have shown that the response rate of cortical bone allografts through delivery of rAAV encoding angiogenesis, including VEGF and RANKL factors, is uncertainly improved. (Jin, et al., Molecular Therapy 9: 519-299 (2004)). Furthermore, the delivery of osteogenic factors, including PDGF, has improved processing of tooth support structures in rat models (Ito, et al., Nat. Med. 11 (3): 291-297 (2005). ).

  Another example of a TNF-related disorder is an oral buccal disease (eg, periodontitis). Periodontitis is the most common inflammatory disease in humans. This disease is a leading cause of tooth loss in adults and is associated with glucose management in systemic diseases such as atherosclerosis, heart failure, and diabetes. The disease affects more than 50% of the US population and is treated primarily by local debridement or surgery of the affected area (Oliver, et al., J. Peridodontol, 69, 269-278, 1998). These treatment modalities are effective for many individuals with rapidly progressive periodontitis, but some patients may remain disconnected from connective tissue after mechanical or surgical procedures. is there. Therefore, the pathogenesis of periodontitis has been explored to identify potential new targets for pharmaceutical interventions that seek to stop disease progression.

  Various immune related cell populations are involved in the pathogenesis of periodontal disease, including specific CD + T cells. The onset process is most likely a response to exogenous periodontal pathogens such as P. gingivalis associated with plaque biomembranes (Darveau, et al., Periodontol 14, 12-32, 2000). As a result, the replenished monocytes, macrophages and fibroblasts produce cytokines such as tumor necrosis factor α (TNF) and interleukin-1β (IL-1) in the periodontal lesion. These cytokines have been found to localize in periodontal tissues as well as in gingival exudates (GCF) associated with lesions. These cytokines are central to the injury cascade and ultimately trigger the production of matrix metalloproteinases (MMPs), prostaglandins and osteoclasts. This cascade ultimately leads to irreversible damage to the tooth-supporting soft tissue and alveolar bone (Graves, Clin Infect Dis 28: 482-490, 1999). Although this disease is induced by specific oral pathogens that inhabit and invade oral tissues, the host's inflammatory response is a major factor in the progression of the disease.

  Patients with periodontal disease have high concentrations of TNF in both synovial fluid and GCF. Examination of an experimental model of periodontal disease reveals a very high association of active bone resorption incidence, consistent with high local levels of TNF at the disease site. Both IL-1 and TNF-α have been found to be significantly elevated in diseased periodontal sites rather than healthy or non-active sites (Ejeil, et al., J Periodontol, 74, 196-). 201, 2003; Gamonal, et al., J Periodontol, 71, 1535-1545, 2000; Gorska, et al., J Clin Periodonto, 30, 1046-1052, 2003; 548-554, 1991). IL-1 has also been forward-correlated with increasing search depth and loss of connectivity (Delima, et al., J Infect Dis, 186, 511-516, 2002). Furthermore, IL-β has a synergistic activity with TNF-α or lymphotoxin in stimulating bone resorption. Incubation of IL-1 with TNF or lymphotoxin in an in vitro model resulted in a 2-fold increase in IL-1 bone resorption activity and a 100-fold increase in activity of TNF or lymphotoxin (Stashenko, et al., J Immunol, 138, 1464-1468, 1987). Reduction of bacteria and its metabolic byproducts by periodontal treatment also results in a decrease in both IL-1β and TNF-α (Al-Shammari, et al., J Periodontol, 72, 1045-1051, 2001; , Et al., J Periodontol, 73, 835-842, 2002; Gamonal, et al., J Periodontol, 71, 1535-1545, 2000; Iwamoto, et al., J Periodontol, 74, 1231-1236, 2003) . Thus, improvements in clinical parameters are paralleled by a decrease in these cytokines, suggesting their importance in the pathogenesis of periodontitis.

  Recent cross-sectional and case-control studies have shown that in patients with periodontitis, the risk of cardiovascular disease and low-weight premature birth is increased two-fold and seven-fold. The local periodontal cascade of TNF-related inflammatory events is believed to trigger many systemic vascular and fetal placental events, increasing the risk of the association of these periodontal and systemic diseases (Paquette). , Et al., J. Contemporary Dental Practice 1: 1-18, 1999).

  Studies on soluble protein delivery of antagonists to IL-1 and TNF in a primate model of periodontitis have shown promising results (Delima, et al., J. Infect Dis, 186, 511-516, 2002) Delima, et al., J Clin Periodontol, 28, 233-240, 2001; Oates, et al., J Clin Periodontol, 29, 137-143, 2002). Histological studies have shown that connective tissue loss of connection is reduced by 51% and alveolar bone loss is reduced by 91% (Delima, et al., J Clin Periodontol, 28, 233-240). , 2001). To date, the only available therapy that functions as a host-regulatory agent for periodontal disease is a low-dose formulation of the antibiotic doxycycline that requires administration twice a day (1); 2; Non-patent document 3). However, the harsh enzymatic environment in the affected periodontal region may destroy the soluble cytokine antagonist before reaching its maximum activity, thus requiring more frequent administration of the active agent to the defect.

  Treatment of TNF-related disorders or conditions such as bone loss, wound healing and bone healing disorders, and buccal diseases of the buccal cavity, particularly treatments that can provide sustained and epidemic therapy, such as the delivery of protein TNF antagonists There is a need for new and effective forms of treatment that result in longer-term localized neutralization of TNF to the periodontal structure compared to neutralization. Treatment of these tumor-related disorders or conditions such as periodontitis may be an important key to reducing the risk of periodontal disease-induced systemic disease conditions such as cardiovascular disease and low weight premature birth .

All publications and references cited herein are hereby incorporated by reference in their entirety.
Caton, et al. , J Periodontol, 71, 521-523, 2000 Gapski, et al. , J Periodontol, 75, 441-452, 2004. Novak, et al. , J Periodontol, 73, 762-769, 2002.

The present invention is directed to methods of treating or preventing bone loss, wound healing and bone healing disorders, and TNF-related disorders or conditions such as periodontitis in mammals. These methods generally use rAAV vectors to deliver a polynucleotide encoding a TNF antagonist to a mammal, resulting in a decrease in the level of TNF in the affected area and alleviation of several TNF-related cascade events. . Examples of TNF-related cascade events include bone regeneration and / or loss of bone resorption, wound healing and bone healing (eg, bone graft healing and fracture repair) defects, and periodontitis. Decreasing TNF may result in lower levels of other disease-causing or contributing substances, such as other inflammatory cytokines, or may eliminate its cascade action.

  The present invention improves or accelerates the repair rate of a mammalian wound or bone tissue by reducing or reducing TNF at the site of wound healing or bone healing, improving the repair rate, Alternatively, a method for increasing the repair range is targeted.

  The present invention is a method for improving wound healing and bone healing in a mammal by administering an effective amount of rAAV to a wound, fracture or bone graft site in a mammal, wherein said rAAV vector encodes a TNF antagonist A method comprising a polynucleotide is provided. In some embodiments, the rAAV vector of the invention is a pulse such as an osteogenic growth factor such as PDGF, or a receptor activator of nuclear factor κB ligand (RANKL) or vascular endothelial growth factor (VEGF). It is administered in combination with a tube formation promoting factor. In some embodiments, osteogenic or angiogenic factors are also delivered by rAAV vectors encoding these factors.

  The present invention is directed to a method of treating a mammalian TNF-related buccal buccal disease. These methods generally use rAAV vectors to deliver a polynucleotide encoding a TNF antagonist to a mammal, resulting in a decrease in the level of TNF at the periodontal site, and several TNF-related oral buccal surfaces Alleviates disorders such as chronic periodontitis, rapidly progressive periodontitis, and gingivitis. Lowering TNF may result in lower levels of other disease-inducing or contributing substances, such as other inflammatory cytokines.

  The present invention is a method for treating or preventing TNF-related buccal diseases in a mammal, comprising administering an effective amount of a rAAV vector to a periodontal region of the mammal, wherein the rAAV vector encodes a TNF antagonist. A method comprising a polynucleotide is provided.

  The present invention also provides a method for reducing the incidence of TNF-related oral buccal disease in a mammal, ameliorating or alleviating TNF-related oral buccal disease, and / or delaying the onset or progression of TNF-related oral buccal disease Also provided is a method comprising administering an effective amount of a rAAV vector to a periodontal region of a mammal, wherein the rAAV vector comprises a polynucleotide encoding a TNF antagonist.

  The present invention also includes a method of reducing the level of TNF in a periodontal region of a mammal having a TNF-related oral buccal disease, comprising administering an effective amount of a rAAV vector to the periodontal region of the mammal, Also provided is a method wherein the rAAV vector comprises a polynucleotide encoding a TNF antagonist.

  The present invention also provides a method for reducing an inflammatory response in a periodontal region of a mammal having a TNF-related oral buccal disease, comprising administering an effective amount of an rAAV vector to the periodontal region of the mammal, Also provided are methods wherein the rAAV vector comprises a polynucleotide encoding a TNF antagonist.

  The present invention also relates to a method for reducing the recurrence of an inflammatory response in a periodontal region of a mammal having a TNF-related oral buccal disease, comprising administering an effective amount of a rAAV vector to the periodontal region of the mammal. Also provided are methods wherein the rAAV vector comprises a polynucleotide encoding a TNF antagonist.

  The present invention also provides a method for improving the regeneration of tooth support structures (eg, alveolar bone, periodontal ligament and cementum) induced by TNF-related oral buccal disease, wherein A method wherein the rAAV vector comprises a polynucleotide encoding a TNF antagonist. Improved regeneration of loss of tooth support structure may be achieved through regenerative procedures such as osteoconductive biomaterials and cell occlusive barrier membranes. In some embodiments, the rAAV vectors of the invention are administered in combination with an osteogenic growth factor (eg, PDGF) or an angiogenic factor (eg, RANKL or VEGF). In some embodiments, osteogenic and / or angiogenic factors are also delivered in the rAAV vector.

  In some embodiments, the polynucleotide in the rAAV vector described herein encodes a tumor necrosis factor receptor (TNFR). Since TNFR can bind to soluble TNF, the introduction of TNFR tends to reduce the level of TNF in affected tissues. In some embodiments, the rAAV vector comprises a polynucleotide encoding a p75 TNFR polypeptide. In other embodiments, the rAAV vector comprises a polynucleotide encoding an Fc (constant domain of an immunoglobulin molecule): p75 fusion polypeptide. In other embodiments, the rAAV vector comprises a polynucleotide encoding a fusion polypeptide in which the extracellular domain of TNFR is fused to Fc.

  In some embodiments, the rAAV vectors of the present invention further comprise a polynucleotide encoding an IL-1 antagonist, such as an IL-1 receptor type II polypeptide.

  The rAAV vector was diagnosed before, when, and / or diagnosed as a mammal having a TNF-related disorder or condition described herein (eg, wound healing, bone healing and buccal disease) May be administered later. The rAAV vector may be administered locally, for example by injection into the periodontal, gingival tissue, connective tissue or epithelium; or may be administered by systemic administration, for example by intramuscular injection. The rAAV vector may be delivered directly or locally via a coated delivery device known in the art (eg, a mouthguard or tray), or a gel matrix or microsphere (eg, an FDA approved polylactic acid glycol). In some cases, it may be delivered by non-surgical procedures known in the art including but not limited to acid microspheres. The rAAV vector may be used to transduce ex vivo gingival tissue that is surgically implanted at the wound site. The rAAV vector may be administered with another agent, such as a TNF antagonist, osteogenic growth factor (eg, PDGF), and pro-angiogenic factors (eg, RANKL and VEGF).

  The present invention also provides a syringe dedicated to periodontal injection, containing an rAAV vector comprising a polynucleotide encoding a TNF antagonist.

  The present invention also provides kits for use in any of the methods described herein. In some embodiments, the kit relates to any of the rAAV vectors described herein in combination with a pharmaceutically acceptable carrier and the use of the rAAV vector in any of the methods described herein. Includes instructions.

Detailed Description of the Invention The invention described herein is a method of preventing or treating a TNF-related disorder or condition (e.g., bone loss, wound healing and bone healing disorders, and periodontitis) in a mammal, comprising: A method is provided comprising administering an effective amount of an rAAV vector comprising a polynucleotide encoding a TNF antagonist at the site of a mammalian disorder or condition. A TNF antagonist expressed in a mammal reduces the level of TNF at the site of the disorder or condition, resulting in relief of the TNF-related disorder or condition. In some embodiments, the TNF antagonist is a fusion polypeptide in which the extracellular domain of TNFR is fused to Fc (the constant domain of an immunoglobulin molecule).

Definitions As used in this specification and in the claims, the singular includes the plural unless specifically stated otherwise. For example, “cell” includes a plurality of cells including a mixture thereof.

  As used herein, a “TNF antagonist” refers to a polypeptide that binds TNF and suppresses and / or masks TNF activity reflected in TNF binding to the TNF receptor, which includes (a TNFR, preferably endogenous (ie, individual or host specific) cell membrane bound TNFR; (b) the extracellular domain of TNFR; and / or (c) the TNF binding domain of TNFR (which may be part of the extracellular domain). Is included). TNF antagonists include, but are not limited to, a TNF receptor (or an appropriate portion thereof as described herein) and an anti-TNF antibody. As used herein, “biological activity” of a TNF antagonist refers to binding to TNF, where TNF is (a) TNFR, preferably endogenous cell membrane bound TNFR; (b) the extracellular domain of TNFR; And (c) inhibiting and / or blocking binding to any of the TNF binding domains of TNFR, which may be part of the extracellular domain. A TNF antagonist can be shown to exhibit biological activity using tests known in the art that measure the activity of TNF and its inhibition, as exemplified herein.

  A “TNF-related disorder or condition” is a disorder or condition that is associated with, caused by, and / or caused in response to elevated TNF levels. Such disorders are thought to be associated with transient or chronic increases in TNF activity and / or local or systemic increases in TNF activity. Such disorders include, but are not limited to, inflammatory diseases such as buccal diseases of the oral cavity, arthritis and inflammatory bowel disease, and congestive heart failure. Examples of oral buccal diseases include chronic periodontitis, rapidly progressive periodontitis and gingivitis. TNF-related disorders also include bone loss and wound healing and bone healing disorders.

  As used herein, “TNF receptor polypeptide” and “TNFR polypeptide” refer to polypeptides derived from TNFR (derived from any species) capable of binding TNF. Two different TNFRs have been reported for cell surface TNFR: type II TNFR (ie p75 TNFR or TNFRII) and type I TNFR (ie p55 TNFR or TNFRRI). Mature full-length human p75 TNFR is a glycoprotein having a molecular weight of about 75-80 kilodaltons (kD). Mature full-length human p55 TNFR is a glycoprotein having a molecular weight of about 55-60 kD. In some embodiments, the TNFR polypeptides of the present invention are derived from type I TNFR and / or type II TNFR.

  A TNFR polypeptide, such as “INFR”, “INFR: Fc”, etc., when considered in the context of the present invention and compositions thereof, respectively, is an intact polypeptide (eg, TNFR undamaged) or the desired biology. Refers to any fragment or derivative thereof (eg, an amino acid sequence derivative) that exhibits a specific activity (ie, binding to TNF). A “TNFR polynucleotide” is any polynucleotide that encodes a TNFR polypeptide (eg, a TNFR: Fc polypeptide).

  As used herein, the “extracellular domain” of TNFR refers to the portion of TNFR that exists between the amino terminus of TNFR and the amino terminus of the TNFR transmembrane region. The extracellular domain of TNFR binds TNF.

  An “IL-1 antagonist” as used herein binds interleukin 1 (IL-1) and inhibits IL-1 activity reflected in IL-1 binding to the IL-1 receptor and Refers to a polypeptide that masks: (a) an IL-1 receptor (IL-1R), preferably endogenous (ie individual or host specific) cell membrane bound IL-1R; (b) IL Either the extracellular domain of -1R; and / or (c) the IL-1 binding domain of IL-1R, which may be part of the extracellular domain. IL-1 antagonists include, but are not limited to, the IL-1 receptor (or an appropriate portion thereof as described herein) and an anti-IL-1 antibody. As used herein, “biological activity” of an IL-1 antagonist refers to binding to IL-1, wherein IL-1 is (a) IL-1R, preferably endogenous cell membrane bound IL-1R. Inhibiting (b) binding to any of the extracellular domains of IL-1R; and / or (c) the IL-1 binding domain of IL-1R, which may be part of the extracellular domain; Or shielding. IL-1 antagonists can be shown to exhibit biological activity using tests known in the art, such as the IL-1 inhibition tests described herein and in the art. .

  As used herein, “IL-1 receptor polypeptide” refers to a polypeptide derived from IL-1R (derived from any species) capable of binding IL-1. An IL-1R polypeptide, when considered in the context of the present invention and its compositions, is an intact polypeptide (eg, intact IL-1R), or the desired biological activity (ie, IL-1R, respectively). Any fragment or derivative thereof (eg, amino acid sequence derivative) exhibiting An “IL-1R polynucleotide” is any polynucleotide that encodes an IL-1R polypeptide.

  As used herein, the “extracellular domain” of IL-1R refers to the portion of IL-1R that exists between the amino terminus of IL-1R and the amino terminus of the IL-1R transmembrane region. . The extracellular domain of IL-1R binds IL-1.

  The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein and refer to a polymer of amino acids of any length. These terms also encompass modified amino acid polymers, such as amino acid polymers that have been subjected to disulfide bond formation, glycosylation, lipidation, or conjugation with a labeling component.

  A “chimeric polypeptide” or “fusion polypeptide” is a polypeptide that includes a region at a position different from that in which it occurs in nature. These regions are usually present in separate proteins and incorporated together in the chimeric or fusion polypeptide, or these regions are usually present in the same protein, but in a new arrangement in the chimeric or fusion polypeptide. Placed. A chimeric or fusion polypeptide may also result from either a linear or branched polymeric form of a TNFR polypeptide.

  The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymeric form of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If there is a modification of the nucleotide structure, this may be imparted before or after assembly of the polymer. As used herein, the term polynucleotide is used interchangeably to refer to double-stranded and single-stranded molecules. Unless otherwise stated or required, any of the embodiments of the invention described herein that are polynucleotides are each known to be expected to constitute a double-stranded form and a double-stranded form. Includes both complementary single-stranded forms.

  A “chimeric polynucleotide” or “fusion polynucleotide” is a polynucleotide that includes a region at a position different from that in which it occurs in nature. These regions are usually present in separate genes and incorporated together in the chimeric or fusion polynucleotide, or these regions are usually present in the same gene, but in a new arrangement in the chimeric or fusion polynucleotide. Placed.

  “AAV” is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or a derivative thereof. Unless otherwise required, the term covers all subtypes, serotypes, pseudotypes, and both natural and recombinant forms.

  As used herein, an “rAAV vector” refers to an AAV vector comprising a polynucleotide sequence that is not derived from AAV (ie, a polynucleotide that is heterologous to AAV), generally a sequence that is subject to cell transformation. Point to. A heterologous polynucleotide is flanked by at least one, preferably two AAV inverted terminal repeats (ITRs). As described herein, rAAV vectors may take any of several forms, including but not limited to plasmids, linear artificial chromosomes, lipid complexes, liposome capsule types, most preferably viruses. It may be encapsulated in particles, particularly AAV.

  “RAAV virus” or “rAAV viral particle” refers to a viral particle consisting of at least one AAV capsid protein (preferably due to all of the capsid proteins of wild type AAV) and encapsidated rAAV.

  “Packaging” refers to a series of intracellular events that result in the assembly and encapsidation of AAV or rAAV particles.

  The AAV “rep” and “cap” genes refer to polynucleotide sequences encoding adeno-associated virus replication and encapsidation proteins. These genes are found in all AAV serotypes examined and are described below and in the art. As used herein, AAV rep and cap are referred to as AAV “packaging genes”.

  AAV “helper virus” refers to a virus capable of replicating and packaging AAV by mammalian cells. A variety of such AAV helper viruses are known in the art, including adenoviruses, herpesviruses and poxviruses (eg vaccinia). Although adenoviruses encompass several different subgroups, subgroup C adenovirus type 5 is most commonly used. Many adenoviruses derived from humans, non-human mammals and birds are also known and can be obtained from depositaries such as ATCC. Herpes family of viruses include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV); these are also deposits such as ATCC Available from

  An “infectious” virus or virus particle is one that includes a polynucleotide component that can be delivered to cells in which the virus species is vegetative. This term does not necessarily imply the ability of a virus to replicate. Assays for counting infectious viral particles have been described in the art.

  A “replicating ability” virus (eg, AAV having a replication ability; sometimes abbreviated as “RCA”) is infectious and within an infected cell (in the presence of sandpaper helper virus or helper virus function). Phenotype refers to a wild-type virus that can replicate. In the case of AVV, replication capacity generally requires the presence of a functional AAV packaging gene. Preferred rAAV vectors described herein lack the ability to replicate in mammalian cells, particularly human cells, because they lack one or more AAV packaging genes. Preferably, such rAAV vectors lack the AAV packaging gene sequence to minimize the likelihood that RCA will be generated by recombination between the AAV packaging gene and the rAAV vector.

  “Gene” refers to a polynucleotide containing at least one open reading frame that can be transcribed and translated to encode a particular protein.

  “Recombinant” when applied to a polynucleotide is the product of various combinations of cloning, restriction or ligation procedures, and other treatments that result in a construct that differs from the naturally occurring polynucleotide. Means that. A recombinant virus is a viral particle comprising a recombinant polynucleotide. Each of these terms includes replication of the original polynucleotide construct and the progeny of the original viral construct.

  A “regulatory element” or “control sequence” is a nucleotide sequence that participates in molecular interactions that contribute to the functional regulation of a polynucleotide, eg, replication, duplication, transcription, splicing, translation or degradation of the polynucleotide. Modulation may affect the frequency, rate or specificity of the process and may have a promoting or inhibitory nature. Regulatory elements known in the art include, for example, transcriptional regulatory sequences (such as promoters and enhancers). A promoter is a DNA region capable of binding RNA polymerase under specific conditions and inducing transcription of a coding region usually located downstream (3 'direction) from the promoter.

  “Operatively linked” or “operably linked” refers to the juxtaposition of genetic elements, which are in a relationship such that each can be operated in the expected manner. For example, a promoter is operably linked to a coding region if the promoter supports the induction of transcription of the coding sequence. As long as this functional relationship is maintained, there may be intervening residues between the promoter and the coding region.

  “Heterologous” means derived from an entity whose genotype differs from that of the remainder of the entity being compared. For example, a polynucleotide introduced into a plasmid or vector derived from a different species by genetic engineering techniques is a heterologous polynucleotide. A promoter that is removed from a natural coding sequence and operably linked to a coding sequence that does not naturally exist in a linked state is a heterologous promoter.

  “Gene modification” refers to the process by which genetic elements are introduced into cells without mitosis or meiosis. This element may be heterologous to the cell, or it may be an additional copy or enhancement of an element already present in the cell. Genetic modification can be achieved by transfecting cells with recombinant plasmids or other polynucleotides, eg, via any process known in the art, such as electroporation, calcium phosphate precipitation, or contact with polynucleotide liposome complexes. It may be done by doing. Genetic modification may also be performed by transduction or infection with, for example, DNA or RNA viruses or viral vectors. Preferably, the genetic element is introduced into a chromosome or mini-chromosome within the cell; any modification that alters the phenotype and / or genotype of the cell and its progeny is included in this term.

  A cell is said to be “stable” modified, transduced or transformed by a gene sequence when the sequence is available to perform its function during long-term culture of the cell in vitro. In a preferred embodiment, such cells are “genetically” modified because a genetic modification that is also inherited by the modified cell's progeny is introduced.

  “Stable incorporation” of a polynucleotide into a cell means that the polynucleotide is incorporated into a replicon that tends to be stably maintained in the cell. Although episomes such as plasmids can sometimes be maintained for many generations, the genetic material carried by the episome is generally more likely to be lost than chromosomal integration material. However, maintenance of the polynucleotide is accomplished by maintaining cells carrying the polynucleotide under selective pressure after incorporating a selectable marker within or adjacent to the polynucleotide. In some cases, the sequence may not be effectively and stably maintained unless it is integrated into the chromosome, so the selection to retain the sequence containing the selectable marker ensures that the marker is stably integrated into the chromosome. The selected cells can be selected. As is well known in the art, antibiotic resistance genes can be conveniently used as such selectable markers. In general, a stably integrated polynucleotide is expected to be maintained on average over at least about 20 generations, preferably at least about 100 generations, and more preferably is maintained permanently. The chromatin structure of eukaryotic chromosomes can also affect the expression level of the integrated polynucleotide. Having a stably maintained episomally carried gene can be particularly useful when it is desired to have multiple stably maintained copies of a particular gene. The selection of stable cell lines with particularly desirable properties in the sense of the present invention is described and illustrated below.

  An “isolated” plasmid, virus or other material is any other material that may be present where the material or similar material exists in nature, or even if it is the original source of production. Refers to a formulation of a substance without at least some of the ingredients. Thus, for example, isolated material may be prepared using purification techniques that concentrate from a raw material mixture. Concentration can be measured based on absolute values, such as weight per solution volume, or relative to a second potential interferent present in the raw material mixture. It is said that the concentration enhancement of the embodiment of the present invention is even more preferable. That is, for example, 2-fold concentration is preferable, 10-fold concentration is more preferable, 100-fold concentration is more preferable, and 1000-fold concentration is still more preferable.

The rAAV formulation has a ratio of infectious rAAV particles to infectious helper virus particles of at least about 10 ² : 1; preferably at least about 10 ⁴ : 1; more preferably at least about 10 ⁶ : 1; even more preferably at least about A helper virus is said to be “substantially free” when it is 10 ⁸ : 1. The formulation also contains an equal amount of helper virus protein (ie, protein if present as a result of such a level of helper virus if the helper virus particle impurities were present in a disrupted form). It is preferably not included. Viral and / or cellular protein contamination is generally observed as the presence of a Coomassie-stained band on an SDS gel (eg, the appearance of bands other than those corresponding to AAV capsid proteins VP1, VP2, and VP3).

  A “host cell” includes an individual cell or cell culture that can be or has been a recipient for a vector or for the incorporation of polynucleotides and / or proteins. A host cell includes the progeny of a single host cell, which progeny is either naturally, accidentally or intentionally mutagenized with the original parent cell (in form or in the genome of the total DNA complement). It need not be completely identical. Host cells include cells that have been transfected in vivo with a polynucleotide of the invention.

  “Transformation” or “transfection” refers to the insertion of an endogenous polynucleotide into a host cell, regardless of the method used for the insertion, eg, lipofection, transduction, infection or electroporation. Endogenous polynucleotides may be maintained as non-integrating vectors, such as plasmids, or may be integrated into the host cell genome.

  An “individual” or “subject” refers to a mammal, which includes livestock animals, sport animals, rodents, primates, pets, horses, dogs, cats, and even humans.

  An “effective amount” is an amount sufficient to produce a beneficial or desired clinical result. An effective amount may be administered in one or more administrations. In the present invention, an “effective amount” is an amount that achieves any of the following: a decrease in TNF level; a decrease in inflammatory response; and / or a reduction, reduction, stabilization, reversal, progression of disease state, Slow or delayed.

  As used herein, “in combination” refers to administration of one treatment modality in addition to another treatment modality, eg, delivery of rAAV to the same subject in addition to administration of a TNF antagonist to the subject. To do, or to administer two different rAAV vectors to the same subject. Thus, “in combination” refers to administering one treatment modality before, during or after delivery of another treatment modality to a subject.

  As used herein, “treatment” is an approach for obtaining beneficial or desired clinical results. The beneficial or desired clinical results in the present invention include amelioration of symptoms, attenuation of disease range, stabilization of disease state (ie, prevention of deterioration), prevention of disease expansion, delay or slowing of disease progression, and remission of disease state. Or mitigation and remission (partial or complete), whether they are detectable or impossible. “Treatment” can also mean improving, accelerating or expanding the healing and regeneration of bone tissue or wound site to which an effective amount of a rAAV vector comprising a polynucleotide encoding a TNF antagonist is administered. For example, treatment of an individual may be associated with a condition associated with elevated TNF levels (congenital or induced genetic defects, infection with viruses, bacteria or parasites, neoplastic or dysplastic diseases, or immune systems such as autoimmunity). May be performed to reduce or limit (including but not limited to dysfunction). Treatment may be either prophylactic or therapeutic; ie, before or after initiation of contact with a pathological event or etiological agent.

  A “biological sample” encompasses various types of samples obtained from an individual and can be used in a diagnostic or monitoring assay. This definition includes blood and other liquid samples from organisms, solid tissue samples (eg, biopsy specimens or tissue cultures or cells derived therefrom), and their progeny. This definition also includes samples that have been manipulated in any way after collection, such as by treatment with reagents, solubilization, or concentration of specific components such as proteins or polynucleotides. The term “biological sample” includes clinical samples and also includes cultured cells, cell supernatants, cell lysates, serum, plasma, biological fluids and tissue samples.

  “Alleviating” a disease means that the range of disease states and / or undesirable clinical signs are reduced and / or the time course of progression is slowed or reduced compared to not administering the rAAV vector of the invention. It means being extended.

General Techniques In practicing the present invention, conventional techniques of molecular biology, virology, animal cell culture and biochemistry, which are within the skill of the art, are used unless otherwise stated. Such techniques are explained fully in the literature. For example, “Molecular Cloning: A Laboratory Manual”, Second Edition (Sambrook, Fritsch & Maniatis, 1989); “Animal Cell Culture” (R. I. Fresney, ed., 1987); J. Miller & MP Calos, eds., 1987); “Current Protocols in Molecular Biology” (FM Ausubel, et al., Eds., 1987); “Current Protocols in Protein Sc” John E Coligan, et al.ed .Wiley and Sons, 1995); and "Protein Purification: Principles and Practice" (Robert K.Scopes, Springer-Verlag, 1994), incorporated herein by reference.

RAAV Vectors for Delivery of TNF Antagonists The present invention provides a recombinant AAV (rAAV) vector for reducing the level of TNF at a periodontal, wound or bone healing site in a subject. In general, these rAAV vectors contain a polynucleotide encoding a TNF antagonist. In some embodiments, the TNF antagonist is a TNFR or TNFR polypeptide, including derivatives having their biological activity. In the present invention, the preferred TNFR is derived from p75 TNFR.

  The rAAV vectors of the present invention comprise a heterologous (ie non-AVV) polynucleotide of interest in place of the AAV rep and / or cap genes that normally comprise the majority of the AAV genome. However, as in the wild type AAV genome, the heterologous polynucleotide is preferably flanked by at least one, more preferably two AVV inverted terminal repeats (ITRs). Mutations in which the rAAV construct is flanked by only a single (usually modified) ITR have been described in the art and can be used in the context of the present invention.

TNF antagonist In the present invention, a TNF antagonist is supplied to an individual, preferably a mammal, most preferably a human, as an expression product of a polynucleotide encoding the TNF antagonist. The polynucleotide encoding the TNF antagonist is delivered to the mammal in the form of an rAAV vector. As defined, such TNF antagonists can be any polypeptide that binds to TNF, including but not limited to TNFR polypeptides and anti-TNF antibodies.

  The TNF antagonist is secreted by the cell to which the rAAV vector is administered; preferably the TNF antagonist is soluble (ie does not bind to the cell). For example, soluble TNF antagonists lack a transmembrane region and are secreted from the cell. Techniques for identifying and removing polynucleotide sequences encoding transmembrane domains are known in the art.

  Preferably, the TNF antagonist is a TNFR polypeptide. The TNFR polypeptide may be intact TNFR (preferably derived from the same species to which rAAV is administered) or a suitable fragment of TNFR. US Pat. No. 5,605,690 provides examples of TNFR polypeptides (including soluble TNFR polypeptides) suitable for use in the present invention. Preferably the TNFR polypeptide comprises the extracellular domain of TNFR. More preferably, the TNFR polypeptide is a fusion polypeptide comprising the extracellular domain of TNFR linked to the constant domain of an immunoglobulin molecule that does not fix complement in the host to which the molecule was delivered. This constant domain may be natural. Alternatively, the constant domain may be modified or mutated so that it does not bind complement or activate the complement system. Preferably, the TNFR polypeptide is a fusion polypeptide comprising the extracellular domain of p75 TNFR linked to the constant domain of an IgG1 or IgG3 molecule. Preferably, when intended for human administration, the Ig used for the fusion protein is human, preferably human IgG1 or IgG3.

  Monovalent and multivalent forms of TNFR polypeptides may be used in the present invention. Multivalent forms of TNFR polypeptides possess multiple TNF binding sites. Multivalent forms of TNFR polypeptides may be encoded in the rAAV vector, eg, via repeated ligation of a polynucleotide encoding a TNF binding domain, each repeat separated by a linker region. Preferably, the TNFR of the present invention is a divalent or dimeric form of TNFR. See, for example, US Pat. No. 5,605,690 and Mohler, et al. 1993, J. et al. Immunol. 151: 1548-1561, a chimeric antibody polypeptide having a TNFR extracellular domain substituted in place of either an immunoglobulin heavy chain or light chain variable domain or both is a TNFR polypeptide of the invention I will provide a. In general, when such a chimeric TNF: antibody polypeptide is produced by a cell, it forms a bivalent molecule through disulfide bonds between immunoglobulin domains. Such a chimeric TNF: antibody polypeptide is referred to as TNFR: Fc.

  In some embodiments, the TNF antagonist is a TNFR polypeptide construct sTNFR (p75): Fc. The polypeptide sequence of sTNFR (p75): Fc is illustrated in FIG. 5 herein and FIG. 1 of US Pat. No. 5,637,540. The coding sequence for this TNF antagonist is present in the plasmid pCAVDHFRhuTNFRFc as described in US Pat. Nos. 5,605,690 and 5,637,540. Any polynucleotide encoding this sTNFR (p75): Fc polypeptide is suitable for use in the present invention. The polynucleotide sequence encoding sTNFR (p75): Fc is illustrated in FIG. 2 of US Pat. No. 5,637,540.

  In the present invention, additional TNF polypeptide sequences include, but are not limited to, those shown in FIGS. 2 and 3 of US Pat. No. 5,395,760.

  A polynucleotide encoding a TNFR polypeptide can be formed from a TNFR polynucleotide sequence known in the art using methods known in the art. In the present invention, preferred polynucleotide sequences encoding TNFR polypeptides include the TNFR polynucleotide sequences described in US Pat. Nos. 5,395,760 and 5,605,690 and GenBank accession number M32315 (human TNFR) and M59378 (murine TNFRI). Polynucleotides suitable for use in the present invention can be synthesized using standard synthetic and recombinant methods.

  Methods for assessing the activity of TNF antagonists are as known in the art and are exemplified herein. For example, the activity of a TNF antagonist may be assessed by a cell-based competitive binding assay. In such an assay, radiolabeled TNF is mixed with serially diluted TNF antagonists and cells expressing cell membrane bound TNFR. A portion of the suspension is centrifuged to separate free and bound TNF and the amount of radioactivity in the free and bound fractions is measured. The activity of a TNF antagonist is assessed by inhibiting TNF binding to cells in the presence of the TNF antagonist.

  As another example, TNF antagonists are analyzed for their ability to neutralize TNF activity in vitro by biological assays using cells sensitive to the cytotoxic activity of TNF as target cells, such as L929 cells. (See, eg, US Pat. No. 5,637,540). In such assays, cell lysis is examined after administering various amounts of TNF antagonist to target cells cultured with TNF. The activity of a TNF antagonist is assessed by a decrease in TNF-induced target cell lysis in the presence of the TNF antagonist.

  The present invention also provides an rAAV vector comprising a polynucleotide encoding an interleukin 1 (IL-1) antagonist. Cytokine IL-1 has been implicated as a mediator that is the main axis in periodontitis (Delima, et al., J Infect Dis, 186, 511-516, 2002). In RA, IL-1 is thought to be involved in inflammatory cell infiltration and cartilage degradation in affected joints. Clinical trials using IL-1 antagonists in patients with RA have shown that blocking IL-1 activity may result in remission of RA symptoms (Camion, et al., 1996, Arthritis Rheum. 39: 1092-1101; Bresnihan, et al., 1996, Arthritis Rheum. 39: S73). In a murine arthritis model, combined administration of anti-TNFα / anti-IL-1 resulted in both attenuation of inflammation and attenuation of articular cartilage damage (Kuiper, et al., 1998, Cytokine 10: 690-702).

  The present invention provides a rAAV vector comprising a TNF antagonist (eg, sTNFR (p75): Fc) and a polynucleotide encoding an IL-1 antagonist (alternatively, the rAAV vector is a polynucleotide encoding a TNF antagonist and an IL-1 antagonist. Including nucleotides). The present invention also provides rAAV vectors comprising a polynucleotide encoding an IL-1 antagonist. Preferably, the IL-1 antagonist is an IL-1 receptor (IL-1R) or IL-1R polypeptide (having their biological activity) that exhibits the desired biological activity (ie, binding to IL-1) Derivatives). In some embodiments, IL-1R is derived from IL-1R type II. IL-1R polypeptide sequences include those shown in FIG. 3 of US Pat. No. 5,637,540, and sequences described in IL-1R GenBank Accession No. U74649 and US Pat. No. 5,350,683. Including, but not limited to. Any polynucleotide encoding an IL-1R polypeptide is suitable for use in the present invention. For example, a polynucleotide sequence encoding a preferred IL-1R polypeptide is illustrated in FIG. 3 of US Pat. No. 5,637,540. Polynucleotides suitable for use in the present invention can be synthesized using standard synthetic and recombinant methods.

  Methods for assessing the activity of IL-1 antagonists are known in the art. For example, the activity of an IL-1 antagonist may be assessed by a cell-based competitive binding assay, as described herein for TNF antagonists. As another example, the activity of an IL-1 antagonist may be assessed for its ability to neutralize IL-1 activity in vitro in an IL-1 biological assay. Such an assay uses a cell line (eg, EL-4 NOB-1) that produces interleukin 2 (IL-2) in response to IL-1 administration. This IL-1 responsive cell line is used in combination with an IL-2 sensitive cell line (eg, CTLL-2). The proliferation of IL-2 sensitive cell lines is dependent on IL-1 responsive cell lines producing IL-2 and is therefore used as a measure of IL-1 stimulation of IL-1 responsive cell lines. The activity of an IL-1 antagonist is believed to be assessed by its ability to neutralize IL-1 activity in such an IL-1 biological assay (Gearing, et al., 1991, J. Am. Immunol. Methods 99: 7-11; Kuiper, et al., 1998).

  In some embodiments, the vectors of the invention are encapsidated within rAAV virions. Accordingly, the present invention includes rAAV virions (recombinant because they contain a recombinant polynucleotide) comprising any of the vectors described herein. Methods for producing such particles are described below.

  The present invention also provides compositions containing any of the rAAV vectors described herein (and / or rAAV viral particles comprising rAAV vectors). These compositions are particularly useful for administration to individuals who may benefit from reduced levels of TNF.

  In general, the compositions of the invention used in reducing the level of TNF comprise an effective amount of a rAAV vector encoding a TNF antagonist, preferably in a pharmaceutically acceptable excipient. As is well known in the art, a pharmaceutically acceptable excipient facilitates administration of a pharmacologically effective substance, and as a liquid solution or suspension, as an emulsion, or into a liquid prior to use. Delivered as a solid form suitable for dissolving or suspending or as a gel, gel matrix or lyophilized composition that can be used to coat or fill solid delivery devices such as dental trays, mouth guards or bone pins It is a relatively inert substance that can. For example, the excipient can provide shape or consistency, or can be a diluent. Suitable excipients include, but are not limited to, stabilizers, wetting and emulsifying agents, osmotic pressure adjusting salts, encapsulating agents, buffering agents, and microspheres such as polyglycol lactic acid. For excipients and formulations for parenteral and enteral drug delivery, see Remington's Pharmaceutical Sciences 19th Ed. Mack Publishing (1995).

  In general, these rAAV compositions are formulated for administration by local injection (eg, intramuscular or periodontal). Accordingly, these compositions are preferably combined with a pharmaceutically acceptable vehicle such as saline, Ringer's balanced salt solution (pH 7.4), dextrose solution, and the like. Although not required, the pharmaceutical composition may optionally be supplied in a unit dosage form suitable for precise dosage administration.

  The present invention also includes any of the above vectors (or compositions comprising such vectors) for use in the treatment of periodontitis and TNF-related buccal diseases such as bone and wound healing disorders. The present invention also includes any of the vectors (or compositions comprising the vectors) used in reducing the level of TNF in a site such as the periodontal, bone or wound healing site of an individual. The present invention further provides the use of any of the vectors (or compositions comprising the vectors) in the manufacture of a medicament for the treatment of TNF-related disorders or conditions such as periodontitis or bone or wound healing disorders. To do. The present invention also provides the use of any of the vectors (or compositions comprising the vectors) in the manufacture of a medicament that reduces the level of TNF activity at a site of injury, such as the periodontal, wound or bone healing site of an individual. To do.

Host cells containing the rAAV of the present invention Host cells containing the rAAV vectors are used to generate the rAAV vectors described herein. Eukaryotic host cells include yeast, insect, avian, plant and mammalian cells. Preferably the host cell is a mammal. For example, host cells include HeLa and 293 cells, both of human origin and readily available.

  The development of host cells capable of expressing rAAV vector sequences provides an established source of material that is expressed at a reliable level. Methods and compositions for introducing an rAAV vector into a host cell and determining whether the host cell contains an rAAV vector are discussed in the sections below, but have already been described in the art and are widely available. it can.

  Included in these embodiments and discussed in the following sections are “producer cells” that are used as the basis for producing so-called packaged rAAV vectors.

Preparation of rAAV of the Invention The rAAV vectors of the invention may be prepared using standard methods in the art. Since various serotypes are functionally and structurally related even at the genetic level, any serotype of adeno-associated virus is suitable (eg, Blacklow, pp. 165-174 of “Parvoviruses and Human Diseases”). "J. Pat Patson, ed. (1988); and Rose, Comprehensive Virology 3: 1, 1974). All AAV serotypes apparently show similar replication characteristics mediated by homologous rep genes; generally all carry three related capsid proteins, such as those expressed in AAV2. ing. The degree of association is further suggested by heteroduplex analysis, which indicates extensive cross-hybridization between serotypes across the genome length; and the presence of similar self-annealing segments at the ends corresponding to ITRs. It has been revealed. Similar infectivity patterns also suggest that the replication function of each serotype is under similar regulatory control. Of the various AAV serotypes, AAV2 is most commonly used. For a general review of the biological and genetic characteristics of AAV, see, eg, Carter, “Handbook of Parvoviruses”, Vol. I, pp. 169-228 (1989); and Berns, "Virology", pp. 1743-1764, Raven Press, (1990). The general principles of rAAV vector construction are known in the art. See, for example, Carter, 1992, Current Opinion in Biotechnology, 3: 533-539; and Muzyczka, 1992, Curr. Top. Microbiol. Immunol. 158: 97-129.

  As described above, the rAAV vector of the present invention comprises a heterologous polynucleotide encoding a TNF antagonist. The rAAV vector may also encode additional polypeptides, such as IL-1 receptor type II. Alternatively, the rAAV vector may also contain a heterologous polynucleotide encoding an IL-1 antagonist such as IL-1R. Such heterologous polynucleotides will generally be of sufficient length to provide the coding sequence and the desired function. Due to encapsidation within the AAV particle, the heterologous polynucleotide is preferably less than about 5 kb, but other serotypes and / or modifications are used to package larger sequences into the AAV virus particle. There is. For example, a preferred polynucleotide encodes TNFR: Fc as shown in FIG. 5 and is approximately 1.5 kb in length.

  Since transcription of the heterologous polynucleotide is desired at the intended target sequence, it may be either as such or heterologous depending on the desired level and / or specificity of transcription within the target cell, eg, as is known in the art. Operably linked to a promoter and / or enhancer. There are various types of promoters and enhancers suitable for use in this situation. For example, Feldhaus (US patent application Ser. No. 09 / 171,759, filed Oct. 20, 1998) describes a modified ITR that includes a promoter that regulates expression from rAAV. A constitutive promoter is preferred when it provides a continuous level of gene transcription and it is desired that the therapeutic polynucleotide be essentially continuously expressed. Inducible or regulatable promoters generally exhibit low activity in the absence of inducers and are upregulated in the presence of inducers. These promoters may be preferred when expression is desired only at specific times or at specific locations, or when it is desirable to use an inducing agent to regulate the level of expression. Promoters and enhancers can also be tissue specific, ie they are only active in certain cell types, probably due to genetic regulatory elements that are inherently present in these cells. Such tissue specific promoters and enhancers are known in the art. Illustratively, examples of tissue specific promoters include various myosin promoters for expression in muscle. Another example of a tissue-specific promoter and enhancer is that of a cellular and / or tissue type regulatory element present in periodontal tissue.

  Preferred inducible or regulated promoters and / or enhancers include those that are physiologically responsive, such as those that are responsive to inflammatory signals and / or disease states. For example, use of promoters and / or enhancers that are activated in response to mediators that induce inflammation, such as those derived from pro-inflammatory cytokine genes (eg, TNFα, IL-1β and IFNγ) may It is thought to result in the expression of TNF antagonists (Varley, et al., 1998, Mol. Med. Today 4: 445-451). The TNFα promoter region is approximately 1.2 kb and the sequence is described in Takashiba, et al. 1993, Gene, 131: 307-308.

  Further representative examples of promoters include SV40 late promoter from simian virus 40, baculovirus polyhedron enhancer / promoter element, herpes simplex virus thymidine kinase (HSVtk), immediate early promoter from cytomegalovirus (CMV), And there are various retroviral promoters including, for example, LTR elements. Additional inducible promoters include heavy metal inducible promoters (eg, mouse mammary tumor virus (mMTV) promoter or various growth hormone promoters) and promoters from T7 phage that are active in the presence of T17 RNA polymerase. Many other types of promoters are known and are generally available in the art, and the sequences of many such promoters are available in sequence databases such as the GenBnk database.

  Since translation is desired in the intended target cell, the heterologous polynucleotide encoding the TNF antagonist also preferably includes regulatory elements that facilitate translation (eg, ribosome binding sites, ie, “RBS” and polyadenylation signals). become. Thus, a heterologous polynucleotide will generally include at least one coding region operably linked to a suitable promoter, and may include, for example, an operably linked enhancer, ribosome binding site, and polyA signal. A heterologous polynucleotide may contain one coding region under the control of the same or different promoters, or it may contain multiple coding regions. The entire unit containing a combination of control elements and coding regions is often referred to as an expression cassette.

  Heterologous polynucleotides encoding TNF antagonists are integrated into the AAV genomic coding region by recombinant techniques, or preferably in place of this region (ie, in place of the AAV rep and cap genes), but generally AAV ITRs Is flanked on either side. This is because the ITR appears directly in juxtaposition both upstream and downstream of the coding sequence, preferably (but not necessarily) without any intervening sequences from AAV, so that it has replication competent AAV ("RCA ") This means that the possibility of recombination to regenerate the genome is reduced. Recent observations suggest that one ITR is sufficient to perform the functions associated with an arrangement that typically consists of two ITRs (US Pat. No. 5,478,745), ie , Vector constructs having only one ITR can be used in combination with the packaging and production methods described herein. The resulting rAAV vector is said to be “deficient” in AAV function when a particular AAV coding sequence is deleted from the vector.

  Given the limited size of the relative encapsidation of different AAV genomes, when inserting large heterologous polynucleotides into the genome, a portion of the AAV genome must be removed, particularly packaging genes. One or more of the may be removed. Removal of one or more AAV genes is desirable in any case to reduce the likelihood of forming RCA. Thus, the rep, cap or both coding or promoter sequences are preferably removed because the functions provided by these genes can be provided in trans.

  rAAV vectors are provided in various forms, such as bacterial plasmids, AAV particles, liposomes, or any combination thereof. In other embodiments, the rAAV vector sequence is provided in a eukaryotic cell that is transfected with the rAAV vector.

When rAAV is used in the form of packaged rAAV particles, there are at least three features of rAAV viral formulations that are desirable for use in gene transfer. First, the rAAV virus is preferably formed at a titer high enough to transduce an effective proportion of cells in the target tissue. In general, gene transfer in vivo requires a large number of rAAV virions. For example, some therapeutic agents may require more than 10 ⁸ particles. Secondly, the rAAV viral preparation should be essentially free of replicating AAV (ie, a phenotype wild type AAV capable of replicating in the presence of helper virus or helper virus function). Third, the rAAV virus formulation is essentially free of other viruses as a whole (eg, helper viruses used to produce AAV) and helper viruses and cellular proteins, and other components such as lipids and carbohydrates. Is necessary to minimize or eliminate the risk of generating an immune response in gene transfer. This latter point is that AAV is a “helper-dependent” virus and is co-infected with helper virus (generally adenovirus) or other in order to be effectively replicated and packaged during the AAV production process It is particularly important in AAV because of the need to provide helper virus function; furthermore, as noted above, adenoviruses have been observed to generate host immune responses in gene transfer applications (eg, Le Byrnes, et al., 1995; Neuroscience, 66: 1015; McCoy, et al., 1995, Human Gene Therapy, 6: 1553; and Barr, et al., 1995, Gene Therapy, 2 : 151).

  When the rAAV vector is packaged within an AAV particle, one or more packages that together encode the functions required for various missing rep and / or cap gene products in order to replicate and package the rAAV vector Supplements the lost function with the gene. The packaging gene or gene cassette is preferably not flanked by the AAV ITR and preferably does not share any substantial homology with the rAAV genome. That is, it is desirable to avoid duplication of two polynucleotide sequences in order to minimize homologous recombination during replication between the vector sequence and the individually provided packaging gene. The level of homology and the corresponding number of recombination increases with increasing length of homologous sequences, as well as their shared level of identity. The level of homology that will present a problem in a given system can be theoretically measured and confirmed experimentally as is known in the art. However, in general, recombination is less than about 25 nucleotide sequences when the overlapping sequence is at least 80% identical in full length, or less than about 50 nucleotide sequences when at least 70% identical in full length, Can be substantially reduced or eliminated. Of course, a lower level of homology is preferred because it further reduces the possibility of recombination. Even if there is no overlapping homology, the number of RCA generations remains somewhat. A further reduction in the number of RCA occurrences (eg, due to heterologous recombination) can be achieved by “dividing” the replication and encapsidation function of AAV, as described by Allen et al. In US Pat. No. 6,541,258. can get.

  The rAAV vector construct and the complementary packaging gene construct can be implemented in several different forms in the present invention. Viral particles, plasmids and stably transformed host cells can all be used to introduce such constructs transiently or stably into packaging cells.

  That is, a variety of different genetically modified cells can be used in the present invention. Illustratively, mammalian host cells may be used with at least one intact copy of a stably integrated rAAV vector. An AAV packaging plasmid containing at least an AAV rep gene operably linked to a promoter can be used to provide replication function (as described in US Pat. No. 5,658,776). Alternatively, a stable mammalian cell line having an AAV rep gene operably linked to a promoter can also be used to provide replication functions (eg, US Pat. No. 5,837,484 of Trempe, et al .; Burstein, et al., WO 98/27207; and Johnson, et al., US Pat. No. 5,658,785). As described above, AAV cap genes that provide encapsidated proteins can be provided with or separately from the AAV rep gene (see, eg, the applications and patents cited above, and Allen, et al. (WO 96/17947). reference). Other combinations are possible.

  As known in the art, and as illustrated in the references cited above and in the examples below, the genetic material is transferred to cells (eg, mammalian “producer” cells used to produce AAV). Such cells can be introduced using any of a variety of means for transforming or transducing cells. Illustratively, such techniques include transfection with bacterial plasmids, infection with viral vectors, electroporation, calcium phosphate precipitation, and introduction using any of a variety of lipid-based compositions (often referred to as “lipofection”). Process). Methods and compositions for implementing these techniques have been described in the art and are widely available.

  Selection of suitably modified cells may be performed by any technique in the art. For example, a polynucleotide sequence used for cell modification may be introduced or operably linked to one or more detection or selection markers known in the art. Illustratively, drug resistance genes can be used as selectable markers. The drug resistant cells can then be fished and cultured before testing for expression of the desired sequence (ie production of the heterologous polynucleotide). Testing for acquisition, localization and / or maintenance of the introduced polynucleotide should be performed using DNA hybridization based techniques (eg, Southern blotting and other techniques known in the art). Can do. Testing for expression can be easily performed by Northern analysis of RNA extracted from genetically modified cells or by indirect immunofluorescence of the relevant gene product. Testing and confirmation of packaging ability and efficiency is made possible by introducing the remaining functional components of AAV and helper virus into the cells to test for the production of AAV particles. If the cells are to be inheritably modified by multiple polynucleotide constructs, it is generally more convenient (but not essential) to introduce them individually into the cells and verify each procedure sequentially.

  The rAAV viral vector is a transient transfection technique described in US Pat. Nos. 6,001,650 and 6,258,595; a stable cell line technique described in WO95 / 34670; or WO96 / 13589. Known in the art, including the use of the shuttle vector approach comprising the adenoviral hybrid vector described in or the rep-cap cell line described in WO 99/15685 with respect to the adenovirus-AAV hybrid vector system (Ad hybrid system). It can be produced by a method. An rAAV vector production system generally has three common elements: 1) host cells that allow replication, including standard host cells known in the art; 2) helper virus function; and 3 ) Has a transpackaging rep-cap construct. Examples of host cells used in the rAAV vector production system include 293-A, 293-S (obtained from BioReliance), VERO and Hela cell lines applicable to the three vector production systems described herein. Helper virus function may be provided by a wild type adenovirus type 5 virus, an adenovirus hybrid vector system, or in a transfection production system when used in the production of a stable cell line. When used, it may be provided by the plasmid pAd helper 4.1 which expresses the adenovirus type 5 (Ad5) E2a, E4-orf6 and VA genes.

  For AAV2 serotype capsid vectors, a rep-cap transpackaging cell line is described in WO 95/34670. For AAV-5 capsids, the AAV2 5 ′ and 3 ′ ITR pseudotype constructs and the AAV-2 rep gene and AAV5 cap sequences under the control of the AAV2 p5 promoter rep-cap cell line (AAV-2 / AAV, respectively) A transpackaging rep-cap plasmid containing the pseudotyped transpackaging construct of -5) is formed as described (Sandalon, et al., J. Virol. 78: 12355-12365 (2004)). For the transfection system, the p5 promoter of the rep-cap (each AAV2 / 5) pseudo-type transpackaging construct is replaced with a minimal heat shock promoter consisting essentially of a TATA box.

  The stable cell lines used for the production of rAAV viral vectors can be transfected with the above cell lines and can be propagated repeatedly, the rep-cap packaging construct as well as the stably integrated AAV ITR serotype expression plasmid. Can be generated by screening to obtain a stable cell line containing. rAAV vectors are produced and purified by any method known in the art and described in WO99 / 11764 and WO00 / 14205.

  Ad hybrid production of rAAV vectors can be performed using the rep-cap cell line described in WO99 / 15685 as described in WO96 / 13598. First, T.A. C. The system developed by He et al. And disclosed in US Pat. No. 5,922,576 creates a recombinant adenovirus / AAV hybrid (Ad / AAV hybrid). This approach is competent E.I. Two plasmid vector systems (transfer or shuttle vector) that cause bacterial recombination in E. coli resulting in a recombinant Ad / AAV hybrid plasmid that is used to derive Ad / AAV hybrid virus plaques for the generation of virus stocks And adenovirus genome-containing vectors).

  Optionally, the rAAV virus formulations can be further processed to concentrate rAAV particles, deplete helper virus particles, or otherwise make them suitable for administration to a subject. For representative techniques, see Atkinson, et al. (WO99 / 11764, WO00 / 14205 and US Pat. No. 6,566,118). Purification techniques may include isodensity gradient centrifugation and chromatographic techniques. Reduction of infectious helper virus activity may include inactivation by heat treatment or pH treatment, as is known in the art. Other processes may include concentration, filtration, diafiltration, or mixing with suitable buffers or pharmaceutical excipients. The formulation can be divided into unit doses or multi-dose aliquots for sale, but these retain the essential characteristics of the batch, such as uniformity of antigen and gene content and relative proportions of contaminating helper viruses .

  Various methods for measuring the infectious titer of a viral formulation are known in the art. For example, one method of titration includes Atkinson, et al. There is a high-throughput titration test as shown in (WO99 / 11764). Viral titers measured by this rapid and quantitative method are in good agreement with those measured by more traditional techniques. However, this high-throughput method is also suitable for many other uses, such as screening for cell lines that allow or do not allow virus replication and infection, since they allow simultaneous processing and analysis of many virus replication reactions.

Methods of Using the rAAV of the Invention The present invention provides methods for treating or preventing a TNF-related disorder or condition in a subject. Examples of such disorders or conditions include bone loss, wound healing and bone healing disorders, bone allograft or autograft healing disorders, and periodontitis. The present invention provides methods wherein administration of the rAAV vectors described herein is used to treat or prevent a TNF-related disorder or condition.

  The present invention also provides a method wherein administration of the rAAV vectors described herein is used to reduce the level of TNF at the site of injury (eg, periodontal, wound and bone graft sites) in the subject. provide. Such a method may be particularly beneficial for individuals having a TNF-related disorder as described herein. It is understood that the TNF level is reduced relative to the TNF level of the subject prior to administration of the rAAV encoding the TNF antagonist or compared to the TNF level of an individual not receiving the rAAV encoding the TNF antagonist. It is understood that TNF level refers to the level of free (uncomplexed or unbound) TNF or active TNF. A method for detecting the level of TNF is described below.

  For example, the treatments described herein may be beneficial for bone grafts in an individual. Treatment may mean prolonging survival of the bone graft relative to expected survival if not receiving treatment.

  The invention also provides a method wherein administration of a rAAV vector (or a composition comprising a rAAV vector) described herein is used to reduce an inflammatory response at a site of injury (eg, a periodontal site) in a subject. To do. It is understood that the inflammatory response is reduced compared to the inflammatory response of the subject prior to administration of the rAAV encoding the TNF antagonist, or compared to the inflammatory response of an individual not administered the rAAV encoding the TNF antagonist. Is done.

  In some embodiments, the rAAV vector (or composition comprising the rAAV vector) is delivered (eg, by intramuscular injection or between adjacent teeth) to a lesion site, such as a periodontal region of a mammal. Thus, the raw material for the TNF antagonist is supplied at the site of inflammation. In some embodiments, the rAAV vector comprises a polynucleotide encoding sTNFR (p75): Fc.

  In some embodiments, the rAAV vector is administered in combination with administration of another agent that reduces inflammation at the site (eg, periodontal site). In some embodiments, the agent is a TNF antagonist, such as TNFR or an anti-TNF antibody. In some embodiments, the other agent administered in combination with the rAAV vector is an IL-1 antagonist. Other agents, such as TNF antagonists or IL-1 antagonists, preferably included in the composition together with a physiologically acceptable carrier, excipient or diluent may be administered by any suitable technique.

  In some embodiments, at least two different rAAV vectors (or a composition comprising at least two different rAAV vectors) deliver a source of TNF antagonist and IL-1 antagonist to the periodontal region of the mammal at the site of inflammation. To be administered. Preferably, one of the rAAV vectors includes a polynucleotide encoding TNFR, and the other of the rAAV vectors includes a polynucleotide encoding IL-1R. In these two different rAAV vectors, the heterologous polynucleotide is a transcription promoter and / or enhancer that is active under similar conditions, or a transcription promoter that is active under different conditions, eg, independently regulated, and In some cases, it may be operably coupled to an enhancer. In various dosage adjustments, two different rAAV vectors (ie, those containing a polynucleotide encoding TNFR and those containing a polynucleotide encoding IL-1R) may be the same or different times at the same time or at different times. And / or in the same or different amounts.

  It will be appreciated that for any of the methods described above, one or more of the one or more rAAV vectors may be administered. For example, as described above, a vector encoding a TNF antagonist, such as a TNF receptor (most preferably sTNFR (p75): Fc) may be administered. Alternatively, additional vectors encoding IL-1 antagonists such as IL-1 receptor polypeptides may be administered. Alternatively, a single vector encoding both a TNF antagonist and an IL-1 antagonist may be administered. This single vector may have the coding sequence under the control of the same or different transcriptional regulatory elements. If multiple vectors are used, they may be administered simultaneously or at different times and / or at the same or different times.

  Furthermore, for any of the methods described above, in some embodiments, the individual to be administered the rAAV vector will have cells containing the rAAV vector (after administration), most preferably the rAAV vector is intracellular. You will have cells that integrate into the genome. Stable integration of rAAV is a significant advantage since it allows for sustained expression over episomal vectors. Thus, in a preferred embodiment, the cells in the individual (ie, at least one cell) will contain stably integrated rAAV. In other words, for any of the methods described above, administration of rAAV results in integration of rAAV into the intracellular genome (although it is not necessary for all rAAV vectors to be integrated, as will be appreciated by those skilled in the art). Methods for measuring and / or distinguishing between embedded and non-embedded types, such as the Southern detection method, are well known in the art.

  Administration of the rAAV vector (preferably packaged as AAV particles) to the periodontal site may be via any of several routes. One mode of administration is via intramuscular delivery. Intramuscular delivery of rAAV vectors can reduce the level of TNF both in the tissue and inter-tissue space near the injection site and in the circulatory system. Another mode of administration of the rAAV compositions of the invention is by injecting the composition directly into a tissue or anatomical site. Administration is performed, for example, by injecting the composition into the area between adjacent teeth.

  An effective amount of rAAV (preferably in the form of AAV particles) is administered depending on the purpose of the treatment. An effective amount may be given in a single dose or in divided doses. If a low percentage of transduction can achieve a therapeutic effect, the therapeutic objective is generally to meet or exceed this level of transduction. In some cases, this level of transduction can be achieved by transduction of only about 1-5% of the target details, but more typically about 20% of cells of the desired tissue type, usually at least about 50%, preferably Is at least 80%, more preferably at least about 95%, even more preferably at least about 99% of cells of the desired tissue type.

As a guide, the number of rAAV particles administered per injection is generally from about 1 × 10 ⁶ to about 1 × 10 ¹⁴ , preferably from about 1 × 10 ⁷ to about 1 × 10 ¹³ , and more The number is preferably about 1 × 10 ⁹ to about 1 × 10 ^12, more preferably about 1 × 10 ¹¹ .

  The present invention also provides methods in which an ex vivo approach to delivering a polynucleotide to a mammal is used in administering the rAAV vectors described herein. Such methods and techniques are known in the art. See, for example, US Pat. No. 5,399,346. Generally, cells are transduced in vitro with an rAAV vector, and the transduced cells are transduced into a mammal, eg, a joint. Suitable cells are known to those skilled in the art, and these cells include autologous cells such as stem cells.

  The effectiveness of rAAV delivery can be monitored by several criteria. For example, samples taken by biopsy or surgical removal (eg, gingival tissue samples) to detect in situ hybridization, PCR amplification using vector-specific probes, and / or rAAV DNA and / or rAAV mRNA May be analyzed by protection of RNAse. Further, for example, collected tissue and / or serum samples may be used using immunoassays including, but not limited to, ELISA, immunoblotting, immunoprecipitation, immunohistological examination and / or immunofluorescent cell counting, or A function-based biological assay based on TNF antagonist-mediated suppression of TNF activity can be used to monitor for the presence of a TNF antagonist encoded by rAAV. For example, if the TNF antagonist encoding rAAV is a TNFR polypeptide, the presence of the encoded TNFR in the collected sample is a function-based organism based on TNFR immunoassay or TNFR-mediated suppression of TNF killing of mouse L929 cells. Can be monitored by pharmacological assays. Examples of such tests are known in the art and are described, for example, in US Pat. No. 6,537,540.

  The effectiveness of rAAV delivery can also be monitored by periodontal tissue destruction and alveolar bone loss histomorphometry and radiographic analysis.

  The effectiveness of the methods described herein can be monitored, for example, by testing the relative levels of TNF in harvested tissue, joint fluid and / or serum following delivery of the rAAV vectors described herein. There is. Assays for assessing levels of TNF are known in the art and include TNF immunoassays (eg, immunoblot and / or immunoprecipitation tests), and TNF cytotoxicity. This includes but is not limited to cytotoxicity assays using sensitive cells. For example, Khabar, et al. , 1995, Immunol. Lett. 46: 107-110.

  Treated subjects may be monitored for clinical features associated with TNF-related oral buccal disease. For example, the subject may be monitored for a decrease in symptoms associated with inflammation. For example, after treating a subject's buccal disease using the methods of the present invention, the subject is evaluated for improvement in a number of clinical parameters including, but not limited to, edema, pain and tooth loss. There is a case.

  Selection of a particular composition, dosing regimen (ie, dose, timing and repeatability), and route of administration includes several different factors including, but not limited to, the subject's medical history, and the condition and characteristics of the subject to be treated. Varies depending on It is ultimately the responsibility of the attending physician to evaluate such features and design an appropriate treatment regimen. The specific dosing regimen may be determined by experience.

  The foregoing description provides, inter alia, compositions and methods for treating TNF-related buccal diseases to reduce TNF levels in mammals. It will be understood that various changes may be applied to these methods by those skilled in the art without departing from the spirit of the invention.

  The following examples serve as a further guide for those skilled in the art and are not intended to be limiting in any way.

Example 1: Experimental Periodontitis Disease Model Isolation of Liposaccharide (LPS) from Porphyromonas gingivalis strain W83 A procedure to isolate LPS from Porphyromonas gingivalis strain W83 has been previously reported. (Darveau & Hancock, 1983). In short, LPS extraction uses a modified Brucella broth medium specific for anaerobic bacteria, and P.P. gingivalis strain W83 was cultured. After this incubation process, each batch was centrifuged at 5,000 rpm for 30 minutes and the supernatant was discarded. These cells were resuspended in sterile water and then centrifuged. The final pellet was frozen at −20 ° C. until the required amount of bacteria for the entire experiment was obtained. In a three step procedure, liposaccharides (“LPS”) were extracted and isolated by sequentially treating bacteria with lysozyme, DNAse, RNAse and protease.

  The isolated LPS was put into SDS-PAGE, and the result is shown in FIG. The first 6 lanes are E.D. in SDS-PAGE. A serial dilution of E. coli LPS is shown, the last lane is P. coli with the appearance of general laddering. gingivalisLPS is shown. Arrow indicates isolated LPS.

Induction of alveolar bone loss in rats The following protocol using gingivalis endotoxin is described in E. E. coli endotoxin was used to fit from a previously reported model (Ramamurthy, et al., 2002). In short, LPS was injected every 3 weeks into the gingival adjacent interdental papilla region for 8 weeks. Experimental periodontitis was induced in adult male Sprague-Dawley rats (each weighing about 250 g) by delivering endotoxin of gingivalis W83 strain (Bramanti, et al., 1989) (10 μL of 1.0 mg / mL formulation). Induced. Injections were made between the first (M1), second (M2) and third (M3) molars of the maxilla and the M1 mesial side. This model was used to generate a more “chronic” disease that resulted in a significant amount of bone loss. The results are shown in FIGS. This method resulted in approximately 50% linear bone loss along the root, including destruction of the root surface cementum and associated periodontal ligament (PDL) by week 8. .

Digital X-ray analysis was performed to monitor the progression of the disease. Standardized intraoral radiographs of maxillary molars were taken at baseline and after 2, 4, 6 and 8 weeks. Previously reported (Braegger, et al., J Periodontal Res, 22, 227-229, 1987; Jeffcoat, J Periodontol, 63, 367-372, 1992) and recently adopted for use in rat teeth ( Taba Jr., et al., Implant Dent, 12, 252-258, 2003), by measuring the difference in radiographic density between the baseline radiograph and the corresponding radiograph at each time point. Densitometric image analysis (CADIA) was performed. In summary, radiographs were digitized using a scanner (UMAX Technologies, Texas, USA) in 600 dpi resolution, grayscale format. The experimental area of interest was defined in the baseline and the area adjacent to the bone crest of the final image. Subsequent radiographic images are aligned for geometric analysis with the baseline image for analysis. Quantitative changes in density between images were generated by a computer at selected pixel regions. The overall density change was determined by multiplying the average gray level change of all pixel areas by the area size in mm ² affected by the change. CADIA values were expressed as mean ± standard error as previously reported (Oates, et al., J Clin Periodontol, 29, 137-143, 2002).

The area showing changes in the bone crest was measured at both the mesial and distal surfaces of each test tooth in the baseline and subsequent images. The image selection tool of the image analysis software (Image Pro-Plus software ^™ (Media Cybernetics, Silver Spring, Md.)) Was used for the measurement of the target area. The dimensional change was determined in mm ² units by dividing from the measured value determined using.

  FIG. 2 shows LPS-mediated bone resorption by radiographic images of representative rat maxilla obtained from LPS injection (A9) and control (B) animals. The arrow in image A (LPS administration) It shows about 50% bone loss 8 weeks after local administration of gingivalisLPS. Image B (saline control) Eight weeks after topical administration of gingivalis LPS shows minimal to no changes in adjacent bone.

FIG. 3 shows the quantitative measurement of LPS-mediated bone loss in an experimental rat model of periodontitis. Digital subtraction radiography was performed to confirm the amount of alveolar bone resorption (mm ² ) that occurred over time. As shown in FIG. 3, induction of LPS disease resulted in a substantial loss (about 50%) of alveolar bone support.

  FIG. 4 shows LPS-mediated bone resorption by sagittal histological micrographs of the adjacent area in M1 to M2 of rat maxillary teeth. Hematoxylin and eosin staining was used for staining. Hematoxylin and eosin staining is a simple method for examining common histological features. In general, basophil nuclei, bacteria, calcium, etc. are stained “blue” by hematoxylin, whereas eosinophils, connective tissue and all other tissues are “red” by eosin. Backstained. As described above, the sterilized solution was administered to the control group (A), and P. gingivalis LPS was administered to the experimental group (B) for 8 weeks. A difference in bone level between the groups shown with a line is observed between groups A and B. Furthermore, in group B, in addition to the loss of alveolar bone support, significant destruction of root cementum and connective tissue connections was also observed.

Example 2 Generation of rAAV Vector Encoding TNFR: Fc Fusion Protein The AAV-TNFR: Fc construct used in the following examples is described in US Pat. No. 6, 537,540, previously described. Briefly, the extracellular domain (ECD) of rat p80 TNFR (type II) was isolated from rat spleen cDNA and the TNFR ECD was fused to the rat IgG1 Fc hinge region. 5A-C show the nucleotide and amino acid sequences of the TNFR: Fc fusion. These sequences are also described in FIGS. 8A-C of US Pat. No. 6,537,540.

  The TNFR: Fc fusion was subcloned into an AAV vector lacking the rep and cap genes. FIG. 6 shows the resulting rAAV vector in which the rat TNFR-Fc fusion polynucleotide is located between and operably linked to the human immediate early CMV enhancer promoter and the synthetic poly A addition signal. ing. The transcription unit containing the TNFR-Fc fusion gene is stored between the AAV-2 ITRs. This rAAV vector plasmid is designated pAAVCMVrTNFR-Fc.

A. Generation of recombinant AAV 2/2 TNFR: Fc Plasmid pAAVCMVrTNFR-Fc (30 μg) was transfected into the Hela C12 packaging cell line by electroporation (Potter, et al., 1984, Proc. Natl. Acad. Sci. USA 79: 7161-7165). The C12 cell line contains AAV2 rep and cap genes whose transcription is paused by induction upon infection with adenohelper (Clark, et al., 1995; Clark, et al., 1996, Gene). Therapy 3: 1121-2132). Twenty-four hours after transfection, cells were trypsinized and re-plated into 100 mm plates at a density of 5 × 10 ^{3 to} 5 × 10 ⁴ plates per plate. The cells were subjected to selection in DMEM containing 10% fetal bovine serum and 300 μg / ml hygromycin B. Drug resistant cell clones are isolated and expanded and tested for their ability to produce infectious AAVCMVrTNFR-Fc vectors, Atkinson, et al. 1998, Nucleic Acid Res. 26: 2821-2823. One such producer cell clone (C12-55) was further used to produce the AAVCMVrTNFR-Fc vector. Production, purification and titration are basically as described herein and are described in Atkinson, et al. (WO 99/11764).

B. Generation of Recombinant AAV 2/5 TNFR: Fc For stable cell lines and rep-cap transpackaging cell lines utilized in the Ad hybrid production process described below, see WO95 / 34670 for AAV-2 serotype vectors and rep- The cap construct has been described. For the AAV-5 capsid, 5 ′ and 3 ′ ITR pseudotype constructs of AAV2, see Sandalon, et al. , J .; Virol. 78: 12355-12365, 2004, a new transpackaging rep-cap plasmid designated pBSHSPRC2C5 containing the AAV-2 rep gene and the AAV5 cap sequence was constructed. Briefly, AAV5cap sequences were amplified by PCR from plasmid pACK2 / 5 using Pfx polymerase (Invitrogen). The AAV-2 rep sequence was then excised from the AAV2 helper plasmid pBSHSPRC2.3 and replaced with an amplified AAV5 capsid sequence with SwaI / XbaI restriction sites. Plasmid pAd helper 4.1 expressed E2a, E4-orf6 and VA genes of adenovirus type 5 (Ad5). For transfection systems, using standard molecular techniques known in the art, the p5 promoter of the rep-cap (each AAV2 / 1) pseudo-type transpackaging construct is transferred to a minimal heat consisting essentially of a TATA box. Replaced with shock promoter.

The cells were grown by seeding 201 flasks at a density of 1.5 × 10 ⁶ cells per flask. The 293 cells were placed in 50 mL of DMEM containing 4 mM L-glutamine and 10% FBS, and the flask was incubated at 37 ° C. for 96 hours under 5% CO ₂ . After 96 hours of incubation, the cells were transfected using calcium phosphate. On the day of transfection, flasks were generally 80-90% confluent. 750 μg of AAV helper plasmid containing 1250 μg of AAV5 capsid, Ad helper plasmid and 375 μg of cis plasmid was added to 104 mL of 300 mM calcium chloride. Then, the plasmid containing calcium chloride was slowly poured into 104 mL of 2X HBS buffer and mixed for 30 seconds. Immediately thereafter, 8 mL of the precipitate was added to each flask and the flasks were placed at 37 ° C. for 6-8 hours under 5% CO ₂ . The remaining flasks were trypsinized and cells were counted for use in productivity calculations. After 6-8 hours, the flasks were removed from the incubator, the medium was aspirated from each flask, and replaced with 50 ml of DMEM containing 4 mM L-glutamine. The flasks were incubated for 72 hours at 37 ° C. under 5% CO ₂ . After 72 hours, the flask was removed from the incubator, the flask was tapped to detach the cells, and the contents of each flask were collected. Based on the capacity of each container, the amount of 100 mM MgCl ₂ and 10% DOC was calculated, resulting in final concentrations of 1.8 mM and 0.5%, respectively. The containers were then placed in a 37 ° C. water bath for 10-20 minutes and then benzonase was added to each container to a final concentration of 10 units / mL. These containers were placed in a 37 ° C. water bath for 60 minutes and inverted every 15 minutes. Next, polysorbate 20 (Tween) was added to each container to make the final concentration 1%. Again, these containers were placed in a 37 ° C. water bath for 60 minutes and inverted every 15 minutes. Then, 5M NaCl was added to each container to bring the final salt concentration to 1M. The lysed material was filtered using two filters, a Polygard CR Optivap XL 10 filter (0.3 μm nominal) and a Milligard Opticap Capsule (0.5 μm nominal). The filtered material was concentrated using tangential flow filtration (TFF). Three TFF membranes with a nominal molecular weight cut-off (NMWCO) of 100 Kda were used for TFF. The filtered material was concentrated to a volume of 500 mL and after concentration, the material was diafiltered at 10 dialysis volume. The diafiltered material was filtered through a Supercap 50 filter (0.45 μm) and allowed to stand at −70 ° C. until purification.

rAAV was purified as follows. The purified producer cell lysate has already been described in Clark, et al. , Were prepared by resuspending producer cells (5 × 10 ⁶ cells / ml) in 0.5% deoxycholate and benzonase nuclease (35 units / ml). Equilibrated POROS HE-20 resin (12.5 mM Tris, pH 8.0; 0.5 mM MgCl ₂ ; 100 mM NaCl) is added to the clarified lysate (2 ml / L) and the mixture is rotated at 4 ° C. for 16 hours. This provided sufficient time for the bonding of the particles. HE-20 resin was pelleted at 2500 xg for 30 minutes and resuspended in 12 ml equilibration buffer per ml resin used. The resin was washed 3 times with gentle inversion using equilibration buffer and pelleted at 2,000 rpm for 10 minutes between each wash. When the final wash was completed, rAAV-2 was eluted by resuspending the pellet in 20 ml of elution buffer (20 mM Tris, pH 8.0; 1 nM MgCl ₂ ; 600 mM NaCl) for 10 minutes. To maximize recovery, vector elution was repeated two more times (3 times total). The eluted virus was filtered through a 0.45 μm membrane filter to remove the resin powder, and then subjected to PI column chromatography. This virus eluate was diluted 6-fold with water to reduce the final salt concentration to 100 mM or less. The final purified product was generated by using a Biocad Sprint HPLC system (PerSeptive Biosystems) in combination with a POROS PI-50 column (bead size 50 μm). After equilibrating the 1.7 ml column with 10 column volumes of 20 mM Tris, pH 7.0; 100 mM NaCl, the batched vector was applied at a flow rate of 5 ml / min. After loading the sample, the column was washed with 10 ml of equilibration buffer. The bound material was then eluted by applying a NaCl step gradient (0.6 M) at a flow rate of 3 ml / min, collecting 1 ml gradient fraction. After column purification, a small amount (20 μl) of the protein-rich fraction was analyzed by SDS-PAGE and SYPRO-Orange (Molecular Probes, Inc.) staining to visualize the eluted protein. Fractions containing the most virus were combined, dialyzed against multiple changes of ₂₀ mM Tris, pH 8.0, 1 mM MgCl ₂ , 200 mM NaCl and stored in small portions at −80 ° C. in 10% glycerol. The total protein content in the vector preparation was measured using a NanoOrange protein quantification kit according to the manufacturer's instructions (Molecular Probes, Inc.). Clark, et al. As described in detail above, the DRP titer of purified rAAV was measured by real-time PCR using a Prism 7700 Taqman sequence detection system (PE Applied Biosystems).

Example 3 Serum Levels of AAV-TNFR: Fc After Intramuscular Injection Lewis rats were treated with 100 μl of 1.0 × 10 ¹² DRP AVV rat TNF: Fc vector pseudotyped with type 2 or type 5 capsids. And administered by intramuscular (quadriceps) injection. Then, serum concentrations at various time points were measured by ELISA as described in the manufacturer's instructions (Biosource International).

  FIG. 7 shows the long-term expression of TNFR: Fc as indicated by serum concentration after delivery of AAV2 / 2 or AAV2 / 5 vector via quadriceps. Type 5 capsids correlated with higher levels of TNFR: Fc protein. Maximum expression of TNFR protein was observed after 60 days and therapeutic levels were demonstrated for up to 1 year.

Example 4: Protection of AAV-TNFR: Fc against LPS-mediated bone loss Rats with LPS-mediated bone loss were induced as described in Example 1. In short, P.I. "Chronic" alveolar bone resorption was induced by injecting gingivalisLP strain SW83 three times a week for 8 weeks. Control solution (PBS) or P.I. g. Endotoxin (LPS) 10 μL was injected at a concentration of 1 mg / ml in the interdental area. The disease induction phase before 21 days to start to, viral vectors 4 the interdental site and first molars mesial surface two places, rAAV-TNFR: the local delivery at a dose of 6 × ¹⁰ 11 DRP 15μl of Fc.

  FIG. 8 shows micro-CT images scanned from fixed, non-demineralized specimens from control, LPS injection, and LPS injection + AAV-TNFR: Fc treated animals. Fixed non-demineralized samples from the biopsy area were evaluated. All specimens were scanned with a micro CT system and reconstructed into 3D images with a mesh size of 18 μm × 18 μm × 18 μm using GEMS microview software. The image was rotated to a standardized position to allow comparison between groups. Next, mineralized voxels were identified from non-mineralized voxels by setting thresholds on these images and determining the optimal gray scale value from the image histogram. A standard algorithm was used for bone measurement. Images were taken 8 weeks after LPS injection. The left image is an overview of three maxillary molars, and the right image is a cross-sectional view of the second molar region. FIG. 8 is a bone anatomy of control (top), disease progression (middle) and blockage of disease progression using AAV-TNFR: Fc.

  The line dimensions of bone resorption were collected and averaged at the interdental site from the CEJ (cementum-enamel-junction) obtained from the cross section to the bone crest. The number of animals was 12 per group. Data was obtained by micro CT analysis. As shown in FIG. 9, there was no significant difference between the PBS injection control group (0.69 ± 0.17 mm) and the AAV-TNFR: Fc administration + LPS group (0.65 ± 12 mm). The LPS alone group showed alveolar bone resorption progression (1.26 ± 0.58 mm; ANOVA, p <0.0001 compared to all groups).

  The data shown in FIGS. 8 and 9 indicate that AAV-TNFR: Fc protected against LPS-mediated bone damage.

Example 5: Kinetic characteristics of TNFR: Fc expression and efficacy of local and systemic delivery of rAAV-TNFR: Fc Rats with LPS-mediated bone loss were as described in Example 1 and of FIG. Guide as shown in the diagram. In short, P.I. Injecting “chronic” alveolar bone resorption by injecting gingivalisLP strain SW83 three times a week for 8 weeks. Control solution (PBS) or P.I. g. Endotoxin (LPS) 10 μL is injected at a concentration of 1 mg / ml in the interdental area. Twenty-one days before the start of the disease induction phase, viral vectors were placed in 4 interdental sites and 2 mesial surfaces of the first molar, 90 × l 8 × 10 ¹¹ DRP of rAAV-TNFR: Fc (6 sites with a volume of 15 μl / site). Local delivery) to assess local delivery. Also, 21 days before the disease induction phase begins, the viral vector is delivered intramuscularly for systemic delivery at a dose of 100 μl of rAAV-TNFR: Fc 7.5 × 10 ¹¹ DRP.

  The test is conducted in two stages: Test I (local delivery) and Test II (systemic delivery). The following two tables show the distribution of the groups and the treatments performed at each time point. In each table, each group has three numbers at each day and week time point. The three numbers are arranged as xyz, where x is the total number of animals surviving at that time; y is the number of animals from which serum samples were taken; z is sacrificed at that time Represents the number of animals.

  Study I AAV-TNFR: Local delivery of Fc

＊補足：各群は各評価時点において動物３組に時差をつけ、順次番号付けする。各組は個別の群として試験期間を通して処置するものとする。

* Supplement: Each group will be numbered sequentially, with a time difference for 3 groups of animals at each evaluation point. Each set shall be treated as a separate group throughout the study period.

  Study II AAV-TNFR: systemic delivery of Fc

Ａ．ＴＮＦＲ：Ｆｃ ＤＮＡレベル、ＴＮＦ−αタンパク質レベルの測定、及び歯周患部におけるサイトカイン産生のＲＮＡ分析
尾部出血により得た血清を、ＴＮＦタンパク質の発現に関して評価する。血清中ＴＮＦは、製造元の説明書（Ｂｉｏｓｏｕｒｃｅ Ｉｎｔｅｒｎａｔｉｏｎａｌ）に記載の通り、ＥＬＩＳＡにより測定する。ＴＮＦ発現のレベルは、Ｍｕｌｔｉｓｃａｎ Ａｓｃｅｎｔ（登録商標）光度計マイクロプレートリーダーと共にＡｓｃｅｎｔ（登録商標）ソフトウェア（Ｔｈｅｒｍｏ Ｌａｂｓｙｓｔｅｍｓ）を使用して定量化する。ラットの血液試料は、ベースライン時並びに２、４、６及び８週に収集する。血液は尾部の出血により採取する。採取した４００〜５００μｌの血液の量では、約２００μｌの血清が得られる。血清を分離するに当たっては、血液を血清分離試験管（Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ Ｎｏ．３６５９５６）に移し、室温で１５分間インキュベートした後、室温で４，０００ｒｐｍにて１０分間回転させる。そして血清試料をクリオバイアル（Ｎｕｎｃ製１．５ｍｌ管）に移し、−７０℃に冷凍する。このＥＬＩＳＡ用の血清試料を、冷凍したままＴＡＲＧＥＴＥＤ ＧＥＮＥＴＩＣＳ Ｃｏｒｐ．に送付する。

A. Measurement of TNFR: Fc DNA levels, TNF-α protein levels, and RNA analysis of cytokine production in periodontal lesions Serum obtained by tail bleeding is evaluated for TNF protein expression. Serum TNF is measured by ELISA as described in the manufacturer's instructions (Biosource International). The level of TNF expression is quantified using Ascent® software (Thermo Labsystems) with a Multiscan Ascent® photometer microplate reader. Rat blood samples are collected at baseline and at 2, 4, 6, and 8 weeks. Blood is collected by tail bleeding. A volume of 400-500 μl of blood collected will yield about 200 μl of serum. In separating the serum, the blood is transferred to a serum separation test tube (Becton Dickinson No. 36556), incubated at room temperature for 15 minutes, and then rotated at 4,000 rpm for 10 minutes at room temperature. The serum sample is then transferred to a cryovial (Nunc 1.5 ml tube) and frozen at -70 ° C. Serum samples for this ELISA were frozen, and TARGETED GENETICS Corp. Send to.

B. In vivo RNA / DNA isolation and analysis Tissue samples taken from rats are immediately snap frozen in dry ice. The samples are then transferred to a 50 mL triangular test tube and stored at −70 ° C. until processed. These tissues are pulverized into a fine powder by a freeze pulverizer (freezer mill, model 6750, Spex Certi-Prep) frozen with liquid nitrogen. The ground powder is returned to the test tube and placed on dry ice. Next, a sample of each organ powder consistent with the amount required by the RNA isolation protocol is metered into a 1.5 mL tube. These tubes are stored on dry ice until processed using the Qiagen RNeasy mini kit. The amount of rat TNF: Fc mRNA contained in each sample of total RNA is quantified using the Perkin Elmer 7700 Sequence Detection System (SDS).

  Gingival tissue samples were collected at baseline and at 4 and 8 weeks according to the schedule shown in FIG. 2, and are described in Xie, et al. , Biotechniques 11: 324-327; 1991. Total RNA is collected as previously described. The amount of TNFR, IL-1β and IL-6 in these samples is analyzed using real-time PCR. In short, PCR including 10 μM detection limit PCR for quantitative evaluation of tgAVV-rat TNFR: Fc DNA seeding in rat tissues is performed in a total volume of 100 μl.

  TNFR: Fc primer: pAAV-rat TNFR: Fc plasmid 1 pg is added to rat genomic DNA as a positive control. The forward primer is 5'-ACTGTGCCTTGAAATTGC-3 ', and the reverse primer is 5'-ACAAGGCTTGCAATCACC-3'. PCR continues for 94 ° C for 30 seconds, 61 ° C for 30 seconds, 72 ° C for 1 minute 28 cycles, and 72 ° C for 1 / 8th cycle. The expected PCR product of 434 bp is separated on an 8% TBE / agarose gel and visualized using EtBr staining.

C. Histomorphological and radiological analysis of periodontal tissue destruction a. Histological examination Rats were sacrificed 8 weeks after surgery, and block biopsy samples were placed in Bouin's fixative (Polysciences, Warrington, PA), 10% v / v acetic acid, 4% v / v formaldehyde, 0% Demineralize with 85% NaCl for 2-3 weeks, then embed in paraffin. The specimen is cut into 4-5 μm sections and hematoxylin and eosin (H & E) staining or toluidine blue staining is performed for visualization by optical or polarizing microscope.

b. Histomorphological examination Giannobile, et al. , J Periodontol, 65: 1158-1168, 1994, using computer-aided image analysis to suppress periodontal tissue destruction in a histomorphological manner by rAAV-rat TNFR: Fc gene transfer Measure ability. Samples were Nikon Eclipse equipped with a SPOT-2 camera (Diagnostic Instruments, Inc. [Sterling Heights, Michigan, USA]) for analysis using Image Pro Plus ^™ software (Media Cybernetics [Silver Spring, Md., USA]). Capture using an E8000 microscope (Nikon, Inc. [Melville, NY, USA]). Coded specimen images are captured at 2, 4, 10 and 20 × magnification for histomorphological analysis. Inspecting all slides with a single masking and calibrated inspection device reveals less than 5% error between and within the inspection devices before and after the test compared to the standard inspection device. become. Measure several parameters of periodontal tissue destruction (from the sample outlined in FIG. 2) 1) M1 measured as the total length of mineral-like tissue on the exposed root surface in mm at M1 / M2 and M2 / M3 Cementum length on M2 distal root and M2 and M3 mesial roots; 2) Bone length measured from cement enamel joint in mm to alveolar crest; and 3) Adjacent molar area The bone density associated with the tooth-supporting alveolar bone in the three predetermined 0.5 mm ² regions of interest is included. And the average value of each evaluated group is calculated | required. The coded specimens are then analyzed by ANOVA and Fisher's PSLD multiple comparison method to determine statistical differences between groups.

c. Micro computed tomography (micro CT)
Micro CT analysis is performed with an EVS micro CT system. Assess fixed non-demineralized samples from the biopsy area. All specimens are scanned on a micro CT system and reconstructed into 3D images with a mesh size of 18 μm × 18 μm × 18 μm using GEMS microview software. The image is rotated to a standardized position to allow comparison between groups. The mineralized voxels are then identified from the non-mineralized voxels by setting a threshold for these images and determining the optimal grayscale value from the image histogram. Bone measurements utilize standard algorithms and include: geometric and morphological evaluation; cross-sectional area analysis and volume measurement; volume fraction of mineralized and non-mineralized tissue; and cortical and cancellous Make measurements on bone morphological characteristics.

  The foregoing invention has been described in some detail using illustrations and examples for the purpose of clarity of understanding, but these illustrations and examples should not be construed as limiting the scope of the invention.

FIG. 1 shows SDS-PAGE of LPS isolated from P. gingivalis W83. FIG. 2 shows radiographs of rat maxilla from LPS injected (A) and control (B) animals. FIG. 3 shows a quantitative measurement of LPS-mediated bone loss in a rat model of experimental periodontitis. The gingiva of Sprague-Dawley rats was injected with P. gingivalis W83 strain LPS (10 mg of 1 mg / ml for each adjacent interpapillary papilla) 3 times a week for 8 weeks, and the bone change area over time (mm ² ) Was measured by digital subtraction radiography. Bars represent mean ± SEM; n = 3 / group. FIG. 4 shows LPS-mediated bone resorption by sagittal histological micrographs of the adjacent area in M1 to M2 of rat maxillary teeth. The control group received a sterile solution and the experimental group received P. gingivalis LPS for 8 weeks. Hematoxylin and eosin staining; magnification 10 times. Figures 5A, 5B and 5C show the nucleotide and amino acid sequences of the rat TNFR: Fc fusion construct. Figures 5A, 5B and 5C show the nucleotide and amino acid sequences of the rat TNFR: Fc fusion construct. Figures 5A, 5B and 5C show the nucleotide and amino acid sequences of the rat TNFR: Fc fusion construct. FIG. 6 shows a diagram of the rAAV vector plasmid pAAVCMVrTNFRFc including the rat TNFR (p80) ECD-IgG1 Fc fusion polynucleotide, operably linked control elements (eg, AAV ITR). FIG. 7 shows the serum levels of rat AAV-TNFR: Fe protein after intramuscular administration of AAV2 / 2 or AAV2 / 5 vectors. Protein in serum from Lewis rats after intramuscular administration of 100 μL of 1.0 × 10 ¹² DRP AVV-rat TNF: Fc vector pseudotyped with either type 2 or type 5 capsids obtained from different time points (rat TNFR: Fc) expression was measured by ELISA. FIG. 8 shows micro CT images showing bone anatomy of control (top) animals, LPS injected (middle) animals, and LPS injected and AAV-TNFR: Fc treated (local delivery) (bottom) animals. The image on the left is an overview of the three maxillary molars, and the image on the right is a cross-sectional view of the second molar. FIG. 9 shows quantitative measurements of bone resorption in control animals, LPS injected animals, and LPS injected and AAV-TNFR: Fc treated (local delivery) animals. Bone resorption measurements were taken at M1, M2 and M3. Bars represent mean ± SEM. FIG. 10 shows a timeline of rAAV-TNFR: Fc delivery, periodontitis induction and sampling.

Claims (20)

  A method of treating or preventing a TNF-related disorder of bone healing in a mammal, comprising administering an effective amount of a recombinant adeno-associated virus (rAAV) vector to the site of the disorder, wherein the rAAV vector comprises tumor necrosis factor A method comprising a polynucleotide encoding a fusion polypeptide comprising an extracellular domain of a receptor (TNFR) and a constant domain of an immunoglobulin molecule.   A method of treating or preventing a TNF-related disorder of wound healing in a mammal comprising administering an effective amount of a recombinant adeno-associated virus (rAAV) vector to the site of the disorder, the rAAV vector comprising a tumor necrosis factor A method comprising a polynucleotide encoding a fusion polypeptide comprising an extracellular domain of a receptor (TNFR) and a constant domain of an immunoglobulin molecule.   A method of treating or preventing a TNF-related buccal buccal disease in a mammal, comprising administering an effective amount of a recombinant adeno-associated virus (rAAV) vector to a periodontal site of the mammal, wherein the rAAV vector comprises a tumor A method comprising a polynucleotide encoding a fusion polypeptide comprising an extracellular domain of necrosis factor receptor (TNFR) and a constant domain of an immunoglobulin molecule.   A method for improving regeneration of tooth support structure loss induced by TNF-related oral buccal disease in a mammal, comprising administering an effective amount of a recombinant adeno-associated virus (rAAV) vector to the periodontal region of the mammal Wherein the rAAV vector comprises a polynucleotide encoding a fusion polypeptide comprising the extracellular domain of tumor necrosis factor receptor (TNFR) and the constant domain of an immunoglobulin molecule.   A method for reducing an inflammatory response in a periodontal region of a mammal having a TNF-related oral buccal disease, comprising administering an effective amount of a recombinant adeno-associated virus (rAAV) vector to the periodontal region of the mammal. The rAAV vector comprises a polynucleotide encoding a fusion polypeptide comprising an extracellular domain of tumor necrosis factor receptor (TNFR) and a constant domain of an immunoglobulin molecule.   A method for alleviating a TNF-related buccal buccal disease in a mammal, wherein an effective amount of a recombinant adeno-associated virus (rAAV) vector is applied to the periodontal region of the mammal to alleviate the buccal condition of the buccal disease of the mammal Wherein the rAAV vector comprises a polynucleotide encoding a fusion polypeptide comprising an extracellular domain of tumor necrosis factor receptor (TNFR) and a constant domain of an immunoglobulin molecule. .   The method according to any one of claims 3 to 6, wherein the periodontal region is selected from the group consisting of periodontal tissue, gingival tissue, connective tissue and epithelium.   7. The method of any one of claims 1-6, further comprising administering a TNF antagonist.   The method according to any one of claims 1 to 6, wherein the TNFR extracellular domain is derived from p75 TNFR.   The method according to any one of claims 1 to 6, wherein the immunoglobulin molecule is a human immunoglobulin.   The method according to claim 1, wherein the immunoglobulin molecule is human IgG1.   The method according to any one of claims 1 to 6, wherein the immunoglobulin molecule is human IgG3.   The method according to any one of claims 1 to 6, wherein the fusion polypeptide comprises the peptide sequence shown in Figs. 5A and 5B.   7. The method of any one of claims 1-6, wherein the polynucleotide encoding the fusion polypeptide is operably linked to a heterologous promoter.   7. The method of any one of claims 1-6, wherein the polynucleotide encoding the fusion polypeptide is operably linked to a constitutive promoter.   7. The method of any one of claims 1-6, wherein the polynucleotide encoding the fusion polypeptide is operably linked to an inducible promoter.   The method of claim 16, wherein the inducible promoter is derived from a TNFα gene.   7. The method of any one of claims 1-6, wherein the polynucleotide further encodes a polypeptide comprising an interleukin-1 (IL-1) antagonist.   19. The method of claim 18, wherein the IL-1 antagonist is an IL-1 receptor polypeptide and the IL-1 receptor polypeptide binds IL-1.   20. The method of claim 19, wherein the IL-1 receptor polypeptide is an IL-1 receptor type II polypeptide.

Images