AU729873B2 - Systemic gene treatment of connective tissue diseases - Google Patents
Systemic gene treatment of connective tissue diseases Download PDFInfo
- Publication number
- AU729873B2 AU729873B2 AU96132/98A AU9613298A AU729873B2 AU 729873 B2 AU729873 B2 AU 729873B2 AU 96132/98 A AU96132/98 A AU 96132/98A AU 9613298 A AU9613298 A AU 9613298A AU 729873 B2 AU729873 B2 AU 729873B2
- Authority
- AU
- Australia
- Prior art keywords
- vector
- dna sequence
- irap
- hematopoietic cell
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
1 P/00/011 Regulation 3.2
AUSTRALIA
Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT
ORIGINAL
S. 0 0 *0.00 0 0 0 @000 0e
S
0@ S. S 0S 0 0S
S
S
Name of Applicant: Actual Inventor(s): Address for Service: Invention Title: TO BE COMPLETED BY APPLICANT University of Pittsburgh of the Commonwealth System of Higher Education Christopher H Evans Paul D Robbins A.P.T. Patent and Trade Mark Attorneys GPO Box 772, Adelaide, SA 5001 Systemic Gene Treatment of Connective Tissue Diseases Details of Associated Divisional Application No: 15134/95 The following statement is a full description of this invention, including the best method of performing it known to us:- SYSTEMIC GENE TREATMENT OF CONNECTIVE TISSUE DISEASES 1. INTRODUCTION Gene therapy of certain connective tissue diseases would require delivery of a gene product to various sites, depending upon the disease, to promote comprehensive therapeutic coverage and the potential for prolonged availability throughout the patient.
The present invention discloses methods of treating diseases or disorders comprising local or systemic delivery of a nucleic acid sequence which expresses a therapeutic or prophylactic gene product such that the expressed gene product is systemically delivered throughout the patient and available for prolonged periods.
2. BACKGROUND OF THE INVENTION There are two main contexts in which genes or DNA sequences encoding a portion of that gene may be used in a therapeutic or prophylactic manner. The first is to compensate for heritable defects in the genome of the patient which lead to such diseases as Gaucher disease, hemophilia, and osteogenesis imperfecta. In the second context, S• °ogenes are transferred for therapeutic reasons to individuals who may well be genetically normal. In this case, gene transfer is used as a drug delivery system for the protein(s), S •antisense RNA, ribozymes or a biologically active fragment thereof encoded by the transferred DNA sequence.
For example, treating arthritis locally with gene therapy based techniques has S been described. Although arthritis is not, for the most part, a genetic disease, it has been shown to be a good candidate for gene treatment. Both in vivo and ex vivo gene therapy techniques for local treatment of joint disease protocols have been forwarded (see Bandara, et al., 1992, DNA Cell Biol. 11: 227-231). The transfer of a potentially therapeutic gene to a joint for treatment of inflammation and cartilage destruction associated with arthritis has been accomplished Application Serial No. 027,750, by Glorioso, et al., filed March 8, 1993).
Gene therapy in its barest sense addresses the chronicity of diseases such as arthritis, which can endure for the lifetime of the patient. Unlike traditional methods of drug delivery, gene therapy allows for the potential of a permanence of therapeutic effect S. to match the persistence of the disease. Furthermore, so far as antisense RNA, ribozymes 35 and protein or biologically active fragments thereof are concerned, there are likely to be cost advantages. A DNA sequence should be inherently less expensive than its cognate gene product since it can be expressed many millions of times into the gene product.
Interleukin-1 (IL-1) is now known to play a role in numerous diseases and disorders, in particular disorders accompanied by inflammation. IL-1 is a growth factor for. synoviocytes and promotes their synthesis of matrix, two properties suggesting the involvement of IL-1 in the synovial hypertrophy that accompanies arthritis. IL-1 both enhances cartilaginous matrix breakdown and inhibits cartilaginous matrix synthesis by chondrocytes, thereby causing loss of cartilage while inhibiting its repair. IL-1 also induces bone resorption and thus may be involved in the loss of bone density seen in rheumatoid arthritis. IL-1 is inflammatory, serves as a growth factor for lymphocytes, is a chemotactic factor and a possible activator of polymorphonuclear leukocytes (PMNs).
When present in a sufficient concentration, interleukin-1 may cause fever, muscle wasting and sleepiness. This being so, agents which antagonize the specific biological activity that IL-1 imparts during the course of that disorder might have therapeutic potential.
Although biological preparations have been disclosed that are able to block one or more of the actions of IL-1 (Larrick, 1989, Immunol. Today 10: 61-66), there are few instances in which an active principle has been purified to homogeneity and characterized, a prerequisite for any pharmacologic use of such an antagonist. Arend, et al. (1991, J. Clin. Invest. 88: 1445-1451) noted an IL-1 inhibitory activity in culture medium conditioned by macrophages. Balavoine, et al. (1986, J. Clin. Invest. 78: 1120-1124) found a similar activity in the urine of febrile patients. Subsequent studies showed these activities to result from the same protein which acted as a competitive 20 inhibitor of the binding of IL-1 to its receptor. The gene encoding this antagonist of IL-1 •has since been described (Hannum, et al., 1990, Nature 343: 336-340; Eisenberg, et al., 1990, Nature 343: 341-346: designated the IL-1 receptor antagonist [IL-lra]; and Carter, et al., 1990, Nature 344: 633-638: designated the IL-1 receptor antagonist protein
[IRAP]).
IL-ira (hereinafter referred to as "IRAP") was originally identified as a product of activated macrophages. Additional cell types have been shown to synthesize this molecule; including synoviocytes, keratinocytes, chondrocytes and polymorphonuclear .leukocytes. IRAP production is increased by a variety of cytokines and other stimuli, including IL-1, interleukin-3 interleukin-4 interleukin-10 (IL-10) and 30 tumor necrosis factor-or (TNF-a). Several of these cytokines are generally considered to go be pro-inflammatory. IRAP is only a weak inhibitor of the biological activities of IL-1.
This reflects the ability of IL-1 to provoke a biological response in sensitive cells when V. only a few percent of its receptors are occupied. As the affinity of IRAP for these receptors does not exceed that of IL-1, the antagonist needs to be present at a molar excess of 10-1,000 fold to reduce responses to IL-1 by 50%. This may be a considerable impediment to the pharmacologic use of IRAP.
IRAP inhibits cellular responses to IL-1 under in vitro conditions, including induction of matrix metalloproteinases, induction of nitric oxide synthase, increased
C-
prostaglandin synthesis, cartilage degradation, bone resorption, increased expression of endothelial cell adhesion proteins, induction of interleukin-6 TNF-a, and other cytokines, and cell division.
IRAP inhibits cellular responses to IL-1 under in vivo conditions, including increased IL-6, death in adrenalectomized animals, hypotension, fever, slow-wave sleep, cartilage breakdown, joint inflammation, hypoglycemia, acute phase response and increased corticosterone. In addition, IRAP may have potential therapeutic properties in a number of animal models of disease (for a review, see Dinarello and Thompson, 1991, Immunol. Today 12: 404-410). In vivo administration of IRAP is well tolerated. Normal animals, including humans, can be infused intravenously with high doses of this protein without any change in physiological or metabolic parameters. For example, human volunteers infused with 133 mg/h IL-Ira for 72 hours exhibited no change in clinical or laboratory values (Dinarello, et al., 1993, J. Amer. Med. Assoc. 269: 1829-1835).
IL-4 is a 20 kDa glycoprotein produced from T-lymphocytes. This cytokine binds to receptors on hematopoietic stem cells, B and T lymphocytes, mast cells and macrophages. Interleukin-4 also induces IgE production and B-cell surface antigens.
Essner, et al. (1989, J. Immunol. 142: 3857-3861) determined that IL-4 down-regulates S. IL-1 and TNF-a at the level of mRNA transcription. Vannier, et al. (1992, Proc. Natl.
Acad. Sci. USA 89: 4076-4080) confirmed the down-regulation of IL-1 by IL-4.
20 Additionally, IL-4 was shown to up-regulate IRAP.
Raz, et al. (1993, Proc. Natl. Acad. Sci. USA 90:4523-4527) injected mice S• intramuscularly with a DNA plasmid encoding IL-4. Total IgG levels increased and a **delayed-type hypersensitivity reaction to an antigen challenge was decreased. The systemic in vivo effects were consistent with a proposed role of 1L-4 in stimulating the
TH
2 lymphocyte subset-mediated immune response. The study also disclosed intramuscular injection and in vivo expression of IL-2 and TGF-1. However, the study S. did not monitor the possible regulatory effect of intramuscular injection and in vivo expression of the IL-4 gene on IL-1 or IRAP activity.
is a 35-40 kDa acid sensitive cytokine produced by helper T-cells, B-cells, 30 monocytes and macrophages. IL-10 has been shown to possess immunosuppressive properties in vitro, as indicated by an ability to suppress cytokine production and antigen S* specific proliferation of cultured clones of TH 1 cell when activated in an accessory cell-dependent manner (for a review, see Howard and O'Garra, 1992, Immunol. Today 13(6): 198-200). IL-10, in a similar fashion as seen with IL-4, inhibits macrophage/monocyte production of IL-1B, IL-6, IL-8, GM-CSF, G-CSF and TNF-a and upregulates IRAP (de Waal Malefyt, et al., 1991, J. Exp. Med. 174: 1209-1220; Fiorentino, et al., 1991, J. Immunol. 147: 3815-3822; Bogdan, et al., 1991, J. Exp. Med.
174: 1549-1555).
Tumor necrosis factor alpha (TNF-a) is a cytokine produced primarily by activated macrophages or lymphocytes. This cytokine is involved in numerous biological phenomena such as inflammation, endotoxic shock, anti-tumor and anti-viral activities. Soluble TNF-ca binding proteins have been identified in normal human urine (Englemann, et al., 1989, J. Biol. Chem. 264: 11974-11980) and in serum of cancer patients (Gatanga, et al., 1990, Lymphokine Res. 9: 225-229).
Both the TNF-a and IL-1 soluble receptors inhibit receptor binding and biological activity of their respective cytokine in vitro. However, their in vivo role remains unclear. Fanslow, et al. (1990, Science 248: 739-742) disclose that direct administration of IL-1 soluble receptor prolongs allograft survival in mice. No mention is made regarding an ability to mount a prolonged biological response to the causal agent(s) of connective tissue disorders.
Connective tissue cells in monolayer culture produce low or undetectable levels of collagenase and other tissue metalloproteinases, such as stromelysin and gelatinase.
However, high levels of these proteinases can be expressed in the presence of IL-1 (Dayer, et al., 1986, J. Clin. Invest. 77: 645-648) and TNF-at (Dayer, et al., 1985, J. Exp.
Med. 162: 2163-2168). Evidence indicates that these proteins promote degradation of extracellular matrix constituents associated with osteoarthritis and rheumatoid arthritis.
2 0 Collagenase, stromelysin, and gelatinase are inhibited by the protein TIMP ("Tissue Inhibitor of MetalloProteinases"). The mechanism of TIMP inhibition of these S metalloproteinases is not fully understood (see Docherty and Murphy, 1990, Ann.
Rheum. Dis. 49: 469-479). There are two distinct TIMP genes encoding TIMP-1 and TIMP-2.
Leukocyte infiltration during an inflammatory response includes margination of these cells within microvessels, followed by adhesion to the vascular endothelial cells.
Increases in leukocyte adhesion during inflammation involves specific interactions between cell surface adhesion molecules on both the leukocyte and endothelium cell surface. Examples of endothelium cell surface adhesion molecules are ICAM-1 and ELAM-1. Synthesis of both ICAM-1 and ELAM-1 are stimulated by IL-1. ICAM-1 is a 30 receptor for the leukocyte adhesion protein, LFA-1. Anti-ICAM-1 antibodies have been shown to suppress pathogenesis of adjuvant arthritis in rats via inhibition of cell to cell adhesion (ligo, et al., 1991, J. Immunol. 147: 4167-4171). Additional leukocyte localized proteins include CR3 and CR4 (which bind the complement degradation product C3bi). LFA-1, CR3 and CR4 are heterodimer integrin receptor proteins which possess the same B polypeptide, designated CD18. Additionally, CD44, which acts both as a lymphocyte homing receptor and as a receptor for certain matrix macromolecules such as glycosaminoglycan hyaluronic acid as well as certain types of collagen, mediates leukocyte binding to endothelial cells (Streeter, et. al., 1988, Nature, 331: 41-46). CD44 also up-regulates IL-1 production (Denning, et al., 1989, FASEB J. 3: A765).
Reactive oxygen intermediates (ROI's) formed in response to inflammatory signals have been implicated in the destruction of extracellular matrix components such as hyaluronic acid and the proteoglycans collagen and elastin. The collagens are composed of a family of fibrous proteins which are secreted by connective tissue cells.
Collagen is the major protein of the extracellular matrix. Elastin, also an extracellular matrix protein expressed in connective tissue cells, forms a cross-linked network possessing both elasticity and tensile strength. Skaleric, et al. (1991, J. Immunol. 147: 2559-2564) suggests that interaction of potential ROI inhibitors such as superoxide dismutase during an inflammatory episode may reduce the erosive effects of ROI's in connective tissue disorders Cartilage is comprised of chondrocytes embedded within an extensive extracellular matrix consisting primarily of collagen fibrils and various proteoglycans.
Pathogenesis of connective tissue diseases such as arthritis includes erosion of proteoglycans. Two cartilage growth factors, insulin-like growth factor 1 (IGF-1) and transforming growth factor-B (TGF-B) positively regulate proteoglycan synthesis (McQuillan, et al., 1986, Biochem. J. 240: 423-430; Morales and Roberts, 1989, J. Biol.
Chem. 263: 12828-12831). Additionally, TGF-B has been shown to regulate expression 20 of adhesion molecules such as fibronectin and fibronectin receptors (Ignotz and Massague, 1986, J. Biol. Chem. 261: 4337-4345; Ignotz and Massague, 1987, Cell 51: 189-197).
Autoimmune diseases are characterized by an immune reaction against self antigens. In other words, the stricken individual has lost immunologic self-tolerance.
Autoimmune antibodies may act against a single cell or organ, such as seen in Hashimoto's thyroiditis. Conversely, systemic autoimmune diseases such as rheumatoid S. arthritis (RA) and systemic lupus erythematosus (SLE) result in exposure and activity of autoimmune antibodies at multiple sites within the patient.
Mechanisms of autoimmune disease are thought to involve any one of a number, 30 or combination thereof, of immunologic phenomenon which include, bypass of T-helper cell function, cross reaction with infectious agents, direct activation of B-cells, idiotype bypass mechanisms, and imbalance of T-suppressor-helper function.
A number of systemic autoimmune and non-autoimmune diseases involving pathogenesis of connective tissue promote inflammation, cytokine mediation and tissue destruction. Therefore, it would be extremely useful to be able to employ antagonists of specific mediators of these processes so as to therapeutically or prophylactically treat these disorders.
Although proteins can be infused into patients during acute conditions, they are difficult to deliver as drugs in chronic diseases. It is difficult to promote a sustained therapeutic response via administration of a protein. Because the oral and transdermal routes are unavailable, proteins need to be delivered by injection. In view of their short systemic half-life, most need to be administered at frequent intervals. Another difficulty in treating a systemic disease is a requirement that the therapeutic or prophylactic antagonist be delivered to multiple sites within the patient, in contrast to local treatment of diseases such as arthritis. Therefore, it would be extremely useful to not only apply antagonists of respective systemic connective tissue disease mediators to patients, but to do so in a fashion which promotes multiple delivery sites and prolonged availability of the antagonist within the patient.
3. SUMMARY OF THE INVENTION The present invention relates to methods of gene treatment of connective tissue disease which require systemic delivery within a mammalian host of a therapeutic or prophylactic protein, RNA, ribozyme or any biologically active fragment thereof.
Systemic delivery delivery to multiple sites within the patient) of the gene product may involve either systemic or local application of the nucleic acid sequence encoding •the therapeutic or prophylactic gene product or biologically active fragment thereof.
S 20 Viral or non-viral mediated delivery and sustained in vivo expression of the nucleic acid •sequence of interest followed by systemic transport of the respective gene product results in a perpetuating systemic drug delivery system within the patient. The present invention discloses strategies providing for nucleic acids which encode therapeutic or prophylactic gene products or biologically active fragments for the treatment of connective tissue diseases and the insertion of these nucleic acid sequences into a viral or non-viral vector molecule via recombinant DNA techniques. Therefore, the present invention also discloses various recombinant vectors for use in systemic treatment of connective tissue diseases or disorders.
30 3.1. CONNECTIVE TISSUE DISEASES TREATED BY SYSTEMIC DELIVERY OF S°THERAPEUTIC OR PROPHYLACTIC GENE PRODUCTS The present invention discloses methods of treating autoimmune and non-autoimmune diseases pathogenically related to the connective tissue which involves systemic delivery within a mammalian host of a therapeutic or prophylactic protein, RNA, ribozyme or any biologically active fragment thereof.
Systemic autoimmune diseases pathologically related to connective tissue amenable to treatment by the methods disclosed in the present invention include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, Sj6irgen's syndrome, 8 polymyositis-dermatomyositis, systemic sclerosis (scleroderma), vasculitis syndromes, juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis and inflammatory bowel disease.
Additional non-immune diseases or disorders pathogenically related to connective tissue which are disclosed for treatment in the present invention include, but not limited to, osteoporosis, osteogenesis imperfecta, and Paget's disease.
3.2. NUCLEIC ACID SEQUENCES UTILIZED IN TREATING CONNECTIVE TISSUE DISEASES The present invention discloses in vivo expression of a specific gene product or biologically active fragment thereof so as to provide therapeutic or prophylactic relief to an autoimmune or non-autoimmune disease pathogenically related to the connective tissue, thereby generating an acceptable level of resistance. The present invention is based in part on the strategy of local or systemic delivery of nucleic acid sequences which may provide a comprehensive approach to treating such diseases. In particular, these nucleic acid sequences should encode gene products which address one or more of the inflammatory, hypertrophic and erosive components of the disease. Nucleic acid sequences which combat one or more of these pathological components may be utilized in practicing the invention.
20 A nucleic acid sequence encoding a cytokine or a biologically active fragment thereof may be used to practice the present invention. Therapeutic or prophylactic o cytokines of the present invention which reduce systemic inflammation include, but are in no way limited to, the IL-1 receptor antagonist gene (IL-lra or IRAP) or a biologically active fragment thereof.
A nucleic acid sequence encoding an anticytokine or a biologically active fragment thereof may be used to practice the present invention. Therapeutic or prophylactic anticytokines, which will antagonize their cognate cytokine and in turn act in an anti-inflammatory manner include, but are in no way limited to a soluble receptor 6 of IL-1 or a biologically active fragment thereof.
3.3. VIRAL AND NON-VIRAL MEDIATED DELIVERY SYSTEMS The invention is also based on the use of various systems for gene transfer of the recombinant vector containing a nucleic acid sequence to be expressed within the mammalian host. The nucleic acid sequence of interest may be prepared for delivery by viral or non-viral mediated strategies.
Virus vectors utilized in the present invention include, but are not limited to retroviral vectors, including but not limited to vectors derived from Moloney murine leukemia virus (MoMLV); adenovirus vectors; adeno-associated vectors; herpes simplex virus vectors; SV40 vectors; polyoma virus vectors; papilloma virus vectors; picornavirus vectors; and vaccinia virus vectors.
Depending on the virus vector system chosen, techniques available to the skilled artisan are utilized to infect the target cell of choice with the recombinant virus vector.
A non-viral vector may be utilized in the present invention, such as, but not solely limited to, plasmid DNA. The use of plasmid DNA will be especially useful in non-viral vector mediated local delivery systems, such as direct injection of naked DNA into the skeletal muscle.
To this end, non-viral mediated delivery systems utilized in the present invention include, but are not limited to direct injection of naked DNA; liposome mediated transduction; calcium phosphate [Ca 3 (P0 4 2 mediated cell transfection, the genetically transformed cells then returned extraarticularly to the mammalian host; mammalian host cell transfection by electroporation, the genetically transformed cells then returned extraarticularly to the mammalian host; DEAE-dextran mediated cell transfection, the genetically transformed cells then returned extraarticularly to the mammalian host; polybrene mediated delivery; protoplast fusion; microinjection; and polylysine mediated transduction.
*S*
3.4. SYSTEMIC DELIVERY OF THERAPEUTIC OR 20 PROPHYLACTIC EXPRESSION PRODUCTS o The invention is based further on promoting systemic delivery of the therapeutic S or prophylactic expression product of the nucleic acid sequence within the mammalian host. Preferred systemic delivery of the gene products entails use of a recombinant viral or non-viral vector followed by in vitro transfection of specific mammalian cell populations, recovery and purification of the transfected cells and administration to the patient. The specific cell populations utilized as targets for transfection by the recombinant vector containing a nucleic acid sequence of interest may include, but are not limited to, bone marrow cell populations containing hematopoietic progenitor cells; peripheral blood leukocyte populations, preferably CD34 blood leukocyte populations, which are enriched for hematopoietic cells and can be utilized to repopulate the transfected hematopoietic cells upon introduction into the patient without ablation; peripheral blood lymphocyte populations; myoblast cells, which may be transplanted back into the host subsequent to in vitro transfection of the nucleic acid ***sequence of interest; and delivery of the recombinant viral or non-viral vector containing the nucleic acid sequence of interest, or the nucleic acid sequence itself, by intramuscular injection into skeletal muscle.
Various combinations of the hereinbefore disclosed steps to treat a specific disease or disorder may be utilized. The malady to be treated may respond more favorably to in vivo expression of a specific systemically delivered gene or gene fragment, the gene or gene fragment being amenable to delivery via one or several modes of vector construction and cellular delivery such that the gene is expressed and the prophylactic or therapeutic protein or protein fragment is systemically delivered throughout the mammalian host. Upon review of this disclosure, it will be within the purview of the skilled artisan to pick and choose between the therapeutic genes and modes of delivery disclosed in the present invention so as to select an efficient method of treatment. Various combinations and preferred embodiments are disclosed and expanded upon.
TREATMENT OF RHEUMATOID ARTHRITIS In one embodiment of the invention, a DNA sequence encoding IRAP or a biologically active portion thereof is administered to a patient so as to promote systemic and prolonged distribution of the expressed IRAP gene product for treatment, either therapeutic or prophylactic in nature, of rheumatoid arthritis.
In a further embodiment of the invention relating to the treatment of rheumatoid arthritis, any of the viral vectors, non-viral vectors or methods of delivering the nucleic acid sequence of interest to the target cell may be utilized to provide systemic application of the gene product or biologically active fragment thereof.
20 In a specific embodiment regarding the IRAP induced treatment of rheumatoid arthritis, the DNA sequence encoding IRAP or a portion thereof is subcloned into a retroviral vector prior to administration to the patient.
Regarding use of a retroviral vector in IRAP induced treatment of rheumatoid arthritis, the retroviral vector may be a MoMLV retroviral vector, resulting in an MoMLV-IRAP construction.
In a specific embodiment regarding the recombinant MoMLV-IRAP construction in the treatment of rheumatoid arthritis, the recombinant MoMLV-IRAP construction is MFG-IRAP (Figure 1 and Figure 2 [SEQ ID NO:3] for IRAP cDNA clone utilized in construction of MFG-IRAP).
30 In regards to promoting systemic delivery of the MFG-IRAP construction in the treatment of rheumatoid arthritis, this retroviral vector is transferred into a standard retroviral packaging cell line, the recovered viral particles used to transfect a mammalian cell population line, including but not limited to bone marrow cell populations containing hematopoietic progenitor cells; peripheral blood leukocyte populations, preferably CD34+ blood leukocyte populations, which are enriched for hematopoietic cells; peripheral blood lymphocyte populations; and myoblast cells, which may be transplanted back into the host subsequent to in vitro transfection. The in vitro transfected cell populations of and are then reintroduced intravenously into the circulatory system of the patient.
In a preferred embodiment of the invention regarding the systemic delivery of the MFG-IRAP construction in the treatment of rheumatoid arthritis, this retroviral vector is transferred into a standard retroviral packaging cell line, the recovered viral particles used to transfect bone marrow cell populations containing hematopoietic progenitor cells and these in vitro transfected cell populations are then reintroduced intravenously into the circulatory system of the patient.
In an additional embodiment regarding treatment of rheumatoid arthritis, a viral or non-viral recombinant vector molecule may be transferred directly into the skeletal muscle by intramuscular injection. In a preferred mode of this technique, the recombinant vector molecule is a plasmid, the IRAP gene or gene fragment thereof being subcloned downstream of a regulatory sequence(s) such that continual promotion of expression of the IRAP gene or gene fragment thereof will occur subsequent to intramuscular injection.
In a further embodiment regarding treatment of rheumatoid arthritis, any of the strategies disclosed within the specification may be utilized for targeting a nucleic acid of interest to the appropriate cell type so as to promote expression of the therapeutic agent, including but not necessarily limited to a nucleic acid sequence encoding a soluble 20 receptor of IL-1 or a biologically active fragment thereof.
3.6. TREATMENT.OF SYSTEMIC LUPUS ERYTHEMATOSUS In another embodiment of the invention, a DNA sequence encoding IRAP or a S* biologically active portion thereof is administered to a patient so as to promote systemic and prolonged distribution of the expressed IRAP gene product for treatment, either therapeutic or prophylactic in nature, of systemic lupus erythematosus.
In a further embodiment of the invention relating to the treatment of systemic lupus erythematosus, any of the viral vectors, non-viral vectors or methods of delivering the nucleic acid sequence of interest to the target cell may be utilized to provide systemic 30 application of the therapeutic gene product of biologically active fragment thereof.
In a specific embodiment regarding the IRAP induced treatment of systemic lupus erythematosus, the DNA sequence encoding IRAP or a portion thereof is subcloned into a retroviral vector prior to administration to the patient.
Regarding use of a retroviral vector in IRAP induced treatment of systemic lupus erythematosus, the retroviral vector may be a MoMLV retroviral vector, resulting in an MoMLV-IRAP construction.
In a specific embodiment regarding the recombinant MoMLV-IRAP construction in the treatment of systemic lupus erythematosus, the recombinant MoMLV-IRAP construction is MFG-IRAP (Figure 1).
In regards to promoting systemic delivery of the MFG-IRAP construction in the treatment of systemic lupus erythematosus, this retroviral vector is transferred into a standard retroviral packaging cell line, the recovered viral particles used to transfect a mammalian cell population line, including but not limited to bone marrow cell populations containing hematopoietic progenitor cells; peripheral blood leukocyte populations, preferably CD34+ blood leukocyte populations, which are enriched for hematopoietic cells; peripheral blood lymphocyte populations; and myoblast cells, which may be transplanted back into the host subsequent to in vitro transfection.
The in vitro transfected cell populations of and are then reintroduced intravenously into the circulatory system of the patient.
In a preferred embodiment of the invention regarding the systemic delivery of the MFG-IRAP construction in the treatment of systemic lupus erythematosus, this retroviral vector is transferred into a standard retroviral packaging cell line, the recovered viral particles are used to transfect bone marrow cell populations containing hematopoietic progenitor cells and these in vitro transfected cell populations are then reintroduced into the circulatory system of the patient.
20 In an additional embodiment regarding treatment of systemic lupus erythematosus, a viral or non-viral recombinant vector molecule may be transferred locally into the skeletal muscle by direct intramuscular injection. In a preferred mode of this technique, the recombinant vector molecule is a plasmid, the IRAP gene or gene fragment thereof being subcloned downstream of a regulatory sequence(s) such that continued expression of the IRAP gene or gene fragment thereof will occur within the transfected tissue.
In a further embodiment regarding treatment of systemic lupus erythematosus, any of the strategies disclosed within the specification may be utilized for targeting a nucleic acid of interest to the appropriate cell type so as to promote expression of the 30 therapeutic agent, including but not necessarily limited to a nucleic acid sequence S* encoding a soluble receptor of IL-1 or a biologically active fragment thereof.
3.7. TREATMENT OF ADDITIONAL CONNECTIVE TISSUE DISEASES In additional embodiments of the invention, treatment of other autoimmune disease which affect connective tissue, including but not limited to Sjargen's syndrome, polymyositis-dermatomyositis, systemic sclerosis (scleroderma), vasculitis syndromes, juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis and inflammatory bowel disease. Additional non-immune diseases or disorders pathogenically related to 13 the connective tissue which are disclosed for treatment in the present invention include, but not limited to, osteoporosis, osteogenesis imperfecta, and Paget's disease. Treatment of these diseases involves the prolonged, systemic delivery of therapeutic or prophylactic expression products encoded by nucleic acid sequences which include but are not solely limited to IRAP or a biologically active fragment thereof; a soluble receptor of IL-1 or a biologically active fragment thereof. Any of the strategies disclosed within the specification may be utilized for the systemic delivery of these DNA sequences to a mammalian host in treating the hereinbefore mentioned diseases.
It is an object of the invention to provide prolonged relief against autoimmune and non-autoimmune diseases affecting connective tissue by systemic delivery of a therapeutic or prophylactic amount of a protein, protein fragment, antisense RNA or ribozyme within a mammalian host; these gene products encoded by a nucleic acid sequence delivered to the mammalian host in a local or systemic manner.
It is another object of the invention to locally or systemically deliver the IRAP gene or gene fragment capable of prolonged expression to a mammalian host so as to provide systemic relief against various autoimmune and non-autoimmune diseases that elicit pathological degradation of connective tissue.
It is a further object of the invention to locally or systemically deliver the IRAP gene or a biologically active gene fragment capable of prolonged expression to a mammalian host so as to provide systemic relief against rheumatoid arthritis or systemic lupus erythematosus.
It is a specific object of the invention to locally or systemically deliver the IRAP gene or a biologically active gene fragment to a mammalian host by transfecting hematopoietic cell containing populations so as to systemically repopulate the mammalian host with the IRAP-transfected hematopoietic cells; allowing for treatment of autoimmune diseases affecting connective tissue including but not limited to rheumatoid arthritis or systemic lupus erythematosus as well as hereinbefore disclosed non-autoimmune diseases affecting connective tissue.
These and other objects of the invention will be more fully understood from the 30 following description of the invention in context of additional gene treatment of autoimmune and non-autoimmune diseases affecting connective tissue, the referenced figure attached hereto and the claims appended hereto.
3.8. DEFINITIONS The terms listed below, as used herein, will have the meanings indicated.
IRAP Interleukin-1 Receptor Antagonist Protein IL-lra Interleukin- 1 Receptor Antagonist Protein RNA Ribonucleic Acid DNA Deoxyribonucleic acid LTR Long Terminal Repeats IL-1 Interleukin-1 IL-2 Interleukin-2 IL-3 Interleukin-3 IL-4 Interleukin-4 IL-6 Interleukin-6 TNF-a Tumor Necrosis Factor alpha MoMLV Moloney Murine Leukemia Virus PDGF Platelet Derived Growth Factor SSc Systemic Sclerosis SLE Systemic Lupus Erythematosus RA Rheumatoid Arthritis NSAID Nonsteroidal Anti-inflammatory Drug TIMP Tissue Inhibitor of Metalloproteinase TGB-3 Type 1 Transforming Growth Factor As used herein, the term "patient" includes members of the animal kingdom including but not limited to human beings.
As used herein, the term "mammalian host" includes members of the animal kingdom including but not limited to human beings.
As used herein, the term "treatment" includes promotion of either therapeutic relief or prophylactic resistance, depending upon the specific connective tissue disease or disorder to be treated, from proteins, peptides or chemical compounds which induce inflammatory or erosive responses during pathogenesis of said connective tissue disease or disorder.
As used herein, the term "biologically active fragment" refers to any portion of derivative of the corresponding wild-type molecule exhibiting biological activity by promoting therapeutic relief or prophylactic resistance from proteins, peptides or 30 chemical compounds which induce inflammatory or erosive responses during *pathogenesis of a connective tissue disease or disorder.
4. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows a schematic representation of the provirus form of Moloney murine leukemia virus (MoMLV) that is used for the construction of retroviral vectors.
The MFG retroviral vector is a simplified MoMVL retroviral vector in which the DNA sequences encoding the pol and env proteins have been deleted. A cDNA encoding human IRAP (Figure 2) was inserted into the NcoI and BamHI cloning sites of the retroviral vector MFG. Specifically, a Pstl to BamHI fragment from the IRAP cDNA was linked to a synthetic oligonucleotide adapter from the NcoI site (representing the start site of translation for IRAP) to the Pstl site (approximately 12 base pairs downstream from the NcoI site) to the MFG backbone digested at NcoI and BamHI in a three part ligation reaction. The straight arrow and the crooked arrow represent unspliced and spliced messenger RNAs respectively. IRAP is encoded by the spliced message. The BAG vector is an MFG derivative. In BAG, the, 3-gal gene is expressed from a non-spliced LTR-driven message whereas the neo gene is expressed from a promoter. Both MFG-IRAP and BAG have the psi site required for packaging of the recombinant RNA into virions.
Figure 2 shows the cloned IRAP cDNA sequence (SEQ ID NOS:3 and 4) utilized in construction of MFG-IRAP, namely a HindII fragment comprising the entire coding region of human IRAP, as described in detail in Example Section 6.1.1.
5. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to methods of gene treatment involving local or systemic delivery within a mammalian host of a nucleic acid sequence which expresses a •therapeutic or prophylactic protein or protein fragment, antisense RNA, ribozyme or •other biologically active fragment. Construction and utilization of a viral or non-viral recombinant vector and the ability of the locally or systemically delivered nucleic acid sequence within the recombinant vector for sustained expression within the mammalian host is tantamount to a perpetual systemic drug delivery system. The methods of the ooo present invention disclose connective tissue diseases or disorders targeted for therapeutic or prophylactic treatment, DNA sequences available for delivery to the mammalian host, strategies to prepare and deliver the DNA sequence so as to optimize expression and availability of the biologically a ctive product, as well as preferred combinations of these S" strategies in treating these systemic connective tissue disorders. The present invention also discloses recombinant viral and non-viral vectors for utilization in treating the connective tissue disorders discussed throughout the specification.
5.1. NUCLEIC ACID SEQUENCES UTILIZED IN TREATING CONNECTIVE TISSUE DISEASES SoThe present invention discloses in vivo expression of a specific gene product or S biologically active fragment thereof so as to provide therapeutic or prophylactic relief to an autoimmune or non-autoimmune disease pathogenically related to a connective tissue, thereby generating an acceptable level of resistance. The present invention is based in part on the strategy of local or systemic delivery of a nucleic acid sequence which may provide a comprehensive approach to treating such diseases. In particular, these nucleic acid sequences should encode gene products which address one or more of the inflammatory, hypertrophic and erosive components of the disease. A nucleic acid sequence which combats one or more of these pathological components may be utilized in practicing the invention. It will become evident upon review of this specification that more than one nucleic acid sequence may be simultaneously administered to the patient to promote systemic treatment of the respective connective tissue disease.
A nucleic acid sequence encoding a cytokine or a biologically active fragment thereof may be used to practice the present invention. Therapeutic or prophylactic cytokines of the present invention which reduce systemic inflammation include, but are in no way limited to, the IL-1 receptor antagonist gene (IL-ra or IRAP) or a biologically active fragment thereof.
A nucleic acid sequence encoding an anticytokine or a biologically active fragment thereof may be used to practice the present invention. Therapeutic or prophylactic anticytokines, which will antagonize their cognate cytokine and in turn act in an anti-inflammatory manner include, but are in no way limited to a soluble receptor of IL- 1 or a biologically active fragment thereof.
5.2. VIRAL AND NON-VIRAL MEDIATED DELIVERY SYSTEMS •The invention is also based on the use of various systems for gene transfer of the 20 recombinant vector containing a nucleic acid sequence to be expressed within the S mammalian host. The nucleic acid sequence of interest may be prepared for delivery by viral or non-viral mediated strategies.
0@*B S•Virus vectors utilized in the present invention include, but are not limited to retroviral vectors, including but not limited to vectors derived from Moloney murine leukemia virus (MoMLV); adenovirus vectors; adeno-associated vectors; herpes simplex virus vectors; SV40 vectors; polyoma virus vectors; papilloma virus vectors; picornavirus vectors; and vaccinia virus vectors.
Depending of the virus vector system chosen, techniques available to the skilled artisan are utilized to infect the target cell of choice with the recombinant virus vector.
30 In a specific embodiment of the invention, a Moloney murine leukemia virus (MoMLV) retroviral vector is utilized to generate a construct for transfection of mammalian cells. Although MoMLV is a RNA virus, it has a DNA intermediate form that stably integrates into the genome of the host cell. The virus has two long terminal repeats (LTRs) at the 5' and 3' ends of proviral DNA that contain promoter, polyadenylation, and integration sequences required for the viral life cycle. A packaging sequence, termed psi, is also required in cis or the production of infectious virus. The virus encodes three proteins, gag, pol, and env, that are required in trans for viral replication. The gag and pol proteins are expressed from a non-spliced message whereas the env protein is expressed from a spliced message generated using the 5' and 3' splice sites shown in Figure 1. To generate a recombinant retroviral vector, the gag, pol, and env genes were removed and the cDNA encoding IRAP (MFG-IRAP) or the genes for B-gal and neo (BAG) were inserted. In MFG-IRAP, the gene is expressed from a LTR-driven spliced message. In BAG, the, B-gal gene is expressed from a non-spliced LTR-driven message whereas the neo gene is expressed from a SV40 promoter. Both MFG-IRAP and BAG have the psi site required for packaging of the recombinant RNA into virions. To generate infectious virus, the proviral DNA is transfected into a packaging line that constitutively produces gag, pol, and env proteins. Figure 1 shows the structure of the cDNA encoding the human interleukin-1 receptor antagonist protein (IRAP) gene inserted into the NcoI and BamHI cloning sites of the retroviral vector MFG (for a review of retroviral vectors, see Miller, 1992, Current Topics in Microbiology and Immunology 158: 1-24).
Any non-viral vector may be utilized in the present invention, such as, but not solely limited to, plasmid DNA. The use of plasmid DNA will be especially useful in non-viral vector mediated systemic delivery systems, such as direct injection of naked DNA into skeletal muscle. Any eukaryotic promoter and/or enhancer sequence available to the skilled artisan which is known to up-regulate expression of the nucleic acid of interest may be used in plasmid vector constructions, including but not limited to a S 20 cytomegalovirus (CMV) promoter, a Rous Sarcoma virus (RSV) promoter, a Murine Leukemia Virus (MLV) promoter, a B-actin promoter, as well as any cell-specific eukaryotic promoter sequence that would be known to be active in the cell targeted for transduction. To this end, non-viral mediated delivery systems utilized in the present invention include, but are not limited to direct injection of naked DNA; liposome mediated transduction; calcium phosphate [Ca 3
(PO
4 2 mediated cell transfection, the genetically transformed cells then returned extraarticularly to the mammalian host; mammalian host cell transfection by electroporation, the genetically transformed cells then returned extraarticularly to the mammalian host; DEAE-dextran mediated cell transfection, the genetically transformed cells then returned extraarticularly to the 30 mammalian host; polybrene mediated delivery; protoplast fusion; microinjection; and polylysine mediated transformation.
5.3. SYSTEMIC DELIVERY OF THERAPEUTIC OR PROPHYLACTIC EXPRESSION PRODUCTS The invention is based further on promoting systemic delivery of the therapeutic or prophylactic expression product of the nucleic acid sequence within the mammalian host. Preferred systemic delivery of the gene products entails use of a recombinant viral or non-viral vector followed by in vitro transfection of specific mammalian cell populations, recovery and purification of the transfected cells and administration to the patient. The specific cell populations utilized as targets for transfection by the recdmbinant vector containing a nucleic acid sequence of interest may include, but are not limited to, bone marrow cell populations containing hematopoietic progenitor cells; peripheral blood leukocyte populations, preferably CD34 blood leukocyte populations, which are enriched for hematopoietic cells and can be utilized to repopulate the transfected hematopoietic cells upon introduction into the patient without ablation; peripheral blood lymphocyte populations; myoblast cells, which may be transplanted back into the host subsequent to in vitro transfection of the nucleic sequence of interest; and delivery of the recombinant viral or non-viral vector containing the nucleic acid sequence of interest, or the nucleic acid sequence itself, by intramuscular injection.
The use of bone marrow containing hematopoietic progenitor cells is described in detail in Example Section 6. Briefly, bone marrow cells are removed, infected with the recombinant virus, and reintroduced back into the mammalian host. Thus, a gene or gene fragment is systemically distributed within the mammalian host, expression of this DNA sequence resulting in systemic delivery of the gene product within the mammalian host.
Peripheral blood is a source of mammalian cells for infection by viral mediated °vectors. More specifically, blood leukocytes, especially the CD34 population which contain the circulating hematopoietic stem cells, may be used as target cells for viral infection, followed by repopulation of the mammalian host marrow without ablation (Karlsson, et al., 1993, Bone Marrow Transplant. 1 (supp. 124-127). Additionally, lymphocytes may also be utilized as target cells to promote systemic delivery of the nucleic acid sequence of interest. The lymphocytes may be removed from the peripheral blood of the host, cultured by known techniques and used as the target cell population for infection by viral vectors containing the nucleic acid sequence of interest. The infected 0 *0 lymphocytes may then be injected into the mammalian host see Anderson, et al., 1990, Human Gene Therapy 1: 331-361).
An additional target cell population are myoblasts. Blood flow into the relatively 30 large mass of skeletal muscle renders this tissue a localized repository for the nucleic acid sequence of interest. Therefore, in vitro infection and reintroduction of myoblast cells into the mammalian host provides a local target within the host which results in systemic delivery of the therapeutic product. Myoblast culture, infection and reintroduction into the host can be achieved by known techniques see Dai, et al., 1992, Proc. Natl. Acad. Sci. USA 89: 10892-10895).
To this end, direct injection into skeletal muscle is an additional method for local delivery of the vector molecule. Direct injection ultimately results, upon expression of the gene product, in systemic delivery of the therapeutic product within the mammalian 19 host (Wolff, et al, 1990, Science 247: 1465-1468; Raz, et al., 1993, Proc. Natl. Acad.
Sci. USA 90:4523-4527). Direct injection of naked DNA, preferably a non-viral vector such as plasmid DNA, is utilized in this mode of localized delivery. Any eukaryotic promoter and/or enhancer sequence available to the skilled artisan which is known to up-regulate expression of the nucleic acid of interest may be used in plasmid vector constructions, including but not limited to a cytomegalovirus (CMV) promoter, a Rous Sarcoma virus (RSV) promoter, a Murine Leukemia Virus (MLV) promoter, a B-actin promoter, as well as any cell-specific eukaryotic promoter sequence that would be known to be active in the cell targeted for transduction.
5.4. TREATMENT OF CONNECTIVE TISSUE DISEASES 5.4.1. SYSTEMIC TREATMENT OF RHEUMATOID ARTHRITIS Rheumatoid arthritis is a multisystem, chronic disease widely held to be an autoimmune disease. Approximately 80% of patients are positive for an autoantibody referred to as rheumatoid factor Rheumatoid factor is raised against the Fc portion of autologous IgG. Additional autoantibodies to nuclear elements, collagen and cytoskeletal filamentous proteins may also be detected. Rheumatoid arthritis most often results in a severe form of chronic synovitis. In addition to this chronic inflammation of the synovial tissue, cell-mediated immunity is also activated, resulting in cartilaginous destruction. It is not known what triggers the autoimmune reaction, but IL-1 may well be a major mediator of rheumatoid arthritis. First, IL-1 is known to activate synovial cells and promote cartilaginous breakdown. Second, IL-1 acts as a chemoattractant for lymphocytes and macrophages, which are thought to play a major role in the Spathogenesis of rheumatoid arthritis.
The present invention discloses and teaches a first line systemic defense encompassing utilization of a DNA sequence encoding a protein or protein fragment which antagonizes IL-1 IRAP or a soluble receptor to IL-1), downregulates IL-1 (IL-4 and IL-10) or acts to induce IRAP in vivo (IL-4 and IL-10). Administration and delivery of these respective nucleic acid sequences may be directed by viral or non-viral mediated methods as disclosed throughout the specification.
30 One embodiment of the invention teaches the delivery of a DNA sequence encoding IRAP or a biologically active protein fragment thereof. The delivery and continuous expression of the DNA sequence of interest promotes prolonged distribution of the expressed IRAP protein or protein fragment for therapeutic relief from rheumatoid arthritis.
In a further embodiment regarding the IRAP induced systemic treatment of rheumatoid arthritis, the DNA sequence encoding IRAP or a portion thereof is subcloned into a MoMLV retroviral vector prior to systemic delivery to the patient. Specifically, a recombinant MoMLV-IRAP construction that may be utilized in the treatment of rheumatoid arthritis is MFG-IRAP (Figure wherein the DNA sequence encoding IRAP or a portion thereof is SEQ ID NO:3 (Figure 2).
In regards to promoting systemic delivery of the MFG-IRAP construction in the treatment of rheumatoid arthritis, this retroviral vector is transferred into a standard retroviral packaging cell line, the recovered viral particles used to transfect a mammalian cell population line, including but not limited to bone marrow cell populations containing hematopoietic progenitor cells; peripheral blood leukocyte populations, preferably CD34+ blood leukocyte populations, which are enriched for hematopoietic cells; peripheral blood lymphocyte populations, which are then reintroduced intravenously into the circulatory system of the patient; and myoblast cells, which may be transplanted back into the host subsequent to in vitro transfection of the nucleic acid sequence of interest.
In a preferred embodiment of the invention regarding systemic delivery of the MFG-IRAP construction for treatment of rheumatoid arthritis, this retroviral vector is transferred into a standard retroviral packaging cell line and the recovered viral particles are used to transfect bone marrow cell populations containing hematopoietic progenitor cells. These in vitro transfected cell populations are then reintroduced into the circulatory system of the patient as described in Example Section 6.
9 5.4.2. SYSTEMIC TREATMENT OF SYSTEMIC LUPUS ERYTHEMATOSUS S Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by injury to the skin, joints, kidney and serosal membrane. A vast array of autoantibodies, especially antinuclear antibodies, are present in the SLE patient. Pathogenesis of SLE includes damage to tissues and cells from deposition of autoantibodies and immune complexes. A major immunologic factor in the pathogenesis of SLE is thought to be Bcell hyperactivity. While no cure currently exists, treatment of SLE has been directed at controlling inflammation, usually by administration of salicylates, NSAIDs or steroids.
The present invention discloses, in part, the ability to control or substantially reduce SLE induced inflammation and tissue damage by systemic or local delivery, 30 prolonged exposure, and expression within the patient of DNA sequences encoding an IL-1 antagonist. As discussed hereinbefore in regards to treating rheumatoid arthritis, examples of such IL-1 antagonists include, but are not limited to, DNA sequence V encoding IRAP, soluble receptor of IL-1, or biologically active protein fragments thereof.
One embodiment of the invention teaches the delivery of a DNA sequence encoding IRAP or a biologically active protein fragment thereof. The delivery and continuous expression of the DNA sequence of interest promotes prolonged distribution of the expressed IRAP protein or protein fragment for therapeutic relief from SLE.
In a further embodiment regarding the IRAP induced systemic treatment of rheumatoid arthritis, the DNA sequence encoding IRAP or a portion thereof is subcloned into a MoMLV retroviral vector prior to systemic delivery to the patient. Specifically, a recombinant MoMLV-IRAP construction that may be utilized in the treatment of SLE is MFG-IRAP (Figure wherein the DNA sequence encoding IRAP or a portion thereof is SES ID NO:3 (Figure 2).
In regards to promoting systemic delivery of the MFG-IRAP construction in the treatment of SLE, this retroviral vector is transferred into a standard retroviral packaging cell line, the recovered viral particles used to transfect a cell population include but are not limited to bone marrow cell populations containing hematopoietic progenitor cells; peripheral blood leukocyte populations, preferably CD34 blood leukocyte populations, which are enriched for hematopoietic cells, which are then reintroduced intravenously into the circulatory system of the patient; and myoblast cells, which may be transplanted back into the host subsequent to in vitro transfection of the nucleic acid sequence of interest. In a preferred embodiment of the invention regarding the systemic delivery, of the MFG-IRAP construction in the treatment of SLE, this retroviral vector is transferred into a standard retroviral packaging cell line, the recovered viral particles used to transfect bone marrow cell populations containing hematopoietic progenitor cells, these in vitro transfected cell populations are then introduced into the 4 S 20 bone marrow of the patient.
5.4.3. TREATMENT OF SYSTEMIC SCLEROSIS Systemic sclerosis (SSc) is an inflammatory based disease characterized by multisystem disorders. The primary pathogenic event is endothelial cell injury followed by eventual proliferation, excessive fibrosis, and vessel obliteration. The cause of SSc is unknown. However, the disease is associated with a variety of immunologic events.
Fibroblast activation, a cause of eventual fibrosis, is thought to involve stimulation by *0 IL-1, TNF-a, TGF-13, PDGF, fibroblast growth factor, and other cytokines.
In regard to treatment of systemic sclerosis, any of the strategies disclosed within o 30 the specification may be utilized for a targeting nucleic acid of interest to the appropriate cell type so as to promote expression of the therapeutic agent, including but not necessarily limited to a nucleic acid sequence encoding IRAP or a biologically active fragment thereof; a soluble receptor of IL-1 or a biologically active fragment thereof.
5.4.4. SYSTEMIC TREATMENT OF POLYMYOSOTIS-DERMATOMYOSITIS Polymyositis is a chronic inflammatory myopathy initially characterized by muscle weakness and eventually resulting in motor disability. The disease is associated with dermatomyositis in approximately 50% of cases. The cause of the disease is 22 unknown, but patients tend to accumulate autoantibodies to tRNA synthases. It has also been documented that cell-mediated immune injury via activation of CD4+ and CD8+ Tlymphocytes is involved in progression of the disorder.
Interleukin-1 is involved in T-cell activation. Therefore, another embodiment of the invention involves methods of treating polymyositis-dermatomyositis both locally and systemically. DNA sequences expressing antagonists of IL-1, as hereinbefore described, are delivered to sites of myopathy either systemically via in vitro transfection of bone marrow or CD34+ cell populations followed by administration of the transfected cells to the patient) or locally (intramuscular injection or in vitro transfection and reintroduction of host myoblast cell populations).
In regard to treatment of polymyositis-dermatomyositis, any of the strategies disclosed within the specification may be utilized for targeting nucleic acid of interest to the appropriate cell type so as to promote expression of the therapeutic agent, including but not necessarily limited to a nucleic acid sequence encoding IRAP or a biologically active fragment thereof; a soluble receptor of IL-1 or a biologically active fragment thereof.
5.4.5. SYSTEMIC TREATMENT OF SJOGREN'S SYNDROME Sj6gren's syndrome is identified clinically by dry eyes and mouth resulting from a lymphocytic infiltration and destruction of the lacrimal and salivary glands. While a number of autoantibodies have been detected during pathogenesis of Sj6gren's syndrome, the most prevalent are autoantibodies against ribonucleoprotein antigens. Most of the infiltrating immune cells are CD4+ T-cells, which are stimulated by IL-1.
Therefore, an additional embodiment of the invention relates to treatment of Sj6gren's syndrome by generation and delivery of DNA sequences encoding an IL-1 antagonist, as disclosed and taught hereinbefore.
Preferred modes of systemic delivery in treating Sjigren's syndrome would be reintroduction of transduced bone marrow cells, leukocytes or lymphocytes. The preferable mode of local delivery would be intramuscular injection.
30 In a further embodiment regarding treatment of Sjogren's syndrome, any of the strategies disclosed within the specification may be utilized for targeting nucleic acid of interest to the appropriate cell type so as to promote expression of the therapeutic agent, including but not necessarily limited to a nucleic acid sequence encoding IRAP or a biologically active fragment thereof; a soluble receptor of IL-1 or a biologically active fragment thereof.
5.4.6. SYSTEMIC TREATMENT OF VASCULITIS SYNDROMES Vasculitis syndromes are characterized by inflammation of and ultimate damage to blood vessels, followed by ischemia. Evidence exists, that along with pathogenic involvement of immune-complex-mediated mechanisms in vasculitis syndromes, cell-mediated immune injury may also play a major role. Specifically, vascular endothelial cells secrete IL-1, which may then activate T lymphocytes and promote inflammation within the blood vessel.
Therefore, the present invention also relates to treatment of various vascular syndromes, including but not limited to, polyarteritis nodosa, allergic angiitis, polyangiitis overlap syndrome and giant cell arteritis (temporal arteritis and Takayasu's arteritis). Treatment involves systemic delivery of DNA sequences encoding IL-1 antagonists, as described hereinbefore for treatment of rheumatoid arthritis and systemic lupus erythematosus.
In a further embodiment regarding treatment of vascular syndromes, any of the strategies disclosed within the specification may be utilized for targeting nucleic acid of interest to the appropriate cell type so as to promote expression of the therapeutic agent, including but not necessarily limited to a nucleic acid sequence encoding IRAP or a biologically active fragment thereof; a soluble receptor of IL-1 or a biologically active fragment thereof.
5.4.7. SYSTEMIC TREATMENT OF ANKYLOSING SPONDYLYTIS *0 Ankylosing spondylytis is a systemic rheumatic disorder characterized by inflammation of the axial skeleton and large peripheral joints. An additional embodiment of the invention indicates treatment of this type of inflammatory arthritis in the same manner as disclosed for rheumatoid arthritis.
As disclosed in embodiments regarding treatment of rheumatoid arthritis, any of .these strategies may be utilized for treating ankylosing spondylytis by targeting the nucleic acid of interest to the appropriate cell type so as to promote expression of the therapeutic agent, including but not necessarily limited to a nucleic acid sequence 30 encoding IRAP or a biologically active fragment thereof; a soluble receptor of IL-1 or a biologically active fragment thereof.
5.4.8. SYSTEMIC TREATMENT OF JUVENILE RHEUMATOID ARTHRITIS Juvenile rheumatoid arthritis is a form of rheumatoid arthritis which manifests itself prior to age 16. Three subtypes (systemic, pauciarticular and polyarticular) have been designated. The disease tends to affect larger joints that may interfere with growth and development.
9. 9 S @699r **Ob 9* 9. 6 9 99 9 9.
SO
0*
S
@00009 This embodiment of the invention dictates use of systemic forms of delivering anti-arthritic proteins or protein fragments as treatment for the disorder. As disclosed above for ankylosing spondylytis, treatment of juvenile rheumatoid arthritis in the same manner as disclosed for rheumatoid arthritis is embraced in the present invention.
As disclosed in embodiments regarding treatment of rheumatoid arthritis, any of these strategies may be utilized for treating juvenile rheumatoid arthritis by targeting the nucleic acid of interest to the appropriate cell type so as to promote expression of the therapeutic agent, including but not necessarily limited to a nucleic acid sequence encoding IRAP or a biologically active fragment thereof; a soluble receptor of IL-i or a biologically active fragment thereof.
5.4.9. SYSTEMIC TREATMENT OF PSORIATIC ARTHRITIS Psoriatic arthritis is a rheumatoid-like arthritis associated with psoriasis of the skin or nails. The majority of patients are affected either at two or three joints or in a systemic fashion. Present forms of treatment include NSAIDs or steroid injections to treat inflammation.
Therefore, it is an additional embodiment of the invention to systemically treat patients suffering from psoriatic arthritis in one or more of the therapeutic strategies hereinabove disclosed for rheumatoid arthritis.
As disclosed in embodiments regarding treatment of rheumatoid arthritis, any of these strategies may be utilized for treating psoriatic arthritis by targeting the nucleic acid of interest to the appropriate cell type so as to promote expression of the therapeutic agent, including but not necessarily limited to a nucleic acid sequence encoding IRAP or a biologically active fragment thereof; a soluble receptor of IL- or a biologically 25 active fragment thereof.
5.4.10. SYSTEMIC TREATMENT OF OSTEOPOROSIS Osteoporosis is a disorder characterized by a decrease in bone mass such that the 30 skeleton is rendered fragile and therefore susceptible to fracture. The pathogenesis of the disease is based on increased osteoclastic resorption coupled with decreased bone formation, resulting in the net loss of bone mass. Increases in resorption are promoted by systemic (such as decreases in estrogen and calcitonin and an increase in PTH) and local factors (such as IL-1, IL-6 and TNF-ct produced locally by macrophages and osteoclasts).
Therapeutic treatment of osteoporosis includes embodiments regarding treatment of rheumatoid arthritis, including but not necessarily limited to a nucleic acid sequence encoding IRAP or a biologically active fragment thereof; a soluble receptor of IL-1 or a biologically active fragment thereof.
Nucleic acid sequences encoding anti-inflammatory therapeutic gene products or biologically active fragments thereof which decrease local concentration of IL-1 and/or TGF-1 are preferred in the treatment of osteoporosis. Delivery of the nucleic acid sequence of interest may include any of the strategies for local or systemic delivery disclosed in this specification, preferably utilizing systemic delivery by way of retroviral mediated infection of either hematopoietic stem cell populations such as bone marrow cells or CD34 cell populations.
5.4.11. SYSTEMIC TREATMENT OF PAGET'S DISEASE Paget's disease (Osteitis deformans) is a skeletal disease marked initially by osteoclastic bone resorption which in turn triggers replacement of normal marrow with vascular fibrous connective tissue. Thus, the resorbed bone is replaced by course-fibered, dense trabecular bone arranged in a disordered fashion. Paget's disease may well be caused by a viral infection. Possible causal viral agents are a slow-virus infection by paramyxovirus, measles virus, a respiratory syncytial virus or a canine virus.
Thus, it is possible that different viruses may be responsible for Paget's disease in different patients.
Present therapeutic indications include aspirin, NSAIDs and glucocorticoids, calcitonins, cytotoxic drugs such as plicamycin and dactinomycin, and etidronate, a diphosphonate compound.
As disclosed in embodiments regarding treatment of rheumatoid arthritis, any of these strategies may be utilized for treating Paget's disease by targeting the nucleic acid of interest to the appropriate cell type so as to promote expression of the therapeutic agent, including but not necessarily limited to a nucleic acid sequence encoding IRAP or a biologically active fragment thereof; a soluble receptor of IL-1 or a biologically active fragment thereof.
Nucleic acid sequences encoding anti-inflammatory therapeutic gene products or 0* 30 biologically active fragments thereof, systemically delivered by way of retroviral S* o mediated infection of either hematopoietic stem cell populations such as bone marrow cells or CD34+ cell populations are preferred in the treatment of Paget's disease.
5.4.12. SYSTEMIC TREATMENT OF INFLAMMATORY BOWEL DISEASE Inflammatory bowel disease is a generic term relating to a group of chronic inflammatory disorders of unknown etiology involving the gastrointestinal tract. Chronic inflammatory bowel disease is divided into two major groups, ulcerative colitis and Crohn's disease. While the cause of inflammatory bowel disease is unknown, possible etiologic factors include infectious, immunologic, familial or psychological factors.
Secondary extraintestinal manifestations such as arthritis and pericholangitis often times occur during chronic inflammatory bowel disease. Drug therapy of inflammatory bowel disease includes administration of anti-inflammatory drugs, sulfasalazine (the active compound thought to be 5-aminosalicylate, most likely by inhibiting prostaglandin synthesis) and glucocorticoids.
Therefore, the present invention relates to systemic therapeutic treatment of inflammatory bowel disease which include embodiments discussed for rheumatoid arthritis, including but not limited to IRAP or a biologically active fragment thereof; a soluble receptor of IL-1 or a biologically active fragment thereof.
Nucleic acid sequences encoding therapeutic gene products or biologically active fragments thereof which act as anti-inflammatory agents are preferred in the treatment of inflammatory bowel disease and can be delivered by any of the strategies disclosed in this specification.
The following examples are offered by way of illustration of the present invention, and not by way of limitation.
6. EXAMPLE: EFFICIENT SYSTEMIC TRANSFER AND PROLONGED EXPRESSION OF MFG-IRAP IN MICE 20 6.1. MATERIALS AND METHODS :1 IRAP cDNA ISOLATION AND RETROVIRAL VECTOR CONSTRUCTION The MFG vector is a simplified MoMVL vector in which the DNA sequences encoding the pol and env proteins have been deleted so as to render it replication defective. Sequences in the gag gene up to base 1035 have been utilized to increase the packaging efficiency of the unspliced transcript. The cDNA encoding the human interleukin-1 receptor antagonist (IRAP) was inserted into the NcoI and BamHI cloning sites of the retroviral vector MFG shown in Figure 1.
A human monocyte cDNA library, in lambda gtl0, was purchased from Clontech (catalog no. HL1036a). The cDNA library was derived from the human monocyte cell 30 line U937, which had been stimulated with lOnM phorbol 12-myristate 13-acetate for 48 •hours before mRNA isolation. The library was screened for IRAP cDNA clones using a 32P-end labelled oligonucleotide encompassing base pairs 309-391 (as indicated by Carter, et al., 1990, Nature 344: 633-638 [see Figure 1 at p. 634]). The probe was antisense. Positive clones were plaque purified from agar plates to nitrocellulose filters.
Two positive phage clones with EcoRI flanking sites were isolated and subcloned into the EcoRI site of pUC18 and were shown to have identical restriction maps. One of the clones was then sequenced. The 5' end of the positive cDNA was truncated at bp 93 and replaced with 81 bp of nonsense DNA (most likely a cloning artifact). DNA downstream from about bp 110 was also missing. The missing 5' sequences were replaced via two sequential PCR reactions. In the first reaction, the 5' sense primer covered bp 43-199.
The 3' antisense primer covered bp 553-577 and added a HindIII site to the 3' end. The product of this reaction was taken for a second PCR reaction using the same 3' primer and a 5' primer covering bp 1-67 which also added a HindIII site to the 5' end. The second PCR product was digested with HindlII, subcloned into pUC18, and sequenced.
The resulting IRAP insert had the following structure (as indicated in SEQ ID NOS:3 and 4, and Figure a 5' HindIII site followed immediately by bp 1 (again, utilizing the numbering of Carter, et al., 1990, Nature 344: 633-638) the entire IRAP coding sequence, 3' flanking DNA from bp 543-577, followed by a 3' HindIII site. This insert was subcloned into the HindIII site of pSV2cat. One of ordinary skill in the art will be aware that any of a multitude of vector molecules, especially DNA plasmid vector molecules with a unique HindIII site, can be substituted for pSV2cat at this stage of the construction of MFG-IRAP. This cDNA for human IRAP (SEQ ID NO:3 as subcloned into pSV2cat) was inserted into MFG by first BamHI-linkering the 3' HindIII site downstream of the stop codon of the IRAP gene, followed by digestion with Pstl and BamHI. This Pstl/BamHI IRAP fragment was ligated to Ncol/BamHI digested MFG, •with the addition of a synthetic oligonucleotide adapter 0 5'-CATGGAAATCTGCA-3' [SEQ ID NO:1] S" 20 3'-CTITAG-5' [SEQ ID NO:2]; from the NcoI site (representing the start site of translation for IRAP [initiating Met underlined) to the Pstl site (approximately 12 base pairs downstream from the NcoI site) in a three part ligation reaction, resulting in MFG-IRAP (Figure The initiation codon of IRAP is underlined in SEQ ID NO: 1. The resulting plasmid, termed MFG-IRAP, contains the entire coding region of IRAP. This three part ligation involving a synthetic oligo and two DNA fragments is well known by those skilled in the art of cloning. The straight arrow and the crooked arrow in Figure 1 represent unspliced and spliced a. messenger RNAs respectively. IRAP is encoded by the spliced message.
30 6.1.2. ISOLATION OF VIRUS PRODUCING CELLS °The retrovirus vector carrying the IRAP gene (MFG-IRAP) is cotransfected into the CRIP cell packaging line (Danos and Mulligan, 1988, Proc. Natl. Acad. Sci. USA 6460-6464) with pSVneo (conferring neomycin resistance) into NIH 3T3 cells using a standard CaPO 4 transfection procedure. NIH 3T3 transfected cells are selected on the basis of resistance to the antibiotic G418. The viral vector containing the neomycin resistant (neo-r) gene is capable of imparting resistance of the cell line to G418. The CRIP cell line expresses the three viral proteins required for packaging the vector viral RNAs into infectious particles. Moreover, the viral particles produced by the CRIP cell line are able to efficiently infect a wide variety of mammalian cell types including human cells. All retroviral particles produced by this cell line are defective for replication but retain the ability to stably integrate into mammalian cells, thereby becoming an heritable trait of these cells. Virus stocks produced by this method are substantially free of contaminating helper-virus particles and are also non-pathogenic.
6.1.3. COCULTURE WITH HEMATOPOIETIC STEM CELLS Bone marrow cells were harvested and cocultured with MFG-IRAP containing viral produced cells essentially as described in Ohashi, et al. (Proc. Natl. Acad. Sci. USA 89: 11332-11336). Briefly, bone marrow cells were harvested from limbs of C57 BL/6J- Gpi-la-Gpi-la-Hbbd-Hbbd female mice 3 days after injection with 5-fluorouracil (150 mg/kg of body weight). Bone marrow cells were precultured for 2 days in Fischer's medium supplemented with 15% fetal calf serum, 2mM L-glutamine, 100 units of penicillin per ml, 100 grams of streptomycin per ml, and cytokines. Recombinant rat stem cell factor (Amgen Biologicals) was used in both preculture and coculture at 100 ng/ml. The concentration of cytokines was 100 units of IL-3 and 100 ng of recombinant IL-6. Preculture was followed by 2 days of coculture with 20 Gy-irradiated viral producer cells in DMEM supplemented with 10% calf serum, antibiotics, Polybrene (8 4, tg/ml) and cytokines. After coculture, lethally irradiated (9.5Gy)B6-(Gpi-lb)/(Gpi-lb) 20 mice were injected with 2 x 106 bone marrow cells for long-term hematopoietic reconstitution studies.
9.
The techniques described in the previous paragraph were utilized to transfer the E. coli LacZ (B-galactosidase) gene to the bone marrow of mice for use as controls. The LacZ gene was inserted at the viral gag gene and the neomycin-resistance gene, neo, resulting in BAG (Figure 1).
e* e* 0 *Sm 6.1.4. ASSAY FOR IRAP ACTIVITY IN HEMATOPOIETIC RECONSTITUTED MICE IRAP activity in reconstituted mice was determined by a human IRAP immunoassay (a quantitative sandwich ELISA) following the manufacturers directions (marketed by Research Diagnostic Systems, Minneapolis, MN; under the trademark Quantikine).
6.2. RESULTS Table 1 shows the results of four separate long-term hematopoietic reconstitution experiments in mice. In all cases, high levels of IRAP expression were obtained.
Furthermore, these levels of expression were maintained for at least 13 months.
Therefore, these studies provide evidence for the ability to generate a prolonged availability of an IL-1 antagonist in a systemic manner in a mammalian host.
As depicted in Table 2, four IRAP+ mice and four control mice (LacZ+) were injected subcutaneously with 10ng of human recombinant IL-11. Three hours later, the mice were bled and sera analyzed for the presence of murine IL-6 using a commercial ELISA kit (Endogen, Boston, MA). In the control mice, IL-1B induced a large increase in serum IL-6 (from 20 pg/ml in mice injected with saline to 28,721 11,090 pg/ml in LacZ+ mice). In the IRAP mice, IL-6 levels only reached 8,553 3,785 pg/ml, an 20 inhibition of 70% These data demonstrate that the human IRAP produced systemically in the mice by the disclosed methods is biologically active and able to suppress an IL-1 induced biological response.
As discussed hereinbefore, other IL-1 antagonists, viral or non-viral strategies for preparing the nucleic acid sequence of interest, and modes of systemic delivery to the 25 mammalian host may be utilized to practice the disclosed invention. Therefore, whereas particular embodiments of this invention have been described above for purposes of illustration, it will be evident to those persons skilled in the art that numerous variations of the details of the piesent invention may be made without departing from the invention as defined in the appended claims.
Oe 0 S S 0 0 0 0.
0 0000
SOS.
0O S 0
SOSOSS
Expt.
I
IV
Table 1 Post Transplant Time (Months) 2 1 3.5 2 3 4 7 Number Serum IRAP (ngLml 104 13.5 513 280.6 154 111.3 381 80.5 306 65.7 175 27.7 271 59 583 247 470 165.6 Table 2
S
S S
S.
S. 0 0
S
000S@e
S
5* 0.55 0 S
S
Serum IL-6 (pg/mi) LacZ+ IRAP+ In-jection Mouse carrier 30 lOng IL-18 lOng IL-lB lOng IL-lB lOng IL-lB 33,102 18,859 42,358 20,562 28,721 ±11,090 6,505 14,125 7,662 5.920 8,553 ±3,785 @0 S S 00
SOSS
0S *5 0 S. S 0 S
S.
OS0SSS
S
S.
S S 505 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: University of Pittsburgh of the Commonwealth System of Higher Education (ii) TITLE OF INVENTION: Systemic Gene Treatment of Connective Tissue Diseases (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Lewis F. Gould, Jr.
STREET: 1700 Market Street Suite 3232 CITY: Philadelphia STATE: PA COUNTRY: US ZIP: 19103 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: 30 CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: NAME: Gould, Jr., Lewis F.
REFERENCE/DOCKET NUMBER: 119131-2 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 215/575-6000 TELEFAX: 215/575-6015 40 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid S 45 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CATGGAAATC TGCA 14 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 6 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GATTTC
6 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 589 base pairs TYPE: nucleic acid STRANDEDNESS: double 30 TOPOLOGY: linear 0 MOLECULE TYPE: cDNA S. (vi) ORIGINAL SOURCE: CELL TYPE: Human Monocyte CELL LINE: U937 (vii) IMMEDIATE SOURCE: LIBRARY: Clontech (HL1036a) S* 40 CLONE: Human IRAP (ix) FEATURE: NAME/KEY: CDS LOCATION: 15..548 5
S..
c (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: AAGOTTOCGA CAGA ATG GAA ATO Met Giu Ile 1 OTO TTC CTG TTO Leu Phe Leu Phe TGC AGA Cys Arg 5 GGC OTO CGC AGT Giy Leu Arg Ser CAC CTA ATO His Leu Ile CGA 000 TCT Arg Pro Ser ACT OTO CTC Thr Leu Leu TCA GAG ACG ATC Ser Glu Thr Ile GGG AGA Gly Arg AAA TCC AGC AAG Lys Ser Ser Lys CAA GOC TTO AGA Gin Ala Phe Arg TGG GAT GTT AAO Trp Asp Val Asn cAG Gin AAG AGO TTC TAT Lys Thr Phe Tyr AGG AAC AAO CAA Arg Asn Asn Gin
CTA
Leu 55 GTT GCT GGA TAO Val Aia Giy Tyr
TTG
Leu CAA GGA OCA AAT GTC AAT TTA GAA GAA Gin Gly Pro Asn Val Asn Leu Giu Giu ATA GAT GTG GTA Ile Asp Val Vai COO ATT Pro Ile GAG OCT OAT Giu Pro His TOO TGT GTO Ser Cys Vai 95
GOT
Aia OTG TTO TTG GGA Leu Phe Leu Giy OAT GGA GGG AAG His Gly Giy Lys ATG TGO OTG Met Oys Leu GAG GOA GTT Giu Ala Val AAG TOT GGT GAT Lys Ser Giy Asp 0* S 0 0 *0 S. OS
S
0 0
S
*0 S S
OOS@
eSS.
0b S. S
GAG
Giu 100 ACC AGA OTO OAG Thr Arg Leu Gin AAO ATO Asn Ilie 110 ACT GAO OTG AGO Thr Asp Leu Ser AAO AGA AAG OAG Asn Arg Lys Gin AAG OGO TTO GOO Lys Arg Phe Aia 35 TTO Phe 125 ATO OGO TOA GAO Ile Arg Ser Asp
AGT
Ser 130 GGO 000 ACC ACC Gly Pro Thr Thr
AGT
Ser 135 TTT GAG TOT GOO Phe Giu Ser Ala TGO 000 GGT TGG Cys Pro Gly Trp OTO TGO ACA GOG Leu Cys Thr Ala GAA GOT GAO OAG Glu Ala Asp Gin 000 GTO Pro Val 155 *0 *500 AGO OTO ACC Ser Leu Thr
AAT
Asn 160 ATG OOT GAO GAA Met Pro Asp Giu GTO ATG GTO ACC Val Met Val Thr AAA TTO TAO Lys Phe Tyr 170 TTO CAG GAG GAO GAG Phe Gin Giu Asp Giu 175 TAGTACTGCC CAGGCCTGCC TGTTOOOATT OTTGCATGAA
GOTT
INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 177 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NOA4 Met Giu Ile Cys Phe Ser Tyr Val Leu Ser Leu Asp 30 Phe 145 Met Glu Leu Lys Leu Asn Phe Gly Ser Ser 130 Leu Pro His Gin Asn Giu G iy Giu 100 Asn Pro Thr Giu Arg Ser Aia Asn Giu Ile Thr Arg Thr Ala Gly 165 Giu Phe Gin Lys 70 His Arg Ly s Thr Met 150 Val1 Thr Arg Leu 55 Ile G ly Leu Gin Ser 135 Giu Met Gly Leu Arg Ser His Leu Ile Thr Leu Leu Leu Cy s Trp Ala Val1 Lys Leu 105 Ly s Giu Asp Thr Pro Vali Tyr Pro 75 Cys Ala Phe Aia Pro 155 Phe Ser Asn Leu Ile Leu Val1 Ala Ala 140 Val1 Tyr Gly Gin Gin Giu Ser Asn Phe 125 Cy s Ser Phe Lys Thr Pro His Val1 Thr Arg Gly Thr Giu 175 Ser Phe Asn Ala Lys Asp Ser Trp Asn 160 Asp
S
S S
SS
S. *S S S
S
S
555555
S
S.
5 5
*S
*5 S S S *5 55..
S S S. S 5* S S 5*
S
*SS'S.
S
S.
S S S 555
Claims (79)
1. A method for reducing pathology of connective tissue in a mammalian host which comprises delivery of a DNA sequence within a mammalian host, said DNA sequence expressing a biologically active gene product such that said biologically active gene product imparts systemic relief from at least one symptom associated with pathology of connective tissues, said DNA sequence encoding a protein or protein fragment that antagonizes interleukin-1.
2. The method of claim 1 wherein the connective tissue pathology is caused by rheumatoid arthritis.
3. The method of claim 2 wherein said DNA sequence is delivered systemically within said mammalian host.
4. The method of claim 2 wherein said DNA sequence is delivered locally within said mammalian host. 0 0
5. The method of claim 3 wherein said DNA sequence is transfected into a hematopoietic cell-containing population.
6. The method of claim 5 wherein said hematopoietic cell-containing population comprises bone marrow cells.
7. The method of claim 5 wherein said hematopoietic cell-containing population comprises CD34 blood leukocytes.
8. The method of claim 2 wherein the DNA sequence is transduced into 30 peripheral blood cells.
9. The method of claim 8 wherein said peripheral blood cells are lymphocytes. *0 The method of claim 2 wherein said DNA sequence is subcloned into a viral vector selected from the group consisting of a retroviral vector, an adenovirus vector, an adeno-associated vector, a herpes simplex virus vector, an SV40 vector, a polyoma virus vector, a papilloma virus vector, a picornavirus vector, and a vaccinia virus vector. S36
11. The method of claim 10 wherein said DNA sequence is transduced into a hematopoietic cell-containing population.
12. The method of claim 11 wherein said hematopoietic cell-containing population comprises bone marrow cells.
13. The method of claim 11 wherein said hematopoietic cell-containing population comprises CD34+ blood leukocytes.
14. The method of claim 10 wherein the DNA sequence is transduced into peripheral blood cells. The method of claim 14 wherein said peripheral blood cells are lymphocytes.
16. The method of claim 10 wherein said viral vector is a retroviral vector.
17. The method of claim 16 wherein said retroviral vector is transduced into a hematopoietic cell-containing population. 2
18. The method of claim 17 wherein said hematopoietic cell-containing population comprises bone marrow cells.
19. The method of claim 17 wherein the hematopoietic cell-containing population comprises CD34 blood leukocytes.
20. The method of claim 16 wherein the DNA sequence is transduced into peripheral blood cells. 30 21. The method of claim 20 wherein said peripheral blood cells are lymphocytes.
22. The method of claim 16 wherein said retroviral vector is MFG-IRAP.
23. The method of claim 22 wherein MFG-IRAP is used to transduce a hematopoietic cell-containing population. C C 0 0O S C
24. The method of claim 23 wherein said hematopoietic cell-containing population comprises bone marrow cells. The method of claim 23 wherein said hematopoietic cell-containing population comprises CD34+ blood leukocytes.
26. The method of claim 22 wherein the DNA sequence is transduced into peripheral blood cells. 27 The method of claim 26 wherein said peripheral blood cells are lymphocytes.
28. The method of claim 4 wherein said DNA sequence is transfected into in vitro cultured myoblast cells and transplanted into said mammalian host.
29. The method of claim 28 wherein said DNA sequence is subcloned into a non-viral vector.
30. The method of claim 29 wherein said non-viral vector is a plasmid DNA vector.
31. The method of claim 28 wherein said DNA sequence is subcloned into a viral vector.
32. The method of claim 31 wherein said DNA sequence is subcloned into a retroviral vector.
33. The method of claim 32 wherein said retroviral vector is MFG-IRAP.
34. The method of claim 4 wherein said DNA sequence is injected directly into skeletal muscle of said mammalian host.
35. The method of claim 34 wherein said DNA sequence is subcloned into a non-viral vector.
36. The method of claim 35 wherein said non-viral vector is a plasmid DNA vector. S. *r C C.. SO OS 0 S. C 0 0 S. C OC
37. The method of claim 34 wherein said DNA sequence is subcloned into a viral vector.
38. The method of claim 37 wherein said DNA sequence is subcloned into a retroviral vector.
39. The method of claim 38 wherein said retroviral vector is MFG-IRAP. The method of claim 1 wherein the connective tissue pathology is caused by systemic lupus erythematosus.
41. The method of claim 40 wherein said DNA sequence is delivered systemically within said mammalian host.
42. The method of claim 40 wherein said DNA sequence is delivered locally within said mammalian host.
43. The method of claim 41 wherein said DNA sequence is transduced into a hematopoietic cell-containing population.
44. The method of claim 43 wherein said hematopoietic cell-containing population are bone marrow cells. The method of claim 43 wherein said hematopoietic cell-containing population comprise CD34 blood leukocytes.
46. The method of claim 41 wherein the DNA sequence is transduced into peripheral blood cells. 30 47. The method of claim 46 wherein said peripheral blood cells are lymphocytes.
48. The method of claim 41 wherein said DNA sequence is subcloned into a viral vector selected from the group consisting of a retroviral vector, an adenovirus vector, an adeno-associated vector, a herpes simplex virus vector, an SV40 vector, a polyoma virus vector, a papilloma virus vector, a picornavirus vector, and a vaccinia virus vector. 39
49. The method of claim 48 wherein said DNA sequence is transduced into a hematopoietic cell-containing population. The method of claim 49 wherein said hematopoietic cell-containing population comprises bone marrow cells.
51. The method of claim 49 wherein said hematopoietic cell-containing population comprises CD34+ blood leukocytes.
52. The method of claim 48 wherein the DNA sequence is transduced into peripheral blood cells.
53. The method of claim 52 wherein said peripheral blood cells are lymphocytes.
54. The method of claim 48 wherein said viral vector is a retroviral vector.
55. The method of claim 54 wherein said retroviral vector is transfected into a hematopoietic cell-containing population. S
56. The method of claim 55 wherein said hematopoietic cell-containing population comprises bone marrow cells.
57. The method of claim 55 wherein the hematopoietic cell-containing population comprises CD34+ blood leukocytes.
58. The method of claim 54 wherein the DNA sequence is transduced into peripheral blood cells. 30 59. The method of claim 58 wherein said peripheral blood cells are lymphocytes.
60. The method of claim 54 wherein said retroviral vector is MFG-IRAP.
61. The method of claim 60 wherein MFG-IRAP is used to transduce a hematopoietic cell-containing population.
62. The method of claim 61 wherein said hematopoietic cell-containing population comprises bone marrow cells.
63. The method of claim 61 wherein said hematopoietic cell-containing population comprises CD34 blood leukocytes.
64. The method of claim 60 wherein the DNA sequence is transfected into peripheral blood cells.
65. The method of claim 64 wherein said peripheral blood cells are lymphocytes.
66. The method of claim 42 wherein said DNA sequence is transfected into in vitro cultured myoblast cells and transplanted into said mammalian host.
67. The method of claim 66 wherein said DNA sequence is subcloned into a non-viral vector. S
68. The method of claim 67 wherein said non-viral vector is a plasmid DNA 20 vector. °69. The method of claim 66 wherein said DNA sequence is subcloned into a viral vector selected from the group consisting of a retroviral vector, an adenovirus vector, an adeno-associated vector, a herpes simplex virus vector, an SV40 vector, a polyoma virus vector, a papilloma virus vector, a picornavirus vector, and a vaccinia virus vector. .e 70. The method of claim 69 wherein said DNA sequence is subcloned into a retroviral vector.
71. The method of claim 70 wherein said retroviral vector is MFG-IRAP.
72. The method of claim 42 wherein said DNA sequence is injected directly into skeletal muscle of said mammalian host.
73. The method of claim 72 wherein said DNA sequence is subcloned into a non-viral vector. 41
74. The method of claim 73 wherein said non-viral vector is a plasmid DNA vector. The method of claim 72 wherein said DNA sequence is subcloned into a viral vector selected from the group consisting of a retroviral vector, an adenovirus vector, an adeno-associated vector, a herpes simplex virus vector, an SV40 vector, a polyoma virus vector, a papilloma virus vector, a picornavirus vector, and a vaccinia virus vector.
76. The method of claim 75 wherein said DNA sequence is subcloned into a retroviral vector.
77. The method of claim 76 wherein said retroviral vector is MFG-IRAP.
78. The method of claim 1 wherein the connective tissue pathology is caused by osteoporosis.
79. The method of claim 78 wherein said DNA sequence is delivered systemically within said mammalian host. S
80. The method of claim 79 wherein said DNA sequence is subcloned into a viral vector selected from the group consisting of a retroviral vector, an adenovirus vector, an adeno-associated vector, a herpes simplex virus vector, an SV40 vector, a polyoma virus vector, a papilloma virus vector, a picornavirus vector, and a vaccinia virus vector.
81. The method of claim 80 wherein said viral vector is a retroviral vector.
82. The method of claim 1 wherein the connective tissue pathology is caused by 30 a connective tissue disease or disorder selected from the group consisting of Sjorgen's syndrome, polymyositis-dermatomyositis, systemic sclerosis, vasculitis syndromes, juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, osteoporosis, osteogenesis imperfecta, Paget's disease and inflammatory bowel disease.
83. The method of claim 82 wherein said DNA sequence is subcloned into a viral vector selected from the group consisting of a retroviral vector, an adenovirus vector, an adeno-associated vector, a herpes simplex virus vector, an SV40 vector, a polyoma virus vector, a papilloma virus vector, a picornavirus vector, and a vaccinia virus vector.
84. The method of claim 83 wherein said viral vector is a retroviral vector. The method of claim 1, wherein said DNA sequence encodes a protein selected from the group of IRAP, sIL-1R, 11-4 and
86. A mammalian cell comprising a recombinant vector wherein said recombinant vector comprises a DNA sequence encoding IRAP or a biologically active fragment thereof. 15 87. A mammalian cell of claim 86 wherein said recombinant vector is retroviral and is derived from a Moloney murine leukemia virus.
88. A mammalian cell of claim 87 wherein said DNA sequence encoding IRAP or a biologically active fragment thereof consists essentially of SEQ ID NO:2. "o 89. A mammalian cell of claim 88 wherein said recombinant vector is retroviral and is MFG-IRAP. The mammalian cell of claim 86 which is a hematopoietic cell. S
91. The mammalian cell of claim 87 which is a hematopoietic cell.
92. The mammalian cell of claim 88 which is a hematopoietic cell.
93. The mammalian cell of claim 89 which is a hematopoietic cell.
94. The hematopoietic cell of claim 90 which is a bone marrow cell. The hematopoietic cell of claim 91 which is a bone marrow cell. 43
96. The hematopoietic cell of claim 92 which is a bone marrow cell.
97. The hematopoietic cell of claim 93 which is a bone marrow cell. Dated this 28th day of November 2000 UNIVERSITY OF PITTSBURGH OF THE COMMONWEALTH SYSTEM OF HIGHER EDUCATION By their Patent Attorneys, A. P. T. Patent and Trade Mark Attorneys 0 00 00 0
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU96132/98A AU729873B2 (en) | 1993-12-14 | 1998-12-07 | Systemic gene treatment of connective tissue diseases |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16764293A | 1993-12-14 | 1993-12-14 | |
US167642 | 1993-12-14 | ||
AU15134/95A AU1513495A (en) | 1993-12-14 | 1994-12-12 | Systemic gene treatment of connective tissue diseases |
AU96132/98A AU729873B2 (en) | 1993-12-14 | 1998-12-07 | Systemic gene treatment of connective tissue diseases |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU15134/95A Division AU1513495A (en) | 1993-12-14 | 1994-12-12 | Systemic gene treatment of connective tissue diseases |
Publications (2)
Publication Number | Publication Date |
---|---|
AU9613298A AU9613298A (en) | 1999-02-04 |
AU729873B2 true AU729873B2 (en) | 2001-02-15 |
Family
ID=25615846
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU96132/98A Ceased AU729873B2 (en) | 1993-12-14 | 1998-12-07 | Systemic gene treatment of connective tissue diseases |
Country Status (1)
Country | Link |
---|---|
AU (1) | AU729873B2 (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU651596B2 (en) * | 1990-06-05 | 1994-07-28 | Immunex Corporation | Type II interleukin-1 receptors |
-
1998
- 1998-12-07 AU AU96132/98A patent/AU729873B2/en not_active Ceased
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU651596B2 (en) * | 1990-06-05 | 1994-07-28 | Immunex Corporation | Type II interleukin-1 receptors |
Also Published As
Publication number | Publication date |
---|---|
AU9613298A (en) | 1999-02-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5766585A (en) | Systemic gene treatment of connective tissue diseases with IRAP-1 | |
EP0815245B1 (en) | Bioactive il-12 fusion proteins | |
RU2279441C2 (en) | Isolated soluble il-20 receptor (variants) | |
EP1763538B1 (en) | Genetically engineered cells for therapeutic applications | |
EP2520302B1 (en) | Stromal cell-derived factor-1 mediates stem cell homing and tissue regeneration in ischemic cardiomyopathy | |
Ghivizzani et al. | Direct retrovirus-mediated gene transfer to the synovium of the rabbit knee: implications for arthritis gene therapy | |
US20020098168A1 (en) | Gene transfer for studying and treating a connective tissue of a mammalian host | |
JP2003526325A (en) | Interleukin 17-like receptor protein | |
AU2821699A (en) | Therapeutic chemokine receptor antagonists | |
CA2690399C (en) | Il-6 antagonist peptides | |
IE61563B1 (en) | Interleukin-1 receptors | |
Gouze et al. | A comparative study of the inhibitory effects of interleukin-1 receptor antagonist following administration as a recombinant protein or by gene transfer | |
Evans et al. | Progress toward the treatment of arthritis by gene therapy | |
JPH0678772A (en) | Alpha chain or portion of human interleukin-5 acceptor, particularly dna for coding shil5r alpha, vector containing said dna, host cell transformed by said vector and method for production and use thereof | |
AU729873B2 (en) | Systemic gene treatment of connective tissue diseases | |
US20040161416A1 (en) | Systemic gene treatment of connective tissue diseases | |
WO1997000318A1 (en) | Osteoclast growth regulatory factor | |
US7374911B2 (en) | Interleukin-1 related gene and protein | |
EP1252187A2 (en) | Fil-1 theta dnas and polypeptides | |
Thornton et al. | Gene therapy for arthritis | |
US20030104518A1 (en) | FIL-1 theta DNAs and polypeptides | |
Gene | GENE THERAPY OR BONE, CONNECTIVE TISSUE AND SKIN TISSUES | |
AU2008200822A1 (en) | Stromal cell-derived factor-1 mediates stem cell homing and tissue regeneration in ischemic cardiomyopathy |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FGA | Letters patent sealed or granted (standard patent) | ||
MK14 | Patent ceased section 143(a) (annual fees not paid) or expired |