EP1871799A2 - Procedes d'abaissement de niveau de facteur tnf dans les troubles associes a ce facteur - Google Patents

Procedes d'abaissement de niveau de facteur tnf dans les troubles associes a ce facteur

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Publication number
EP1871799A2
EP1871799A2 EP06740114A EP06740114A EP1871799A2 EP 1871799 A2 EP1871799 A2 EP 1871799A2 EP 06740114 A EP06740114 A EP 06740114A EP 06740114 A EP06740114 A EP 06740114A EP 1871799 A2 EP1871799 A2 EP 1871799A2
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EP
European Patent Office
Prior art keywords
tnf
raav
tnfr
mammal
vector
Prior art date
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Application number
EP06740114A
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German (de)
English (en)
Inventor
Haim Burstein
William V. Giannobile
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Ampliphi Biosciences Corp
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Targeted Genetics Corp
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Publication date
Application filed by Targeted Genetics Corp filed Critical Targeted Genetics Corp
Publication of EP1871799A2 publication Critical patent/EP1871799A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This invention relates to methods of using adeno-associated virus (AAV) vectors encoding a tumor necrosis factor (TNF) antagonist to reduce the levels of TNF in an individual for treating TNF-associated diseases.
  • AAV adeno-associated virus
  • TNF tumor necrosis factor
  • Tumor necrosis factor- ⁇ (TNF ⁇ ) and tumor necrosis factor- ⁇ (TNF ⁇ ) are homologous multifunctional cytokines; the great similarities in structural and functional characteristics of which have resulted in their collective description as tumor necrosis factor or "TNF.” Activities generally ascribed to TNF include: release of other cytokines including IL-I, IL-6, GM-CSF, and IL-10, induction of chemokines, increase in adhesion molecules, growth of blood vessels, release of tissue destructive enzymes and activation of T cells. See, for example, Feldmann et al., 1997, Adv. Immunol, 64:283-350, Nawroth et al., 1986, J. Exp.
  • TNF initiates its biological effect through its interaction with specific, cell surface receptors on TNF -responsive cells.
  • TNFR tumor necrosis factor receptor
  • p75 or Type II
  • p55 or Type I
  • TNFR Type I and TNFR Type II each bind to both TNF ⁇ and TNF ⁇ . Soluble, truncated versions of the TNFRs 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).
  • TNF-associated disorders are congestive heart failure, inflammatory bowel diseases (including Crohn's disease), arthritis, and asthma.
  • U.S. Patent No. 6,537,540 and PCT WO 00/65038 disclose administration of a recombinant AAV vector comprising a polynucleotide encoding a TNF antagonist to reduce levels of TNF and treat TNF-associated diseases.
  • Other examples of TNF associated disorders or conditions are impairment of wound healing and bone fracture repair, and bone allo- and auto-graft healing. This class of TNF associated disorders may be triggered by infection with a microbial, bacterial, fungal, or viral agent or initiated by a host immune mediated defect that triggers a cascade of inflammatory cytokines including TNF.
  • TNF-associated disorders is oral buccal disease (such as periodontitis).
  • Periodontitis is one of the most common inflammatory diseases in man.
  • the disease is the major cause of tooth loss in adults and is associated with systemic diseases such as atherosclerosis, heart failure, and diabetic glucose control.
  • the disease affects >50% of the US population and is treated mainly by local debridement or surgery of the affected sites. Oliver et al., J Periodontal, 69, 269-278, 1998. Although these treatment modalities are effective for many individuals exhibiting aggressive periodontitis, some patients may continue to lose connective tissue attachment even after mechanical or surgical treatment.
  • periodontitis is explored to identify possible new targets for pharmaceutical intervention to attempt to halt the progression of disease.
  • a variety of immune-associated cell populations are responsible for the pathogenesis of periodontal diseases including specific CD+ T cells.
  • the pathogenesis process is most likely a response to exogenous periodontal pathogens such as P. gingivalis associated with plaque biofilms.
  • P. gingivalis associated with plaque biofilms.
  • Darveau et al. Periodontal 14, 12-32, 2000. Consequently, recruited monocoytes, macrophages, and fibroblasts produce cytokines such as tumor necrosis factor alpha (TNF) and interleukin-1 beta (IL-I) within periodontal lesions.
  • TNF tumor necrosis factor alpha
  • IL-I interleukin-1 beta
  • cytokines can be found to be localized in periodontal tissues as well as in gingival crevicular fluid (GCF) associated with the lesion. These cytokines are central to the damaging cascade, ultimately triggering the production of matrix metalloproteinases (MMPs), prostaglandins, and osteoclasts. The end result of this cascade is an irreversible damage to tooth-supporting soft tissues and alveolar bone. Graves, Clin Infect Dis 28: 482-490, 1999. Although the disease is initiated by specific oral pathogens that colonize and invade the oral tissues, the host inflammatory response is a major factor in the progression of disease.
  • MMPs matrix metalloproteinases
  • IL-I has also been positively correlated to increased probing depth and attachment loss. Delima et al., J Infect Dis, 186, 511-516, 2002.
  • IL-lbeta has synergistic activity with TNF-alpha or lymphotoxin in stimulating bone resorption.
  • TNF in a primate model of periodontitis have shown promising results.
  • Histological investigations revealed a 51% reduction in connective tissue attachment loss and a 91% reduction in alveolar bone loss.
  • Delima et al., J Clin Periodontal, 28, 233-240, 2001 the only therapy available that acts as a host modulatory agent for periodontal disease is a low dose formulation of the antibiotic doxycycline which requires twice daily administration.
  • TNF-associated disorders or conditions such as bone loss, impairment of wound healing and bone healing, and oral buccal diseases, particularly treatments that can provide sustained, controlled therapy, e.g., provide prolonged local regional neutralization of TNF to the periodontal structures compared to a neutralization of TNF by delivery of a protein TNF antagonist.
  • Treatment of these TNF-associated disorders or conditions such as periodontitis may provide a key in reducing the risk of periodontal disease triggered systemic disease conditions such as cardiovascular disease and premature birth weight.
  • All publications and references cited herein are hereby incorporated by reference in their entirety. BRIEF SUMMARY OF THE INVENTION
  • the present invention is directed to methods for treating or preventing
  • TNF associated disorders or conditions such as bone loss, impairment of wound healing and bone healing, and periodontitis in a mammal.
  • the methods generally employ an rAAV vector to deliver a polynucleotide encoding a TNF antagonist to the mammal, which in turn reduces the levels of TNF at the site and results in palliation of a number of TNF-associated cascade events.
  • TNF-associated cascade events are loss of bone regeneration and/or bone resorption, impairment of wound healing and bone healing (e.g., bone graft healing and bone fracture repair), and periodontitis.
  • Lowering TNF may in turn reduce levels or eliminate a cascade effect of other disease causing or contributing agents, such as other inflammatory cytokines.
  • the present invention is directed to methods of reducing or lowering TNF at the site of wound healing or bone healing thereby increasing the rate of, accelerating the rate of, increasing the percent of, or increasing the extent of repair of the wound or bone tissue of a mammal.
  • the invention provides methods of improving wound healing and bone healing in a mammal by administering an effective amount of an rAAV vector to the wound, bone fracture or bone graft site of the mammal, wherein said rAAV vector comprises a polynucleotide encoding a TNF antagonist.
  • the rAAV vectors of the invention are administered in conjunction with osteogenic growth factors such as PDGF, or vascularization promoting factors such as receptor activator of nuclear factor KB ligand (RANKL) or vascular endothelial growth factor (VEGF).
  • the osteogenic or vascularization factors are also delivered by an rAAV vector encoding those factors.
  • the present invention is directed to methods for treatment of TNF- associated oral buccal diseases of a mammal.
  • the methods generally employ an rAAV vector to deliver a polynucleotide encoding a TNF antagonist to the mammal, which in turn reduces the levels of TNF at the periodontal site and results in palliation of a number of TNF-associated oral buccal disorders, such as chronic periodontitis, aggressive periodontitis, and gingivitis. Lowering TNF may in turn reduce levels of other disease causing or contributing agents, such as other inflammatory cytokines.
  • the invention provides methods for treating or preventing a TNF- associated oral buccal disease in a mammal, which comprise administering an effective amount of an rAAV vector to a periodontal site of the mammal, wherein said rAAV vector comprises a polynucleotide encoding a TNF antagonist.
  • the invention also provides methods for reducing incidence of a TNF- associated oral buccal disease, ameliorating or palliating a TNF-associated oral buccal disease, and/or delaying the development or progression of a TNF-associated oral buccal disease in a mammal, which comprise administering an effective amount of an rAAV vector to a periodontal site of the mammal, wherein said rAAV vector comprises a polynucleotide encoding a TNF antagonist.
  • the invention also provides methods for reducing TNF levels at a periodontal site in a mammal having a TNF-associated oral buccal disease, which comprise administering an effective amount of an rAAV vector to the periodontal site of the mammal, wherein said rAAV vector comprises a polynucleotide encoding a TNF antagonist.
  • the invention also provides methods for reducing an inflammatory response at a periodontal site in a mammal having a TNF-associated oral buccal disease, which comprise administering an effective amount of an rAAV vector to the periodontal site of the mammal, wherein said rAAV vector comprises a polynucleotide encoding a
  • the invention also provides methods for reducing the re-occurrence of an inflammatory response at a periodontal site in a mammal having a TNF-associated oral buccal disease, which comprise administering an effective amount of an rAAV vector to the periodontal site of the mammal, wherein said rAAV vector comprises a polynucleotide encoding a TNF antagonist.
  • the invention also provides methods of improving the restoration of lost tooth supporting structures (such as alveolar bone, periodontal ligament and cementum) caused by TNF associated oral buccal disease, which comprise administering an effective amount of an rAAV vector to the periodontal site of the mammal, wherein said rAAV vector comprises a polynucleotide encoding a TNF antagonist.
  • the improvement of the restoration of lost tooth supporting structures may be achieved via regenerative procedures such as osteoconductive biomaterials and cell occlusive barrier membranes.
  • the rAAV vectors of the invention are administered in conjunction with an osteogenic growth factor (such as PDGF) or a vascularization promoting factor (such as RANKL or VEGF).
  • the osteogenic and/or vascularization factor are also delivered in an rAAV vector.
  • the polynucleotide in the rAAV vectors described herein is one encoding a tumor necrosis factor receptor (TNFR). Since TNFR is capable of binding to soluble TNF, the introduction of TNFR tends to reduce the levels of TNF in the affected tissues.
  • the rAAV vector comprises a polynucleotide encoding a p75 TNFR polypeptide.
  • the rAAV vector comprises a polynucleotide encoding an Fc (constant domain of an immunoglobulin molecule):p75 fusion polypeptide.
  • the rAAV vector comprises a polynucleotide encoding a fusion polypeptide in which the extracellular domain of TNFR is fused to Fc.
  • the rAAV vectors of the invention further comprise a polynucleotide encoding an IL-I antagonist, such as an IL-I receptor type II polypeptide.
  • the rAAV vector may be administered prior to, during, and/or after a mammal has been diagnosed with a TNF-associated disorders or conditions described herein (such as wound healing, bone healing, and oral buccal disease).
  • the rAAV vector can be administered locally, such as injected to the peridontium, the gingival tissue, the connective tissue, or the epithelium; or systemically, such as by intramuscular injection.
  • the rAAV vector may be delivered topically either directly or through coated delivery , devices known in the art (for example, mouth guards or trays), or by nonsurgical procedures know in the art, including without limitation gel matrixes and microspheres (such as the FDA approved polylactic glycolic acid microspheres).
  • the rAAV vector may be used to transduce gingival tissues ex vivo which may be implanted surgically at the wound site.
  • the rAAV vector may be administered with another agent, such as a TNF antagonist, an osteogenic growth factor (such as PDGF), and a vacularization promoting factor (such as RANKL and VEGF).
  • a TNF antagonist such as a TNF antagonist, an osteogenic growth factor (such as PDGF), and a vacularization promoting factor (such as RANKL and VEGF).
  • RANKL and VEGF vacularization promoting factor
  • kits for use in any of the methods described herein comprises any of the rAAV vectors described herein in combination with a pharmaceutically acceptable carrier, and instructions for use of the rAAV vectors in any of the methods described herein.
  • Figure 1 depicts an SDS-PAGE of LPS isolated from P. gingivalis W83.
  • Figure 2 depicts radiographic images from the rat maxillae from LPS injected (A) and control (B) animals.
  • Figure 3 depicts a quantitative measure of LPS-mediated bone loss in the rat model of experimental periodontitis.
  • Sprague-Dawley rats were subject to a three- times weekly injection of strain W83 P.
  • gingivalis LPS (10 ⁇ l of 1 mg/ml to each interproximal papilla) to gingivae over a period of 8 weeks.
  • Figure 4 depicts LPS-mediated bone resorption via sagittal histologic photomicrographs of the interproximal region between Ml and M2 of the rat maxillary teeth.
  • the control group received sterile solution and the experimental group received P. gingivalis LPS for 8 weeks. Hematoxylin & Eosin, 1OX magnification.
  • Figures 5A, 5B and 5C depict the nucleotide and amino acid sequences of rat TNFR: Fc fusion construct.
  • Figure 6 depicts a diagram of the rAAV vector plasmid pAA VCMVrTNFRFc, including the rat TNFR(p80)ECD-IgGlFc fusion polynucleotide, operatively linked control elements, including AAV ITRs.
  • Figure 7 depicts serum levels of rat AAV-TNFR:Fc protein following intramuscular administration of AAV2/2 or AAV2/5 vectors.
  • Protein (rat TNFR:Fc) expression in the serum from Lewis Rats following intramuscular administration of lOO ⁇ L of an 1.0 x 10 12 DRP AVV-rat TNF:Fc vector pseudotyped with either type 2 or type 5 capsids from different time-points were determined by ELISA.
  • Figure 8 depicts MicroCT images diaplaying bone anatomy of control
  • Figure 9 depicts a quantitative measurement of bone resorption of control
  • Figure 10 depicts a timeline for r AA V-TNFR:Fc delivery, periodontitis induction, and sample collection.
  • the invention described herein provides methods for preventing or treating
  • TNF-associated disorders or conditions such as bone loss, impairment of wound healing and bone healing, and oral buccal diseases
  • said method comprising administering an effective amount of an rAAV vector comprising a polynucleotide encoding a TNF antagonist to the site of the disorder or condition of the mammal.
  • the TNF antagonist expressed in the mammal reduces the levels of TNF at the site of the disorder or condition and results in palliation of TNF-associated disorders or conditions.
  • the TNF antagonist is a fusion polypeptide in which the extracellular domain of TNFR is fused to an Fc (constant domain of an immunoglobulin molecule).
  • a "TNF antagonist” as used herein refers to a polypeptide that binds TNF and inhibits and/or hinders TNF activity as reflected in TNF binding to a TNF-receptor including any of the following: (a) TNFR, preferably endogenous ⁇ i.e., native to the individual or host), cell membrane bound TNFR; (b) the extracellular domain(s) of TNFR; and/or (c) the TNF binding domains of TNFR (which may be a portion of the extracellular domain).
  • TNF antagonists include, but are not limited to, TNF receptors (or appropriate portions thereof, as described herein) and anti-TNF antibodies.
  • the "biological activity" of a TNF antagonist is to bind to TNF and inhibit and/or hinder TNF from binding to any of the following: (a) TNFR, preferably endogenous, cell membrane bound TNFR; (b) the extracellular domain(s) of TNFR; and (c) the TNF binding domains of TNFR (which may be a portion of the extracellular domain).
  • a TNF antagonist can be shown to exhibit biological activity using assays known in the art to measure TNF activity and its inhibition, an example of which is provided herein.
  • TNF-associated disorders or conditions are those disorders or conditions that are associated with, result from, and/or occur in response to, elevated levels of TNF. Such disorders may be associated with episodic or chronic elevated levels of TNF activity and/or with local or systemic increases in TNF activity. Such disorders include, but are not limited to, inflammatory diseases, such as oral buccal diseases, arthritis and inflammatory bowel disease, and congestive heart failure. Examples of oral buccal diseases are chronic periodontitis, aggressive periodontitis, and gingivitis. TNF- associated disorders also include bone loss and impairment of wound healing and bone healing.
  • TNF receptor polypeptide and "TNFR polypeptide” refer to polypeptides derived from TNFR (from any species) which are capable of binding TNF.
  • TNFR Two distinct cell-surface TNFRs have been described: Type II TNFR (or p75 TNFR or TNFRII) and Type I TNFR (or p55 TNFR or TNFRI).
  • Type II TNFR or p75 TNFR or TNFRII
  • Type I TNFR or p55 TNFR or TNFRI
  • the mature full-length human p75 TNFR is a glycoprotein having a molecular weight of about 75-80 kilodaltons (kD).
  • the mature full-length human p55 TNFR is a glycoprotein having a molecular weight of about 55-60 kD.
  • TNFR polypeptides of this invention are derived from TNFR Type I and/or TNFR type II.
  • TNFR polypeptides such as “TNFR”, “TNFR:Fc” and the like, when discussed in the context of the present invention and compositions therefor, refer to the respective intact polypeptide (such as, TNFR intact), or any fragment or derivative thereof (such as, an amino acid sequence derivative), that exhibits the desired biological activity (i.e., binding to TNF).
  • a "TNFR polynucleotide” is any polynucleotide which encodes a TNFR polypeptide (such as a TNFR:Fc polypeptide).
  • an extracellular domain" of TNFR refers to a portion of
  • IL-I antagonist refers to a polypeptide that binds inter leukin 1 (IL-I) and inhibits and/or hinders IL-I activity as reflected in IL-I binding to an IL-I receptor including any of the following: (a) IL-I receptor (IL-IR), preferably endogenous (i.e., native to the individual or host), cell membrane bound IL-IR; (b) the extracellular domain(s) of IL-IR; and/or (c) the IL-I binding domains of IL-IR (which may be a portion of the extracellular domain).
  • IL-IR IL-I receptor
  • IL-I antagonists include, but are not limited to, IL-I receptors (or appropriate portions thereof, as described herein) and anti- IL-I antibodies.
  • biological activity of an IL-I antagonist is to bind to IL-I and inhibit and/or hinder IL-I from binding to any of the following: (a) IL-IR, preferably endogenous, cell membrane bound IL-IR; (b) the extracellular domain(s) of IL-IR; and/or (c) the IL-I binding domains of IL-IR (which may be a portion of the extracellular domain).
  • An IL-I antagonist can be shown to exhibit biological activity using assays known in the art, including IL-I inhibition assays, which are described herein as well as in the art.
  • IL-I receptor polypeptide refers to polypeptides derived from EL-I receptor (from any species) which are capable of binding IL-I .
  • IL-IR polypeptides when discussed in the context of the present invention and compositions therefor, refer to the respective intact polypeptide (such as intact IL-IR), or any fragment or derivative thereof (such as, an amino acid sequence derivative), that exhibits the desired biological activity (i.e., binding to IL-I).
  • a "IL-IR polynucleotide” is any polynucleotide which encodes a IL-IR polypeptide.
  • an "extracellular domain" of IL-IR refers to a portion of
  • polypeptide that is found between the amino-terminus of IL-IR and the amino-terminal end of the IL-IR transmembrane region.
  • the extracellular domain of IL-IR binds IL-I.
  • polypeptide polypeptide
  • peptide and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, or conjugation with a labeling component.
  • a "chimeric polypeptide” or “fusion polypeptide” is a polypeptide comprising regions in a different position than occurs in nature.
  • the regions may normally exist in separate proteins and are brought together in the chimeric or fusion polypeptide, or they may normally exist in the same protein but are placed in a new arrangement in the chimeric or fusion polypeptide.
  • a chimeric or fusion polypeptide may also arise from polymeric forms, whether linear or branched, of TNFR polypeptide(s).
  • polynucleotide and “nucleic acid”, used interchangeably herein, refer to a polymeric form of nucleotides of any length, including 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 present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • polynucleotide refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • a "chimeric polynucleotide” or “fusion polynucleotide” is a polynucleotide comprising regions in a different position than occurs in nature. The regions may normally exist in separate genes and are brought together in the chimeric or fusion polynucleotide, or they may normally exist in the same gene but are placed in a new arrangement in the chimeric or fusion polynucleotide.
  • AAV is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes, serotypes, psuedotypes and both naturally occurring and recombinant forms, except where required otherwise.
  • rAAV vector refers to an AAV vector comprising a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for the genetic transformation of a cell.
  • the heterologous polynucleotide is flanked by at least one, preferably two, AAV inverted terminal repeat sequences (ITRs).
  • an rAAV vector can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes and, most preferably, encapsidated in a viral particle, particularly an AAV.
  • rAAV virus or "rAAV viral particle” refers to a viral particle composed of at least one AAV capsid protein (preferably by all of the capsid proteins of a wild-type AAV) and an encapsidated rAAV.
  • Packaging refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle or rAAV particle.
  • AAV "rep” and “cap” genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. They have been found in all AAV serotypes examined, and are described below and in the art. AAV rep and cap are referred to herein as AAV "packaging genes”.
  • a "helper virus” for AAV refers to a virus that allows AAV to be replicated and packaged by a mammalian cell.
  • helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia.
  • the adenoviruses encompass a number of different subgroups, although
  • Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.
  • HSV herpes simplex viruses
  • EBV Epstein-Barr viruses
  • CMV cytomegaloviruses
  • PRV pseudorabies viruses
  • infectious virus or viral particle is one that comprises a polynucleotide component which it is capable of delivering into a cell for which the viral species is trophic.
  • the term does not necessarily imply any replication capacity of the virus.
  • Assays for counting infectious viral particles are described in the art.
  • a "replication-competent" virus ⁇ e.g. , a replication-competent AAV, sometimes abbreviated as "RCA" refers to a phenotypically wild-type virus that is infectious, and is also capable of being replicated in an infected cell (i.e., in the presence of a helper virus or helper virus functions).
  • replication competence generally requires the presence of functional AAV packaging genes.
  • Preferred rAAV vectors as described herein are replication-incompetent in mammalian cells (especially in human cells) by virtue of the lack of one or more AAV packaging genes.
  • such rAAV vectors lack any AAV packaging gene sequences in order to minimize the possibility that RCA are generated by recombination between AAV packaging genes and an rAAV vector.
  • a "gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated.
  • Recombinant as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature.
  • a recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.
  • control element or "control sequence” is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature.
  • Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers.
  • a promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) from the promoter.
  • “Operatively linked” or “operably linked” refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.
  • Heterologous means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.
  • a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide.
  • a promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter.
  • Genetic alteration refers to a process wherein a genetic element is introduced into a cell other than by mitosis or meiosis.
  • the element may be heterologous to the cell, or it may be an additional copy or improved version of an element already present in the cell.
  • Genetic alteration may be effected, for example, by transfecting a cell with a recombinant plasmid or other polynucleotide through any process known in the art, such as electroporation, calcium phosphate precipitation, or contacting with a polynucleotide-liposome complex.
  • Genetic alteration may also be effected, for example, by transduction or infection with a DNA or RNA virus or viral vector.
  • the genetic element is introduced into a chromosome or mini-chromosome in the cell; but any alteration that changes the phenotype and/or genotype of the cell and its progeny is included in this term.
  • a cell is said to be “stably” altered, transduced, or transformed with a genetic sequence if the sequence is available to perform its function during extended culture of the cell in vitro.
  • such a cell is "inheritably” altered in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell.
  • “Stable integration" of a polynucleotide into a cell means that the polynucleotide has been integrated into a replicon that tends to be stably maintained in the cell.
  • episomes such as plasmids can sometimes be maintained for many generations, genetic material carried episomally is generally more susceptible to loss than chromosomally- integrated material.
  • maintenance of a polynucleotide can often be effected by incorporating a selectable marker into or adjacent to a polynucleotide, and then maintaining cells carrying the polynucleotide under selective pressure.
  • sequences cannot be effectively maintained stably unless they have become integrated into a chromosome; and, therefore, selection for retention of a sequence comprising a selectable marker can result in the selection of cells in which the marker has become stably-integrated into a chromosome.
  • Antibiotic resistance genes can be conveniently employed as such selectable markers, as is well known in the art.
  • stably-integrated polynucleotides would be expected to be maintained on average for at least about twenty generations, preferably at least about one hundred generations, still more preferably they would be maintained permanently.
  • the chromatin structure of eukaryotic chromosomes can also influence the level of expression of an integrated polynucleotide.
  • An "isolated" plasmid, virus, or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture.
  • Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this invention are increasingly more preferred. Thus, for example, a 2-fold enrichment is preferred, 10-fold enrichment is more preferred, 100-fold enrichment is more preferred, 1000-fold enrichment is even more preferred. [0071] A preparation of rAAV is said to be "substantially free" of helper virus if the ratio of infectious rAAV particles to infectious helper virus particles is at least about 10 2 :l; preferably at least about 10 4 :l, more preferably at least about 10 6 :l; still more preferably at least about 10 8 :l.
  • Preparations are also preferably free of equivalent amounts of helper virus proteins (i.e., proteins as would be present as a result of such a level of helper virus if the helper virus particle impurities noted above were present in disrupted form).
  • helper virus proteins i.e., proteins as would be present as a result of such a level of helper virus if the helper virus particle impurities noted above were present in disrupted form.
  • Viral and/or cellular protein contamination can generally be observed as the presence of Coomassie staining bands on SDS gels (e.g. the appearance of bands other than those corresponding to the AAV capsid proteins VPl, VP2 and VP3).
  • a "host cell” includes an individual cell or cell culture which can be or has been a recipient for vector(s) or for incorporation of polynucleotides and/or proteins.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic of total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
  • a host cell includes cells transfected in vivo with a polynucleotide(s) of this invention.
  • Transformation or “transfection” refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, for example, lipofection, transduction, infection or electroporation.
  • the exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome.
  • An "individual” or “subject” refers to a mammal, and includes, but is not limited to, domestic animals, sports animals, rodents, primates, pets, horses, dogs, cats, including humans.
  • an “effective amount” is an amount sufficient to effect beneficial or desired clinical results.
  • An effective amount can be administered in one or more administrations.
  • an “effective amount” is an amount that achieves any of the following: reduction of TNF levels; reduction of an inflammatory response; and/or palliation, amelioration, stabilization, reversal, slowing or delay in the progression of the disease state.
  • TNF antagonist to a subject in addition to the delivery of an rAAV to the same subject, or administration of two different rAAV vectors to the same subject.
  • "in conjunction with” refers to administration of one treatment modality before, during or after delivery of the other treatment modality to the subject.
  • treatment is an approach for obtaining beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean an improvement of, increase rate of, or increased extent of healing and regeneration of tissue of a bone or wound site receiving an effective amount of an rAAV vector comprising a polynucleotide encoding a TNF antagonist.
  • treatment of an individual may be undertaken to decrease or limit the pathology associated with elevated levels of TNF, including, but not limited to, an inherited or induced genetic deficiency, infection by a viral, bacterial, or parasitic organism, a neoplastic or aplastic condition, or an immune system dysfunction such as autoimmunity. Treatment may be performed either prophylactically or therapeutically; that is, either prior or subsequent to the initiation of a pathologic event or contact with an etiologic agent.
  • a "biological sample” encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay.
  • the definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom, and the progeny thereof.
  • the definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides.
  • biological sample encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.
  • “Palliating" a disease means that the extent and/or undesirable clinical manifestations of a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not administering rAAV vectors of the present invention.
  • This invention provides recombinant AAV (rAA V) vectors for reducing levels of TNF at a periodontal, wound or bone healing site in a subject.
  • these rAAV vectors comprise a polynucleotide encoding a TNF antagonist.
  • the TNF antagonist is a TNFR, or a TNFR polypeptide (including biologically active derivative(s) thereof).
  • a preferred TNFR is derived from the p75 TNFR.
  • An rAAV vector of this invention comprises a heterologous (i.e. non-
  • the heterologous polynucleotide is preferably flanked by at least one, more preferably two, AAV inverted terminal repeats (ITRs). Variations in which an rAAV construct is flanked by a only a single (typically modified) ITR have been described in the art and can be employed in connection with the present invention.
  • a TNF antagonist is supplied to an individual, preferably a mammal, most preferably a human, as an expressed product of a polynucleotide which encodes a TNF antagonist.
  • the polynucleotide encoding the TNF antagonist is delivered to the mammal in the form of an rAAV vector. As defined, such a
  • TNF antagonist may be any polypeptide which binds to TNF including, but not limited to, a TNFR polypeptide and an anti-TNF antibody.
  • the TNF antagonist is secreted by the cell that receives the rAAV vector; preferably the TNF antagonist is soluble ⁇ i.e., not attached to the cell).
  • soluble TNF antagonists are devoid of a transmembrane region and are secreted from the cell. Techniques to identify and remove polynucleotide sequences which encode transmembrane domains are known in the art.
  • the TNF antagonist is a TNFR polypeptide.
  • TNFR polypeptide may be an intact TNFR (preferably from the same species that receives the rAAV) or a suitable fragment of TNFR.
  • U.S. Patent 5,605,690 provides examples of TNFR polypeptides, including soluble TNFR polypeptides, appropriate for use in the present invention.
  • the TNFR polypeptide comprises an extracellular domain of TNFR. More preferably, the TNFR polypeptide is a fusion polypeptide comprising an extracellular domain of TNFR linked to a constant domain of an immunoglobulin molecule that does not fix complement in a host delivered the molecule. The constant domain may be naturally occurring.
  • the constant domain may be modified or mutated so that it does not bind to complement or activate complement system.
  • the TNFR polypeptide is a fusion polypeptide comprising an extracellular domain of the p75 TNFR linked to a constant domain of an IgGl or IgG3 molecule.
  • an Ig used for fusion proteins is human, preferably human IgGl or IgG3.
  • TNFR polypeptides may be used in the present invention.
  • Multivalent forms of TNFR polypeptides possess more than one TNF binding site.
  • Multivalent forms of TNFR polypeptides may be encoded in an rAAV vector, for example, through the repeated ligation of polynucleotides encoding TNF binding domains, each repeat being separated by a linker region.
  • the TNFR of the present invention is a bivalent, or dimeric, form of TNFR.
  • a chimeric antibody polypeptide with TNFR extracellular domains substituted for the variable domains of either or both of the immunoglobulin heavy or light chains would provide a TNFR polypeptide for the present invention.
  • a chimeric TNFR:antibody polypeptide when such a chimeric TNFR:antibody polypeptide is produced by cells, it forms a bivalent molecule through disulfide linkages between the immunoglobulin domains.
  • TNFR:Fc Such a chimeric TNFR:antibody polypeptide is referred to as TNFR:Fc.
  • the TNF antagonist is a TNFR polypeptide construct sTNFR(p75):Fc.
  • polypeptide sequence of sTNFR(p75):Fc is depicted in Fig. 5 herein and Fig.l of U.S. Pat. No. 5,637,540.
  • the coding sequence for this TNF antagonist is found in plasmid pCAVDHFRhuTNFRFc as described in U.S. Patent Nos. 5,605,690 and 5,637,540. Any polynucleotide which encodes this sTNFR(p75):Fc polypeptide is suitable for use in the present invention.
  • a polynucleotide sequence encoding sTNFR(p75):Fc is depicted in Fig. 2 of U.S. Pat. No. 5,637,540.
  • additional TNFR polypeptide sequences include, but are not limited to, those indicated in Figures 2 and 3 of U.S. Patent No. 5,395,760.
  • Polynucleotides which encode TNFR polypeptides can be generated using methods known in the art from TNFR polynucleotide sequences known in the art.
  • preferable polynucleotide sequences which encode TNFR polypeptides include, but are not limited to, TNFR polynucleotide sequences found in U.S. Patent Nos. 5,395,760 and 5,605,690 and GenBank entries M32315 (human TNFR) and M59378 (murine TNFRI).
  • TNF antagonist activity may be assessed with a cell- based competitive binding assay.
  • radiolabeled TNF is mixed with serially diluted TNF antagonist and cells expressing cell membrane bound TNFR. Portions of the suspension are centrifuged to separate free and bound TNF and the amount of radioactivity in the free and bound fractions determined. TNF antagonist activity is assessed by inhibition of TNF binding to the cells in the presence of the TNF antagonist.
  • TNF antagonists may be analyzed for the ability to neutralize TNF activity in vitro in a bioassay using cells susceptible to the cytotoxic activity of TNF as target cells, such as L929 cells (see, for example, Example 3 of U.S. Pat. No. 5,637,540).
  • target cells cultured with TNF, are treated with varying amounts of TNF antagonist and subsequently are examined for cytolysis.
  • TNF antagonist activity is assessed by a decrease in TNF-induced target cell cytolysis in the presence of the TNF antagonist.
  • the invention also provides rAAV vectors comprising a polynucleotide encoding an interleukin 1 (IL-I) antagonist.
  • IL-I interleukin 1
  • cytokine IL-I has been implicated as a pivotal mediator in periodontitis. Delima et al., J Infect Dis, 186, 511-516, 2002. In RA, IL-I appears to be involved in infiltration of inflammatory cells and cartilage destruction in the affected joint. A clinical trial with an IL-I antagonist in patients with RA indicated that blocking IL-I activity may result in amelioration of RA symptoms (Campion et al., 1996, Arthritis Rheum. 39:1092-1101; Bresnihan et al., 1996, Arthritis Rheum. 39:S73). In a murine arthritis model, a combined anti-TNF ⁇ /anti-IL-1 treatment led to both diminished inflammation and to diminished joint cartilage damage (Kuiper et al., 1998, Cytokine 10:690-702).
  • the present invention provides rAAV vectors comprising a polynucleotide encoding a TNF antagonist (such as sTNFR(p75):Fc) and an IL-I antagonist (or, the rAAV vector comprises a polynucleotide which encodes a TNF antagonist and an IL-I antagonist).
  • the present invention also provides rAAV vectors comprising a polynucleotide encoding an IL-I antagonist.
  • the IL-I antagonist is an IL-I receptor (IL-IR), or an IL-IR polypeptide (including biologically active derivatives(s) thereof), that exhibits the desired biological activity (i.e., binding to IL-I).
  • the IL-IR is derived from IL-IR type II.
  • IL-IR polypeptide sequences include, but are not limited to, that depicted in Fig. 3 of U.S. Pat. No. 5,637,540 and those found in IL-IR GenBank entry U74649 and U.S. Patent 5,350,683. Any polynucleotide which encodes an IL-IR polypeptide is suitable for use in the present invention.
  • a polynucleotide sequence encoding a preferred IL-IR polypeptide is depicted in Fig. 3 of U.S. Pat. No. 5,637,540.
  • Suitable polynucleotides for use in the present invention can be synthesized using standard synthesis and recombinant methods.
  • Methods to assess IL-I antagonist activity are known in the art.
  • IL-I antagonist activity may be assessed with a cell-based competitive binding assay as described herein for TNF antagonists.
  • IL-I antagonist activity may be assessed for the ability to neutralize IL-I activity in vitro in a bioassay for IL-I.
  • a cell line for example, EL-4 NOB-I
  • IL-2 interleukin 2
  • This IL-I responsive cell line is used in combination with a IL-2 sensitive cell line (for example, CTLL-2). Proliferation of the IL-2 sensitive cell line is dependent on the IL-I responsive cell line producing IL-2 and thus, is used as a measure of 11-1 stimulation of the IL-I responsive cell line.
  • IL-I antagonist activity would be assessed by its ability to neutralize IL-I activity in such a IL-I bioassay (Gearing et al., 199I 5 J. Immunol. Methods 99:7-11; Kuiper et al., 1998).
  • the vector(s) of the invention are encapsidated into an rAAV virus particle.
  • the invention includes an rAAV virus particle (recombinant because it contains a recombinant polynucleotide) comprising any of the vectors described herein. Methods of producing such particles are described below. [0096]
  • the present invention also provides compositions containing any of the rAAV vectors (and/or rAAV virus particles comprising the rAAV vectors) described herein. These compositions are especially useful for administration to individuals who may benefit from a reduction in the level of TNF.
  • compositions of the invention for use in reducing TNF levels comprise an effective amount of an rAAV vector encoding a TNF antagonist, preferably in a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable excipients are relatively inert substances that facilitate administration of a pharmacologically effective substance and can be supplied as liquid solutions or suspensions, as emulsions, or as solid forms suitable for dissolution or suspension in liquid prior to use or as gels, gels matrixes or freeze dried compositions that can be used to coat or fill solid delivery devises such as dental tray, mouth guards, or bone pins,.
  • an excipient can give form or consistency, or act as a diluent.
  • Suitable excipients include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers and microspheres such polyglycolic lactic acid.
  • Excipients as well as formulations for parenteral and nonparenteral drug delivery are set forth in Remington 's Pharmaceutical Sciences 19th Ed. Mack Publishing (1995).
  • these rAAV compositions are formulated for administration by local injection (such as intramuscularly or at the peridontium). Accordingly, these compositions are preferably combined with pharmaceutically acceptable vehicles such as saline, Ringer's balanced salt solution (pH 7.4), dextrose solution, and the like. Although not required, pharmaceutical compositions may optionally be supplied in unit dosage form suitable for administration of a precise amount.
  • the invention also includes any of the above vectors (or compositions comprising the vectors) for use in treatment of TNF-associated oral buccal disorders, such as periodontitis and impairment of bone and wound healing .
  • the invention also includes any of the above vectors (or compositions comprising the vectors) for use in reducing TNF levels at the site, such as periodontal , bone or wound healing sites in an individual.
  • the invention further provides use of any of the above vectors (or compositions comprising the vectors) in the manufacture of a medicament for treatment of TNF-associated disorders or conditions, such as periodontitis or bone or wound healing .
  • the invention also provides use of any of the above vectors (or compositions comprising the vectors) in the manufacture of a medicament for reducing TNF activity levels at the site of the disorder, such as periodontal, wound, or bone healing site in an individual.
  • Host cells comprising rAAV vectors are used for generation rAAV vectors described herein.
  • eukaryotic host cells are yeast, insect, avian, plant and mammalian cells.
  • the host cells are mammalian.
  • host cells include, but are not limited to, HeLa and 293 cells, both of human origin and both readily available.
  • the rAAV vectors of this invention may be prepared using standard methods in the art.
  • Adeno-associated viruses of any serotype are suitable, since the various serotypes are functionally and structurally related, even at the genetic level (see, e.g., Blacklow, pp. 165-174 of "Parvoviruses and Human Disease” J.R. Pattison, ed. (1988); and Rose, Comprehensive Virology 3:1, 1974). All AAV serotypes apparently exhibit similar replication properties mediated by homologous rep genes; and all generally bear three related capsid proteins such as those expressed in AA V2.
  • the rAAV vectors of this invention comprise a heterologous polynucleotide that encodes a TNF antagonist.
  • the rAAV vectors may also encode additional polypeptides, such as an IL-I receptor type II.
  • the rAAV vectors may comprise a heterologous polynucleotide that encodes an IL-I antagonist, such as an IL-IR.
  • heterologous polynucleotide will generally be of sufficient length to provide the encoding sequence and desired function.
  • the heterologous polynucleotide will preferably be less than about 5 kb although other serotypes and/or modifications may be employed to allow larger sequences to packaged into the AAV viral particles.
  • a preferred polynucleotide encodes a TNFR:Fc as represented in Fig. 5, is about 1.5 kb in length.
  • heterologous polynucleotide Since transcription of the heterologous polynucleotide is desired in the intended target cell, it can be operably linked to its own or to a heterologous promoter and/or enhancer, depending for example on the desired level and/or specificity of transcription within the target cell, as is known in the art.
  • a heterologous promoter and/or enhancer Various types of promoters and enhancers are suitable for use in this context.
  • Feldhaus U.S. patent application 09/171,759 , filed 20 Oct. 1998) describes a modified ITR comprising a promoter to regulate expression from an rAAV. Constitutive promoters provide an ongoing level of gene transcription, and are preferred when it is desired that the therapeutic polynucleotide be expressed on an ongoing basis.
  • Inducible or regulatable promoters generally exhibit low activity in the absence of the inducer, and are up- regulated in the presence of the inducer. They may be preferred when expression is desired only at certain times or at certain locations, or when it is desirable to titrate the level of expression using an inducing agent. Promoters and enhancers may also be tissue-specific, that is, they exhibit their activity only in certain cell types, presumably due to gene regulatory elements found uniquely in those cells. Such tissue-specific promoters and enhancers are known in the art. By way of illustration, an example of tissue-specific promoters includes various myosin promoters for expression in muscle. Another example of tissue-specific promoters and enhancers are of regulatory elements for cell and/or tissue types that are in the peridontium.
  • 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 conditions.
  • promoters and/or enhancers that are activated in response to mediators that drive inflammatory flares including, but not limited to, those from proinflammatory cytokine genes (e.g., TNF ⁇ , IL- l ⁇ and IFN ⁇ ), would result in the expression of a TNF antagonist during the period of inflammatory flare (Varley et al., 1998, MoI Med. Today 4:445-451).
  • the TNF ⁇ promoter region is approximately 1.2 kb, and the sequence has been reported by Takashiba et al., 1993, Gene, 131:307-308.
  • promoters are the SV40 late promoter from simian virus 40, the Baculovirus polyhedron enhancer/promoter element, Herpes Simplex Virus thymidine kinase (HSV tk), the immediate early promoter from cytomegalovirus (CMV) and various retroviral promoters including LTR elements.
  • Additional inducible promoters include heavy metal ion inducible promoters (such as the mouse mammary tumor virus (mMTV) promoter or various growth hormone promoters), and the promoters from T7 phage which are active in the presence of T7 RNA polymerase.
  • mMTV mouse mammary tumor virus
  • T7 phage which are active in the presence of T7 RNA polymerase.
  • a large variety of other promoters are known and generally available in the art, and the sequences for many such promoters are available in sequence databases such as the GenBank database.
  • the heterologous polynucleotide encoding a TNF antagonist will preferably also comprise control elements that facilitate translation (such as a ribosome binding site or "RBS" and a polyadenylation signal).
  • the heterologous polynucleotide will generally comprise at least one coding region operatively linked to a suitable promoter, and can also comprise, for example, an operatively linked enhancer, ribosome binding site and poly-A signal.
  • the heterologous polynucleotide can comprise one encoding region, or more than one encoding region under the control of the same or different promoters. The entire unit, containing a combination of control elements and encoding region, is often referred to as an expression cassette.
  • a heterologous polynucleotide encoding a TNF antagonist is integrated by recombinant techniques into or preferably in place of the AAV genomic coding region (i.e., in place of the AAV rep and cap genes), but is generally flanked on either side by AAV ITRs.
  • an ITR appears both upstream and downstream from the coding sequence, either in direct juxtaposition, preferably (although not necessarily) without any intervening sequence of AAV origin in order to reduce the likelihood of recombination that might regenerate a replication-competent AAV ("RCA") genome.
  • RCA replication-competent AAV
  • Recent evidence suggests that a single ITR can be sufficient to carry out the functions normally associated with configurations comprising two ITRs (U.S.
  • Patent 5,478745 and vector constructs with only one ITR can thus be employed in conjunction with the packaging and production methods described herein.
  • the resultant rAAV vector is referred to as being "defective" in AAV functions when specific AAV coding sequences are deleted from the vector.
  • insertion of a large heterologous polynucleotide into the genome necessitates removal of a portion of the AAV genome, in particular, one or more of the packaging genes may be removed. Removal of one or more AAV genes is in any case desirable, to reduce the likelihood of generating RCA. Accordingly, encoding or promoter sequences for rep, cap, or both, are preferably removed, since the functions provided by these genes can be provided in trans.
  • the rAAV vectors are provided in a variety of forms, such as in the form of bacterial plasmids, AAV particles, liposomes or any combination thereof.
  • the rAAV vector sequence is provided in the eukaryotic cells transfected with the rAAV vector.
  • the rAAV is to be used in the form of a packaged rAAV particle, there are at least three desirable features of an rAAV virus preparation for use in gene transfer.
  • the rAAV virus should be generated at titers sufficiently high to transduce an effective proportion of cells in the target tissue. High number of rAAV viral particles are typically required for gene transfer in vivo. For example, some treatments may require in excess of 10 8 particles.
  • the rAAV virus preparations should be essentially free of replication-competent AAV ⁇ i.e., phenotypically wild-type AAV which can be replicated in the presence of helper virus or helper virus functions).
  • the rAAV virus preparation as a whole be essentially free of other viruses (such as a helper virus used in AAV production) as well as helper virus and cellular proteins, and other components such as lipids and carbohydrates, so as to minimize or eliminate any risk of generating an immune response in the context of gene transfer.
  • viruses such as a helper virus used in AAV production
  • helper virus and cellular proteins such as lipids and carbohydrates
  • AAV is a "helper-dependent" virus that requires co-infection with a helper virus (typically adenovirus) or other provision of helper virus functions in order to be effectively replicated and packaged during the process of AAV production; and, moreover, as described above, adenovirus has been observed to generate a host immune response in the context of gene transfer applications (see, e.g., Le et al., 1997; 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).
  • helper virus typically adenovirus
  • an rAAV vector is to be packaged in an AAV particle, in order to replicate and package the rAAV vector, the missing functions are complemented with a packaging gene, or a plurality thereof, which together encode the necessary functions for the various missing rep and/or cap gene products.
  • the packaging genes or gene cassettes are preferably not flanked by AAV ITRs and preferably do not share any substantial homology with the rAAV genome.
  • the level of homology and corresponding frequency of recombination increase with increasing length of the homologous sequences and with their level of shared identity.
  • the level of homology that will pose a concern in a given system can be determined theoretically and confirmed experimentally, as is known in the art. Generally, however, recombination can be substantially reduced or eliminated if the overlapping sequence is less than about a 25 nucleotide sequence if it is at least 80% identical over its entire length, or less than about a 50 nucleotide sequence if it is at least 70% identical over its entire length. Of course, even lower levels of homology are preferable since they will further reduce the likelihood of recombination. It appears that, even without any overlapping homology, there is some residual frequency of generating RCA.
  • rAAV vector construct, and the complementary packaging gene constructs can be implemented in this invention in a number of different forms. Viral particles, plasmids, and stably transformed host cells can all be used to introduce such constructs into the packaging cell, either transiently or stably.
  • a variety of different genetically altered cells can thus be used in the context of this invention.
  • a mammalian host cell may be used with at least one intact copy of a stably integrated rAAV vector.
  • An AAV packaging plasmid comprising at least an AAV rep gene operably linked to a promoter can be used to supply replication functions (as described in U.S. Patent 5,658,776).
  • a stable mammalian cell line with an AAV rep gene operably linked to a promoter can be used to supply replication functions (see, e.g., Trempe et al., U.S. Patent 5,837,484; Burstein et al., WO 98/27207; and Johnson et al., U.S. Patent 5,658,785).
  • the AAV cap gene providing the encapsidation proteins as described above, can be provided together with an AAV rep gene or separately (see, e.g., the above-referenced applications and patents as well as Allen et al. (WO 96/17947). Other combinations are possible. [0116] As is described in the art, and illustrated in the references cited above and in Examples below, genetic material can be introduced into cells (such as mammalian "producer" cells for the production of rAAV) using any of a variety of means to transform or transduce such cells.
  • such techniques include, but are not limited to, transfection with bacterial plasmids, infection with viral vectors, electroporation, calcium phosphate precipitation, and introduction using any of a variety of lipid-based compositions (a process often referred to as "lipofection")- Methods and compositions for performing these techniques have been described in the art and are widely available.
  • Selection of suitably altered cells may be conducted by any technique in the art.
  • the polynucleotide sequences used to alter the cell may be introduced simultaneously with or operably linked to one or more detectable or selectable markers as is known in the art.
  • Drug resistant cells can then be picked and grown, and then tested for expression of the desired sequence (i.e., a product of the heterologous polynucleotide).
  • Testing for acquisition, localization and/or maintenance of an introduced polynucleotide can be performed using DNA hybridization-based techniques (such as Southern blotting and other procedures as known in the art).
  • RNA extracted from the genetically altered cells can be readily performed by Northern analysis of RNA extracted from the genetically altered cells, or by indirect immunofluorescence for the corresponding gene product. Testing and confirmation of packaging capabilities and efficiencies can be obtained by introducing to the cell the remaining functional components of AAV and a helper virus, to test for production of AAV particles. Where a cell is inheritably altered with a plurality of polynucleotide constructs, it is generally more convenient (though not essential) to introduce them to the cell separately, and validate each step seriatim. References describing such techniques include those cited herein. [0118] rAAV viral vectors can be made by methods known in the art, including transient transfection strategies as described in U. S. Pat. No.
  • rAAV vector production system generally has three common elements: 1) a permissive host cell for replication, including standard host cells known in the art ; 2) a helper virus function; and 3) a transpackaging rep-cap construct.
  • host cells used in rAAV vector production system are 293-A, 293-S (obtained from BioReliance), VERO, and HeLa cell lines which are applicable for the three vector production systems described herein.
  • Helper virus function may be provided by a wild type adenovirus type 5 virus when utilized in stable cell line manufacture, an adenovirus hybrid vector system, or a plasmid pAd Helper 4.1 expressing the E2a, E4-orf6 and VA genes of adenovirus type 5 (Ad5) when utilized in transfection production systems.
  • Ad5 adenovirus type 5
  • AAV2 5' and 3' ITR pseudotyped constructs and a transpackaging rep-cap plasmid containing an AAV-2 rep gene and the cap sequences of AAV 5 (AAV-2/AAV-5, respectively) pseudotyped transpackaging construct under the control of the AA V2 p5 promoter rep-cap cell line are generated as described.
  • the p5 promoter of the rep-cap (AAV2/5 respectively) pseudotyped transpackaging construct is replaced with a minimal heat shock promoter consisting of essentially a TATA box.
  • Stable cell lines for rAAV viral vector production can be generated by transfecting cell lines described above and screening for stable cell lines which can be repeatedly propagated and which contain the rep-cap packaging construct as well as the AAV ITR serotype expression plasmids stably integrated.
  • rAAV vectors are produced and purified by any methods know in the art and described in WO99/11764 and WO00/14205
  • Ad hybrid production of rAAV vectors can be performed as described in
  • WO96/13598 using a rep-cap cell line described in WO99/15685 The system originally developed by T.C. He et al and disclosed in US Pat. No. 5,922,576 is used to produce recombinant Adenovirus/AAV hybrids (Ad/AAV hybrids).
  • Ad/AAV hybrids Adenovirus/AAV hybrids
  • This approach utilizes two plasmid vector systems (a transfer or shuttle vector and an adenovirus genome containing vector) that undergo bacterial recombination in competent E.coli yielding a recombinant Ad/AAV hybrid plasmid which is utilized to derive Ad/AAV hybrid viral plaques for generation of a viral stock.
  • rAAV virus preparations can be further processed to enrich for rAAV particles, deplete helper virus particles, or otherwise render them suitable for administration to a subject. See Atkinson et al. for exemplary techniques (WO 99/11764, WO 00/14205, and U.S. Pat. No. 6,566,118).
  • Purification techniques can include isopynic gradient centrifugation, and chromatographic techniques.
  • Reduction of infectious helper virus activity can include inactivation by heat treatment or by pH treatment as is known in the art.
  • Other processes can include concentration, filtration, diafiltration, or mixing with a suitable buffer or pharmaceutical excipient.
  • Preparations can be divided into unit dose and multi dose aliquots for distribution, which will retain the essential characteristics of the batch, such as the homogeneity of antigenic and genetic content, and the relative proportion of contaminating helper virus.
  • Various methods for the determination of the infectious titer of a viral preparation are known in the art. For example, one method for titer determination is a high-throughput titering assay as provided by Atkinson et al. (WO 99/11764). Virus titers determined by this rapid and quantitative method closely correspond to the titers determined by more classical techniques. In addition, however, this high-throughput method allows for the concurrent processing and analysis of many viral replication reactions and thus has many others uses, including for example the screening of cell lines permissive or non-permissive for viral replication and infectivity.
  • the invention provides methods for treating or preventing TNF-associated disorders or conditions in a subject.
  • disorders or conditions are bone loss, impairment of wound healing and bone healing, bone allo-and auto-graft healing, and periodontitis.
  • the invention provides methods in which administration of rAAV vectors described herein is used to treat or prevent TNF-associated disorders or conditions.
  • the invention also provides methods in which administration of rAAV vectors described herein is used to reduce levels of TNF at the site of disorder (e.g., a periodontal site, a wound site, and a bone grafting site) in a subject. Such methods may be particularly beneficial to individuals with a TNF-associated disorders described herein. It is understood that TNF levels are reduced when compared to TNF levels of a subject prior to receiving rAAV encoding a TNF antagonist or when compared to TNF levels of an individual that does not receive rAAV encoding a TNF antagonist. It is understood that TNF levels refers to levels of free (uncomplexed or unbound) or active TNF. Methods to detect TNF levels are described below.
  • the treatment described herein may be beneficial to bone graft in an individual.
  • Treatment may mean prolonging survival of a bone graft as compared to expected survival if not receiving treatment.
  • the invention also provides methods in which administration of rAAV vectors described herein (or compositions comprising an rAAV vector(s)) is used to reduce an inflammatory response at the site of disorder (e.g., periodontal site) in a subject. It is understood that an inflammatory response is reduced when compared to an inflammatory response in a subject prior to receiving rAAV encoding a TNF antagonist or when compared to an inflammatory response in an individual that does not receive rAAV encoding TNF antagonist.
  • the rAAV vector (or compositions comprising an rAAV vector(s)) is delivered to a site of disorder such as a periodontal site (e.g., by intramuscular injection or to the interproximal area) of a mammal thus providing a source of the TNF antagonist at the site of inflammation.
  • the rAAV vector comprises a polynucleotide encoding sTNFR(p75):Fc.
  • rAAV vectors are administered in conjunction with administration of another agent that reduces inflammation at the site (such as periodontal site).
  • the agent is a TNF antagonist, such as a TNFR or an anti- TNF antibody.
  • the other agent administered in conjunction with the rAAV vectors is an IL-I antagonist.
  • the other agent, such as TNF antagonist or IL-I antagonist, preferably in composition with physiologically acceptable carriers, exicipients or diluents, may be administered by suitable techniques.
  • At least two different rAAV vectors are administered to deliver the source of TNF antagonist and IL-I antagonist to the periodontal site of a mammal at the site of inflammation.
  • one of the rAAV vectors comprises a polynucleotide encoding a TNFR and another one of the rAAV vectors comprises a polynucleotide encoding an IL-IR.
  • the heterologous polynucleotides may be operably linked to transcriptional promoters and/or enhancers which are active under similar conditions or to transcriptional promoters and/or enhancers which are active under different conditions, e.g., independently regulated.
  • the two different rAAV vectors i.e., one comprising a polynucleotide encoding a TNFR and one comprising a polynucleotide encoding IL-IR
  • one or more rAAV vectors may be administered.
  • a vector may be administered that encodes a TNF antagonist, such as TNF receptor (most preferably sTNFR(p75):Fc).
  • an additional vector may be administered that encodes an IL-I antagonist, such as an IL-I receptor polypeptide.
  • a single vector encoding both a TNF antagonist and an IL-I antagonist may be administered. This single vector may have the coding sequences under control of the same or different transcriptional regulatory elements. If more than one vector is used, it is understood that they may be administered at the same or at different times and/or frequencies.
  • the individual receiving rAAV vector(s) will have cells which contain the rAAV vector (after administration), and most preferably will have cells in which the rAAV vector(s) is integrated into the cellular genome.
  • Stable integration of rAAV is a distinct advantage, as it allows more persistent expression than episomal vectors.
  • cells (i.e., at least one cell) in the individual will comprise stably integrated rAAV.
  • rAAV(s) results in integration of the rAAV(s) into cellular genomes (although, as is understood by those in the art, not all rAAV vectors need be integrated). Methods of determining and/or distinguishing integrated vs. non-integrated forms, such as Southern detection methods, are well known in art.
  • Administration of rAAV vectors (preferably packaged as AAV particles) to a periodontal site may be through any of a number of routes.
  • One mode of administration is through intramuscular delivery. Intramuscular delivery of the rAAV vectors can reduce TNF levels both in tissue and inter-tissue spaces near the site of injection and also in circulation.
  • Another mode of administration of rAAV compositions of the invention is through injection of the composition(s) directly to the tissue or anatomical site. For example, administration is by injection of the composition to the interproximal area.
  • An effective amount of rAAV (preferably in the form of AAV particles) is administered, depending on the objectives of treatment.
  • An effective amount may be given in single or divided doses.
  • the objective of treatment is generally to meet or exceed this level of transduction.
  • this level of transduction can be achieved by transduction of only about 1 to 5% of the target cells, but is more typically about 20% of the cells of the desired tissue type, usually at least about 50%, preferably at least about 80%, more preferably at least about 95%, and even more preferably at least about 99% of the cells of the desired tissue type.
  • the number of rAAV particles administered per injection will generally be between about 1 x 10 6 and about 1 x 10 14 particles, preferably, between about 1 x 10 7 and 1 x 10 13 particles, more preferably about 1 x 10 9 and 1 x 10 12 particles and even more preferably about 1 x 10 ⁇ particles.
  • the invention also provides methods in which administration of rAAV vectors described herein use ex vivo strategies for delivery of polynucleotides to the mammal. Such methods and techniques are known in the art. See, for example, U.S. Patent 5,399,346. Generally, cells are transduced by the rAAV vectors in vitro and then the transduced cells are introduced into the mammal, for example, into an arthritic joint. Suitable cells are known to those skilled in the art and include autologous cells, such as stem cells.
  • samples removed by biopsy or surgical excision may be analyzed by in situ hybridization, PCR amplification using vector-specific probes and/or RNAse protection to detect rAAV DNA and/or rAAV mRNA.
  • harvested tissue and/or serum samples can be monitored for the presence of TNF antagonist encoded by the rAAV with immunoassays, including, but not limited to, ELISA, immunoblotting, immunoprecipitation, immunohistology and/or immunofluorescent cell counting, or with function-based bioassays dependent on TNF antagonist-mediated inhibition of TNF activity.
  • the rAAV encoded TNF antagonist is a TNFR polypeptide
  • the presence of the encoded TNFR in harvested samples can be monitored with a TNFR immunoassay or a function-based bioassay dependent on TNFR- mediated inhibition of TNF killing of mouse L929 cells. Examples of such assays are known in the art and described, for example in U.S. Pat. No. 6,537,540.
  • the effectiveness of rAAV delivery can also be monitored by histomorphometric and radiographic analysis of the periodontal tissue destruction and alveolar bone loss.
  • the effectiveness of the methods provided herein may, for example, be monitored by assessment of the relative levels of TNF in harvested tissue, joint fluid and/or serum subsequent to delivery of the rAAV vectors described herein.
  • Assays for assessing TNF levels are known in the art and include, but are not limited to, immunoassays for TNF, including, but not limited to, immunoblot and/or immunoprecipitation assays, and cytotoxicity assays with cells sensitive to the cytotoxic activity of TNF. See, for example, Khabar et al., 1995, Immunol. Lett. 46:107-110.
  • the treated subject may also be monitored for clinical features which accompany the TNF-associated oral buccal disorder.
  • subjects may be monitored for reduction is symptoms associated with inflammation.
  • the subject may be assessed for improvements in a number of clinical parameters including, but not limited to, swelling, pain, and tooth loss.
  • compositions, dosage regimen ⁇ i.e., dose, timing and repetition
  • route of administration will depend on a number of different factors, including, but not limited to, the subject's medical history and features of the condition and the subject being treated.
  • the assessment of such features and the design of an appropriate therapeutic regimen is ultimately the responsibility of the prescribing physician.
  • the particular dosage regimen may be determined empirically.
  • compositions and methods for treating TNF-associated oral buccal diseases for reducing the levels of TNF in a mammal provide, inter alia, compositions and methods for treating TNF-associated oral buccal diseases for reducing the levels of TNF in a mammal. It is understood that variations may be applied to these methods by those of skill in this art without departing from the spirit of this invention.
  • LPS isolation was as previously described (Darveau & Hancock, 1983).
  • LPS extraction consisted of culturing the P. gingivalis strain W83 via an anaerobic chamber using a modified Brucella-Broth Medium specific for anaerobes. After the growing process, each batch was centrifuged at 5000 rpm for 30 min. The supernatant was discarded, the cells were resuspended in sterile water, then centrifuged. The final pellet was frozen at -20 0 C until the necessary amount of bacteria was obtained for the entire experiment.
  • LPS liposaccharide
  • Figure 1 The first six lanes show a serial dilution of E. coli LPS on the SDS-PAGE, and the last lane shows the P. gingivalis LPS with typical laddering appearance. An arrow indicates the isolated LPS.
  • Standardized intraoral radiographs of maxillary molar teeth were taken on all animals at baseline and after 2, 4, 6 and 8 weeks.
  • Computer-assisted densitometric image analysis was performed by measuring radiographic density differences between baseline radiographs and paired radiographs from each time point as previously described (Braegger et al., J Periodontal Res, 22, 227-229, 1987; Jeffcoat, J Periodontal, 63, 367- 372, 1992) and recently adapted (Taba Jr. et al., Implant Dent, 12, 252-258, 2003) for use with rat teeth.
  • radiographs was digitized using a scanner (UMAX Technologies, Texas, USA) at 600 dpi image resolution over a grayscale format.
  • FIG. 1 shows LPS-mediated bone resorption via radiographic images from representative rat maxillae from LPS injected (A) and control (B) animals. Arrows in Image A (LPS treatment) show approximately 50% bone loss after 8 weeks of local P. gingivalis LPS administration.
  • FIG. 3 shows a quantitative measure of LPS-mediated bone loss in the rat model of experimental periodontitis. Digital subtraction radiography was performed to ascertain the amount of alveolar bone resorption that occurred over time (in mm 2 ). As shown in Figure 3, the LPS-disease induction lead to a significant (approximately 50%) loss of alveolar bone support.
  • FIG. 4 shows LPS-mediated bone resorption via sagittal histologic photomicrographs of the interproximal region between Ml and M2 of the rat maxillary teeth.
  • Hematoxylin & Eosin staining was used.
  • Hematoxylin-eosin stain is a simple method for general histology. Generally, basophilic nuclei, bacteria, calcium, and so on are stained "blue” with hematoxylin, while eosinophilic cytoplasm, connective, and all other tissues are counterstained “red” with eosin.
  • the control group (A) received sterile solution
  • the experimental group (B) received P. gingivalis LPS for 8 weeks as described above. The difference in the bone level between groups indicated by the lines was noted between group A and group B. In addition to the loss of alveolar bone support, significant destruction of tooth root cementum and connective tissue attachment was noted in group B.
  • Example 2 Generation of rAAV vectors encoding TNFR:Fc fusion protein
  • the AAV-TNFR:Fc construct used in the examples below was previously described in U.S. Pat. No. 6,537,540, which is incorporated by reference in its entirety. Briefly, the extracellular domain (ECD) of the rat p80 TNFR (Type II) was isolated from rat spleen cDNA and the TNFR ECD was fused to the rat IgGlFc hinge region. The nucleotide and amino acid sequences of the TNFR:Fc fusion are shown in Figures 5A-C. These sequences are also in Figures 8A-C of U.S. Pat. No. 6,537,540.
  • the TNFR:Fc fusion was subcloned into an AAV vector whose rep and cap genes were deleted.
  • Figure 6 depicts 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 a synthetic polyA addition signal.
  • the transcription unit containing the TNFR-Fc fusion gene is enclosed between the AAV-2 ITRs.
  • This rAAV vector plasmid was denoted pAA VCMVrTNFR-Fc.
  • Plasmid pAAVCMVrTNFR-Fc (30 ⁇ g) was transfected into the HeIa C12 packaging cell line by electroporation (Potter et al., 1984, Proc. Natl. Acad. ScL USA 79:7161-7165).
  • the C12 cell line contains the AA V2 rep and cap genes that are transcriptionally quiescent until induction upon infection with adenovirus helper (Clark et al., 1995; Clark et al., 1996, Gene Therapy 3:1124-1132). Twenty four hours post- transfection, the cells were trypsinized and replated in 100 mm plates at densities ranging from 5x10 3 to 5x10 4 cells per plate.
  • the cells were subjected to selection in DMEM containing 10% fetal bovine serum and 300 ⁇ g/ml hygromycin B. Drug-resistant cell clones were isolated, expanded and their ability to produce infectious AAVCMVrTNFR- Fc vectors was tested and compared in an infectivity assay as described in Atkinson et al., 1998, Nucleic Acid Res. 26:2821-2823. One such producer cell clone (C12-55) was further used for production of AAVCMVrTNFR-Fc vector. Production, purification and titration were carried out essentially as described herein and as generally described in Atkinson et al. (WO 99/11764).
  • the AAV-2 serotype vector and the rep-cap construct was described in WO95/34670.
  • a new transpackaging rep-cap plasmid designated pBSHSPRC2C5 was constructed containing an AAV-2 rep gene and the cap sequences of AAV 5 as described in Sandalon et al., J. Virol.78:12355-12365, 2004. Briefly, the AAV5 cap sequence was amplified by PCR from plasmid pACK2/5 with Pfx polymerase (Invitrogen).
  • the AA V2 cap sequence was excised from the AA V2 helper plasmid pBSHSPRC2.3 and replaced with the amplified AA V5 capsid sequence with a Swal/Xbal restriction site.
  • the plasmid pAd Helper 4.1 expressed the E2a, E4-orf6, and VA genes of adenovirus type 5 (Ad5).
  • the p5 promoter of the rep-cap (AAV2/1 respectively) pseudotyped transpackaging construct was replaced with a minimal heat shock promoter consisting of essentially a TATA box, using standard molecular techniques know in the art.
  • Cells were grown by seeding 201 flasks at a cell density of 1.5 x 10 6 cells per flask. The 293 cells were placed into 5OmL of DMEM with 4mM L-glutamine and 10% FBS. The flasks were incubated for 96 hours at 37°C with 5% CO 2 . After 96 hours of incubation, the cells were transfected using calcium phosphate. The flasks were typically 80 - 90% confluent on the day of transfection.
  • AAV Helper plasmid containing the AA V5 capsid 1250 ⁇ g of Ad Helper plasmid, and 375 ⁇ g of the cis plasmid were added to 104 mL of 300 mM Calcium Chloride.
  • the plasmid containing calcium chloride was slowly poured into 104 mL of 2X HBS buffer and allowed to mix for 30 seconds. 8 mL of the precipitate was immediately added to each flask. The flasks were placed at 37°C with 5% CO 2 for 6 to 8 hours. The remaining flask was trypsinized, and cells were counted to be used to calculate productivity.
  • the flasks were removed from the incubator and the media was removed from each of the flasks by aspiration and replaced with 50 mL of DMEM media with 4mM L- glutamine.
  • the flasks were incubated for 72 hours at 37 0 C with 5% CO 2 . After 72 hours, the flasks were removed from the incubator and tapped to release the cells. The contents of each of the flasks were collected. Based on the volume of each container, the amount of 100 mM MgCl 2 and 10% DOC were calculated to achieve a final concentration of 1.8 mM and 0.5%, respectively.
  • the containers were placed in a 37 0 C water bath for 10 - 20 minutes.
  • Benzonase was added to each container to achieve a final concentration of 10 units per mL.
  • the containers were placed in a 37 0 C water bath for 60 minutes, inverting every 15 minutes.
  • Polysorbate 20 (Tween) was added to each container to achieve a final concentration of 1%.
  • the containers were placed in a 37°C water bath for 60 minutes, inverting every 15 minutes.
  • 5M NaCl was added to each container to achieve a final salt concentration of IM.
  • the lysed material was filtered through two filters, Polygard CR Optivap XL 10 filter (0.3 ⁇ m nominal) and Milligard Opticap Capsule (0.5 ⁇ m nominal). Once filtered, the material was concentrated using tangential flow filtration (TFF).
  • TFF membranes Three TFF membranes were used with a 100 Kda nominal molecular weight cut off (NMWCO). The filtered material was concentrated to a volume of 500 mL. Once concentrated, the material was diafiltered with 10 diavolumes. The diafiltered material was filtered through a Suporcap 50 filter (0.45 ⁇ m) and placed at -70 0 C until purification.
  • NMWCO nominal molecular weight cut off
  • rAAV was purified as follows.
  • the clarified producer cell lysate was prepared by resuspending producer cells (5x10 6 cells/ml) in 0.5% deoxycholic acid and benzonase nuclease (35 units/ml) as described previously in Clark et al., supra.
  • Equilibrated POROS HE-20 resin (12.5 raM Tris, pH 8.0; 0.5 mM MgCl 2 ; 100 mM NaCl) was added to the clarified lysate (2 ml/liter) and the mixture was rotated for 16 hr at 4° C to allow sufficient time for particle binding.
  • the HE-20 resin was pelleted at 2,500xg for 30 minutes and resuspended in 12 ml of equilibration buffer per ml of resin used. The resin was washed three times using equilibration buffer by gentle inversion and pelleted at 2,000 rpm for 10 min between each wash. After the final wash, rAAV-2 was eluted by 10 minute resuspension of the pellet in 20 ml of elution buffer (20 mM Tris, pH 8.0; 1 mM MgCl 2 ; 600 mM NaCl). Vector elution was repeated two more times (3 times total) for maximal recovery.
  • the eluted virus was filtered through a 0.45 ⁇ m membrane filter to remove resin fines prior to PI column chromatography.
  • the virus eluate was diluted 6-fold with water to reduce the final salt concentration to ⁇ 100 mM.
  • a Biocad Sprint HPLC system PerSeptive Biosystems
  • POROS PI-50 column 50 ⁇ m bead size
  • a 1.7 ml column was equilibrated with 10 column volumes of 20 mM Tris, pH 7.0; 100 mM NaCl prior to application of the batched vector at a flow rate of 5 ml/min. After sample loading, the column was washed with 10 ml equilibration buffer.
  • Bound material was eluted by application of a NaCl step gradient (0.6 M) at a flow rate of 3 ml/min, and 1 ml gradient fractions were collected. Following column purification, a small aliquot (20 ⁇ l) of peak protein containing fractions was analyzed by SDS-PAGE and SYPRO-Orange (Molecular Probes Inc.) staining to visualize the eluted proteins. Peak virus containing fractions were pooled, dialyzed against multiple changes of 20 mM Tris, pH 8.0, 1 mM MgCl 2 , 200 mM NaCl, and stored in aliquots at -8O 0 C in 10% glycerol.
  • the total protein content in vector preparations was determined using the NanoOrange Protein Quantitation Kit according to the manufacturer's instructions (Molecular Probes, Inc.). DRP titers were determined for purified rAAV by real time PCR methodology utilizing a Prism 7700 Taqman sequence detector system (PE Applied Biosystems) as detailed in Clark et al., supra.
  • Example 3 Serum Levels of AAV-TNFR: Fc After Intramuscular Injection [0158] Lewis Rats were administered lOO ⁇ L of an 1.0 x 10 12 DRP AVV-rat
  • Figure 7 shows the long-term expression of TNFR:Fc as shown by serum levels after delivery of AAV2/2 or AAV2/5 vectors via the quadriceps muscle. Type 5 capsids were correlated with higher levels of TNFR:Fc protein. Peak expression of TNFR protein was noted after 60 days, with therapeutic levels demonstrated for up to one year.
  • Rats with LPS-mediated bone loss were induced as described in Example
  • P. gingivalis LPS strain W83 was injected thrice per week for 8 weeks to induce "chronic" alveolar bone resorption. Injection of 10 ⁇ L of control solution (PBS) or P.g. endotoxin (LPS) were done at interdental areas at a concentration of 1 mg/ml.
  • the viral vector was delivered locally 21 days before starting the disease induction phase at a dosage of 15 ⁇ L of 6x10 11 DRP of rAAV-TNFR:Fc at 4 interdental sites and 2 sites at mesial aspect of the first molar.
  • Figure 8 shows MicroCT images scanned from fixed-non-demineralized specimens from control, LPS injected, and LPS injected and AAV-TNFR:Fc treated animals. Fixed, non-demineralized samples from biopsied areas were evaluated. All specimens were scanned on the MicroCT system and reconstructed into a three- dimensional image at a mesh size of 18 ⁇ mX18 ⁇ mX18 ⁇ m using the GEMS Microview software. Images were rotated into a standardized position to enable comparison across groups. These images were then thresholded to distinguish mineralized from non- mineralized voxels, by determining an optimal grayscale value from image histograms. Standard algorithms were utilized to make the bone measurements. Images were taken after 8 weeks of LPS injections. Left images show overview of the 3 maxillary molar teeth. Right images are cross-section views of the second molar sites. Figure 8 shows the bone anatomy of control (top), disease progression (middle), and disease progression blockage using AAV-TNFR:Fc.
  • Rats with LPS-mediated bone loss are induced as described in Example 1, and as diagrammed in Figure 10. Briefly, P. gingivalis LPS strain W83 is injected thrice per week for 8 weeks to induce "chronic" alveolar bone resorption. Injection of 10 ⁇ L of control solution (PBS) or P.g. endotoxin (LPS) is done at interdental areas at a concentration of 1 mg/ml.
  • PBS control solution
  • LPS P.g. endotoxin
  • the viral vector is delivered locally 21 days before starting the disease induction phase at a dosage of 90 ⁇ L (delivered at 6 sites in a volume of 15 ⁇ l/site) of 8xlO n DRP of rAAV-TNFR:Fc at 4 interdental sites and 2 sites at mesial aspect of the first molar for evaluation of local delivery.
  • the viral vector is delivered intermusculary for systemic delivery as a single dose 21 days before starting the disease induction phase at a dosage of 100 ⁇ L of 7.5x10 11 DRP of rAAV-TNFR:Fc.
  • the groups have three numbers under each day or week timepoint.
  • the three numbers are arranged as x-y-z, where x is the total number of animals alive at that timepoint; y is the number of animals from whom a serum sample is taken, and z is the number of animals sacrificed at that timepoint.
  • Each group is staggered in 3 sets of animals for each time- point of evaluation and serially numbered. Each set will be treated over the study length as individual groups.
  • Serum obtained by tail bleeding is evaluated for TNF protein expression.
  • Serum TNF is determined by ELISA as described by the manufacturer's instructions (Biosource International). Levels of TNF expression are quantified using Ascent® software (Thermo Labsystems) in conjunction with a Multiscan Ascent® photometric microplate reader. Blood samples from rats are collected at baseline, 2, 4, 6 and 8 weeks. Blood collection is by tail bleed. The amount of 400-500 ⁇ l of collected blood provides about 200 ⁇ l serum. To separate the serum, blood is transferred into serum separator tubes (Becton Dickinson # 365956), is incubated for 15' at room temperature, and is spun for 10' at 4000 rpm at room temperature. Serum samples are transferred to cryo vials (1.5 ml tubes by Nunc) and are frozen at -70 0 C. The serum samples for ELISA are sent frozen to the TARGETED GENETICS Corp.
  • Tissue samples are harvested from rats and immediately flash-frozen on dry ice. They are then transferred to 50 mL conical tubes and stored at -7O 0 C until they are processed. The tissues are ground into a fine powder in a liquid nitrogen cooled freezer mill (Freezer Mill, Model 6750 Spex Certi-Prep). The ground powder is then poured back into its tube and placed on dry ice. A sample of each organ powder consistent with the amount called for in the RNA isolation protocol is then weighed out into a 1.5 mL tube. These tubes are kept on dry ice until processed using the Qiagen RNeasy mini kit. The amount of ratTNFR:Fc mRNA contained in each sample of total RNA is quantified using the Perkin Elmer 7700 Sequence Detection System (SDS). [0168] Gingival tissue samples are collected according to the schedule outlined in
  • RNA is harvested as previously described. Xie et al., Biotechniques 11 :324-327; 1991. The samples are analyzed for the amount of TNFR, IL- IB and IL-6 using RealTime PCR. Briefly, PCRs are performed in a total volume of 100 ⁇ l containing 10 ⁇ M Limit-of-Detection PCR for the Qualitative Assessment of tgAAV-ratTNFR:Fc DNA Dissemination in Rat Tissues. [0169] TNFR:Fc primers. One picogram of pAAV-ratTNFR:Fc plasmid 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'.
  • PCRs proceed for 28 cycles of 30 s at 94 0 C, 30s at 61°C, 1 min at 72 0 C and 1 cycle of 8 min at 72 0 C.
  • the projected PCR product of 434 bp is separated on 8% TBE/agarose gels and visualized using EtBr staining.
  • Rats are sacrificed at 8 weeks after surgery and block biopsies are placed into Bouin's fixative (Polysciences, Warrington, PA), decalcified with 10% vol/vol acetic acid, 4% vol/vol formaldehyde, 0.85% NaCl for 2-3 weeks, and then embedded in paraffin.
  • the specimens are cut into 4-5 ⁇ m sections and stained with hematoxylin and eosin (H & E) or toluidine blue for visualization by light or polarized light microscopy.
  • H & E hematoxylin and eosin
  • toluidine blue for visualization by light or polarized light microscopy.
  • a single masked, calibrated examiner examines all of the slides and demonstrates a pre- and post-study calibration inter- and intra-examiner error of ⁇ 5% compared to a standard examiner.
  • Several parameters of periodontal tissue destruction are measured and include (from specimens outlined in Fig.
  • microCT Micro Computed Tomography
  • MicroCT analysis is performed by EVS microCT system. Fixed non- demineralized samples from biopsied areas are evaluated. All specimens are scanned on the MicroCT system and reconstructed into a three-dimensional image with a mesh size of 18 ⁇ mX18 ⁇ mX18 ⁇ m using the GEMS Microview software. Images are rotated into a standardized position to enable comparison across groups. These images are then thresholded to distinguish mineralized from non-mineralized voxels, by determining an optimal grayscale value from image histograms. Standard algorithms are provided to conduct the following bone measurements: geometric and morphologic assessment; cross sectional area analysis and volumetric measurements; volume fractions of mineralized and non-mineralized tissues; and morphologic features of cortical and trabecular bone.

Abstract

Procédés d'utilisation de vecteurs de virus adéno-associés de recombinaison (rAAV) codant un antagoniste vis-à-vis du facteur TNF pour le traitement de troubles ou d'états associés au facteur TNF, du type perte osseuse, mauvaise guérison de blessure ou mauvaise guérison osseuse, et maladies oro-bucales.
EP06740114A 2005-03-31 2006-03-31 Procedes d'abaissement de niveau de facteur tnf dans les troubles associes a ce facteur Withdrawn EP1871799A2 (fr)

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PCT/US2006/011764 WO2006105344A2 (fr) 2005-03-31 2006-03-31 Procedes d'abaissement de niveau de facteur tnf dans les troubles associes a ce facteur

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EP2623978A1 (fr) 2012-02-03 2013-08-07 Charité - Universitätsmedizin Berlin Sous-ensembles de lymphocyte T CD8+ en tant que marqueurs pour la prédiction de la guérison de fractures retardées
NZ629309A (en) * 2012-02-22 2016-03-31 Nvip Pty Ltd Tumour necrosis factor receptor fusion proteins and methods of using the same
IL302282A (en) * 2020-10-29 2023-06-01 Regenxbio Inc Vectored TNF-alpha antagonists for ocular indications

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WO1994006476A1 (fr) * 1992-09-15 1994-03-31 Immunex Corporation Procede de traitement de l'inflammation dependante du facteur necrosant des tumeurs malignes (tnf) a l'aide d'antagonistes dudit tnf
US6537540B1 (en) * 1999-05-28 2003-03-25 Targeted Genetics Corporation Methods and composition for lowering the level of tumor necrosis factor (TNF) in TNF-associated disorders
AU2002324625B2 (en) * 2001-08-07 2008-05-08 Immunex Corporation Interleukin-1 receptors in the treatment of diseases
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WO2006105344A3 (fr) 2007-01-11
CA2603325A1 (fr) 2006-10-05
AU2006230419A1 (en) 2006-10-05
WO2006105344A2 (fr) 2006-10-05
JP2008536826A (ja) 2008-09-11

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