WO1999011292A2 - Compositions and methods for treating arthritis utilizing gene therapy - Google Patents
Compositions and methods for treating arthritis utilizing gene therapy Download PDFInfo
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- WO1999011292A2 WO1999011292A2 PCT/US1998/018308 US9818308W WO9911292A2 WO 1999011292 A2 WO1999011292 A2 WO 1999011292A2 US 9818308 W US9818308 W US 9818308W WO 9911292 A2 WO9911292 A2 WO 9911292A2
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- 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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/45—Transferases (2)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/55—Protease inhibitors
- A61K38/57—Protease inhibitors from animals; from humans
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- 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
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- 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
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
- C12N2740/13051—Methods of production or purification of viral material
- C12N2740/13052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
Definitions
- the present invention relates generally to pharmaceutical compositions and methods, and more specifically, to compositions and methods for treating arthritis utilizing gene therapy.
- rheumatoid arthritis is a debilitating, chronic inflammatory disease affecting 1 to 2% of the world's population. This disease causes pain, swelling and destruction of multiple joints in the body. The disease can also result in damage to other organs such as the lungs and kidneys.
- rheumatoid arthritis is an autoimmune disease and that many different stimuli can trigger an inappropriate immune response of an immunologically susceptible individual. Examples of potential stimuli include both infectious agents (e.g., viruses) and endogenous host proteins (e.g., collagen).
- the stimuli i.e., an antigen
- an antigen is taken up by antigen-presenting cells (macrophages or dendritic cells in the synovial membrane), processed, and presented to T lymphocytes.
- This presentation initiates an immunological cascade mediated by numerous factors, ultimately resulting in the pain, swelling and cartilage destruction which are associated with rheumatoid arthritis.
- Therapeutic regimens which are presently being utilized include both steroidal and non-steroidal anti-arthritic agents.
- anti-cancer drugs such as methotrexate have become the first line of therapy.
- Other drugs such as cyclosporin and azathioprine (alone or in combination) have also been employed. Many of these agents however, are highly toxic, have significant side effects.
- the present invention discloses novel compositions and methods for treating arthritis, and further provides other related advantages.
- the present invention provides compositions and methods for treating or preventing a variety of forms of arthritis, including for example, rheumatoid arthritis, osteoarthritis, and reactive forms of arthritis (e.g., due to a disease process such as Lyme disease, see generally, Goldenberg, Textbook of Rheumatology, and Cooke and Dattwyler, Ann. Rev. Med. 43:93-103, 1992).
- rheumatoid arthritis e.g., osteoarthritis, and reactive forms of arthritis
- reactive forms of arthritis e.g., due to a disease process such as Lyme disease, see generally, Goldenberg, Textbook of Rheumatology, and Cooke and Dattwyler, Ann. Rev. Med. 43:93-103, 1992.
- methods for treating or preventing arthritis comprising the general step of administering to a mammal a therapeutically effective amount of a recombinant retrovirus which directs the expression of an anti-arthritic agent, wherein the recombinant retrovirus is administered at a titer of greater than 10 7 cfu/ml, 10 8 cfu/ml, 10 9 cfu/ml, or, 10 10 cfu/ml.
- a titer of greater than 10 7 cfu/ml, 10 8 cfu/ml, 10 9 cfu/ml, or, 10 10 cfu/ml.
- mammals may be treated utilizing such techniques, including for example, humans, horses, dogs, cats, rats and mice.
- the present invention also provides a variety of anti-arthritic agents that may be utilized, including for example, pro-drug converting enzymes, inhibitors of tumor necrosis factor, IL-1 Receptor antagonists and inhibitors of matrix metalloproteinase expression such as TIMP-1 or TIMP-2.
- the recombinant retrovirus is administered intraarticularly.
- Figure 1 is a description of all modifications carried out on retroviral vector as shown in A), resulting in the cross-less retroviral vector shown in B).
- the cross-less retroviral backbone cloned into a prokaryotic vector is called pBA-5.
- Retroviral vector and “recombinant retroviral vector” refers to a nucleic acid construct which carries, and within certain embodiments, is capable of directing the expression of a nucleic acid molecule of interest.
- the retroviral vector must include at least one transcriptional promoter/enhancer or locus defining element(s), or other elements which control gene expression by other means such as alternate splicing, nuclear RNA export, post-translational modification of messenger, or post-transcriptional modification of protein.
- Such vector constructs must also include a packaging signal, long terminal repeats (LTRs) or portion thereof, and positive and negative strand primer binding sites appropriate to the retrovirus used (if these are not already present in the retroviral vector).
- LTRs long terminal repeats
- the recombinant retroviral vector may also include a signal which directs polyadenylation, selectable markers such as Neomycin resistance, TK, hygromycin resistance, phleomycin resistance histidinol resistance, or DHFR, as well as one or more restriction sites and a translation termination sequence.
- selectable markers such as Neomycin resistance, TK, hygromycin resistance, phleomycin resistance histidinol resistance, or DHFR
- restriction sites and a translation termination sequence typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3' LTR or a portion thereof.
- Retroviral gene delivery vehicle and “retroviral vector particle” as utilized within the present invention refers to a retrovirus which carries at least one gene of interest.
- the retrovirus may also contain a selectable marker.
- the recombinant retrovirus is capable of reverse transcribing its genetic material into DNA and incorporating this genetic material into a host cell's DNA upon infection.
- Prodrug activating enzyme refers to a compound that is capable of activating an otherwise inactive compound into an active compound. Representative examples of prodrug activating enzymes include herpes simplex thymidine kinase and cytosine deaminase.
- Anti-arthritic agent refers to a molecule which is capable of (1) preventing or lessening the pathological and/or clinical symptoms associated with arthritis; (2) downregulating the white blood cell response which initiates the inflammatory cascade and results in pain, swelling and cartilage destruction; (3) inhibiting the proliferation of synoviocytes; (4) decreasing the production/activity of matrix metalloproteinases; or, (5) inhibiting blood vessel formation.
- Such molecules include, for example, proteins (e.g., inhibitors of TNF ⁇ or the IL-1 Receptor), antisense and ribozyme nucleic acid sequences.
- the present invention provides compositions and methods for treating arthritis, comprising the general step of administering to a patient a therapeutically effective amount of a recombinant retrovirus which directs the expression of an anti-arthritic agent, wherein the recombinant retrovirus is administered at a titer of greater than 10 7 cfu/ml. That administration of such recombinant retroviruses can provide a significant therapeutic advantage is surprising, particularly in light of published results which suggest that such a method would be ineffective for the treatment or prevention of arthritis (see Nita et al, Arthritis and Rheumatism 3P(5):820- 828, 1996).
- the present invention provides recombinant retroviruses which are constructed to carry or express a selected nucleic acid molecule of interest.
- numerous retroviral gene delivery vehicles may be utilized within the context of the present invention, including for example those described in EP 0,415,731; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No. 5,219,740; WO 9311230; WO 9310218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and Hart, Cancer Res. 53:962-967, 1993; Ram et al., Cancer Res.
- retroviruses include those described in WO 91/02805, as well as lentiviral vectors based upon, for example, HIV (see, e.g., PCT Application No.
- Retroviral gene delivery vehicles of the present invention may be readily constructed from a wide variety of retroviruses, including for example, B, C, and D type retroviruses as well as spumaviruses and lentiviruses (see RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985).
- Preferred retroviruses for the preparation or construction of retroviral gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Avian Leukosis Virus, Bovine Leukemia Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma Virus.
- retroviruses selected from the group consisting of Avian Leukosis Virus, Bovine Leukemia Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma Virus.
- Particularly preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe, J. Virol. 19:19-25, 1976), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC
- retroviruses may be readily obtained from depositories or collections such as the American Type Culture Collection ("ATCC”; Rockville, Maryland), or isolated from known sources using commonly available techniques.
- retroviruses may be readily utilized in order to assemble or construct recombinant retroviral vectors and recombinant retroviruses given the disclosure provided herein, and standard recombinant techniques (e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Kunkle, N4S 52:488, 1985).
- portions of the recombinant retroviral vectors or viruses may be derived from different retroviruses.
- retroviral vector LTRs may be derived from a Murine Sarcoma Virus, a tR ⁇ A binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.
- a recombinant retroviral vector as discussed above which lacks certain elements such as gag/pol or env that are required for the production of infectious retrovirus.
- Such defective retroviral vectors can then be introduced into a cell (termed a "packaging cell") which contains those elements necessary for production of infectious recombinant retrovirus that are lacking in the retroviral vector. The result is to produce a recombinant retrovirus which is infectious, but which lacks those elements required to required to replicate.
- retroviral vectors may be utilized within the present invention in order to prepare recombinant retroviruses.
- retroviral vector constructs comprising a 5' LTR, a tR ⁇ A binding site, a packaging signal, one or more heterologous sequences, an origin of second strand D ⁇ A synthesis and a 3' LTR, wherein the vector construct lacks gag/pol or env coding sequences.
- LTRs Long Terminal Repeats
- LTRs may be readily identified in the pro virus due to their precise duplication at either end of the genome.
- a 5' LTR should be understood to include a 5' promoter element and sufficient LTR sequence to allow reverse transcription and integration of the DNA form of the vector.
- the 3' LTR should be understood to include a polyadenylation signal, and sufficient LTR sequence to allow reverse transcription and integration of the DNA form of the vector.
- the tRNA binding site and origin of second strand DNA synthesis are also important for a retrovirus to be biologically active, and may be readily identified by one of skill in the art.
- retroviral tRNA binds to a tRNA binding site by Watson-Crick base pairing, and is carried with the retrovirus genome into a viral particle.
- the tRNA is then utilized as a primer for DNA synthesis by reverse transcriptase.
- the tRNA binding site may be readily identified based upon its location just downstream from the 5' LTR.
- the origin of second strand DNA synthesis is, as its name implies, important for the second strand DNA synthesis of a retrovirus. This region, which is also referred to as the poly-purine tract, is located just upstream of the 3' LTR.
- retroviral vector constructs which are provided herein also comprise a packaging signal, as well as one or more nucleic acid molecules (e.g., heterologous sequences), each of which is discussed in more detail below.
- retroviral vector constructs are provided which lack both gag/pol and env coding sequences.
- the phrase "lacks gag/pol or env coding sequences" should be understood to mean that the retroviral vector does not contain at least 20, preferably at least 15, more preferably at least 10, and most preferably at least 8 consecutive nucleotides which are found in gag/pol or env genes, and in particular, within gag/pol or env expression cassettes that are used to construct packaging cell lines for the retroviral vector construct.
- Packaging cell lines suitable for use with the above-described retroviral vector constructs may be readily prepared (see U.S. Serial No. 08/240,030, filed May 9, 1994; see also WO 92/05266 and WO 95/30763), and utilized to create producer cell lines (also termed vector cell lines or "VCLs") for the production of recombinant vector particles.
- producer cell lines also termed vector cell lines or "VCLs”
- packaging cell lines are made from human (e.g., HT1080 cells) or mink parent cell lines, thereby allowing production of recombinant retroviruses that are capable of surviving inactivation in human serum (e.g., they are "complement resistant").
- packaging cell lines that produce greater than recombinant retroviral particles at titers greater than 10 ⁇ cfu/ml may readily be obtained.
- titers are generally obtained from titer assays on HT1080 cells, which produce a three-fold lower titer than titers obtained on murine 3T3 cells.
- anti-arthritic agents can be delivered (and expressed) by the recombinant retroviruses of the present invention, including not only proteins, but antisense and ribozymes sequences.
- the recombinant retrovirus directs the expression of a cytotoxic gene.
- cytotoxic genes include the genes which encode proteins such as ricin (Lamb et al., Ewr.
- recombinant retroviruses which direct the expression of a gene product that activates a compound with little or no cytotoxicity (i.e., a "prodrug activating or converting enzyme" into a toxic product.
- gene products include varicella zoster virus thymidine kinase (VZVTK), herpes simplex virus thymidine kinase (HSVTK) (Field et al, J. Gen. Virol. 49:115-124, 1980; Munir et al., Protein Engineering 7(l):83-89, 1994; Black and Loeb, Biochem 32(43): 11618-11626, 1993), and E.
- VZVTK varicella zoster virus thymidine kinase
- HSVTK herpes simplex virus thymidine kinase
- interleukin- 1 antagonists such as Interleukin- 1 Receptor Antagonist Protein or 'TRAP
- 'TRAP Interleukin- 1 Receptor Antagonist Protein
- ⁇ isenberg et al Nature 343(6256):341-346, 1990, Carter et al, Nature 344(6267):633-638, 1990, Carrier et al, Cytokine 4(2):83- 89, 1992, Eisenberg et al, PNAS USA 55(12):5232-5236, 1991
- type II IL-1 receptors e.g., Genebank Accession Nos.
- Poxvirus "decoy” receptors naturally occurring proteins similar to the type II "decoy” receptor, which are used by the virus to help regulate local immune responses
- anti-IL- 1 antibodies which bind to and neutralize the biological activity of IL-1
- antisense or ribozyme molecules which target interleukin- 1.
- TNF antagonists such as sTNFR; sTNFR/Ig (e.g., a construct of the extracellular portion of the TNF-R which is linked to an immunoglobulin constant region); anti-TNF antibodies (e.g., antibodies which bind to and neutralize the biological activity of TNF); and antisense or ribozyme molecules which target TNF (see generally Nophar et al, EMBO J. 9(10):3269-3278, 1990, Gray et al, PNAS USA 57:7380-7384, 1990; Smith et al, Science 245(4958): 1019- 1023, 1990; Genebank Accession Nos. X55313, M60275, M37764 and M32315).
- gene products which can also be utilized for treating or preventing arthritis include gene products which switch an immune response from helper 1 (inflammatory) to a helper 2 response (e.g., IL-4 (Klein et al, Immunogenetics 41(1):51, 1995, Arai et al, J. Immunol. 742(l):274-282, 1989, Yokota et al, PNAS USA 53(16):5894-5898, 1986, Genebank Accession Nos. X81851, M23442 and M13982), IL-10 (Kube, Immunogenetics 45(l):82-82, 1996, Vieira et al, PNAS USA 55(44): 1172-1176, 1991, Genebank Accession Nos.
- IL-4 Kerbein et al, Immunogenetics 41(1):51, 1995, Arai et al, J. Immunol. 742(l):274-282, 1989, Yokota et al, PNAS USA 53(16):58
- chrodrocyte growth factors and stimulators such as IGF-1 (EP 0155655, Tobin et al, Mol Endocrinol 4(12): 1914- 1920, 1990, Genebank Accession Nos. A29117, E00602, A04811, A04812, AA542914 AA456717 and M37484); TGF- ⁇ (JP 1986219395, EP 0542679, EP 0433225, Kim et al, J. biol Chem. 264(l):402-408, 1989, Genebank Accession Nos. E00973 A18277 and A23751); and human growth hormone.
- IGF-1 EP 0155655, Tobin et al, Mol Endocrinol 4(12): 1914- 1920, 1990, Genebank Accession Nos. A29117, E00602, A04811, A04812, AA542914 AA456717 and M37484
- TGF- ⁇ JP 1986219395, EP 0542679, EP
- Recombinant retrovirus of the present invention is preferably concentrated, purified, and formulated so as to preserve the infectious capability of the virus upon reconstitution (see generally, U.S. Serial No. 08/153,342 and PCT Publication Nos. WO 95/16582 and WO 95/16852). More specifically, recombinant retrovirus of the present invention can be purified and concentrated as generally described in PCT publication No. WO 95/16582, and then preserved by adding a sufficient amount of a formulation buffer to the media containing the recombinant retrovirus, in order to form an aqueous suspension.
- the formulation buffer is an aqueous solution that contains a saccharide, a high molecular weight structural additive, and a buffering component in water.
- a "buffering compound” or “buffering component” should be understood to refer to a substance that functions to maintain the aqueous suspension at a desired pH.
- the aqueous solution may also contain one or more amino acids.
- the recombinant retrovirus can be preserved prior to the addition of the formulation buffer. Briefly, the crude recombinant retrovirus is clarified by passing it through a filter, and then concentrated, such as by a cross flow concentrating system (Filtron Technology Corp., Nortborough, MA). Within one embodiment, DNase is added to the concentrate to digest exogenous DNA.
- the digest is then diafiltrated to remove excess media components and establish the recombinant retrovirus in a more desirable buffered solution.
- the diafiltrate is then passed over a Sephadex S-500 gel column and a purified recombinant retrovirus is eluted.
- a sufficient amount of formulation buffer is added to this eluate to reach a desired final concentration of the constituents and to minimally dilute the recombinant retrovirus, and the aqueous suspension is then stored, preferably at -70°C or immediately dried.
- the formulation buffer can be an aqueous solution that contains a saccharide, a high molecular weight structural additive, and a buffering component in water.
- the aqueous solution may also contain one or more amino acids.
- the crude recombinant retrovirus can also be purified by ion exchange column chromatography. This method is described in more detail in U.S. Patent Application Serial No. 08/093,436.
- the crude recombinant retrovirus is clarified by passing it through a filter, and the filtrate loaded onto a column containing a highly sulfonated cellulose matrix.
- the recombinant retrovirus is eluted from the column in purified form by using a high salt buffer.
- the high salt buffer is then exchanged for a more desirable buffer by passing the eluate over a molecular exclusion column.
- a sufficient amount of formulation buffer is then added, as discussed above, to the purified recombinant retrovirus and the aqueous suspension is either dried immediately or stored, preferably at -70°C.
- the aqueous suspension in crude or purified form can be dried by lyophilization or evaporation at ambient temperature.
- lyophilization involves the steps of cooling the aqueous suspension below the glass transition temperature or below the eutectic point temperature of the aqueous suspension, and removing water from the cooled suspension by sublimation to form a lyophilized retrovirus. Briefly, aliquots of the formulated recombinant retrovirus are placed into an Edwards Refrigerated Chamber (3 shelf RC3S unit) attached to a freeze dryer (Supermodulyo 12K). A multistep freeze drying procedure as described by Phillips et al.
- the recombinant retrovirus which directs the expression of one or more anti-arthritic agents is then administered in a therapeutically effective amount to a patient in order to treat or prevent the arthritis.
- efficacious administration of the recombinant retroviruses described herein may be assessed in several ways, including: (1) by preventing or lessening the pathological and/or clinical symptoms associated with the arthritis; (2) by downregulating the inflammatory response which initiates the inflammatory cascade and results in pain, swelling and cartilage destruction; (3) by inhibiting the proliferation of synoviocytes; (4) by decreasing the production/activity of matrix metalloproteinases; and (5) by inhibiting blood vessel formation.
- assays may be utilized to determine whether a particular therapeutic regimen or anti-arthritic agent will prevent or lessen the pathological and/or clinical symptoms associated with arthritis, prior to use in humans.
- animal models such as adjuvant-induced arthritis (see Kaklamanis, Clin. Rheumatol 11:41-41, 1992; Houri and O'Sullivan, Curr. Opin. Rheumatol 7:201- 205, 1995), rat streptococcal cell wall arthritis, collagen induced arthritis (Trentham et al., J. Exp. Med. 746:857, 1977; Stuart et al., Ann. Rev. Immunol. 2:199-218, 1984; and Wooley et al., J. Exp.
- disease progression may be monitored through measurements of inflammation (e.g., volume of affected paws, or the diameter of affected joints), through radiologic evaluation, or by histological assessment of joint damage.
- inflammation e.g., volume of affected paws, or the diameter of affected joints
- assays for determining efficacious agents and administration may likewise be readily determined prior to human clinical trials.
- downregulation of the inflammatory response may be readily determined by assaying synovial fluid from animals which have been treated with the anti-arthritic agent (or recombinant retrovirus) (e.g., by white cell counts or neutrophil activity).
- inhibition of synoviocyte proliferation may be readily determined by assaying the effect of the anti-arthritic agent or recombinant retroviruson on tritiated thymidine incorporation of synoviocytes, or by directly scoring pannus formation.
- Assessment of matrix metalloproteinase inhibition may be accomplished by measuring inhibition of collagenase activity utilizing commercially available kits (e.g., available from Boehringer Mannheim).
- Inhibition of blood vessel formation may likewise be readily determined based upon standard assays, for example, the Chick Chorioallantoic Membrane or "CAM” assay (see the Examples below).
- the above assays may be readily practiced by one of ordinary skill in the art given the disclosure provided herein (see generally, Kelley et al., eds., Textbook of Rheumatology (4th edition), W.B. Saunders Company, Philadelphia, 1993).
- one or more injections of the recombinant retrovirus is administered at a titer of greater than 10 7 cfu/ml, 10 8 cfu ml, 10 9 cfu ml, or, 10 10 cfu/ml.
- the recombinant retrovirus may be delivered at a variety of sites an locations (e.g., intravenously) in order to systemically treat or prevent the arthritis, or locally (e.g., intraarticularly by direct injection or under artheroscopic guidance) to selected joints.
- the synovial fluid prior to administration of the recombinant retrovirus or anti-arthritic agent, may be aspirated from the joint, and the joint flushed or otherwise treated with one or more enzymes capable of breaking down extracellular matrix proteins.
- enzymes include collagenases, elastase, hyaluronidase, dispase, papain, pronase, and trypsin.
- a complement resistant recombinant retrovirus in order to help prevent or limit the amount of retrovirus that is degraded due to the action of complement.
- the recombinant retroviral preparation be 'clean', in that it should contain low levels of foreign antigens (e.g., fetal calf serum) and low levels of endotoxin.
- This example describes modification of the extended packaging signal ( ⁇ +) by PCR on the template KT-1 (see WO 95/16582) using primers that introduce two stop codons in the extended packaging signal sequence.
- the template pKT-1 contains the modification ATT at the normal ATG start site of gag (position 621-623 of SEQ ID NO: 1).
- the start site was further modified to the stop codon, TAA, and an additional stop codon TGA was added to replace the codon TTA at position 645-647 of SEQ ID NO: 1.
- PCR reactions were carried out on pKTl, each introducing one stop codon.
- the primers for the PCR were designed such that the two PCR products had overlapping regions and a splice-overlap extension PCR (SOE-PCR) was carried out with the two PCR products in order to combine the two introduced stop codons on one strand.
- SOE-PCR splice-overlap extension PCR
- the first set of ohgonucleotides introducing the change from ATT to TAA were 5'-GGG-AGT-GGT-AAC-AGT-CTG-GCC-TTA-ATT-CTC-AG (SEQ ID NO: _) and 5'-CGG-TCG-ACC-TCG-AGA-ATT-AAT-TC (SEQ ID NO: _ and the second set of ohgonucleotides introducing the change from TTA to TGA were 5'CTG-GGA-GAC-GTC-CCA-GGG-ACT-TC (SEQ ID NO: __) and 5'GGC-CAG- ACT-GTT-ACC-ACT-CCC-TGA-AGT-TTG-AC (SEQ ID NO: __).
- the flanking primers of the final 708 base pair PCR product introduced the Aat ⁇ l and the Xhol sites, at the 5' and 3', respectively.
- the ends of the 708 base pair product were blunted and phosphorylated and the product introduced into the Smal and Ec RV digested vector pBluescript SK- (Stratagene, San Diego, Calif.).
- the resulting plasmid was designated pBA-2, and is shown diagrammatically in Figure 1.
- Retroviral envelope sequence was removed upstream of the 3' LTR between the Clal site and the TAG stop codon of the envelope coding sequence.
- the DNA sequence was modified by PCR such that the TAG stop codon was replaced by a Clal site and the 97 nucleotides upstream from this new Clal site to the original Clal site were deleted, as well as the 212 base pairs of retroviral sequence downstream of the 3' LTR.
- the PCR product was cloned into pPCRII (Invitrogen, San Diego, Calif.) using the TA cloning kit (Invitrogen, San Diego, Calif.) and called pBA-1.
- pBA-1 was digested with Clal and Apal resulting in a 640 base pair fragment that was cloned into the Clal and Apal digested pBA-3 resulting in the plasmid pBA-4.
- This plasmid combines the above described 5' LTR and the packaging signal with the 3' LTR.
- pBA-4 was digested with Apal and Ec RI, ends blunted and religated in order to remove extraneous 3' polylinker sites, resulting in plasmid ⁇ BA-5a.
- pBA-5a was cut with NotI (blunted) and EcoRI and introduced into Smal and EcoRI digested pUC18 (GIBCO/BRL, Gaithersburg, MD) resulting in pBA-5b. This construct moved the retroviral vector from a pBluescript into an alternate pUC18 vector.
- HSV-TK Recombinant Vector Containing HSV-TK
- pBH-1 PCT#UU 091-02805
- Xhol and EcoRI Xhol and EcoRI
- BstBI EcoRI and BstBI resulting in a 1.3 kb fragment. Both fragments were cloned into a Xhol and Clal digested pBA-5b resulting in a retroviral vector designated "pMO-TK”.
- the cloning of a human TIMP-1 gene into recombinant retroviral vectors can be accomplished by PCR amplification using two 38-mer ohgonucleotides.
- the ohgonucleotides contain 25 nt corresponding to the TIMP-1 gene template, plus additional 5'-flanking sequences comprising a Pme I recognition site and buffer sequence, as follows:
- TIMP-1 cDNA clone Docherty et al., Nature 318:66, 1985
- Thermalase thermostable DNA polymerase is performed in a reaction containing 1.5 mM MgCl2 provided by the supplier, 5% DMSO, and Hot Start Wax beads according to the following protocol:
- the approximately 700 bp TIMP-I gene amplicon is digested with Pme I (or Xho I and Cla I) and ligated into a retroviral vector backbone (pKT-lL, pKT-3BL, pKT-3BCL or pBA-5b) that have been digested with Pme I and treated with CIAP.
- a retroviral vector backbone pKT-lL, pKT-3BL, pKT-3BCL or pBA-5b
- Proper orientation of the insert sequence is determined by restriction endonuclease digests. Production of packaged vector particles is accomplished using the methods described below. Other related gene products from this group are readily cloned using these approaches and ohgonucleotide primers which contain appropriate restriction sites, and are specific for their respective gene.
- This example describes the gag/pol expression plasmid cassette that contains wobbled non-coding sequences upstream from the gag start site, thereby reducing recombination potential between the gag/pol expression element and the extended packaging signal of a retroviral vector construct, and inhibiting co-packaging of the gag/pol expression element along with the retroviral vector.
- a 406 bp DNA fragment with a Clal site at the 5' end (underlined) and a Narl site at the 3' end (underlined) was synthesized by Operon (Operon Technologies Inc, Alameda CA). The sequence of the 406 bp DNA fragment was verified and is provided in Table 1. The synthesized DNA was transferred to a shuttle plasmid as a Clal-Narl fragment to create the plasmid pWOB.
- the Clal-Narl fragment from pWOB was isolated by Clal-Narl digest, and the 406 bp fragment cloned into the Clal-Narl site of pSCVIO to create the plasmid pSCVlO/wob (-Narl fragment) which resulted in the loss of the 481 bp Narl-Narl fragment just downstream of the wobbled Clal-Narl fragment.
- the PCR fragment was digested with Clal and PstI and the 131 bp fragment cloned into pSCVIO replacing the existing C/ T-Pst/ DNA fragment to create the plasmid pSCV10/5'truncated g/p.
- This example describes the construction of the 5' truncated gag/pol construct analogous to that described under section B above in the pCI (Promega Corp, Madison, WI) vector backbone.
- fragments were prepared for a three-way ligation as follows: pCI was digested with Smal and NotI and the 4 kb fragment was isolated. pSCVIO was digested with Xhol and NotI and the 4.7 kb fragment was isolated. pSCVl 0/5' truncated g/p as described in section B was digested with Clal, filled in with Klenow to blunt, then digested with Xhol and the 0.95 kb fragment was isolated. These three fragments were then ligated together to give the final plasmid pCI/5 'truncated g/p.
- the wobbled gag/pol sequence (0.47 kb) was retrieved from plasmid pSCVlO/wob (-Narl fragment) as described in section A above by digestion with Clal and Xhol. This fragment was cloned into the Clal-Xhol digested pBluescript SK- (Stratagene, San Diego, CA) to create pBluescript/wob (- Narl fragment).
- This plasmid was digested with EcoRI and N ⁇ r7 to retrieve the wobbled gag sequence and the EcoRI-Narl fragment cloned into the EcoRI-Narl digested pCI/5 1 truncated g/p described in section C above in order to create pCI/5 'truncated wob g/p.
- This example describes the construction of the 5' and 3' truncated gag/pol construct in the pCI vector where the 5' truncation as described in section C is combined with the following 3'truncation upstream of the stop codon eliminating the D ⁇ A sequence coding for the last 28 amino acids of the pol protein.
- the plasmid pCI/5'truncated g/p described in section C was linearized with the restriction enzyme Smal which is located 84 bases upstream of the gag/pol termination codon in the open reading frame of gag/pol.
- the linearized plasmid was ligated to the ohgonucleotide 5' TAAGCGGCCG CTTA (SEQ ID NO: ).
- This ohgonucleotide is self-complementary and forms a palindromic duplex containing a TAA termination codon and a NotI restriction endonuclease site.
- the reaction was purified by GeneClean and digested with Smal to recut any vector that did not contain an insert. The reaction was used to transform XL1 Blue E. coli (Stratagene, San Diego, CA) and plasmid DNA from a correct clone was then digested with NotI.
- NotI cuts in the inserted ohgonucleotide as well as just upstream of the SV40 termination/polyadenylation site of the pCI vector.
- the digested plasmids were purified by Geneclean and religated to recircularize. Bacteria were transformed and clones were identified which deleted the sequences between the NotI site introduced by the ohgonucleotide and the Not7 site in the pCI vector. These sequences include sequences encoding the last 28 amino acids of gag/pol as well as MoMLV sequences and vector sequences carried over from pSCVIO.
- the resulting gag/pol construct was named pCI- GPM.
- the identically shortened gag/pol region was cloned by standard techniques into a pSCVIO background expression cassette. This expression plasmid was named pSCV10/5',3'tr.
- This example describes the construction of the 5' and 3' truncated and wobbled gag/pol construct in the pCI vector combining the 5' truncation and wobbled gag/pol sequence from section D above with the 3'truncation described in section E above.
- pCI/5 'truncated wob g/p was linearized with Smal and all further steps leading to the 3'truncation of gag/pol were carried out as described in section E above, leading to the 5' and 3' truncated and wobbled gag/pol construct in pCI named pCI-WGPM.
- PCLs with the gag/pol expression plasmid cassette pCI-WGPM described in Example 3 F and the env expression plasmid pCMV-b/envam result in producer cell lines where sequence overlap between all three areas of homology is completely eliminated.
- the cell lines HT 1080 (ATCC #CCL 121) and D17 (ATCC #CCL 183) were used as parent cell lines to establish the PCLs.
- gag/pol plasmid pCI-WGPM was co-transfected together with a phleomycin r expressing marker plasmid into HT 1080 and D17 cells, selected and dilution cloned as described above.
- HT 1080 and D17 derived clones were isolated and analyzed for intracellular p30 expression levels as described above. Results of the p30 Western are shown below in Table 2.
- Table 2 HT 1080 and D17 derived clones screened for intracellular p30 levels after introduction of gag/pol expression cassette pCI-WGPM
- HT-1080g/p 96 26 3-4 clones have p30 levels (pCI-WGPM) (27%) that are comparable to HTSCV21
- pCI-WGPM 12 HT 1080 and 22 Dl 7 gag/pol intermediates with the highest p30 expression levels were analyzed for titer potential as described above.
- the titer results for the HT 1080 gag/pol intermediates are shown below in Table 3.
- the titer potential measured within the range indicates decreases in titer potential of 10-200 fold in most clones.
- D17 derived PCL clones named HAIIwob and DAIIwob, respectively, were isolated and analyzed for titer potential. Briefly, several rounds of titer potential analysis were carried out using various retroviral vectors. The DA or HA PCL (PCT #WO 92/05266) controls were included as a reference for high titer potential PCLs.
- the PCL clones were transduced in 24-well plates with the ⁇ -gal coding retroviral vector DX/ND7 (WO 95/16852) at an moi of 5-10 in the presence of 8 ⁇ g/ml polybrene, transient supernatants harvested, filtered (0.45 ⁇ m), HT 1080 target cells transduced and transient ⁇ -gal titer determined using a standard Galactolight transfer of expression procedure described previously.
- the same transduction assay as described for the first round was repeated with the top clones from round one using standardized PCL cell numbers.
- the top clones from round two were used to transduce with several retroviral vectors, supernatant from transient and stable pools harvested, filtered, HT 1080 target cells transduced and titers determined.
- Transient ⁇ -gal titer on VCL pools from transduced HAII and DAII PCLs determined by Galactolight readout.
- the best DAIIwob PCL clone shows a 4-fold reduction in titer but most clones show >10-fold reduction.
- the best HAIIwob PCL clone shows a 50-fold reduced titer potential and most HAIIwob clones have >100-fold reduced titer potential.
- the DAII wob and HAIIwob PCL clones gave in average about a 5-50 fold lower titer potential when compared to DAII and HAII PCLs.
- DA amphotropic cell line derived from D 17 cells ATCC No. 183, WO 92/05266
- DA amphotropic cell line derived from D 17 cells ATCC No. 183, WO 92/05266
- cells are seeded at 5.0 x 10 5 cells/10 cm tissue culture dish in 10 ml DMEM and 10% FBS, 4 ⁇ g/ml polybrene (Sigma, St. Louis, MO) on day 1.
- FBS 4 ⁇ g/ml polybrene
- 3 ml, 1.0 ml and 0.2 ml of the freshly collected virus-containing HX media is added to the cells.
- the cells are incubated with the virus overnight at 37°C.
- the retroviral also encodes a selectable marker, e.g., neomycin resistance
- a selectable marker e.g., neomycin resistance
- the media is removed and 1.0 ml DMEM, 10% FBS with 800 ⁇ g/ml G418 is added to the plate. Only cells that have been transduced with the vector and expressing the selectable marker will survive. In the case of neomycin resistance, G418 resistant pools can be generated over a period of a week.
- a pool of cells is then dilution cloned by removing the cells from the plate and counting the cell suspension, diluting the cells suspension down to 10 cells/ml and adding 0.1 ml to each well (1 cell/well) of a 96 well plate (Corning, Corning, NY). Cells are incubated for 14 days at 37°C, 10% CO2- As many as twenty-four clones are selected and expanded up to 24 well plates, 6 well plates then 10 cm plates at which time the clones are assayed for expression and the supernatant are collected and assayed for viral titer.
- the titer of the individual clones is determined by infection of HT 1080 cells, (ATCC No. CCL 121). On day 1, 5.0 x 10 5 HT1080 cells are plated on each well of a 6 well microtiter plate in 3.0 ml DMEM, 10%> FBS and 4 ⁇ g/ml polybrene. On day 2, the supernatant from each clone is serially diluted 10 fold and used to infect the HT1080 cells in 1.0 ml aliquots.
- the media is replaced with fresh DMEM, 10% FBS media, and the cells incubated with the vector overnight at 37°C, 10%o CO2- On day 3, selection of transduced cells is performed (assuming the presence of a selectable marker in the recombinant vector) by replacing the media with fresh DMEM, 10% FBS media containing the appropriate selection agent, for instance, 800 ⁇ g/ml G418 in the case of neomycin resistance.
- Cells are incubated at 37°C, 10% CO2 for 14 days at which time G418 resistant colonies are scored at each dilution to determine the viral titer of each clone as cfu/ml.
- cell lines are derived that produce greater than or equal to 1.0 x 10° " cfu/ml in culture.
- selectable markers other than drug resistance or when no selectable marker is encoded by the recombinant vector, other titer methods, such as antibody-based assays, PCR assays, etc., may be employed.
- the packaging cell line HX is transduced with vector generated from the
- a sample of RIPA lysate containing a total protein concentration of 20 mg and a commercial molecular weight (MW) marker (Amersham, Chicago, IL) are mixed 1 :1 with 2x sample buffer (4% (w/v) SDS, 50 mM Tris-HCl pH 7.0, 24% (v/v) glycerol, 0.1% (w/v) bromophenol blue and 0.05% b-mercaptoethanol) and heated to 65°C for 10 minutes. After heating the sample and MW marker are placed on ice.
- 2x sample buffer 4% (w/v) SDS, 50 mM Tris-HCl pH 7.0, 24% (v/v) glycerol, 0.1% (w/v) bromophenol blue and 0.05% b-mercaptoethanol
- Tris HC1 based minigel BioRad, Hercules, CA
- running buffer 3.0 gm Tris-HCl pH 8.6, 1.0 gm SDS and 14.4 gm glycine to 1.0 L
- sample and MW marker are loaded onto the gel.
- Approximately 70 to 120V is applied to the gel until the marker reaches the bottom of the gel.
- the protein bands are transferred from the gel to an Immobilon-PTM membrane (Millipore, Bedford, MA) by immersing the gel in CAPS buffer pH 11.0 with 5%> (v/v) methanol for 5 minutes.
- the Hoefer HSI TTE transfer apparatus Hoefer Scientific Instruments, San Francisco, CA is used to transfer proteins from the gel to the membrane. Approximately 70V is applied to the gel for 1.5 hours.
- the Immobilon-PTM membrane is blocked for 30 minutes at room temperature with 0.5% BM blocking reagent (Boehringer Mannheim, Chicago, IL) containing 3%> BSA, heated slowly in a microwave.
- the membrane is then probed with a primary mouse antibody that reacts specifically with the protein being detected at a 1 :2000 dilution in BM blocking solution containing a 3% BSA and incubated for 1 hour at room temperature.
- the membrane is washed three times with 40 ml of PBST (0.2% Tween-20 in PBS) for 10 min and a secondary goat anti-mouse, HRP-labeled antibody (Jackson, Bar Harbor, MA) at a 1 :20,000 dilution in 3% BSA solution (Sigma, St.
- the membrane is then washed three times with 40 ml of PBST for 10 minutes. After washing, the membrane is submersed in ECL developing solution (Amersham, Chicago, IL) for 1 minute and then wrapped in plastic wrap and exposed to Hyperfilm (Amersham, Chicago, IL) for 5 seconds. The film is then developed and the protein bands are analyzed.
- ECL developing solution Amersham, Chicago, IL
- Hyperfilm Amersham, Chicago, IL
- the microtiter plate is washed four times with fresh FACS buffer. After the final wash, 150 ⁇ l from each well is transferred to a FACS tube (Falcon 2052 tubes) and 100 ⁇ l of FACS buffer is added. The fluorescent signal is detected by a FACScan counter.
- Frozen tissue sections are dried at room temperature for 30 minutes. The dried tissue sections are fixed with anhydrous acetone for 15 minutes at room temperature and allowed to air dry. The sections are rehydrated with PBS for several minutes. Tissue sections are then incubated in a humid chamber for 45 to 120 minutes at room temperature with 50 ⁇ l of primary antibody that specifically reacts with the protein to be detected (Marek et al., Cancer 67:1377, 1991). The tissue sections are then washed with PBS and allowed to soak in PBS for several minutes.
- the sections are incubated with 50 ⁇ l of HRP conjugated anti-mouse antibody or HRP-conjugated strept-avidin (DAKO, Carpenteria, CA) for 45 to 120 minutes at room temperature.
- the tissue sections are then washed with PBS and allowed to soak in PBS for several minutes.
- the sections are incubated with 60 to 100 ⁇ l substrate solution containing 0.01% H2O2 and 3,3 diaminodenzidine (DAB) for 8 minutes or for 20 minutes with substrate solution containing 100 ⁇ l of a-ethyl carbazole (60 mg of 3-amino-9-ethylcarbazole in 25 ml DMF) in 1 ml of acetate buffer (0.68 g sodium acetate in H2O pH 5.2 adjusted to a volume of 250 mis) and 0.01% H2O2.
- the tissue sections are washed with water for 2-5 minutes and then stained with haemotoxylin for 15 seconds.
- Anti-angiogenic activity of various polypeptides useful in accordance with certain aspects of this invention may be assayed on the chorioallantoic membrane (CAM) as described by Takigawa et al, (Biochem. Int. 14:351, 1987). Briefly, B16 melanoma cells are inoculated subcutaneously into the loins of C57BL/6N mice. When the tumors reach approximately 1 cm in diameter, they are excised, cut into pieces of 2 mg and placed on sterile Whatman GF/B glass fiber filter disks (6 mm in diameter; Reeve- Angel, Clifton, NJ) to which 30 ⁇ l of transduced cells have been added.
- CAM chorioallantoic membrane
- CAM 10-day-old chicken embryos through windows made in the egg shells on day 8 of inoculation.
- the embryos are killed 5 days later by injection of 10% formalin in PBS.
- the CAM is excised, fixed in 10%> formalin in PBS inverted, and examined under a stereo-microscope.
- Angiogenesis is assayed by measuring the number and thickness of capillaries beneath the filter. A thick capillary, a middle sized capillary, a small capillary, and 5 minute capillaries are given 3, 2, 1 and 1 points, respectively, and the average number of points is defined as the angiogenic activity.
- the diameters of tumors on the filters are measured in three dimensions and the tumor size is calculated as (p/6) abc mm- (a, b, and c: length, width, and height, respectively). Low average number of points and decreased tumor size indicates anti- angiogenic activity.
- HSVTK vector transduced cells to gancyclovir may be used to determine the activity of expressed HSVTK expressed in cells treated according to the disclosed methods. Briefly, cells that are transduced with pTK-3 are seeded into six plates at a density of 2.5 x 10° " per plate. In addition, untransduced CT26 and CT26 b-gal (this cell line was transduced with a virus carrying the reporter gene b- galactosidase from E. coll), are also seeded into six plates as controls.
- Crude recombinant retrovirus is obtained from a Celligan bioreactor (New Brunswick, New Brunswick, NJ) containing DA cells transformed with the recombinant retrovirus (U.S.S.N. 07/395,932) bound to the beads of the bioreactor matrix.
- the cells release the recombinant retrovirus into the growth media that is passed over the cells in a continuous flow process.
- the media exiting the bioreactor is collected and passed initially through a 0.8 micron filter then through a 0.65 micron filter to clarify the crude recombinant retrovirus.
- the filtrate is concentrated utilizing a cross flow concentrating system (Filtron, Boston, MA).
- DNase Intergen, New York, NY
- concentrate Approximately 50 units of DNase (Intergen, New York, NY) per ml of concentrate is added to digest exogenous DNA.
- the digest is diafiltrated using the same cross flow system to 150 mM NaCl, 25 mM tromethamine, pH 7.2.
- the diafiltrate is loaded onto a Sephadex S-500 gel column (Pharmacia, Piscataway, NJ), equilibrated in 50 mM NaCl, 25 mM tromethamine, pH 7.4.
- the purified recombinant retrovirus is eluted from the Sephadex S-500 gel column in 50 mM NaCl, 25 mM tromethamine, pH 7.4.
- the formulation buffer containing lactose was prepared at a 2x concentrated stock solution.
- the formulation buffer contains 25 mM tromethamine, 70 mM NaCl, 2 mg/ml arginine, 10 mg/ml human serum albumin (HSA), and 100 mg/ml lactose in a final volume of 100 mis at a pH 7.4.
- the purified recombinant retrovirus is formulated by adding one part 2x lactose formulation buffer to one part S-500 purified recombinant retrovirus.
- the formulated recombinant retrovirus can be stored at -70°C to -80°C or dried.
- the formulated retrovirus is lyophilized in an Edwards Refrigerated Chamber (3 Shelf RC3S unit) attached to a Supermodulyo 12K freeze dryer (Edwards High Vacuum, Tonawanda, NY).
- a Supermodulyo 12K freeze dryer Edwards High Vacuum, Tonawanda, NY.
- the lyophilized recombinant retrovirus is reconstituted with 1.0 ml water.
- the infectivity of the reconstituted recombinant retrovirus is determined by a titer activity assay.
- the assay is conducted on HT 1080 fibroblasts or 3T3 mouse fibroblast cell line (ATCC CCL 163). Specifically, 1.0 x 10 ⁇ cells are plated onto 6 cm plates and incubated overnight at 37°C, 10% CO2- Ten microliters of a dilution series of reconstituted recombinant retroviruses are added to the cells in the presence of 4 mg/mL polybrene (Sigma, St.
- cells that have been transduced with a recombinant vector which encodes the neo resistance gene are selected for neomycin resistance in G418 containing media and incubated for 5 days at 37°C, 10% CO2- Following initial selection, the cells are re- fed with fresh media containing G418 and incubated for 5 to 6 days. After final selection, the cells are stained with Commassie blue for colony detection. The titer of the sample is determined from the number of colonies, the dilution and the volume used.
- Mus dunni NIH NIAID Bethesda, MD
- small scale cocultivations are performed by mixing of 5.0 x 10 ⁇ Mus dunni cells with 5.0 x 10 ⁇ producer cells and seeding the mixture into 10 cm plates (10 ml standard culture media/plate, 4 ⁇ g/ml polybrene) at day 0. Every 3 to 4 days the cultures are split at a 1 :10 ratio and 5.0 x 10 ⁇ Mus dunni cells are added to each culture plate to effectively dilute out the producer cell line and provide maximum amplification of RCR.
- culture supernatant is harvested, passed through a 0.45 ⁇ cellulose-acetate filter, and tested in the MdH marker rescue assay.
- Large scale co-cultivations are performed by seeding a mixture of 1.0 x 10 ⁇ Mus dunni cells and 1.0 x 10 ⁇ producer cells into a total of twenty T-150 flasks (30 ml standard culture media flask, 4 ⁇ g/ml polybrene). Cultures are split at a ratio of 1 :10 on days 3, 6, and 13 and at a ratio of 1 :20 on day 9.
- the final supernatant is harvested, filtered and a portion of each is tested in the MdH marker rescue assay.
- the MdH marker rescue cell line is cloned from a pool of Mus dunni cells transduced with LHL, a retroviral vector encoding the hygromycin B resistance gene (Palmer et al., PNAS 84: 1055-1059, 1987).
- the retroviral vector can be rescued from MdH cells upon infection of the cells with RCR.
- One ml of test sample is added to a well of a 6-well plate containing 1.0 x 10 ⁇ MdH cells in 2 ml standard culture medium (DMEM with 10%> FBS, 1% 200 mM L-glutamine, 1% non-essential amino acids) containing 4 ⁇ g/ml polybrene.
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JP2003528923A (en) * | 2000-04-05 | 2003-09-30 | イーペーエフ ファルマシューティカルス ゲゼルシャフト ミット ベシュレンクテル ハフツング | Drug containing a tissue inhibitor of metalloproteinase-2 (TIMP-2) as a bone synthesis metabolic active substance |
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EP0334301A1 (en) * | 1988-03-21 | 1989-09-27 | Chiron Viagene, Inc. | Recombinant retroviruses |
WO1992011359A1 (en) * | 1990-12-20 | 1992-07-09 | University Of Pittsburgh Of The Commonwealth System Of Higher Education | A truncated interleukin-1 receptor gene for the treatment of arthritis |
WO1994020517A1 (en) * | 1993-03-08 | 1994-09-15 | University Of Pittsburgh Of The Commonwealth System Of Higher Education | Gene transfer for treating a connective tissue of a mammalian host |
WO1995016353A1 (en) * | 1993-12-14 | 1995-06-22 | University Of Pittsburgh Of The Commonwealth System Of Higher Education | Systemic gene treatment of connective tissue diseases |
WO1996006941A1 (en) * | 1994-08-26 | 1996-03-07 | Hoechst Aktiengesellschaft | Genetic therapy of diseases caused by the immune system, said therapy using a cell-specific active substance regulated by the cell cycle |
WO1996021014A2 (en) * | 1994-12-30 | 1996-07-11 | Chiron Corporation | Production and administration of high titer recombinant retroviruses |
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1998
- 1998-09-02 WO PCT/US1998/018308 patent/WO1999011292A2/en not_active Application Discontinuation
- 1998-09-02 JP JP2000508393A patent/JP2001514235A/en not_active Withdrawn
- 1998-09-02 EP EP98945862A patent/EP1009444A2/en not_active Withdrawn
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EP0334301A1 (en) * | 1988-03-21 | 1989-09-27 | Chiron Viagene, Inc. | Recombinant retroviruses |
WO1992011359A1 (en) * | 1990-12-20 | 1992-07-09 | University Of Pittsburgh Of The Commonwealth System Of Higher Education | A truncated interleukin-1 receptor gene for the treatment of arthritis |
WO1994020517A1 (en) * | 1993-03-08 | 1994-09-15 | University Of Pittsburgh Of The Commonwealth System Of Higher Education | Gene transfer for treating a connective tissue of a mammalian host |
WO1995016353A1 (en) * | 1993-12-14 | 1995-06-22 | University Of Pittsburgh Of The Commonwealth System Of Higher Education | Systemic gene treatment of connective tissue diseases |
WO1996006941A1 (en) * | 1994-08-26 | 1996-03-07 | Hoechst Aktiengesellschaft | Genetic therapy of diseases caused by the immune system, said therapy using a cell-specific active substance regulated by the cell cycle |
WO1996021014A2 (en) * | 1994-12-30 | 1996-07-11 | Chiron Corporation | Production and administration of high titer recombinant retroviruses |
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HUNG G L ET AL: "SUPPRESSION OF INTRA-ARTICULAR RESPONSES TO INTERLEUKIN-1 BY TRANSFER OF THE INTERLEUKIN-1 RECEPTOR ANTAGONIST GENE TO SYNOVIUM" GENE THERAPY, vol. 1, no. 1, 1 January 1994, pages 64-69, XP000671119 * |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2003528923A (en) * | 2000-04-05 | 2003-09-30 | イーペーエフ ファルマシューティカルス ゲゼルシャフト ミット ベシュレンクテル ハフツング | Drug containing a tissue inhibitor of metalloproteinase-2 (TIMP-2) as a bone synthesis metabolic active substance |
JP4939715B2 (en) * | 2000-04-05 | 2012-05-30 | イーペーエフ ファルマシューティカルス ゲゼルシャフト ミット ベシュレンクテル ハフツング | Agents containing tissue inhibitors of metalloproteinase-2 (TIMP-2) as osteosynthesis metabolic active substances |
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