DE10025920A1 - Methods and agents for treating immune reactions in the eye - Google Patents

Abstract

The invention relates to an agent and a method for the gene therapy treatment of corneal diseases of the human or animal eye, for the inhibition of transplant rejection. The above comprises an expressed construct, with a sequence coding for an immunomodulatory protein or polypeptide under the control of a functional promoter and a functional polyadenylation signal.

Description

The invention relates to an agent and a method for gene therapy treatment development of corneal diseases of the human or animal eye suppression of transplant rejection.

With some diseases of the eye it is necessary to transplant foreign tissue animals, mostly from other, deceased people or animals comes from. This transferred or transplanted foreign tissue takes over Function of the original diseased or destroyed tissue. The most important and the oldest transplantation procedure on the eye is the corneal transmission. This operation removes the clouded cornea from a human eye and replaced by the clear cornea of a deceased. In this way, a previously blind eyes regain normal visual acuity. From surgical On the one hand, this surgical procedure is largely optimized today and becomes one routine procedures that can hardly be improved. So be annually approximately 5,000 patients operated on in Germany and approximately 50,000 in the USA.

The most important, as yet unsolved problem of this foreign tissue transfer is Immune response, in which the immune system of the transplant recipient over recognizes worn tissues as foreign and develops defense mechanisms that prevent Destruct the transplanted foreign tissue.  

Depending on the likelihood of long-term success of corneal transmission un one divides the patients into those with a good prognosis and those with one bad forecast. 10% of patients with a good prognosis fall ill an immune response and 50% of patients with a poor prognosis have an immune response. The consequence of the immune reaction is the clouding of the trans planted cornea and thus renewed blindness.

To suppress an immune response after the transplant, heu state of the art used immunosuppressive drugs. The important medicines to prevent an immune response after corneal transmission are corticosteroids that are used locally as eye drops on the eye. Next A substantial failure rate of this treatment leads to dangerous consequences This form of therapy, namely infections of the cornea, development of the glaucoma (Glaucoma) and clouding of the lens. These side effects prevent one higher doses and possibly an improved success rate. From the Therefore, other options are sought to modulate the immune response or prevent.

For some important immunosuppressive agents such as B. Cyclosporin A could after Corneal transmission is only a very slight effect, but it has considerable side effects proven, so that they are clinically used only in exceptional cases be set. The ratio of effect to possible is correspondingly unfavorable Side effect when using monoclonal antibodies.

The course of the immune response is partially known. The activity of the immune system can be mediated across multiple populations of helper and effector cells, A distinction is then made between types of immune response as cell-mediated (cytoto xical) type 1 (Th1) or a type 2 mediated by soluble effector molecules Reaction (Th2). The reactions inhibit each other within certain limits and are characterized by the release of response type-specific cytokines. The immune response after organ transplantation is essentially a cell-mediated telte (cytotoxic) response of the type Th1 and is dependent on the expression of the Th1 Cytokines IL-2 and IFN-gamma accompanied. For the formation of tolerance is predominance the Th2 over Th1 cytokine expression is important. In the early phase after organ transplantation leads to increased expression of IL-2  Receptor (CD25), and the cytokines IL-2, IFN-gamma, IL-4 and IL-10. In the anti-CD4 (RIB 5/2) induced, MHC specific graft tolerance goes IFN gamma quickly returned while IL-4 persisted. There is a Th1 / Th2 shift. The Th1 response is blocked (Lehmann 1997, Transplantation 64, 1181-7).

Various factors play a role in the polarization of the Th1 or Th2 path Role, e.g. B. the cytokine profile of natural immunity, the nature of the antigen as well as the activity of co-stimulatory molecules and locally secreted hormones against the respective genetic background. The Th1 answer is opposite intracellular pathogens, in graft rejection and in Autoimmune diseases, the Th2 response to intestinal nematodes, the Graft tolerance and the graft versus host response matter (Romagnani 1997, Int Arch Allergy Immunol 113, 153-6).

For the suppression of the rejection reaction after transplantation is the Indukti on a Th2 immune response and the suppression of a Th1 response advantageous arrested. Since the Th1 and Th2 responses mutually inhibit each other, leads induction of a Th2 response to inhibit the Th1 response, and vice versa returns.

T cell-dependent immune responses are triggered by an interaction of T- Lymphocytes with matching T cell receptor (TcR) with antigen presenting cell len (APC) initiated, the TcR present an antigen peptide with the MHC molecule animals. This interaction of TcR and MHC- is necessary for complete T cell activation. However, the peptide is insufficient and a second, "costimulatory" signal must be generated from an adequate pairing of accessory molecules on both at the In Interaction involved cells result. The co-stimulation system is well defined, at the cell surface protein B7 expressed by the antigen presenting cell (B7.1, B7.2) molecules interact with CD28 molecules that are activated by the T Cell can be expressed (Janeway 1994, Current Biology Ltd., Garland Publishing Inc); this B7-CD28 interaction leads to the expression of CTLA-4 by the T- Lymphocytes, which are sensitive due to their particularly high affinity for B7 the T cell for stimulation by B7 and finally the Enables transduction of an adequate co-stimulatory signal. The preventi on co-stimulation induces and inhibits specific T cell anergy in vitro  Autoimmune and alloimmune responses in vivo (Janeway 1994, Current Biology Ltd, Garland Publishing Inc. Linsley 1992, Science 257: 792-795; Lin 1994, transplant Proc. 26: 3200-3201; Wallace 1994, Transplantation 58: 602-610; Baliga 1994, Transplantation 58: 1082-1090).

The prevention of such co-stimulation can e.g. B. by expression of soluble ligan those for B7 that "saturate" the CD28 molecule and the recognition of the ge prevent common signal presence of TcR and CD28. So a CTLA4-lg Fusion protein produced in which the extracellular part of the murine CTLA4- Molecule and the constant region of human immunoglobulin gamma1 with are fused together, mCTL4-4 gamma1, which functions as a monoclonal Antibody to B7 behaves (Lane et al. Immunology 1993 Sep. 80 (1): 56-61). Blockage of the CD28-B7 T cell co-stimulation pathway by the fusion protein CTLA4-lg induces tolerance to kidney, lung and cardiac allotransplanta rat and pancreatic allograft in mouse and rat and one significant increase in corneal transplant survival in the mouse (Lin 1994, Transplant Proc. 26: 3200-3201; Lenschow 1992, Science 257: 789-792; Yin 1995, J Immunol 155: 1655-1659; Chahine 1995, Transplantation 59: 1313-1318; Matsumura 1995, Transplantation 59: 551-558; Finck 1992, PNAS 89: 102-106; Hoffmann 1997, Graefe's Arch Clin Exp Ophthalmol 235: 535-540).

The co-stimulatory signals B7 / CD28 are for the activation of Th1 cells of great importance. After liver transplantation on the rat, the allograftreak is tion by invading macrophages and CD4 + cells into the graft and characterized a Th1 reaction. After treatment with CTLA4 fusion protein the monocyte invasion is reduced and expression of IL-4 and IL- 10 (Mark 1997, Langenbeck's Arch Chir Suppl Kongressbd 114: 299-302). The CD28 / B7 blockade with CTLA4lg hinders Th1- but not Th2- Cytokine synthesis (Akalin 1996, Transplantation 62: 1942-1945). It leads to never Transplantation on the rat in the graft, among other things, for the expression of the Th2 cytokines IL-4 and IL-10, however, for untreated controls for expression the Th1 cytokines IL-2 and IFN-gamma (Sayegh 1995, J Exp Med 181: 1869-1874).

The inhibition of co-stimulatory signals that are essential for T cell activation has therefore proven to be particularly effective in preventing animal transplant rejection in animal experiments  proven to induce anergic T lymphocytes. These recognize the alloantigen in question, but no longer react to it (Janeway 1994, Current Biology Ltd., Garland Publishing Inc). Such a strategy has the advantage over the treatment with immunosuppresives, only for a rela tiv short period, namely during the initial phase of exposure of the recipient ger immune system against the transplant alloantigens, to be applied have to.

To suppress costimulation, soluble ligands can therefore be used at the Signal mediation between APC and T cell involved surface molecules be set. These molecules can act as proteins for the patient can be administered mixed or locally, but administration is also conceivable suitable expression constructs from DNA on which the sol necessary inhibitors is contained, and that after insertion solution in the body's own cells cause them to produce the inhibitors.

The transport and expression of genetic information in cells is extensive known and described. On the one hand, there are systems of biological origin how to use attenuated or modified viral or bacterial vectors, which have been changed in their genetic "content" in such a way that they express desired information in the infected cell. In addition to Si safety concerns regarding back mutation or recombination to pathogenic Strains have the disadvantage that such vectors have a negative impact on the immune system Numerous potential recognition structures provide what makes them usable in the therapeutic approaches. On the other hand you use gene trans fer physical or chemical methods, such as. B. the lipofection or the voltage-induced transfer (electroporation).

Mashour et al. (Mashour et al. 1994, Gene Ther 1: 122-126) the expression of reporter genes in mice after transfection with adenoviral vectors. Budenz et al. (Budenz et al. 1995, Invest. Ophtalmol Vis Sci 36: 2211-2215). Also is the transfection of Endothelial cells of the cornea of rabbits known by means of adenoviral vectors, the expression of the reporter gene lacZ was observed (Larkin 1996, Transplantation 61: 363-370). Reporter gene expression was also started human corneal endothelial cells after adenoviral transfer and the ex-  Endothelial cells after adenoviral transfer and the expression of CTLA4lg in observed in vitro (Oral et al. 1997, Gene Ther 4: 639-647). A variation of the me The method, which uses the so-called lipoadenofection, has also been published (Aran chibia-Carcamo et al. 1998, Transplation 65: 62-67).

A number of other transfection attempts are described. So the ep Stein-Barr virus-mediated transfer of IL-10 expression vectors to the allograft Rejection in the transplantation of cardiac muscle tissue in rabbits (Brauner et al. 1997, J Thorac Cardiovasc Surg 114: 923-933).

The herpes virus-induced autoimmune reaction (Herpetic Stromal Keratitis) could in mice can be suppressed by the application of CTLA4lg fusion protein. (Gangappa et al. 1998, Clin Immunol Immunopathol 86: 88-94).

Non-viral reporter gene transfer based on a peptide-based system has been published in various species, including humans (Shrewing et al. 1997, Transplantation 64: 763-769).

A number of methods for introducing targets into target cells are known from for example transfection, lipofection, injection, "ballistic transfer" (see WO 91/07487 and DE 44 16 784), viral vectors or electroporation. So it is knows that corneal epithelial cells can be removed by electroporation with reporter genes can be transfected (Oshima et al. 1998, Gene Ther: 1347-54).

Dumbbell-shaped expression constructs are described in WO 98/21322.

It is known from WO 98/56417, WO 96/31229 and EP 0 682 039 that the rejection reaction is suppressed by inhibition of costimulatory signals leaves.

So far, the suppression of a rejection reaction after corneal transplant tion by the expression of suitable genes to the knowledge of the applicant so far has not been described. The previous knowledge about the development of the immune system reaction indicate that it is important in suppressing an immune response is an expression of the within the transplanted tissue as early as possible  Th2 cytokines IL4 and / or the extracellular domain of the CTLA4 ligand or to allow other inhibitors of the co-stimulatory signals.

Based on this prior art, it is the task of those described here Invention to provide suitable means for the postoperative suppression of the Rejection reaction and thus a permanent tolerance of the graft to it possible. Furthermore, it is an object of the invention to transfer from therapeutic effective nucleic acid constructs in the eye tissue, especially the cornea, to enable, especially in a form in which the transferred nucleic acid constructs are operationally active, d. H. can be read.

According to the invention, this object is achieved in that so-called expression con structs, so DNA polymers, especially those that are one for an immune module regulatory protein or polypeptide coding sequence under the control of a radio tional promoter and a functional polyadenylation signal, in the tissue of the cornea or other tissue associations surrounding the eye be introduced that these expression constructs for the synthesis of the immune modulatory protein or peptides, or a plurality thereof.

So-called minimalistic dumbbell constructs are preferred as expression constructs, d. H. dumbbell-shaped, linear double-stranded covalently closed nucleic acid mo leküle used, as described for example in DE 196 48 625. The advantage of this is that they do not contain any sequences that are not immediately serve the purpose of the treatment, and are therefore smaller as well as respect the character of the immune response induced by the DNA itself is more certain. The main problem here is that un methylated sequences of the general base sequence NNCGNN (with N: each base, C: Cytosine; G: guanine) can have a strong immunostimulatory effect, and above all the cytotoxic, i.e. graft rejection mediators Strengthen the arm of the immune response. The said minimalist dumbbell constructions minimize the number of immunostimulatory sequences contained on them elements and thus the strength of the DNA-induced cytotoxic stimulus.

In principle, any method that uses DNA as a transfection method can be used Can express cells, so v. a. Lipofection, peptide-mediated methods,  Electroporation or ballistic transfer, as described, for example, in EP 0 686 697 is described. The latter method, the transfer by Be shot of the tissue to be transfected with strongly accelerated particles is ge Very effective because the DNA is directly in the nucleus of the cells to be transfected is transported. All other non-viral methods suffer from e.g. T. gerin efficiency of this core transport. The nature of the ballistic transfer method However, it also involves part of the tissue from the treatment is damaged, and that particles are deposited in the transfected tissue. It was also found that the direct, postoperative transfect tion of implants using a so-called "gene gun" (Bio-Rad, Munich, WO 95/19799) to significantly delay the rejection reaction in corneal trans planted mice leads, and that in the ophthalmic examination of the treated eye no damage could be detected that the Would exclude use of the method in the patient.

The invention is also suitable for the treatment of stem cells or stem cell transplants, especially the limbus epithelium. Stem cells come everywhere wherever cells have to be renewed continuously. They are the reserve tent len for cell proliferation and divide whenever there is a need for cell innovation exists. The task of cell renewal is to count the number of cells Stant to get, so that on the one hand not too many cells are formed, and on the other hand a sudden cell loss can be compensated. If necessary, individual Stem cells become division cells. The epithelium of the corneal surface is egg subjected to a constant process of cell renewal, in which the peeled off Cells need to be replaced with new ones. Only the lowest cell layer contains Zel len that can share. This creates a vertical migration movement. At the same time, there is a horizontal cell migration movement from the limbus to the Center. Stem cells are only found in the basal limbus epithelium. The regeneration of the The surface of the eye, for example, after burns, is largely determined by the integri affected by the limbal epithelium. A loss of all stem cells leads to the most severe Wound healing disorders. Drug therapy for limbus insufficiency is not possible. Only a limbus transplant improves the situation and leads to wound healing. However, allogeneic grafts offer little prospect Success because vessels grow into the transplanted tissue and are very likely An immune reaction follows. (Kenyon 1989, Ophthalmology 96 (5): 709-22).  

The advantage of treating stem cells with the agent according to the invention is therefore obvious, because it is the rejection of the graft targeted immune defense suppressed without risk of infection is changed.

However, the invention is also suitable for the treatment of virus-induced keratitis. Experimental studies have shown that the lymphocytic infiltrate in the cornea during the development phase of herpes simplex keratitis gig Th1 contains cells that produce IL2 and IFN-gamma, while IL4 pro inducing Th2 cells are involved in the healing process (Niemialtowski 1992, J Immu nol 148: 1864-70). The goal of therapy is to target the immune system to suppress without promoting the infection. This is already the case in animal experiments succeeded. Systemic and intracorneal administration of IL10 before HSV The inflammation of the cornea could be significantly reduced without the infection To weaken the immune system (Tumpey 1994, J Immunol 153: 2258-65). Likewise Inflammation caused by anti-interferon-gamma and anti-IL2 in all weakened immune system (Hendricks 1992, J Immunol 149: 3023-3028).

Preferred as expression constructs for suppression are the transplanes rejection or virus-induced keratitis, those which the cytokines IL-4, IL-10, the extracellular part of CTLA4 or inhibitors of other ko stimulatory signals, for example non-signal-inducing antibodies against Express CD-40 or CD-40 ligand. Preferred promoters are Immediate early promoter of the cytomegalovirus and other viral promoters (SV40) and tissue-specific promoters, especially for corneal cells of the Eye-specific keratin gene promoters.

A major advantage of the invention is that by the expression of the biologically active proteins through the cells of the tissue in which they are supposed to act, the need Maneuverability of stabilization by fusion with foreign peptides, such as z. B. for which CTLA-4 is used by fusion with immunoglobulin peptides. The expressed bioactive proteins are smaller and can be more easily Diffuse tissue. This is a major advantage of using expression vectors  for proteins and peptides versus using recombinant Proteins and peptides.

Further advantageous measures are contained in the remaining subclaims. The invention will be explained in more detail using exemplary embodiments and figures as follows shown:

1. Synthesis of DNA polymers (dumbbell constructs)

Expression vector for murine CTAL4: 10 mg plasmid pG mCTLA4 were labeled with Re restriction enzyme EcoRI (MBI, Leon Rot) and hairpin oligodeoxyribonucleotide AATTCGGCCG GCCGTTTTCG GCCGGCCG (TIB, Berlin) analogous to that in DE 196 48 625 included regulation on dumbbell constructions implemented.

Expression vector for murine interleukin 4: 10 mg plasmid pG mlL4 were also analyzed Restriction enzyme EcoRI (MBI, Leon Rot) and hairpin oligodeoxyribonucleotide AATTCGGCCG GCCGTTTTCG GCCGGCCG (TIB, Berlin) analogous to that in DE 196 48 625 included regulation on dumbbell constructions implemented.

The reporter gene luciferase (for methodology, see the Prome manual ga, Madison, Wisconsin, USA, at http://www.promeqa.com/tbs/tm033/tm033.html ) was used as a marker to successfully treat corneal cells by means of ballistic transfer and thus the successful implementation of the invention to prove.

Expression vector for luciferase: 10 mg of plasmid pMOL Lux were restricted Onsenzym Eco31l (MBI, Leon Rot) and hairpin oligodeoxyribonucleotide AGGGCGGCCG GCCGTTTTCG GCCGGCCG and GGGACGGCCG GCCGTTTTCG GCCGGCCG (TIB, Berlin) analogous to the regulation contained in DE 198 26 758 Dumbbell constructions implemented.

2. Keratoplasty

The technique of corneal transplantation in the mouse model is described in detail ("Orthotopic corneal transplantation in the mouse - a new surgical technique  with minimal endothelial cell loss ": Er-Ping Zhang et al. 1996, Graefe's Arch Clin Exp Ophthalmol 234: 714-719). Briefly, this works as follows: The Trans Corneal plantation is performed in 5 month old BALB / c (H-2d) mice. MHC-different C3h (H-2k) mice serve as donors. Become recipient mice with a mixture of 6 mg / kg body weight xylazine (Rompun, Bayer) and 75 mg / kg Body weight anesthetized ketamine hydrochloride (Ketanest, Parke-Davis). The Donor cornea is removed using Catroviejo scissors. The cornea will sewn into the receiver eye with 8 seams. At the end of the operation, the Eyelids sewn for 24 hours. The mice are given cortisone for 11 days treated (5 times a day, 2 drops of Spersadex solution).

3. Ballistic gene transfer in mouse eyes

The transfection of the cornea takes place on day 11 with a commercially available one Ballistic transfer device (model "Helios", Bio-Rad, Munich). For every Transfection is used 0.1-0.5 mg gold (1.6 µm; Bio-Rad), which according to Manufacturer's specification with 1 µg minimalistic gene expression vectors codie rend for murine CTLA4 and / or murine interleukin 4 (composition of the Loading varies depending on experimental parameters). The 1.6 µm Large gold particles are grafted onto the transplant using helium under pressure (200 psi) Eye accelerated on day 10 after surgery.

To measure the expression of transgenes, minimalistic expression vectors coding for luciferase were introduced into the native corneas of mice using the same methodology as described above for CTLA4 and IL-4. The corneas were removed at the time of the measurement, snap frozen in liquid nitrogen, homogenized and examined for the expression of luciferase by known methods (see above: catalog from Promega Inc. Madison, Wisconsin, USA). The results of the measurements are shown in Fig. 1. From this, a maximum of expression can be seen after only 24 hours. The measured values shown are mean values from three measurements.

Expression vectors for IL-4 and IL-10 were also introduced into mouse corneae. The results of the expression, determined using a commercially available ELISA for the gene products in question, are shown in FIG. 2. Corneae were transfected using the ballistic method as described. Coated gold particles with a size of 1 μm were used with expression vectors for the designated genes; mouse eyes were treated with uncoated gold analogously as a control. One recognizes an expression of the transfected cytokine gene increased in the case of the IL-4 from a low basal level by two magnitudes, as well as an also significantly increased expression of the IL-10, but of an already increased level, presumably due to the transfection method. The measured values shown are mean values of three measurements.

The results of graft survival study are shown in FIGS. 3 and Fig. 4. In Fig. 3, the number of days past until the clinically manifest rejection reaction of the graft is plotted on the Y axis over the various groups; in Fig. 4 on the X-axis.

Claims (14)

1. Means for gene therapy treatment of corneal diseases of the human or animal eye to suppress the transplant ons rejection, consisting of an expression construct with one for one immunomodulatory protein or polypeptide coding sequence below Control of a functional promoter and a functional polyadeny lation signal. 2. Composition according to claim 1, wherein the expression product is the cytokine interleu kin 4 (IL-4) expresses. 3. Composition according to claim 1, wherein the expression product of the cytoknn interleu kin 10 (IL-10) expresses. 4. Composition according to claim 1, wherein the expression product of the extracellular Expresses part of CTLA4 (CD 152). 5. Composition according to claim 1, wherein the expression product inhibitors of ko stimulatory signals, especially non-signal-inducing protein of CD-40 or CD-40 ligand, or B7.1, B-7.2 or CD-28 ter in particular antibodies against CD-40 or CD-40 ligand, or B7.1, B- 7.2 or CD-28, expresses. 6. Composition according to one or more of claims 1 to 5, wherein the Promoto ren are selected from the group of the immediate early promoter of the Cy tomegalievirus (CMV) or the viral promoter (SV40) or for cornea cells of the eye tissue-specific keratin gene promoters.   7. Composition according to at least one of the preceding claims, wherein the Expression constructs dumbbell-shaped, linear double-stranded covalently ge are closed nucleic acid molecules. 8. Composition according to claim 1, wherein this in the tissue of the cornea or tissue surrounding the eye is inserted so that the se expression constructs for the synthesis of the immunomodulatory protein or peptides, or a plurality thereof. 9. Medicament or vaccine containing the agent according to claim 1 to 8 Use in gene therapy. 10. Procedure for the treatment of corneal diseases of the human or animal eye, in which the cornea or parts thereof with a Agent according to claim 1 to 8 is treated. 11. The method according to claim 10, wherein the agent via transfection, lipofection, Injection, ballistic transfer, viral vectors, electroporation or any other means is introduced into cells of the cornea or parts thereof. 12. The method of claim 10 or 11, wherein isolated corneal cells with the agent are treated according to claims 1 to 8. 13. The method according to claim 10 or 11, wherein stem cells of the limbuse epithelium of the human or animal eye by means of the An Proverbs 1 to 8 are treated. 14. Corneal cells treated with an agent according to claims 1 to 8.