CN101972275A - Gene treatment using TGF-beta secretion cell - Google Patents

Abstract

The subject invention is related to a cell-mediated gene therapy treatment for orthopedic disease using a member belonging to the transforming growth factor-beta (TGF-beta) superfamily. TGF- beta gene therapy as a new treatment method for degenerative arthritis is demonstrated. After transfection of TGF- beta cDNA expression vectors into fibroblasts (NIH 3T3-TGF- beta 1), the cells were injected into rabbit achilles tendon and knee joints with artificially-made cartilage defects. Intratendinous injections were performed to determine the optimal concentration for in vivo expression. Partially defected cartilage model was made to simulate degenerative arthritis of the knee joint. The partial cartilage defect treated with the cell-mediated gene therapy procedure was covered by newly formed hyaline cartilage which indicates that the cells survived and stimulated matrix formation in this area. Completely denuded cartilage areas were covered by fibrous collagen.

Description

Use the gene therapy of TGF-β

The application is the CN 00807074.1 that submitted on May 3rd, 2000, is entitled as dividing an application of " using the gene therapy of TGF-β ".

Background technology

1. invention field

The present invention relates to coding transforming growth factor member's at least a gene is introduced at least a mammal connective tissue, be used for the regenerated method of mammalian hosts connective tissue.The invention still further relates to a kind of connective tissue cell is, it carries a kind of dna vector molecule, and this carrier contains coding transforming growth factor member's gene.

2. the summary of association area

In art of orthopedic surgery, degenerative arthritis or osteoarthritis are modal diseases with the cartilage injury.Almost each joint of body all can be suffered from as knee joint, hip, shoulder or even wrist.The pathogeny of this disease is the degeneration (Mankin etc., J.Bone Joint Surg, 52A:460-466,1982) of HAC.The hyaline cartilage distortion in joint, formation fibril, final depression.If the cartilage of degeneration can be regenerated in some way, most patient can enjoy life again, and not make us weak pain.Do not report the method for the damage hyaline cartilage of regenerating so far as yet.

The classical pathway that medicine is sent (as oral, intravenous or intramuscular administration) is invalid for medicine being carried to the joint.The half-life of intra-articular injection medicine is shorter usually.Another shortcoming of intra-articular injection medicine is to need frequent duplicate injection, to obtain the acceptable drug level at articular cavity, is used for the treatment of chronic disease such as arthritis.Because medicine so far is the targeting joint optionally, must make the medicine of mammalian hosts contact general high concentration, realize continuing, the intraarticular therapeutic dose.But not the medicine of target organ contact high concentration has increased the weight of the tendency of anti-arthritic deposits yields serious side effects (as the variation of the gastrointestinal upset of mammalian hosts and hematology, cardiovascular, Liver and kidney system).

In art of orthopedic surgery, considered the drug candidate of some cytokines as treatment orthopedics disease.Think that bone morphogenetic protein is osteoplastic effective stimulus agent (Ozkaynak etc., EMBO J, 9:2085-2093,1990; Sampath and Rueger, Complication in Ortho, 101-107,1994), and reported that TGF-β can be used as osteogenesis and chondrogenetic stimulant (Joyce etc., J Cell Biology, 110:2195-2207,1990).

Think that transforming growth factor-beta (TGF-β) is a kind of multifunctional cytokine (Sporn and Roberts, Nature (London), 332:217-219,1988), and in cell growth, differentiation and extracellular matrix protein are synthetic, play regulating action (Madri etc., J Cell Biology, 106:1375-1384,1988).TGF-β is at growth (Chenu etc., Proc.Natl.Acad.Sci, the 85:5683-5687 of vitro inhibition epidermis cell and skeletonization like cell, 1988), but it stimulates endochondral ossification and final bone (Critchlow etc., the Bone of forming in vivo, 521-527,1995; Lind etc., A Orthop Scand 64 (5): 553-556,1993; With Matsumoto etc., In vivo, 8:215-220,1994).The beta induced bone formation of TGF-is to mediate by its stimulation to the subperiosteum pluripotent cell, the final differentiating cartilage-forming cell of these cells (Joyce etc., J Cell Biology, 110:2195-2207,1990; With Miettinen etc., J Cell Biology, 127-6:2021-2036,1994).

Biological action (Andrew etc., Calcif Tissue In.52:74-78,1993 of TGF-β in orthopedics have been reported; Borque etc., Int J Dev Biol, 37:573-579,1993; Carrington etc., J Cell Biology, 107:1969-1975,1988; Lind etc., A Orthop Scand.64 (5): 553-556,1993; Matsumoto etc., In vivo 8:215-220,1994).In mice embryonic, dyeing shows TGF-β and is closely related derived from mesochymal tissue (as connective tissue, cartilage and bone).Except embry discovery, TGF-β also is present in bone formation and cartilage forms the position.It can also strengthen the healing of rabbit fracture of tibia.Recently, therapeutic value (Critchlow etc., Bone, 521-527,1995 of TGF-β have been reported; With Lind etc., A OrthopScand 64 (5): 553-556,1993), but its shortterm effect and expensively limited clinical practice widely.

It is undesirable that TGF-β intra-articular injection is used for the treatment of arthritis, because the TGF-β acting duration of injection is short, because the TGF degradation in vivo becomes inactive form.Therefore, need the new method of a kind of long-term release TGF-β, be used for hyaline cartilage regeneration.

Reported autograft chondrocyte regeneration articular cartilage (Brittberg etc., New Engl J Med 331:889-895,1994), but this method comprises the operation of twice wide excision soft tissue.If intra-articular injection is enough to treat degenerative arthritis, this will have great benefit economically with on the physiology to patient.

Gene therapy is a kind of specific protein to be transferred to the method for specific part, and it can address this problem, and (Jon A.Wolff compiles, 3-25,1994 for Wolff and Lederberg, Gene Therapeutics; And Jerk, JNatl Cancer Inst, 89 (16): 1182-1184,1997).

United States Patent (USP) 5,858,355 and 5,766,585 disclose virus or the plasmid construction thing of making a kind of IRAP (interleukin 1 receptor antagonist protein) gene; With this construction transfection synovial cell (5,858,355) and medullary cell (5,766,585); With transfectional cell is expelled in the rabbit joint, but unexposed usefulness belongs to the gene regeneration connective tissue of TGF-beta superfamily.

U.S. Patent number 5,846,931 and 5,700,774 disclose the compositions that injection contains the parathyroid hormone-related peptide of bone morphogenetic protein (BMP) (belonging to TGF β " superfamily ") and truncate, and what influence cartilaginous tissue formation keeps and induce cartilaginous tissue.Yet unexposed use BMP gene is made gene therapy methods.

Although the disclosed content of these prior aries, for at least a cell of at least a gene of coded product being introduced the mammalian hosts connective tissue in external or body, the method that is used for the treatment of mammalian hosts still has very reality and basic needs.In addition, also need a kind of method, promptly adopt a kind of gene of coding transforming growth factor member connective tissue of in mammalian hosts, regenerating.More specifically say, need a kind of method, express the gene of coded protein TGF-beta superfamily in vivo in host's connective tissue cell.

The invention summary

The present invention has satisfied the above-mentioned needs of this paper.The invention provides a kind of gene and introduce at least a cell of mammal connective tissue, be used for the treatment of the method for mammalian hosts at least a coded product.This method comprises the dna vector molecule that produces the gene contain this product of encode with recombinant technique, and will contain in the dna vector molecule introducing connective tissue cell of gene of this product of encoding.This dna vector molecule can be any dna molecular that can be transmitted and keep in target cell or tissue, thereby the gene of this product interested of encoding can stably express.Being preferred for dna vector molecule of the present invention is virus or plasmid DNA carrier molecule.This method preferably includes the cell of the gene of this product of coding being introduced the mammal connective tissue, is used for the treatment of.

The present invention relates to a kind of method of treatment of arthritis, comprising:

A) produce a kind of recombinant virus or plasmid vector, this carrier comprises the DNA sequence of coded protein transforming growth factor superfamily member, and this sequence is connected with a promoter navigability;

B), obtain the connective tissue cell of a group transfection with described recombinant vector in-vitro transfection population of cultured connective tissue cells group; With

C) connective tissue cell of transfection is transplanted in the articular cavity of mammalian hosts by intra-articular injection, thereby the expression of this DNA sequence in articular cavity causes connective tissue regeneration.

This recombinant vector can be but be not limited to retroviral vector, preferred retroviral vector, and carrier can also be a plasmid vector.

The connective tissue cell of storage a group transfection before method of the present invention is included in and transplants.Cell can be stored among the 10%DMSO under the liquid nitrogen before transplanting.

Connective tissue cell includes but not limited to fibroblast, mesenchymal cell, osteoblast or chondrocyte.Fibroblast can be NIH 3T3 cell or human foreskin fibroblast.

Connective tissue includes but not limited to cartilage, ligament or tendon.Cartilage can be a hyaline cartilage.

Method of the present invention is used the member of transforming growth factor superfamily, comprises transforming growth factor (TGF-β).The member of transforming growth factor superfamily can be TGF-β 1, TGF-β 2, TGF-β 3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 or BMP-7.Preferably, TGF-β is TGF-β 1, TGF-β 2 or the TGF-β 3 of people or pig.

The invention still further relates to the regenerated method of a kind of hyaline cartilage, comprising:

A) produce a kind of recombinant virus or plasmid vector, comprise the DNA sequence of coded protein transforming growth factor superfamily member in this carrier, this sequence is connected with the promoter navigability;

B), obtain the connective tissue cell of a group transfection with this recombinant vector in-vitro transfection a group population of cultured connective tissue cells; With

C) by intra-articular injection the connective tissue cell of transfection is transplanted in the articular cavity of mammalian hosts, thereby the expression of this DNA sequence in articular cavity causes hyaline cartilage regeneration.

The method of transfection can be undertaken by methods such as liposome, coprecipitation of calcium phosphate, electroporation and the mediations of DEAE-glucosan.

Method of the present invention comprises uses preferred plasmid pmT β 1.

The invention still further relates to a kind of connective tissue cell is, it contains a kind of recombinant virus or plasmid vector, and this carrier contains the DNA sequence of coding transforming growth factor superfamily member.This connective tissue cell system can include but not limited to, fibroblast, mesenchymal cell system, chondrocyte system, osteoblast system or osteocyte system.Fibroblast can be human foreskin fibroblast system or NIH 3T3cells.

Connective tissue cell of the present invention is the member who comprises the transforming growth factor superfamily.Preferably, the member of transforming growth factor superfamily is TGF-β 1, TGF-β 2, TGF-β 3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 or BMP-7.Preferred, this member is TGF-β 1, TGF-β 2 or the TGF-β 3 of people or pig.

Connective tissue cell of the present invention is to contain the cell that carries recombinant vector pmT β 1.

These and other purposes of the present invention will obtain more fully to understand description, accompanying drawing and the claim below the present invention.

The accompanying drawing summary

The expression of Fig. 1-TGF-β 1mRNA.Obtain total RNA from NIH 3T3 cell or with the NIH 3T3 cell separation of pmT β 1 (a kind of TGF-β 1 expression vector) stable transfection.These cells exist or do not exist under the situation at zinc grows.Survey total RNA (15 milligrams) in contrast with TGF-β 1cDNA or β actin cDNA.

The gross examination of skeletal muscle of Fig. 2 A-2B-regeneration of cartilage

2A. on the femur ankle, make the rectangle part cartilage defect, use NIH 3T3 injection cell knee joint without TGF-β 1 transfection.This damaged failing is capped.

2B. the tissue that the damaged quilt in injection NIH 3T3-TGF-β 1 cell 6 week back newly forms covers.Almost with on every side cartilage is identical for the color of regenerating tissues.

The microscopic examination of Fig. 3 A-3D-regeneration of cartilage (X 200)

3A and 3B. inject the hematoxylin-eosin (H﹠amp of the damaged part in 4 and 6 week backs with control cells; E) analyze.Inorganization covers initial defect area.

Hematoxylin-eosin (the H﹠amp of 3C and 3D. injection TGF-β 1- transfectional cell 4 and 6 all backs defect area; E) analyze.When 4 weeks, after injection TGF-β 1 cells transfected, the segmental defect zone is covered by hyaline cartilage.After 4 weeks of injection and 6 weeks, the regenerating tissues thickening, it is highly almost identical with normal cartilage during 6 weeks.On the histology, regenerated cartilage (arrow) is identical with on every side hyaline cartilage.

In Fig. 4 A-4B-rabbit joint/ immunohistochemical analysis (x200) that TGF-β 1 expresses.

Brown (Brown) immunoperoxidase product shows has high-caliber reorganization TGF-β 1 to express (4B) in NIH 3T3TGF-β 1 cell.

4A has shown the hyaline cartilage in the rabbit joint of injecting control cells.

Fig. 5 A-5B-H﹠amp; The microscopic examination (X200) of the regenerating tissues of E dyeing (A) and safranin-O dyeing (B).

5A. at part damage field, H﹠amp; E dyeing (black arrow) has shown regenerated hyaline cartilage.

5B. in the zone of peeling off cartilage completely, regenerating tissues (white arrow) is fibrous collagen.

The plasmid figure of Fig. 6-pmT β 1.

The gross morphology of the rabbit heel string of Fig. 7 A-7D-injection TGF-β 1 transfectional cell.

7A. the tendon of injection control cells.

7B. the tendon of injection TGF-β 1 transfectional cell, injection 6 weeks of back.

7C.7A in the cross-sectional view of tendon.

7D.7B in the cross-sectional view of tendon.

Fig. 8 A-8F-H﹠amp; The microscopic examination of the regenerating tissues in the E dyeing rabbit heel string.

8A, 8B and 8C have shown the tendon in injection 6 weeks of control cells.8A. amplify 50 times.8B. amplify 200 times.8C. amplify 600 times.

8D, 8E and 8F have shown the tendon in 6 weeks behind injection TGF-β 1 transfectional cell.8D. amplify 50 times.8E. amplify 200 times.8F. amplify 600 times.TGF-β 1 transfectional cell that is injected into this tendon be it seems round than endogenous tendon cell.By the autocrine and the fibrous collagen of paracrine mode producing of effect, tendon increases.Tendon increases behind injection TGF-β 1 transfectional cell.

Fig. 9 A-9B-is to using H﹠amp; E dyeing (A) and with the microscopic examination of the regenerating tissues in the rabbit heel string of TGF-beta 1 antibodies immunohistochemical staining (B).Brown immunoperoxidase product shows that high-caliber reorganization TGF-β 1 expresses in NIH3T3-TGF-β 1 cell.

Detailed Description Of The Invention

Term used herein " patient " refers to the member of animal kingdom, includes but not limited to the mankind.

Term used herein " mammalian hosts " comprises the member of animal kingdom, includes but not limited to the mankind.

Term used herein " connective tissue " is the tissue that connects or support its hetero-organization or organ, includes but not limited to ligament, cartilage, tendon, bone and the synovial membrane of mammalian hosts.

Term used herein " phoirocyte " or " cell of connective tissue " are included in the cell of finding in the connective tissue, such as fibroblast, cartilage cell (chondrocyte) and osteocyte (Gegenbaur's cell and osteocyte), they are as the same collagen extracellular matrix of secreting with smooth muscle cell of adipocyte (adipocyte). Preferred phoirocyte is fibroblast, cartilage cell and osteocyte. Preferred phoirocyte is fibroblast. Phoirocyte also comprises mesenchymal cell, and they are also referred to as the prematurity fibroblast. Will be appreciated that the present invention can adopt the mixed culture of phoirocyte and single type of cell to implement.

Term used herein " phoirocyte system " comprises the multiple phoirocyte that originates from the common parent cell.

Term used herein " hyaline cartilage " refers to cover the connective tissue of articular surface. Only as an example, hyaline cartilage includes but not limited to articular cartilage, costal cartilage and nasal cartilages.

Concrete, known hyaline cartilage is self, variation is reacted, and motion is reduced friction and stablize. Find in addition in same joint or between the joint thickness, cell density, matrix form all differently with engineering properties, but keeping identical general structure and function. Some function of hyaline cartilage comprises the ability that to the ability of the disperse weight load that compresses surprising rigidity, elasticity and give prominence to, at utmost reduces the peak stress of subchondral bone, and powerful durability.

Totally on histology, hyaline cartilage looks like a kind of smooth firm surface of energy resistance to deformation. The extracellular matrix of cartilage contains the cartilage cell, but lacks blood vessel, lymphatic vessel and nerve. Keep the interactional exquisiteness between cartilage cell and the matrix and the structure of high-sequential has played the effect of keeping the hyaline cartilage 26S Proteasome Structure and Function, keeping simultaneously low-level metabolic activity. O ' Driscoll, J.Bone Joint Surg., 80A:1795-1812,1998 describe the 26S Proteasome Structure and Function of hyaline cartilage in detail, are incorporated herein for your guidance.

Term used herein " transforming growth factor-beta (TGF-β) superfamily " has comprised the protein of one group of structurally associated, impact atomization miscellaneous in embryo development procedure. This family comprises M ü llerian inhibiting substances (MIS), and it is that normal male is grown essential (Behringer etc., Nature, 345:167,1990), fruit bat 15 pleats (DPP) gene outcome, it is that the dorsoventral axis line forms and necessary (Padgett etc., Nature, 325:81-84 take place the imaginal disk form, 1987), Africa xenopus Vg-1 gene outcome, it is positioned at the plant pole (Weeks etc. of ovum, Cell, 51:861-867,1987), actin (Mason etc., Biochem, Biophys.Res.Commun., 135:957-964,1986), it can induce Africa xenopus embryo's mesoderm and the formation (Thomsen etc. of pre-structure, Cell, 63:485,1990), and bone morphogenetic protein (BMP ' s, such as BMP-2,3,4,5,6 and 7, osteogenin, OP-1), it can induce cartilage and bone from the beginning to form (Sampath etc., J.Biol.Chem., 265:13198,1990). TGF-β gene outcome can affect various atomizations, comprises that Adipogenesis, flesh generation, Chondrogenesis, haemocyte generate and epidermal cell differentiation (for summary, seeing Massague, Cell 49:437,1987), are incorporated herein for your guidance.

Synthesized at first the TGF-'beta ' family protein as larger precursor protein, then through carrying out the proteolysis cutting from about 110=140 amino acid whose cluster alkaline residue place of C-end. The C-end regions of these protein all is structurally associated, and different family members can be divided into different subgroups according to its homology degree. Although the homology scope amino acid sequence homogeny in the concrete subgroup is between 70%-90%, the homology between the subgroup is obviously much lower, usually 20%-50% only. In all cases, it seems that reactive specy be the dimer that C-terminal fragment disulfide bond connects. The member who had studied for these family's great majority, find that homodimer has biologically active, but for other family members, such as inhibin (Ung etc., Nature, 321:779,1986) and TGF-β (Cheifetz etc., Cell, 48:409,1987) as if, also detected heterodimer, these heterodimers have different biological properties from each homodimer.

The member of TGF-β gene superfamily comprises TGF-β 3, TGF-β 2, TGF-β 4 (chicken), TGF-β 1, TGF-β 5 (Africa xenopus), BMP-2, BMP-4, fruit bat DPP, BMP-5, BMP-6, Vgr1,0P-1/BMP-7, fruit bat 60A, GDF-1, Africa xenopus Vgf, inhibin-β A, inhibin-β B, inhibin-α and MIS. At Massague, Ann.Rev.Biochem.67:753-791 has described these genes in 1988, is incorporated herein for your guidance.

The member of preferred TGF-beta superfamily is TGF-β. Preferred member is TGF-β 1, TGF-β 2, TGF-β 3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 or BMP-7. Even preferred member is the TGF-β of people or pig. Preferred member is people or pig TGF-β 1, TGF-β 2 or TGF-β 3. Most preferred member is people or pig TGF-β 1.

Term used herein " but selected marker " comprises the gene outcome of cellular expression, the DNA that this cell energy stable maintenance is introduced, phenotype such as morphologic conversion or enzymatic activity that this DNA causes cellular expression to change. Separate the cell of expressing certain rotaring redyeing gene and can realize that for example introducing has the mark that can give the enzymatic activity of antibiotic or other drug resistance by but the gene of second coding selected marker is introduced same cell. The example of selected marker includes but not limited to: thymidine kinase, dihyrofolate reductase, aminoglycoside phosphotransferase, it is given aminoglycoside antibiotics (such as kanamycins, neomycin and Geneticin) resistance, hygromycin B phosphotransferase, xanthine-guanine phosphoribosyl transferase, CAD (a kind of protein, has from the beginning biosynthetic first three kind enzymatic activity-carbamylphosphate synthetase of uracil, aspartic acid turns to carbamyl enzyme and dihydroora tase (dihydroorotase)), adenine deaminase and asparagine synthetase (Sambrook etc., MolecularCloning, 16 chapters, 1989), be incorporated herein for your guidance.

Term used herein " promoter " can be the activated any dna sequence dna that can control the eucaryote transcription. Promoter can have activity in eukaryotic or prokaryotic. Preferred promoter has activity in mammalian cell. Promoter can be constitutive expression or derivable. Preferred promoter is derivable. So preferred startup can be induced by exogenous stimulation. Preferred promoter is by hormone or metal inducement. Most preferred promoter is the metallothionein gene promoter. Similarly, can insert in this dna vector construction also controlling " enhancer element " of transcribing, be used for the expression that construction of the present invention strengthens gene of interest.

Term used herein " DC-chol " means a kind of cationic-liposome that contains the cation cholesterol derivative. " DC-chol " molecule comprises uncle's amino, the space arm of moderate-length (two atoms) and carbamoyl joint key (Gao etc., Biochem.Biophys.Res, Commun., 179:280-285,1991).

Term used herein " SF-chol " is defined as a cationoid liposome.

The term " BA " relevant with liposome used herein refers to introduce the ability of functional DNA and/or protein in target cell.

Used herein and nucleic acid, protein, the relevant term " BA " of protein fragments or derivatives thereof refer to that the nucleic acid of nucleic acid or amino acid sequence imitation wild type or the known organism that protein causes learn the ability of function.

Term used herein " is kept " when using together with the liposome transmission, guides the DNA that enters to remain on intracellular ability. When other situations of being used for, it refers to that target DNA remains in target cell or the tissue, thereby gives the ability of result for the treatment of.

The invention discloses the technology that in vitro and in vivo interested dna sequence dna is delivered to the phoirocyte of mammalian hosts. In vitro technology relates to cultivation target phoirocyte, external dna sequence dna, dna vector or other interested transmission carriers are transfected into phoirocyte, then the phoirocyte of modifying is transplanted in the target joint of this mammalian hosts, thereby is affected the expression in vivo of this gene of interest product.

Alternative methods as external fibroblast manipulation, the gene of the interested product of coding is introduced liposome, be injected directly into joint area, wherein liposome and connective tissue cell merge, and cause gene expression in vivo to belong to the gene outcome of TGF-beta superfamily.

As the alternative methods that external connective tissue cell is handled, the gene of coding product interested is introduced joint area as naked DNA.Naked DNA enters connective tissue cell, causes gene expression in vivo to belong to the gene outcome of TGF-beta superfamily.

The disclosed a kind of in vitro method for the treatment of connective tissue disease of this description comprises: at first produce a kind of recombinant virus or plasmid vector, it contains the DNA sequence of coded protein or its bioactive fragment.With the connective tissue cell of this recombinant vector infection or transfection a group In vitro culture, obtain the connective tissue cell that a group contains this carrier then.These connective tissue cells are implanted in the target joint chamber of mammalian hosts then, influence the expression subsequently in articular cavity of this albumen or protein fragments.The expression of this interested DNA sequence is useful for alleviating harmful joint pathology variation relevant with connective tissue disease.

Those of ordinary skills should be understood that the preferred source of the cell of treatment human patients is patient's oneself a connective tissue cell, as the fibroblast of self.

More specifically, as gene, this method comprises uses a kind of transforming growth factor member that can encode, but or its biologically active derivatives or its fragment and a selected marker or its biologically active derivatives or segmental gene.

As gene, an alternative embodiment of the invention comprises can the encode member of at least one transforming growth factor of use, or its biologically active derivatives or segmental gene, with as the DNA plasmid vector, use those of ordinary skills known, can be in target cell or tissue, no matter use what transmission method, can both be after transmission any DNA plasmid vector of stable maintenance.

A kind of method is directly to transmit the dna vector molecule to target cell or tissue, no matter it is virus or plasmid DNA carrier molecule.As gene, this method also comprises the use segmental gene of transforming growth factor member or biologically active derivatives or its of encoding.

An alternative embodiment of the invention provides a kind of method, and the gene of at least a coded product is introduced at least a connective tissue cell, is used for the treatment of mammalian hosts.The inventive method comprises uses non-viral material, and the gene of this product of coding is introduced connective tissue cell.More specifically, this method comprises liposome, coprecipitation of calcium phosphate, electroporation or the mediation of DEAE-glucosan, and as gene, but comprise use can encode transforming growth factor superfamily member or biologically active derivatives or its fragment and a selected marker or biologically active derivatives or its segmental gene.

An alternative embodiment of the invention provides another kind of method, and the gene of at least one coded product is introduced at least a connective tissue cell, is used for the treatment of mammalian hosts.This another kind method comprises that use can utilize virus the dna vector molecule to be passed to the biological method of target cell or tissue.Preferably, this virus is a kind of virusoid, and its genome changes, this virusoid only can be transmitted and in target cell stable maintenance, but the ability of not in target cell or tissue, not duplicating.Further handle the viral genome of change with recombinant DNA technology, make viral genome play the effect of dna vector molecule, it contains the interested heterologous gene that will express in target cell or tissue.

Preference of the present invention is a kind of TGF-β to be passed to the method in target joint chamber, and this method is by passing to TGF-β gene the connective tissue of mammalian hosts with disclosed in vitro technology in retroviral vector and this description.In other words, the interested DNA sequence sub-clone of encoding function TGF-β albumen or protein fragments is gone in the selected retroviral vector, make the viral vector of reorganization long then to enough titres, be used for the Infection in Vitro population of cultured connective tissue cells, and preferably the connective tissue cell of transduction is transplanted in the interested joint cell of preferred autograft by intra-articular injection.

Another kind of method for optimizing of the present invention relates to TGF-beta superfamily gene by passing to the mammal connective tissue in use adenovirus vector, adeno-associated virus (AAV) carrier or the direct body of herpes simplex virus (HSV) carrier.In other words, the DNA sequence sub-clone of interested encoding function TGF-beta protein or protein fragments is gone into each viral vector.Make the viral vector that contains TGF-β grow into enough titres then, preferably directly enter articular cavity by intra-articular injection.

Directly the dna molecular that contains gene of interest in intra-articular injection causes the transfection of receptor's connective tissue cell, thereby walked around the fibroblast that recovery, In vitro culture, transfection, selection and transplanting contain this dna vector, promoting the requirement of interested heterologous gene stably express.

Include but not limited to the method for target joint connective tissue the dna molecular submission: dna molecular to be wrapped in the cationic-liposome, interested DNA sequence sub-clone to be gone into retrovirus or plasmid vector, or dna molecular itself is injected directly into the joint.No matter the dna molecular submission is given kneed form, dna molecular is preferably with the form submission of dna vector molecule (recombinant virus dna carrier molecule or recombinant dna plasmid carrier molecule).Will be in eukaryotic cell the promoters active fragment be inserted into the direct upstream of heterologous gene coding region, guarantee interested expression of heterologous genes.Those of ordinary skills can utilize the known method of vector construction and technology to guarantee suitable expression after dna molecular enters connective tissue.

In preference, the fibroblast that will reclaim from knee joint is at In vitro culture, then as the transmission system of gene therapy.Obviously the applicant is not limited to use disclosed specific connective tissue.May use other tissue-derived, be used for the In vitro culture technology.Use the method for gene of the present invention to can be used for arthritic prevention or treatment.Should understand that the applicant is not limited to only treat kneed prevention and therapeutic use.May utilize the arthritis in the preventative or any susceptible of the therapeutic treatment joint of the present invention.

In another embodiment of the present invention, provide a kind of chemical compound with treatment effective dose parenteral, it contains coding proteic gene of TGF-superfamily and suitable pharmaceutical carrier.

An alternative embodiment of the invention provides parenteral to be applied to the chemical compound that patient prevents effective dose, and it contains coding proteic gene of TGF-superfamily and suitable pharmaceutical carrier.

An alternative embodiment of the invention comprises the aforesaid method of this paper, comprises external gene being introduced cell.This method also comprises then goes into mammalian hosts with the cell transplantation that infects.After this method is included in effective transfection connective tissue cell, but before infection cell is implanted into mammalian hosts, the connective tissue cell of storage transfection.The connective tissue cell that it will be understood by those skilled in the art that infection can be frozen in 10%DMSO under liquid nitrogen.This method comprises that use can prevent from the arthritic mammalian hosts of height susceptible arthritic method to take place basically.

An alternative embodiment of the invention comprises a kind of method, the gene of at least a coded product is introduced at least a connective tissue cell of mammalian hosts, be used for the treatment of mammalian hosts as previously mentioned, comprise by the viral vector that will contain this product gene of encoding and directly introduce mammalian hosts, realize the infection of cells in vivo.Preferred this method comprises by the intra-articular injection realization directly introduces mammalian hosts.This method comprises that use can prevent from the arthritic mammalian hosts of height susceptible arthritic method to take place basically.This method also comprises this method is used for the treatment of suffers from arthritic mammalian hosts.This method also comprises as this method reparation of the aforesaid use of this paper and regeneration connective tissue in addition.

It will be understood by those skilled in the art that and use the viral vector of liposome to be not limited to the fissional restriction required, realize the infection and the integration of connective tissue cell as retrovirus.As gene, use method, but comprise gene and the selectable marker gene (as antibiotics resistance gene) that uses coding to belong to the TGF-beta superfamily member as the aforesaid non-viral material of this paper.

An alternative embodiment of the invention is that the DNA sequence with the member of coding TGF-beta superfamily passes to the connective tissue of mammalian hosts by the disclosed arbitrary method of this description, thereby realizes the expression in vivo of collagen, makes connective tissue (as cartilage) regeneration.

Disclosed as an example, rather than in the concrete grammar to the present invention's restriction, the DNA plasmid vector that will contain TGF-β coded sequence is connected to the downstream of metallothionein promoter.

Connective tissue is the organ that is difficult to targeting in the treatment.The drug delivery of intravenous known in the art and oral cavity route is difficult to arrive these connective tissues, and the shortcoming that makes mammalian hosts general contact treatment medicine is arranged.In particular, knownly directly arrive the joint in intra-articular injection albumen mass-energy.Yet, short in the intraarticular half-life with the medicine major part of parcel protein form injection.The connective tissue that the present invention introduces mammalian hosts by the proteinic gene that coding be can be used for treat mammalian hosts solves these problems.More specifically say, the invention provides a kind of method, the proteinic mammalian hosts gene that coding is had anti--arthritis character is introduced connective tissue.

In the present invention, the applying gene treatment has solved the short and high problem of cost of the acting duration relevant with TGF-β administration.Transfectional cell can be survived in tissue culture more than 6 weeks and do not had a metamorphosis.In order to determine the durability and the persistent period of effect, with injection cell in the rabbit heel string.If for cells in vivo nutrition supply abundance, this cell can be survived the sufficiently long time, and produce TGF-β, the cell around stimulating.This cell all has function in tendon He in two kinds of environment of intraarticular.

The concentration of transfectional cell is the key factor of local action.Experiment formerly (Joyce etc., on seeing, 1990) in, the dosage of TGF-β has been determined the types of organization that forms.Particularly, cartilage forms with the interior osteoplastic ratio of film and descends along with the reduction of dosage.TGF-β also has two phasic properties (Centrella etc., Endicrinology, 119:2306-2312,1986) in stimulation osteoblast of former generation and MC3T3 cell.That is, according to concentration, it can be irritating also can be (Chenu etc., Proc.Natl.Acad.Sci, 85:5683-5687,1988) of inhibition.In embodiment provided herein, NIH 3T3-TGF-β 1 cell is with 10 ⁴, 10 ⁵With 10 ⁶The variable concentrations of cells/ml stimulates collagen synthetic.Tendon is 10 ⁶Increase at most under the concentration of cells/ml.

In an embodiment, with 0.3 milliliter 10 ⁶Cells/ml concentration injection of joint.Collect the injection specimen in back 2-6 week.The environment in joint is different with heel string.Cell can move freely at intraarticular.They are movable to the zone that this cell is had specific affinity.Synovial membrane, meniscus and cartilage defect zone are the possible positions of cell adhesion.After injecting for 6 weeks, to regenerating tissues, but do not observe at synovial membrane and meniscus place at the cartilage defect regional observation of partially or completely damage.For the special affinity of damage field another advantage that is clinical practice.If degenerative arthritis can only can be cured with the intra-articular injection cell, can treat patient so easily, without major operation.

The TGF-β of injection emiocytosis may stimulate hyaline cartilage regeneration in two ways.A kind of is that the chondrocyte of staying injured area produces TGF-beta receptor (Brand etc., J Biol Chem, 270:8274-8284,1995 at its cell surface; Cheifetz etc., Cell, 48:409-415,1987; Dumont etc., M CellEndo, 111:57-66,1995; Lopez-Casillas etc., Cell, 67:785-795,1991; Miettinen etc., J Cell Biology, 127:6,2021-2036,1994; With Wrana etc., Nature, 370:341-347,1994).The TGF-β that these receptors may be subjected to adhering to the injection emiocytosis of damage field stimulates.Because with potential form secretion (Wakwfield etc., J Biol Chem, 263,7646-7654,1988), potential TGF-β needs activation process to TGF-β in vivo.Another kind of mode is that potential TGF-β or the excretory TGF-β of transfectional cell may combine (Dallas etc., J Cell Biol, 131:539-549,1995) with TGF-β conjugated protein (LTBT) on the extracellular matrix of the cartilage layers of part damage.

No matter mechanism of action how, the synthetic discovery of hyaline cartilage shows that high TGF-β over a long time can stimulate hyaline cartilage regeneration.This carrier of high local concentrations may not be local irritant key factor, but chondrocyte may be the only carrier (Brittberg etc., New Engl JMed 331:889-895,1994) that TGF-β is passed to the cartilage affected area in theory.The double-deck substrate of collagen is the another kind of possible carrier (Frenkel etc., J Bone J Surg (Br) 79-B:831-836,1997) of local distribution transfectional cell.

Determined the tissue property of new formation with Histological method.At H﹠amp; In the E dyeing, the tissue of new formation and hyaline cartilage identical (Fig. 4) on every side.In order to assess the character of new formative tissue, with Safranin-O dyeing tissue (Rosenburg, J Bone Joint Surg, 53A:69-82,1971).Opposite with the fibrous collagen of white, the new tissue that forms is dyed redness, and pointing out it is hyaline cartilage (Fig. 5).

Cell in the damage field has produced fibrous collagen fully.Because have the bone sample substrate barrier that TGF-β is stimulated, osteoblast may not stimulated on every side.NIH 3T3-TGF-β 1 cell does not stimulate peripheral cell, but produces fibrous collagen by autocrine stimulation.The fact that autocrine and paracrine activate irritation cell has increased the probability for the treatment of degenerative arthritis with the chondrocyte of TGF-β 1 expression constructs stable transfection.

Cell line with TGF-β 1 expression constructs stable transfection can be survived in tendon and knee joint.This cell line is at tendon and damage fully in the cartilage zone and to produce fibrous collagen.Yet this cell line produces hyaline cartilage in the articular cartilage of part damage.The stimulation mechanism of the autocrine of this effect and paracrine pattern shows, carries out the method that gene therapy is a kind of new treatment hyaline cartilage damage with the member of TGF-beta superfamily gene.

The present invention has prepared stable fibroblast (NIH3T3-TGF-β 1 and human foreskin fibroblast TGF-β 1) cell line by transfection TGF-β 1 expression constructs.These cells that produce TGF-β have been kept the active TGF-β of high concentration in vivo for a long time.

Answer about gene therapy, first problem of the probability of particularly cell-mediated gene therapy is a cell survival rate in vivo.Although TGF-β can suppress immunocyte external, cell may not be survived in other species tissues with efficient immune surveillance system.The second, should assess the optium concentration that gene is expressed in vivo.We are expelled to cell in the heel string of rabbit with three kinds of variable concentrations, answer this problem.The optium concentration of injecting in the tendon has been determined the concentration of the intra-articular injection that will use.The 3rd problem is how cell stimulates regenerating bone or cartilage at intraarticular.

The cell of injection has two kinds of binding modes.A kind of is to activate peripheral cell (paracrine activation) (Snyder, Sci Am, 253 (4): 132-140,1985) by secretion TGF-β, and another kind is that self activates (autocrine activation).Cell concentration can influence these approach, but surrounding may be the greatest factor of decisive action pattern.The intraarticular joint fluid is two kinds of different environment with ligament inside, and their blood supply, nutrition supply are all different with the cell that centers on.Cells transfected is expelled in two kinds of varying environments, to seek out the binding mode of cell.The whole purpose of this research is the TGF-β-mediated gene therapy of assessment to orthopedics's disease, and the pattern of effect in the clear and definite body.

The following example explanation the present invention is provided, rather than restriction.

Embodiment

Example I-material and method

Plasmid construction

In order to produce metallothionein expression constructs (pM), made up XbaI and Bam HI restriction site by the polymerase chain amplification at the oligonucleotide that is used for increasing with genomic DNA, produced metallothionein I promoter (660/+63).The amplified fragments sub-clone is gone into pBluescript (Stratagene, La Jolla, Xba I-Bam HI site CA).Go into the Bam HI-Sal I site of pM by containing the 1.2kb Bgl II fragment sub-clone that TGF-β 1 coded sequence and 3 ' end contain growth hormone polyadenylation site, produced plasmid pmT β 1.

Cell culture and transfection-TGF-β cDNA is transfected into fibroblast (NIH 3T3-TGF-β 1) or human foreskin fibroblast/TGF-β 1, containing the Dulbecco improvement Eagle culture medium (GIBCO-BRL of 10% hyclone, Rockville, MD) the middle cultivation.The adding of TGF-β 1cDNA sequence is had in pmT β 1 carrier of metallothionein gene promoter.Also the neomycin resistance gene sequence is inserted this carrier.

With the calcium phosphate precipitation method this carrier is inserted used cell.Cell for the gene order of selecting to have transfection is added to neomycin (300 mcg/ml) in the culture medium.Then, select the bacterium colony of survival and analyze and TGF-β 1 ELISA (R﹠amp by Northern; D system) expression of TGF-β 1 mRNA is confirmed in test.The cell that will have TGF-β 1 expression is stored in the liquid nitrogen, cultivates before the injection.

Northern engram analysis-from cell, isolate total RNA with guanidinium isothiocyanate/phenol/chloroform.Containing electrophoresis 10 microgram RNA on 1.0% agarose gel of 0.66M formaldehyde, transfer on the DURALON-UV film, and crosslinked with UV STRATALINKER (STRATAGENE).Prehybridization trace, and 65 ℃ of hybridization in the solution of 1% bovine serum albumin, 7% (w/v) SDS, 0.5M sodium phosphate and 1mM EDTA.Hybridized trace 20 minutes with 50 ℃ of washings of 0.1%SDS, 1XSSC, egative film then exposes.Make the RNA trace and be used for people TGF-β's 1 ³²The cDNA probe hybridization of P-labelling.Do the contrast of sample load with the beta-actin probe.

Injection cell is gone into the New Zealand white rabbit of the heavy 2.0-2.5 kilogram of rabbit-selection as animal model.After ketamine and roumpon anesthesia, cover white rabbit with disinfecting towel.Exposing heel string, is 10 with the 0.2-0.3 ml concn ⁴, 10 ⁵With 10 ⁶The injection cell of cells/ml is to the position, center of tendon.Zinc sulfate is added in the rabbit drinking water, is used to express the DNA of transfection.After the definite optium concentration of heel string experiment, carry out intra-articular injection.Expose knee joint, with scalpel fabrication portion and cartilage defect fully.Fabrication portion is damaged on the hyaline cartilage layer, notes not exposing subchondral bone.Expose subchondral bone manufacturing complete collyriculum after removing all hyaline cartilage.Behind the suture operation wound, intra-articular injection 10 ⁶The cell of cells/ml concentration adds zinc sulfate in drinking water.

After histology-collection heel string and the knee joint, use the formalin fixed specimen, use the nitric acid decalcification.They are embedded in the paraffin mass, are cut into 0.8 micron slab.With hematoxylin-eosin and the regenerated tissue of Safranin-O dyeing microscopic examination.

Example II-result

Stable cell line-carried out transfection with coprecipitation of calcium phosphate method (Fig. 1).The cell colony express transgenic mRNA of about 80% survival.The cell of these generation TGF-β 1 that select is cultivated in solution of zinc sulfate.When cell was cultivated in 100 μ M solution of zinc sulfate, they produced mRNA.TGF-β secretion rate is about 32 nanograms/10 ⁶Cell/24 hour.

The regeneration of rabbit articular cartilage defect-observed the rabbit heel string to verify the survival rate of NIH 3T3-TGF-β 1 cell.10 ⁶Cells/ml concentration, heel string than other 10 ⁴With 10 ⁵Two kinds of concentration are all thick.After causing part and complete cartilage defect, with 0.3 milliliter 10 ⁶NIH 3T3-TGF-β 1 injection cell of cells/ml is in knee joint.2-6 weekly check joint, injection back.In the cartilage of part damage, we find the new hyaline cartilage that forms; In injection two weeks of back, hyaline cartilage occurs, and in injection 6 weeks of back, cartilage defect is covered (Fig. 2) by hyaline cartilage.The thickness of regeneration of cartilage is along with the time thickens (Fig. 3) in the past.The TGF-β 1 of injection emiocytosis can be by observing (Fig. 3) with TGF-beta 1 antibodies immunohistochemical staining.Using the offside joint of injecting without the normal fibroblast of TGF-β 1 transfection not observe hyaline cartilage covers.In the zone of part damage, regeneration hyaline cartilage take on a red color (Fig. 4) in Safrain-O dyeing.(almost the degree of depth with damaged is identical for the new cartilage degree of depth that forms).This cell of finding the prompting injection is by normal cartilage cell around the paracrine action mode activation.

Regenerating tissues is not hyaline cartilage but fibrous collagen in Sun Shang the cartilage fully.Their color is white in color in Safrain-O dyeing, rather than the redness (Fig. 5) of hyaline cartilage acquisition.Cartilage is covered by bacillar structure, means only by these cells of autocrine mode activation.Can be it seems owing to existing thick calcified bone substrate to stop the stimulation of TGF-β by the activated surrounding bone cell of TGF-β to it.The cell of injection may be because this barrier can not stimulate osteocyte.

TGF-β 1 cells transfected is expelled in the rabbit heel string.The tendon of operation shows that comparison is according to the overall thicker morphology (Fig. 7) of tendon like this.H﹠amp is made in section to this heel string; E dyeing, at test under microscope, NIH3T3-TGF-β 1 cell survival of injection also produces fibrous collagen (Fig. 8) at the heel string of rabbit.Microscopy is made the regenerated tendinous tissue of immunohistochemical staining with the TGF-beta 1 antibodies, shows the expression (Fig. 9) of TGF-β 1 in tendon.

Though described specific embodiments of the invention in order to illustrate, details of the present invention to those skilled in the art obviously can be done many variations, yet these change without prejudice to the determined scope of claim of the present invention.

All lists of references that this paper quotes are incorporated herein for your guidance.

List of references

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Claims (11)

1. the purposes of chondrocyte system, described cell line is connected with the promoter navigability at external quilt, the DNA sequence transfection of coding transforminggrowthfactor-, it is characterized in that, described cell line is used to prepare the medicine of treatment of arthritis or regeneration hyaline cartilage, and described cell realizes directly introducing mammalian hosts by intra-articular injection.

2. purposes as claimed in claim 1 is characterized in that described DNA sequence is in viral vector.

3. purposes as claimed in claim 2 is characterized in that described viral vector is a retroviral vector.

4. purposes as claimed in claim 2 is characterized in that, described viral vector is the related viral vector of gland.

5. purposes as claimed in claim 2 is characterized in that described viral vector is an adenovirus vector.

6. purposes as claimed in claim 2 is characterized in that described viral vector is a herpes simplex virus vector.

7. purposes as claimed in claim 1 is characterized in that described recombinant vector is a plasmid vector.

8. purposes as claimed in claim 1 is characterized in that, the described chondrocyte of storage is earlier before transplanting.

9. purposes as claimed in claim 8 is characterized in that described chondrocyte ties up under the liquid nitrogen and is stored among the 10%DMSO.

10. purposes as claimed in claim 1 is characterized in that, described transfection is finished by liposome, coprecipitation of calcium phosphate, electroporation and the mediation of DEAE-glucosan.

11. purposes as claimed in claim 1 is characterized in that, described promoter is a metallothionein I promoter.

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