Compositions for gene therapy of rheumatoid arthritis including a gene encoding an anti-angiogenic protein or parts thereof

Abstract

The present invention relates to the compositions for a gene therapy of rheumatoid arthritis including a gene encoding an anti-angiogenic protein or parts thereof. More specifically, the present invention provides a gene therapy of rheumatoid arthritis by preparing a recombinant vector that expresses a gene encoding an anti-angiogenic protein such as endostatin or parts thereof, and transplanting the recombinant vector or a cell that is transfected or transduced with the recombinant vector into the affected area of a patient, and also provides the compositions for the gene therapy. The compositions for the gene therapy according to the present invention can be used effectively for the treatment of rheumatoid arthritis, for which effective treating methods have not been developed until now, by providing the anti-angiogenic proteins into the knee of a patient continuously to prevent the synovial tissue hyperplasia and the resulting inflammation.

Description

TECHNICAL FIELD

[0001] The present invention relates in general to compositions for gene therapy of rheumatoid arthritis including a gene encoding an anti-angiogenic protein or parts thereof, and more particularly to a gene therapy for treating rheumatoid arthritis by constructing a recombinant vector including a gene encoding an anti-angiogenic protein or parts thereof and transplanting the recombinant vector or the cells transfected or transduced with the recombinant vector into the affected area of a patient, and compositions for the gene therapy.

BACKGROUND ART

[0002] Rheumatoid arthritis is a chronic inflammatory disease involving multiple joints. The main pathology of the affected synovial tissue consists of the hyperplasia and the subintimal infiltration of T and B lymphocytes. Such inflammation of the synovial tissue is thought to be caused by T lymphocyte reactive to an unknown autoantigen. Nonetheless, the T lymphocyte infiltrated in the almost tissues does not show any indication of activation on the surface of cell and also does not almost express cytokines. In contrast to this, it is observed that both synovial tissue and fluid are enriched with the cytokines derived from macrophage. These cytokines may include interleukin-1 (IL-1) which can accelerate the growth of synovial fibroblast and tumor necrosis factors (TNFs). These experimental results suggest the hypothesis that T lymphocyte is importantly associated with the induction of inflammation to synovial tissues and the inflammation is maintained by the cytokines derived from the activated synovial cells.

[0003] One of the major intents of rheumatoid arthritis treatment is to prevent the synovial tissue hyperplasia, because it forms the pannus tissue that irreversibly destroys the cartilage and bone in the affected joint. Effective drugs for treating rheumatoid arthritis have not been developed until the present time and the developed drugs can exhibit limited efficacies. Once arthritis occurs, it causes economic loss as well as severe pain.

[0004] Medical treatments of rheumatoid arthritis being used presently are as follows. The drugs used often for initial treatment are non-steroidal anti-inflammatory drugs (NSAIDs). These NSAIDs limitedly improve a patient's condition, but cannot prevent the cartilage destruction of joint area or the progress of disease. Moreover, half the patients using this treatment should stop the treatment within one year because of serious side effect. Next, gold drugs such as gold sodium thiomalate and gold sodium thiosulfate, or disease modifying anti-rheumatic drugs (DMARDs) such as penicillamine and anti-malarials are used. These drugs also decrease the progress of rheumatoid arthritis, but after 5 years of the treatment using DMARDs, only 5-15% of the patients adhere to use the drugs because the serious side effect can be accompanied. If the drugs mentioned above are not effective any more, the affected joint area with rheumatoid arthritis should be replaced by artificial joint by surgical operation.

[0005] In this manner, most of the treatments of rheumatoid arthritis used until now were not designed with a particular target molecule and had a limitation of showing slight effects in most cases. In the meantime, it has been reported that therapeutic effects appeared by taking notice of inflammation-inducing cytokines such as TNF as a target molecule for treating the rheumatoid arthritis and introducing an antibody specific to TNF or a soluble TNF-receptor into the affected area of a patient to result in inactivation of the TNF (Maini, R. N. et al., Immunol. Rev. 144:195, 1995; Moreland, L. W. et al., N. Engl. J. Med. 337:141, 1997). In company with this, various gene transfer experiments in vivo are progressing in an animal model of rheumatoid arthritis with continuously expressing the molecules having an immune inhibitory function. Consequently, most of the current treatments are directed to correction of the immune aberration that supposedly drives the synovial cell proliferation.

[0006] Angiogenesis, the formation of new blood vessels, is one of the earliest histopathologic findings in rheumatoid arthritis and appears to be required for pannus development. This neovascularization is thought not only to maintain the chronic architectural changes via delivery of required blood-borne elements to the pannus, but also to play an active role in inflammation as a source of both cytokine and protease activity. The expanded vascular-bed volume resulting from angiogenesis may provide increased access for inflammatory cells to infiltrate the synovium. Although the factors specifically promoting angiogenesis in rheumatoid arthritis have not yet been identified, both synovial tissue and fluid are enriched with angiogenesis-promoting molecules. These include cytokines, such as basic fibroblast growth factor (bFGF), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and soluble adhesion molecules, such as E-selectin. These data suggested a therapeutic potential for using an anti-angiogenic procedure for favorably changing the disease course of rheumatoid arthritis.

[0007] Until now a lot of factors that repress angiogenesis have been found. Most of them are created from the cleavage of protoprotein, and representatively angiostatin, endostatin and platelet factor-4 and the like have been known.

[0008] Angiostatin is composed of 98.sup.th to 440.sup.th amino acids of plasminogen. Angiostatin was initially isolated from mice bearing a Lewis lung carcinoma and was identified as a 38-kDa internal fragment of plasminogen that encompasses the first four kringles of the molecule (O'Relly, M. et al., Cell 79:715, 1994). It was reported that the growth of primary tumors was inhibited effectively by injecting purified angiostatin hypodermically in six cancer model experiments (O'Relly, M. et al., Nat. Med. 2:689, 1996).

[0009] Endostatin consists of C-terminal 183 amino acids of collagen XVIII and has an anti-angiogenic activity. It was reported that the growth of primary tumors was inhibited effectively by injecting purified endostatin hypodermically in four cancer model experiments (O'Relly, M. et al., Cell 88:277, 1997).

[0010] Platelet factor-4 belongs to CXC cytokine family, which consists of chemotactic polypeptides below 10 kDa, and has an anti-angiogenic activity. It was reported that the platelet factor-4 inhibited the growth of cancer, such as B-16 melanoma and HCT-116 colon carcinoma (Maion, T. E. et al., Cancer Research, 51:2077, 1991).

[0011] Angiogenesis is known to be associated with various diseases, such as tumor formation and metastasis, retinitis, angioma, chronic inflammation, intestinal adhesions, atherosclerosis, rheumatoid arthritis and so on, but it has not yet been verified that anti-angiogenic factors were effective to all the diseases listed above actually. Only the tumor inhibitory effects of these factors associated with a particular disease have been reported (U.S. Pat. No. 5,856,315, U.S. Pat. No. 5,733,876, U.S. Pat. No. 5,792,845, U.S. Pat. No. 5,854,205, U.S. Pat. No. 6,024,688). The US patents disclose that the treatment effects for various kinds of diseases, such as ovarian carcinoma (HTB161, A2780S), colon carcinoma (MIP, CACO2), Lewis Lung Carcinoma (LLC), fibrosarcoma (T241), prostate gland carcinoma (PC-3) and breast carcinoma (MDA-MB), were identified by injecting anti-angiogenic factors with the type of recombinant protein.

[0012] Meanwhile, unlike general treating method that applies a toxicity to cells directly, the treating method, which cures diseases by inhibiting angiogenesis by means of supplying anti-angiogenic factors as described above, is based on the principle of inhibiting the cell growth, so anti-angiogenic factors over certain concentration should be supplied continuously to exhibit effects in vivo. But, the method of supplying anti-angiogenic factors with the type of recombinant proteins costs too much for administering proteins continuously, is troublesome and has a problem in that it imparts toxicity to a patient. Therefore, it has been required to develop a method of supplying anti-angiogenic factors to the affected area continuously and locally.

[0013] Accordingly, the present inventors have attempted new approaches for the probability of the treatment of rheumatoid arthritis by anti-angiogenesis in order to replace the prior treatment of rheumatoid arthritis having focused on solving the immunological problems. More particularly, the present inventors obtained the cell lines for producing representative anti-angiogenic factors such as angiostatin, endostatin and platelet factor-4 through inserting their genes into a viral vector and then transplanting them the affected area of mice induced with rheumatoid arthritis. We also performed the histological examinations for the level of hyperplasia in synovial cell and cartilage destruction and the immunological examinations for the concentrations of cytokines associated with the joint inflammation, as well as macroscopic examination for joint swelling to obtain the results for the progressive level of rheumatoid arthritis. The results showed that the incidence of rheumatoid arthritis in our treatment was remarkably reduced in comparison with the control group. This is to show that our gene therapy using an anti-angiogenic gene is effective to treatment of rheumatoid arthritis.

DISCLOSURE OF THE INVENTION

[0014] It is therefore an object of the present invention to provide compositions for gene therapy of rheumatoid arthritis including a DNA encoding an anti-angiogenic protein or parts thereof. More particularly, the present invention provides a recombinant vector including a DNA encoding an anti-angiogenic protein or parts thereof, a cell into which the recombinant vector is introduced and compositions for the gene therapy of rheumatoid arthritis including the recombinant vector or the recombinant cell as an active ingredient.

[0015] To accomplish this object, the present invention provides compositions for gene therapy including a DNA encoding an anti-angiogenic protein or parts thereof, which shows therapeutic effects on rheumatoid arthritis.

[0016] According to the compositions of the present invention, the DNA encoding an anti-angiogenic protein or parts thereof can be provided with an inserted form in retroviral vector, adenoviral vector, adeno-associated viral vector, herpes simplex viral vector or plasmid that can be expressed in an animal cell, or with the form of a recombinant cell that is collected by transfecting or transducing a cell with the recombinant vector including the DNA in order to supply anti-angiogenic factor to the affected area continuously and locally.

[0017] Furthermore, the present invention provides a gene therapy of treating rheumatoid arthritis by delivering the compositions to the affected area of a patient.

[0018] As used herein, the term "anti-angiogenic gene" means a DNA encoding an anti-angiogenic protein or parts thereof. It is not limited to a natural DNA, but may include any forms of proper modifications under maintenance of anti-angiogenic activity and additions of elements for expression regulation if it can be used suitably for the purpose of the present invention, regardless of whether or not it is obtained by genetic engineering method or chemical method.

[0019] Hereinafter, the present invention will be described in detail.

[0020] The present invention is characterized in that the composition for gene therapy of treating arthritis comprises a DNA encoding anti-angiogenic protein or parts thereof as an active ingredient. The anti-angiogenic gene used in the gene therapy for treating arthritis according to the present invention is preferably a gene encoding at least one protein selected from the group consisting of angiostatin, endostatin, platelet factor-4, thrombospondin-1, thrombospondin-2, METH-1, METH-2 (anti-angiogenic proteins having metalloprotease domain and thrombospondin domain; Vanzquez F. et al., J. Biol. Chem. 274(33):23349-57, 1999) and hepatocyte growth factor.

[0021] Particularly, the anti-angiogenic gene included in the composition for gene therapy of treating arthritis according to the invention is preferably the gene encoding the angiostatin including 98.sup.th to 440.sup.th amino acids of human plasminogen and the four kringles, the gene encoding the endostatin including 1334.sup.th to 1516.sup.th amino acids of human collagen XVIII, or the gene encoding the mouse platelet factor-4 protein.

[0022] Although an entire amino acid sequence may be used as the anti-angiogenic factor, a fragment known as having anti-angiogenic activity may also be used. For example, angiostatin kringle 1-3 fragment may be used for this purpose.

[0023] The example of the anti-angiogenic gene included in the composition for gene therapy of treating arthritis according to the invention may preferably be the DNA derived from human, mouse, rhesus, pig or bovine plasminogen, and more preferably the cDNA of human angiostatin. The endostatin gene may also preferably be the DNA derived from human, mouse, rhesus, pig or bovine collagen XVIII, and more preferably the cDNA of human endostatin. The preferable embodiments of the invention identified that the angiostatin and endostatin genes cloned from human foreskin fibroblast (HFF) are effective to gene therapy for treating arthritis.

[0024] The recombinant vector included in the composition for gene therapy for treating arthritis according to the invention also comprises the anti-angiogenic gene. It is preferable that the recombinant vector further comprises a nucleotide sequence encoding a signal peptide required for secretion in the upstream or downstream in order to secrete the protein expressed by the gene out of the cell.

[0025] The signal peptide may include any signal peptides known as associated to protein secretion in eukaryotic cell, and may preferably be 18 of amino-terminal amino acids of human plasminogen represented as the SEQ ID No. 2 or the signal peptide of mouse immunoglobulin .kappa. chain encoded by the nucleotide sequence represented as SEQ ID No. 13.

[0026] For example, as described below in one preferable example according to the invention, the signal sequence can be functionally connected to the anti-angiogenic gene by synthesizing a nucleotide including the nucleotide sequence encoding 18 of the amino-terminal amino acids of human plasminogen and the sequences having restrictive enzyme cleaving sites, hybridizing it into double helical DNA, and treating it with restrictive enzyme to insert it into a certain site of target vector. However, the construction of vector having a signal sequence for producing the secretion type of anti-angiogenic protein is not limited to as mentioned above, but may be accomplished by the method well known in the field of this art.

[0027] The vector used in the composition for gene therapy of treating arthritis according to the invention is a vector in which the anti-angiogenic gene can be inserted to supply the anti-angiogenic factor continuously to the affected area. The vector may include virus-derived vectors such as retroviral vector, adenoviral vector, adeno-associated viral vector, and herpes simplex viral vector, plasmids capable of being expressed in bodies of animals such as pCXN2 (Gene, 108:193-200, 1991) and PAGE207 (Japan Patent Laid-Open No. Sho6-4684), and their modified vectors.

[0028] In the preferable example according to the present invention, a viral vector was prepared, which was to produce angiostatin, endostatin or platelet factor-4 as a anti-angiogenic factor, using MT5 retroviral vector which had been filed before Korean Industrial Property Office (KIPO) by the present inventors (Korean Patent Appl. No. 9748095; KCCM-10205), and the effect for treating arthritis was verified by experimental results as to mouse using the viral vector. The MT5 retroviral vector is a vector based on murine leukemia virus (MLV) including mutant noncoding sequence of human elongation factor 1 (EF 1), without coding sequence derived from virus and is good in both external gene expressing efficiency and viral titer. That is to say, the vector is to enhance stability by completely removing the gag, pol and env sequence of MLV, is to include at the upstream of external gene insertion site, a part of noncoding sequence of EF 1a as a noncoding sequence derived from heterogeneous gene for providing splicing receptor, and is to control splicing efficiency appropriately to maintain gene expressing highly with also maintaining virus producing concentration highly.

[0029] Particularly, in one example of the present invention, angiostatin DNA fragment encoding 93.sup.rd to 368.sup.th amino acids of human plasminogen represented as SEQ ID No. 2 was obtained from human foreskin fibroblast (HFF) through PCR and was inserted into pGEM T easy vector to construct pGEM T easy-hASTi vector. The sequence encoding 18 of amino-terminal amino acids of plasminogen of SEQ ID No. 2 was synthesized as a signal sequence, and was inserted into pGEM T easy-hASTi vector to construct pGEM T easy-hAST vector. The obtained vector was cleaved with BamHI to prepare human angiostatin gene fragment connected with the signal sequence, which was inserted into MT5 vector. In the meantime, to prepare cell lines expressing human angiostatin, MT5-hAST vector DNA was transfected into 293T cell with the plasmid expressing gag-pol and env gene of murine leukemia virus and then non-cellular virus was obtained from the cell culture media.

[0030] The obtained virus was transduced into NIH3T3, and then the cell lines introduced with retrovirus were selected and cultivated to prepare NIH3T3 cells expressing human angiostatin protein. By means of the same method, NIH3T3 cells expressing human endostatin protein and mouse platelet factor-4 protein were prepared respectively.

[0031] Another example of the present invention verified the effect of gene therapy for arthritis using the cell lines respectively expressing anti-angiogenic proteins such as human angiostatin protein, human endostatin protein and mouse platelet factor-4 protein.

[0032] Particularly, a gene therapy to mice without any macroscopic signs of inflammation in collagen-induced arthritis mouse model was performed. Namely, anti-angiogenic protein-producing NIH3T3 cell lines mixed with PBS were transplanted in the knee joint area of rear leg of the arthritis-mouse, and then the progressive level of arthritis was investigated by measuring joint swelling, synovial hyperplasia, destruction of cartilage, and joint inflammation-associated cytokine level.

[0033] The results showed that the swelling level in the knee joint area in case of transplanting angiostatin-producing NIH3T3 cell lines was remarkably decreased to 27% in comparison that the control group in case of injecting only PBS or transplanting the cell lines transferred with only MT5 vector was 47% or 67%. The frequency showing the significant level of IL-1 was also remarkably decreased. The synovial hyperplasia and cartilage destruction in the knee joint area were also remarkably decreased. Furthermore, similar results to the transplant of antiostatin producing NIH3T3 cell lines were obtained when endostatin producing NIH3T3 cell lines and platelet factor-4 producing NIH3T3 cell lines were transplanted. These results show that the gene therapy using anti-angiogenic gene according to the present invention is effective to suppression and treatment of arthritis.

[0034] In another example of the present invention, duration of therapeutic effects was measured. The result showed that therapeutic effect of a single injection lasts for 14 days after treatment.

[0035] The composition of gene therapy for treating arthritis according to the present invention can be prepared by preparing viral particles including recombinant DNA encoding anti-angiogenic protein or cell lines transduced with the viral particles and mixing them with carriers used in gene therapy (Crystal R G et al., Nature Genet. 8:42-51, 1994).

[0036] The carriers used in gene therapy may include any carriers generally used in injection liquid. For example, the carriers may include distilled water, sodium chloride solutions, the mixtures of sodium chloride and inorganic salts or their similar mixtures, the solutions of materials such as mannitol, lactose, dextran, and glucose, amino acid solutions such as glycine and arginine, the mixtures of organic acid solutions or salt solutions and glucose solutions, and their similar solutions. The injection liquid may be prepared in the form of solution, suspension or colloidal solution by adding osmotic modulator, pH controller, vegetable oil such as sesame oil or bean oil, lecithin or surfactant such as non-ionic surfactant to the carriers in the conventional manners. This injection may be prepared in the form of powder or lyophilization and then dissolved in the form of solution before being used.

[0037] The composition of gene therapy for treating arthritis according to the present invention may be dissolved in a sterilized carrier, in case of solid phase, if needed, before treatment of gene therapy, or may be used as it is without further treatment in case of liquid phase.

[0038]

Claims

1-14. (canceled)

15. A method of delaying the progression of one or more rheumatoid arthritis-associated symptoms in a patient having rheumatoid arthritis, wherein said symptoms are selected from the group consisting of: (i) joint swelling, (ii) synovial hyperplasia, (iii) cartilage destruction, and (iv) joint inflammation-associated cytokine level; said method comprising directly injecting into one or more sites of rheumatoid arthritis or sites adjacent thereto within the same limb of the patient a sufficient amount of a composition comprising: (a) a host cell comprising a nucleic acid encoding endostatin or an anti-angiogenic fragment thereof, wherein said nucleic acid is operably linked to a signal sequence encoding a secretion peptide; and (b) a carrier; wherein said host cell is transformed by a retroviral vector comprising said nucleic acid operably linked to said signal sequence, and wherein said host cell is histocompatible with said patient.

16. The method of claim 15, wherein said endostatin is derived from human collagen XVIII, mouse collagen XVIII, rhesus collagen XVIII, pig collagen XVIII, or bovine collagen XVIII.

17. The method of claim 16, wherein said endostatin is derived from human collagen XVIII.

18. The method of claim 15, wherein said retroviral vector is derived from the MT5 plasmid (KCCM Deposit No. 10205).

19. The method of claim 15, wherein said nucleic acid comprises the nucleotide sequence represented by SEQ ID NO:8.

20. The method of claim 15, wherein said composition is injected into a site within the same limb as and adjacent to a site of rheumatoid arthritis.

21. The method of claim 20, wherein said composition is injected into a knee of said patient.

22. The method of claim 21, wherein the progression of joint swelling in an ankle of said patient is delayed.