MXPA00006546A - Use of pigmented retinal epithelial cells for creation of an immune privilege site - Google Patents

Abstract

The present invention relates to a novel in vivo method for creation of a localized immunosuppressive environment in tissue. The method involves the transplanting of pigmented retinal epithelial cells into a mammal thereby producing a localized immunosuppressive environment. The transplanted pigmented retinal epithelial cells may also be used to produce therapeutic proteins or other biologically active molecules that may be useful in treatment of diseases.

Description

USE OF PIGMENTED RETINAL EPITHELIAL CELLS FOR THE CREATION OF AN IMMUNE PRIVILEGE SITE TECHNICAL FIELD The present invention relates to a novel in vi ve method for the creation of a localized immunosuppressive environment in tissues. The method involves the transplantation of retinal, pigmented epithelial cells in a mammal, resulting in a localized immunosuppressive environment. Retinal, pigmented, transplanted epithelial cells can also be used to produce therapeutic proteins or other biologically active molecules that may be useful in the treatment of diseases.

BEFORE THE INVENTION OF THE INVENTION Certain chronic diseases result in the destruction of functional cells in affected organs. Mammals with such diseases are often unable to produce proteins or hormones necessary to maintain normal physiological function. In such REF.121385 cases, the transplantation of healthy organs or cells to the affected mammal can alleviate the symptoms of the disease. Cell and tissue transplantation is being used therapeutically in a wide range of disorders including, but not limited to, cystic fibrosis (lungs), renal failure, degenerative heart disease, diabetes, neurodegenerative disorders, liver failure and pancreatic insufficiency. Unfortunately, such transplants are frequently rejected by the body due to an immune response initiated in response to tissue or foreign cells. Currently, the only recourse to prevent rejection of transplanted tissue is to administer immunosuppressive agents, but the individual is placed at medical risk making immunosuppressive therapy itself more a liability than a benefit in some cases. Therefore, the benefits of transplantation have been limited by the serious side effects of systemic immunosuppression, which is necessary if successful transplantation is to be achieved in humans. It has recently been discovered that there are privileged immune sites in the body where the grafted tissue can survive for prolonged periods of time (Streilein, J. W., 1995, Sci ence 270: 1158-1159). Such sites include, for example, the eye, the testicles, and the brain. The characteristics of the privileged sites include intratissular structural barriers such as the presence of a blood-tissue barrier, absence of efferent lymphatic organs and direct drainage of tissue fluid into the blood. Additional features of the privileged immune sites include the establishment of an immunosuppressive environment through the secretion of immunosuppressive cytokines such as TGFβ or Fas L. It is believed that the Fas L protein is particularly important for the prolonged survival of the grafted tissue and is believes that it acts through the activation of apoptosis in T cells activated by the antigen, Fas +, of the patient (Griffith, TS et al., 1995, Sci ence 270: 1189-1192). The eye, an organ segregated into two anatomically distinct regions, is a particularly interesting example of a privileged immune site. The immune privilege in the anterior chamber is believed to be due to Fas L, whereas in the posterior chamber it is believed to be due to the physical barrier created by the pigmented, retinal epithelial cells (RPE) of the retina, secreting the posterior chamber of the immune cells of the blood. Based on this, it would be surprising of course if isolated RPE cells, no longer in a tight confluent layer, could produce a privileged immune site. The present invention is based on the discovery that pigmented, retinal epithelial cells secrete Fas L and are able to function outside the structural confines of the retina to produce a privileged immune site. The expression of Fas L in the privileged immune site of the eye is believed to directly kill activated lymphocytes that can invade the eye in response to inflammation, and with this destroy the vision when reacting with important structures such as the retina. The expression of Fas L protein by retinal pigmented epithelial cells is surprising given the fact that they also express the receptor for Fas L (Esser et al., 1995, Bi och, Bi ophys Res. Com. 213: 1026-1034) . However, the cells appear resistant to the signals for apoptosis.

Recently, studies have suggested that Sertoli cells, when transplanted simultaneously with pancreatic islet cells in the diabetic rat, act as an effective local immunosuppressant on host tissue (Selawry and Cameron 1993, Cell Transplan ti on 2: 123-129). This cell transplantation protocol is achieved without prolonged systemic immunosuppression, otherwise necessary when the islets are transplanted without the Sertoli cells. As a result, the graft is not rejected and the islets remain viable allowing the cells of the transplanted pancreatic islets to function normally and produce insulin for an indefinite period of time. Graft survival seems to correlate with the constitutive expression of Fas L by Sertoli cells. The development of methods designed to improve productive cell transplantation techniques could be useful for the treatment of diseases such as Parkinson's disease and diabetes. Likewise, it is desirable to avoid systemic immunosuppression with the ability to immunosuppress locally (eg, at the site of the graft) by administering an immunosuppressant that is biologically tolerated by the host. Therefore, identification of cells capable of distributing local immunosuppression and promoting efficient graft acceptance and functional restoration of tissue-related dysfunction is desirable.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a novel method for the creation of an immunologically privileged site in a mammal. The method of the invention comprises the transplantation of retinal pigment epithelial cells (RPE), whereby an immunosuppressive environment located at the transplant site is produced. The present invention relates to the discovery that RPE cells secrete large amounts of the immunosuppressive cytokine referred to as Fas ligand (Fas L). The Fas L protein is believed to exert its immunosuppressive effect by stimulating apoptosis in the T cells activated by the Fas + antigen of the patient. In addition to immunosuppressive cytokines, RPE cells produce additional biological factors such as growth factors, cytokines, and hormones, which may be useful in the treatment of a wide range of different diseases. The invention further relates to the co-administration of RPE cells together with cells that deliver a functionally active therapeutic molecule, as a method for the treatment of diseases resulting from a deficiency of a biological factor in a mammal. In cases where the RPE cells are co-administered with the cells and / or matrices that deliver therapeutic molecules, the RPE cells can be co-administered either as a single composition, or alternatively, as separate compositions. When the RPE cells are administered as a separate composition, the RPE cells can be administered before the co-administration of the cells supplying a therapeutic protein, or the biologically active molecule, in an amount sufficient for the creation of a site of immune privilege. The co-administration of RPE cells has the advantage that the RPE cells create an immunologically privileged site, thereby increasing the survival time of the co-administered cells. Co-administered cells that produce functionally active proteins or biologically active molecules include, but are not limited to, insulin-producing β cells, neural or non-neural cells producing dopamine or hormone-producing endocrine cells. In yet another embodiment of the invention, RPE cells can be engineered to produce a therapeutic protein or a biologically active molecule that can be useful in the treatment of the disease. For example, RPE cells can be engineered to produce a wide range of proteins including, but not limited to, growth factors, cytokines, or biologically active molecules such as hormones. The ability of RPE cells to suppress the normal graft rejection response, ordinarily stimulated in the host patient, increases the development and viability of the transplanted RPE cells. The invention further relates to the in vitro coupling of RPE cells thereto or a different matrix for purposes of increasing the long-term viability of the transplanted cells. In addition, co-administered cells that produce therapeutic proteins or biologically active molecules can be coupled to the same or a different matrix before transplantation. The materials of which the support matrix may be composed include those materials to which the cells adhere after incubation in vi tro, upon which the cells may develop, and which may be implanted in the body of the mammal. without producing a toxic reaction or an inflammatory reaction that could destroy the implanted cells. The invention provides pharmaceutical compositions comprising RPE cells and a pharmaceutically acceptable carrier. The invention further encompasses pharmaceutical compositions comprising RPE cells and cells that produce a functionally active therapeutic protein, or a biologically active molecule, contained in a pharmaceutically acceptable carrier. The compositions of the invention can be used for the treatment of diseases where the creation of an immunologically privileged site and the administration of a functionally active therapeutic protein, or another biologically active molecule is desired. Such diseases include neurological, cardiac, endocrine, hepatic, pulmonary, metabolic or immune system related diseases. For example, neurological disorders such as Parkinson's disease, Huntington's disease, Alzheimer's disease, ALS, stroke and traumatic head and spine damage can be treated. Non-neurological diseases include, but are not limited to, diabetes, blood coagulation disorders, and cystic fibrosis.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. FACS analysis of Fas L-induced apoptosis. The presence of apoptotic cells is demonstrated by the increased intensity in fluorescence. The percentage of apoptotic cells increases in proportion to the level of Fas L present in the medium. Figure 2. FACS analysis of Fas L-induced apoptosis. Increased apoptosis in the presence of Fas L is indicated in the inserts of the attached table presented forward of each FACS analysis.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for producing a localized, sustained immunosuppressant effect in tissues. This is achieved by the general step of transplanting RPE cells into tissue from the host patient. By sustained localized immunosuppressive effect, it is understood that the transplanted RPE cells will suppress the immune response ordinarily assembled by host tissue to foreign entities such as transplanted cells, and that immunosuppression will occur at the (local) graft site rather than the generalized suppression of the whole body (systemic), which occurs with ordinary methods of immunosuppression by agents such as cyclosporine. In a preferred embodiment, transplanted RPE cells (which are intended to replace dysfunctional cells or that in some way alleviate tissue dysfunction) can avoid being rejected and thereby survive and functonally integrate into the host tissue. In addition, the method of the present invention can also be used wherein the RPE cells are co-administered with additional cells or tissues, such as neural cells, endocrine cells, muscle cells, and other cells that produce a functionally active therapeutic molecule. In addition, the RPE cells can be coupled before transplantation to a natural or synthetic matrix that increases the long-term viability of the transplanted cells. The method of the present invention can be used to improve the performance of tissue transplants by providing localized immunosuppression. That is, the RPE cells can be used to facilitate the survival of the transplant and the graft function of the cells that are transplanted. The present invention is based on the discovery that the RPE cells secrete the immunosuppressive cytokine Fas L. The Fas L protein has been shown to prolong the viability of the grafted tissue through the activation of apoptosis in the lymphocytes activated with the Fas + antigen, of the recipient or patient.

With local immunosuppression by an immunosuppressive agent derived from RPE cells, such as Fas L, there could be no cellular immune attack undertaken against the transplanted cells, including the RPE cells themselves. In addition, since immunosuppression is local and by a biologically tolerable agent, the side effects associated with systemic immunosuppression and cytotoxicity of agents such as cyclosporin, could be avoided. Hence, the RPE cell transplantation method provides a significant improvement over the use of systemic immunosuppression with cyclosporine, as the adjunctive therapy necessary for transplantation. Immunosuppression localized by an immunosuppressive agent derived from RPE cells, such as Fas L, can facilitate the survival of xenografts and allografts. With allografts, cotransplantation with RPE cells would provide localized immunosuppression to eliminate the need for systemic immunosuppression. With xenografts, cotransplantation with RPE cells can provide efficient local immunosuppression to eliminate the need for systemic immunosuppression, or RPE cells can be used in combination with a systemic immunosuppressant to prevent rejection, thereby reducing the dose of the systemic immunosuppressant required. When cotrasplanted, RPE cells can not only provide immunosuppression, but can provide regulatory, nutritional, and other factors which support the survival and / or development of the transplanted tissue. Therefore, RPE cells will not only provide inhibition of the immune response, but will also allow increased development and viability of allografts and xenografts by concomitant trophic support.

RPE cell sources The source of RPE cells is by isolation of primary cells from the mammalian retina. The protocols for harvesting RPE cells are well defined (Li and Turner, 1988, Exp. Eye Res. 47: 911-917, Lopez et al., 1989, Invest. Oph thalmol, Vi. Sci 30: 586-588) and consider a routine methodology (see below). In most published reports of cotransplantation of RPE cells, the cells are derived from the rat (Li and Turner, 1988, López et al., 1989), however, it is contemplated that the method of the present invention can be used with RPE cells from any suitable mammalian source. A preferred source of RPE cells for use with mammals, such as humans, are human RPE cells. However, if available and adequate, porcine RPE cells can be used. In addition, for the isolated primary RPE cells, cultured animal and human RPE cell lines can be used, in the practice of the invention. The methods of the invention further encompass the transplantation of engineered RPE cells to express functionally functional proteins. active, enzymes that produce biologically active factors or biologically active molecules. The present methods and compositions can employ RPE cells engineered to produce a wide range of functionally active therapeutic proteins, enzymes that produce biologically active factors or biologically active molecules including growth factors, cytokines, hormone and peptide fragments of hormones, inhibitors of cytokines, peptide growth and differentiation factors, interleukins, chemokines, interferons, colony stimulating factors and angiogenic factors. Examples of such proteins include, but are not limited to, the TGF-β superfamily of molecules, including the five isoforms of TGF-β and bone morphogenetic proteins (BMP), latent TGF-β binding proteins (LTBP); keratinocyte growth factor (KGF); hepatocyte growth factor (HGF); platelet-derived growth factor (PDGF); insulin-like growth factor (IGF); the growth factors of basic fibroblasts (FGF-1, FGF-2 etc.), vascular endothelial growth factor (VEGF); Factor VIII and Factor IX; erythropoietin (EPO); tissue plasminogen activator (TPA); activins and inhibins. Hormones that can be used in the practice of the invention include growth hormone (GH) and parathyroid hormone (PTH). The DNA segment encoding the protein of interest can be obtained using a variety of molecular biological techniques, generally known to those skilled in the art. For example, cDNA or genomic libraries can be selected using primers or probes with sequences based on known nucleotide sequences. The polymerase chain reaction (PCR) can also be used to generate the DNA fragment coding for the protein of interest. Alternatively, the DNA fragment can be obtained from a commercial source. The DNA encoding the translational or transcriptional products of interest can be engineered by recombinant engineering into a variety of vector systems that provide large-scale DNA replication for the preparation of engineered RPE cells. These vectors can be designed to contain the necessary elements to direct the transcription and / or translation of the DNA sequence in the RPE cells. Vectors that can be used include, but are not limited to, those derived from recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA. For example, plasmid vectors such as pBR322, pUC 19/18, pUC 118, 119 and the M13 mp series of vectors can be used. Bacteriophage vectors can include? GtlO,? gtll,? gtld-23,? ZAP / R and the EMBL series of bacteriophage vectors. The cosmid vectors that can be used include, but are not limited to pJB8, pcV 103, pCV 107, pCV 108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16, and the caromid 9 series of vectors. Alternatively, recombinant viral vectors can be manipulated including, but not limited to, those derived from viruses such as herpesviruses, retroviruses, vaccinia viruses, adenoviruses, adeno-associated viruses or bovine papilloma viruses. Methods that are well known to those skilled in the art can be used to construct expression vectors containing the sequence encoding the protein, operatively associated with the appropriate signals for the control of transcription / translation. These methods include recombinant DNA techniques in vi tro and synthetic techniques. See, for example, the techniques described in Sambrook et al., 1992, Mol ecul ar Cl oning, A Labora tory Manual, Col d Spri ng Harbor Laboratory, N.Y. and Ausubel et al., 1989, Current Protocol in Molecular Biology, Greene Publishing Associates &; Wiley Interscience, N.Y. The genes that code for the proteins of interest can be operatively associated with a variety of different proponent elements / autators. The expression elements of these vectors can vary in their strength and specificities. Depending on the host / vector system used, any of a number of appropriate transcription and translation elements may be used. The promoter may be in the form of the promoter that is naturally associated with the gene of interest. Alternatively, the DNA can be placed under the control of the recombinant or heterologous promoter, for example, a promoter that is not normally associated with that gene. For example, specific promoter / enhancer elements of RPE can be used to regulate the expression of the transferred DNA in the RPE cells. In some cases, the promoter elements may be constitutive or inducible promoters and may be used under appropriate conditions to direct the regulated or high level expression of the gene of interest. The expression of genes under the control of constitutive promoters does not require the presence of a specific substrate to induce the expression of the gene, and will occur under all conditions of cell growth. In contrast, the expression of genes controlled by inducible promoters responds to the presence or absence of an inducing agent. Specific start signals are also required for sufficient translation of the inserted sequences encoding the protein. These signals include the ATG start codon and the adjacent sequences. In cases where the entire coding sequence, including the start codon and the adjacent sequences, are inserted into appropriate expression vectors, no additional signals for translation control may be necessary. However, in cases where only a portion of the coding sequence is inserted, exogenous translational control signals, including the ATG start codon, must be provided. In addition, the start codon must be in phase with the structure of reading the coding sequences of the protein to ensure translation of the complete insert. These exogenous signals for translation control and start codons can be from a variety of origins, both natural and synthetic. The efficiency and the control of the expression can be improved by the inclusion of sequences of attenuation of the transcription, increasing elements, etc. It is also within the scope of the invention that multiple genes can be used, combined over a simple genetic construct under the control of one or more promoters, or prepared as separate constructions thereof or of different types. In this way, an almost endless combination of different genes and genetic constructions can be employed. Certain combinations of genes may be designed, or their use may otherwise be successful, the achievement of synergistic effects on cell stimulation, and any and all combinations such as are intended to fall within the scope of the present invention. Of course, many synergistic defects have been described in the scientific literature, so that a person skilled in the art could easily be able to identify the probable combinations of synergistic genes, or even gene-protein combinations.

For the production of high-yield, long-term recombinant proteins, stable expression is preferred. Instead of using expression vectors containing viral origins of replication, host RPE cells can be transformed with DNA controlled by appropriate expression control elements (eg, promoter, enhancer sequences, polyadenylation terminators of transcription terminators, etc.), and a selectable marker. After the introduction of the foreign DNA, engineered RPE cells can be allowed to develop for 1 to 2 days in an enriched medium, and then switch to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and develop to form foci which in turn can be cloned and expanded into cell lines. This method can be advantageously used to manipulate cell lines expressing a therapeutic gene product of interest. To increase the long-term viability of the transplanted cells, for example, the transplanted RPE cells or the co-administered cells, the cells to be transplanted can be coupled to a support matrix before transplantation. The materials of which the support matrix may be comprised include those materials to which the cells adhere after incubation in vi tro, and on whose cells they may develop, and which may be implanted in the body of the mammal without producing a toxic reaction, or an inflammatory reaction that could destroy the implanted cells or otherwise interfere with their biological or therapeutic activity. Such materials can be synthetic or natural chemicals, or substances that have a biological origin. Matrix materials include, but are not limited to, glass and other oxides of silicon, polystyrene, polypropylene, polyethylene, polyvinylidene fluoride, polyurethane, polyalginate, polysulfone, polyvinyl alcohol, acrylonitrile polymers, polyacrylamide, polycarbonate, polypentent, nylon, amylases, natural and modified gelatin and natural and modified collagen, natural and modified polysaccharides, including dextrans and celluloses (for example nitrocellulose), agar, and magnetite. They can be used either resorbable or non-resorbable materials. Extracellular matrix materials are also intended, which are well known in the art. The extracellular matrix materials can be obtained commercially or prepared by developing cells that secrete such a matrix, removing the secretory cells, and allowing the cells to be transplanted to interact with and adhere to the matrix. The matrix material on which the cells to be implanted are developed, or with which the cells are mixed, can be an indigenous product of the RPE cells themselves. Thus, for example, the matrix material may be the extracellular matrix or the basement membrane material that is produced and secreted by the RPE cells that are to be implanted. To improve cell adhesion, survival and function, the solid matrix can optionally be coated on its outer surface with factors known in the art that promote cell adhesion, development or cell survival. Such factors include cell adhesion molecules, extracellular matrix, such as, for example, fibronectin, laminin, collagen, elastin, glucose, inoglucans or proteoglycans or growth factors, such as, for example, nerve growth factor (NGF). Alternatively, if the solid matrix to which the implanted cells are coupled is constructed of porous material, the factor or factors that promote growth or survival can be incorporated into the matrix material., from which it would be slowly released after the implant i n vi. When coupled to the support according to the present invention, the cells used for transplantation are generally on the "outer surface" of the support. The support can be solid or porous. However, even in a porous support, the cells are in direct contact with the external medium without an intervening membrane or other barrier. Thus, according to the present invention, the cells are considered to be on the "outer surface" of the support even when the surface to which they adhere may be in the form of internal folds or convolutions of the porous support material that are not on the outside of the particle or sphere itself. The configuration of the support is preferably spherical, as in a sphere, but may be cylindrical, elliptical, a sheet or flat strip, a needle or spike, and the like. A preferred form of the support matrix is a glass sphere. Another preferred sphere is a polystyrene sphere. The sizes of the spheres can be in the range of about 10 microns to 1 mm in diameter, preferably about 90 microns, up to about 150 microns. For a description of the various microcarrier spheres, see for example, Fi sh er Bi or t ech So urce 87-88, Fisher Co. , 1987, pp. 72-75; If Gma Cell Cul t ure Ca tal og, Sigma Chemical Co. , St., Louis, 1991, pp. 162-163; Ven trex Produc t Ca tal og, Ventrex Laboratories, 1989; these references are incorporated by reference herein. The upper limit of the size of the sphere can be dictated by the stimulation of the sphere of unwanted host reactions, which can interfere with the function of the transplanted cell or cause damage to the surrounding tissue. The upper limit of the size of the sphere can also be dictated by the method of administration. Such limitations are easily determined by a person skilled in the art.

Pharmaceutical Formulations and Methods to Create an Immunologically Privileged Site The present invention encompasses methods and compositions for creating a localized immunosuppressive environment. Pharmaceutical compositions for use in accordance with the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the RPE cells, and any cells, tissues or matrices that are to be cotransplanted with the RPE cells, and physiologically acceptable salts and solvents can be formulated for administration by surgical transplantation or injection. As used herein, a pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and the like. The use of such media and agents is well known in the art.

The present invention also encompasses the equipment of compartments adapted to receive a container adapted to contain RPE cells, and a second container adapted to contain cells that produce a therapeutic molecule. The invention also relates to an article of manufacture comprising a packaging material and the RPE cells contained within the packaging material. The methods of the present invention encompass the administration of RPE cells to a mammal, to reach localization in proximity to the selected tissue. For example, the location can be any site within the mammalian body such as endothelial tissue, muscle tissue, neural tissue and organs, etc. The proximity of the RPE cells to the tissue is determined by the specific tissue that is transplanted and the function that is sought to be restored in a given transplant. The administration of the RPE cells is achieved by conventional techniques. Preferred techniques for the administration of the RPE cells include the injection of RPE cells into the host or the surgical transplantation of the cells into the host. Before transplantation, the recipient mammal can be anesthetized using local or general anesthesia according to conventional techniques. The number of RPE cells necessary to achieve the purposes of the present invention will vary depending on the specific tissue being transplanted and the desired function of the RPE cells. By. example, the RPE cells are administered in an effective amount to provide an immunologically privileged site. In general, such an effective amount is defined as that which prevents immune rejection of subsequently co-administered cells or tissue. The dose range of the RPE cells to be used in the practice of the invention may vary between 103 and 109 cells, although the preferred dose of RPE cells administered will be between 10 5 and 10 7 cells. The immune rejection can be determined, for example, histologically, or by functional evaluation of the cells or tissue transplanted. In one embodiment of the invention comprising the co-administration of producer cells of a functionally active therapeutic protein or another biologically active molecule, together with the RPE cells, the cells are administered in a therapeutically effective amount. In such embodiment of the invention, the RPE cells may be coadministered as a single composition, or alternatively, as two separate compositions. In addition, the RPE cells can be re-administered in an effective amount as necessary, to sustain an immunologically privileged site. Alternatively, co-administered cells that deliver a therapeutic protein, or another biologically active molecule, can be re-administered in an amount effective to sustain a therapeutic effect. In another additional embodiment of the invention, the transplanted cells can be coupled to a matrix before transplantation. The number of cells to be transplanted can be determined by a person skilled in the art by methods known in the art, and will be dependent on the amount of therapeutic protein or other biologically active molecule that is produced by the cells and the amount Therapeutically effective, known, molecule, necessary to treat the disease.

The following examples are provided to illustrate, but not to limit the invention.

EXAMPLE PRODUCTION OF FAS L IMMUNOLOGICALLY AND BIOLOGICALLY ACTIVE BY RPE CELLS The following section describes the experimental results demonstrating that retinal pigmented epithelial cells express biologically active Fas L. Enzyme-linked immunoassays with the anti-Fas L antibody indicated that substantial amounts of Fas L were released into the culture medium by pigmented, retinal epithelial cells. In addition, the secreted Fas L was biologically active in the induction of apoptosis in human fetal thymocytes.

Isolation Culture of Epithelial Cells Pigmented Retinal The primary isolates of RPE cells were made using fetal human eyes, from 18 to 20 weeks of gestation. The fetal eyes are collected within 15 minutes of conception and their outer surface is briefly washed with sterile, cold saline to eliminate as much external contamination as possible. The ocular tissue is transferred to a dissection box containing solution A (culture medium RPMI 1640 (Gibco, Cat. No. 22-400) to which is added a stock solution of penicillin / is treptomycin / fungizone (Gibco , Cat. No. 15240-039) to give a final concentration of 2% vol / vol). Using sterile forceps and scissors, excess fatty tissue is trimmed from the ocular tissue. Using a disposable sterile scalpel, the ocular tissue is sectioned just behind the iris and the front tissue is discarded. The posterior 2/3 of the ocular tissue are sectioned from the top to the bottom with the scalpel, and the internal faces of the two halves face upwards. Each half is then attached to the silicone layer at the bottom of the dissection box using sterile disposable 23 gauge needles, 7.6 x 10 cm (3-4 inches) (Baxter, Cat. No. 23GI). This exposes the pigmented retinal epithelial cell layers, which are gently detached from the choroid membrane to which the sheet of RPE cells is coupled. Usually, two large sheets of RPE cells are recovered from each eye. Once the RPE cell layer is decoupled, it is examined microscopically to determine if there is significant contamination with the choroidal membrane. The RPE cell layer is transferred from the box to 10 ml of a sterile A solution. Filtered collagenase (Liberase ™, Boehringer Mannheim) is added to a final concentration of 1 mg / ml. The RPE fabric is transferred to a water bath at 37 ° C and incubated for 15 minutes. The tube is then centrifuged at 100 x g for 5 minutes at room temperature in a laboratory table centrifuge, Beckman (Beckman, Model No. GPR). The tube is transferred back to the laminar flow hood and the aqueous phase is sucked gently. Ten ml of culture medium (RPMI 1640 containing 10% fetal calf serum, 2 M glutamine, and acid FGF, 10 ng / ml) is added and the RPE tissue in the button or pellet is resuspended. A small aliquot of the suspension is placed on a microscope slide and examined microscopically. The digestion step with collagenase produces a limited fragmentation of the protection or envelope of RPE cells and eliminates the small residual choroidal tissue and the associated cellular contaminants, but does not result in a dissociation of the RPE cell layer in single cells. The RPE cells derived as described above were suspended in 10 ml of Culture Medium to which the stock solution of the antibiotic / antifungal was added to a final concentration of 1%. All culture reagents (medium, serum, FGF, glutamine and trypsin used for subculture) have been qualified for the manufacture of cells by GMP (good manufacturing practices) by Washington Labs. These qualified reagents are supplied by Washington Labs for the initial phase of cellular expansion of primary islets of RPE tissue. The suspension of RPE cells is transferred to 25 ml Falcon culture flasks which are coated with a recombinant coupling protein, Pronectin F (Protein Technologies, Cat. No. 5002-00, Lot No. R0117-c) to facilitate the coupling of cells. Flasks are coated as follows: a 5 mg bottle of sterile Pronectin F was dissolved in 5 ml of sterile diluent solution (lithium perchlorate in water) in a laminar flow hood. Aliquots are mixed with qualified Phosphate Buffered Saline (PBS) (Gibco, Cat. No. 14287) to produce a Pronectin F concentration of 10 μg / ml. Five ml of this solution is transferred sterile to Falcon culture flasks, which are allowed to stand in the laminar flow hood for two hours at room temperature. The solution is removed with a sterile pipette and the flask is rinsed twice with PBS free of sterile Pronectin F. The flasks were allowed to dry in the laminar flow hood after removal of the second rinse solution. The caps are placed hermetically on the flasks and the flasks are stored under refrigeration for up to 4 months for the culture of RPE cells. The coating with Pronectin F facilitates cell division by a factor of 4 to 5 times, compared to that observed with uncoated flasks. The results observed with Pronectin F are approximately equivalent to those observed with mouse laminin (Gibco, Cat. No. L2020) and culture flasks coated with human laminin (Sigma, derived from placenta, Cat. No. L6274). The initial culture of RPE cells is performed in culture medium to which a supplement of the antibiotic / antifungal solution is added, from the reserve solution to a final concentration of 1%. The cultures are uniformly contaminated by microbial agents that are acquired by the tissue during transit through the birth canal, if antibiotic / antifungal solution is added, supplemental to a final concentration of less than 1%. The cultures are evenly contaminated by microbial agents that are acquired by the tissue during transit through the birth canal, if the antibiotic / antifungal supplement is omitted from the culture medium. Antibiotic / antifungal agents are maintained in the RPE cultures for approximately 2 weeks, with medium changes at least once a week. After this, the cultures are changed to antibiotic / antifungal free culture medium for an additional two weeks. Less than one culture in 10 shows evidence of contamination with bacteria, yeast or fungi after switching to antibiotic / antifungal free medium, provided that the antibiotic / antifungal reagent is present from the time of tissue onset. The frequency of changes of the medium during the culture of RPE cells is dictated by the changes in glucose and lactate in the cultures. After initial plating of the RPE cells, aliquots of the medium are removed from the flasks once every two to three days and subjected to glucose and lactate analysis, using a YSI glucose-lactate analyzer.

(YSI, Model No. 2700). The analyzer is standardized in each assay using internal glucose and lactate standards provided by YSI. If the analysis indicates that the crops have consumed more than 1/2 to 1/3 of the glucose, the culture medium is changed. As a minimum, the culture medium is changed once a week, to ensure that the effective concentrations of antibiotic / antifungal agents are maintained. A comparison of the glucose consumed to the lactate produced is also determined. The uninfected culture medium showed a glucose: lactate ratio of 0.80: 1 and more in sparsely populated to almost confluent cultures. Excess production of lactate by scarce crops is observed as an indication of contamination with bacteria, and such cultures are discarded. Excessive glucose consumption in the absence of approximately equivalent lactate accumulation is observed as an indication of fungal or yeast contamination and such cultures are discarded. The yields of RPE cells directly from a single eye are in the range of about 250,000 to 1 million cells. The cells are small, round and filled with melanin granules that give the cells a dark black appearance. After introduction into the culture, the cells migrate out of the fragments of the RPE sheets that are coupled to the flasks. The melanin granules are visible in more than 95% of the cells that migrate, and constitute an index of the purity of the RPE cells in the preparation. Morphologically, the RPE cells change from small, round black cells to larger cuboidal cells, with pigmentation greatly diminished as they diffuse outward from the tissue fragments of RPE. The original morphological appearance is reacquired, after the establishment of the confluence of the crop. At confluence, the 25 cm2 culture flask produces approximately 5 million cells. Cells are recovered from the flask by exposure to 0.2% trypsin (radiation sterilized, graded) for 10 minutes, followed by scraping the cells from the surface of the flask with a sterile spatula (CoStar, Cat. No. 3008) . Scraping is necessary because the cells are very strongly adherent and the long times needed for the dissociation of the cells from the flasks, with digestion with trypsin alone, produces very low cell viability (10% or less). The combination of trypsinization and scraping produces preparations with more than 90% viability as judged by the exclusion of Tripan Blue dye. The RPE cells recovered from the flasks are divided into three aliquots and processed subsequently as follows. Aliquot 1 (approximately 4.5 million cells) and Alícuota 2 (approximately 0.45 million cells) in 1 ml of antibiotic / antifungal free culture medium is adjusted to a final concentration of 7.5% with DMSO (Sigma, Cat. No. D2650, endotoxin-qualified and culture tested) and 20% fetal calf serum. The cells are transferred to cryopreservation flasks and frozen in a controlled-rate cryopreservation apparatus (Nalge, Cryo-l-C, Cat. No. 5100-001). The flasks used are from Corning (Corning, Cat. No. 25704). Aliquot III is used for immunoperoxidase staining, immunofluorescent staining and immunohistochemical staining for known markers for RPE cells. The cells are plated on sterile, multi-well glass slides, coated with Pronectin F, the cells are allowed to attach overnight in the culture incubator and then further evaluated for the presence of markers to judge the purity of the cells. the RPE cells in culture, including the presence of cyteratin, vesicular dopamine transporter protein, and tyrosine hydroxylase.

ELISA Testing of Conditioned Media RPE cells were isolated and cultured as described above, except that the collagenase used was from Sigma Type la (Cat. No. C-9891) and also two culture media were used in different experiments, as described. Initially, the cells were placed either in DMEM-F12 culture medium (Gibco, Cat. No. 12440-20 and 1765-021) or in RPMI 1640 medium (Gibco, Cat. No. 21870-084). Both culture media were supplemented with 2 mM glutamine, 10% fetal calf serum, an antibiotic / antifungal reagent and acid FGF (10 ng / ml). The cells were plated in culture flasks coated with either mouse laminin (Gibco, Cat. No. L2020) or with Pronectin F (Protein Technologies, Cat. No. 5002-00, Lot No. R0117-C). The RPE cells were grown to confluence and transferred to the DMEM / F12 medium containing 10% fetal calf serum or RPMI 1640 medium containing 2% fetal calf serum. When DMEM / F12 was used, the cells were seeded onto plates on laminin-coated flasks. If the RPMI 1640 medium was used, the cells were subcultured on flasks coated with Pronectin. When the RPE cells reached confluency, the culture media were harvested and stored frozen at -80 ° C until assayed for the presence of Fas L by ELISA or bioassay with fetal thymocytes. The ELISA test protocol includes the following steps. Ninety-six well plates (Biologicals quality, Cat. No. 3791) are coated with the human anti-Fe L antibody (Santa Cruz Biotech., At No. SC-956 or Pharmingen, Cat. No. 65431a) by addition of 100 μl / well of a stock antibody solution (10 μg antibody / ml) and allowing the coating to proceed overnight in the cold room. The 96-well plates are then washed three times with 0. 5 ml of phosphate buffered saline (PBS, Irvine Scientific, Cat. No. 9240) which contains 0. 05% Tween (Tween-20, BioRad, Cat. No. 170-6531). The nonspecific binding of the protein was then minimized by coating the unbound sites on the plates with 200 μl of 1% bovine serum albumin (Amersham, cat No. RPN 412) in PBS. After standing for 2 hours at 37 ° C, the blocking solution is decanted and the wells are washed once with 0.5 ml of PBS-Tween. The plates prepared as described above were further incubated either with the peptide Fas L (Santa Cruz, Biotech, Cat. No. SC 956L, 0-100 ng in 100 μl of PBS to generate a standard curve) or with 100 μl of conditioned culture medium, harvested from the RPE cells (pass 0, to pass 9). After the Fas L or Fas L peptide in the conditioned medium had bound to the plates for 1 hour at room temperature, the plates were washed three times with PBS-Tween. A second biotinylated, human anti-Fas L antibody was added to form a sandwich (Biotinylated Nok-I antibody, Pharmingen, Cat. No. 65322, 100 μl of a 5 μg / ml solution). After binding for 1 hour at room temperature, the unbound antibody was washed from the plates with 3 washes of PBS-Tween. The solution of avidin-horseradish peroxidase (ABC Vectrastain, Vector Labs., Cat. No. PK-6100) was then added at 50 μl per well, and the biotin-antibody binding was performed by incubation for 30 minutes at room temperature. ambient. The unbound avidin-horseradish peroxidase was removed with three washes of PBS-Tween. One hundred μl of the OPD solution was then added for color development.

The OPD solution (orthophenylenediamine, Sigma, Cat. No. P6662) was repaired by dissolving OPD at 0.5 mg / ml in 50 M citrate phosphate buffer, pH 5.0 (Sigma, Cat. No. P-4922) containing 1% hydrogen peroxide. After the proper color development had occurred by incubation of the plates at room temperature, the reaction was stopped by the addition of 2 N sulfuric acid solution (Sigma, Cat. No. S-1526). The absorption of the plates was determined on a Bio-Tek Microplate BioKinetics plate reader (Model EL 340) using a 490 nm filter. Standard curves were generated using the synthetic N-terminal 22-amino acid peptide of Fas L (SC0567). The peptide was added to the culture medium with identical supplements to those used for cell culture, to generate a standard curve, with 0 to 60 ng of Fas L peptide per 200 μl of reaction medium.

Apoptosis Bioassays Induced by Fas L To determine whether the cross-reactive material was capable of inducing apoptosis, as is the case with intact Fas L (bound or free surface), bioassays were performed. Apoptosis of lymphocyte populations is inducible after the interaction of Fas bound to the cell surface with its ligand, Fas L. The induction of apoptosis requires, however, that the lymphocytes be activated (for example, by treatment with anti-CD3 antibodies for subsets of T cells). Fetal thymocytes are in a high state of activation and could be used for studies of apoptosis without the requirement of activation. The experimental protocol with fetal thymocytes was as follows: 7.5 million human fetal thymocytes, freshly isolated (ABR, Inc.) were incubated in 5 ml of fresh medium or medium conditioned with RPE cells (DMEM / F12 medium containing 10% serum fetal calf) for 6 to 12 hours. The medium conditioned with RPE cells used in the assays had previously been selected for the content of Fas L by ELISA assays and contained the cross-reacting material Fas L in a concentration range of 0 to 13 ng / 100 μl of conditioned medium.

After incubation, the cells were centrifuged in a centrifuge (5 minutes at 100 rpm) and the cell button was fixed, permeabilized and stained using the APO-DIRECTMR equipment provided by Pharmingen. The staining involved the use of propidium iodide for the total DNA content and the use of FITC-dUTP and the terminal deoxynucleotide-transferase (TdT) to mark breaks in the DNA strand. Two color Fas analyzes were performed to quantify propidium iodide and fluorescence of FITC-dUMP, using a Beckton-Dickinson FACS cell sorter by scanning. Electronic gate was used to eliminate cellular aggregates. The data presented therefore refer to single cells.

RESULTS Results of ELISA Assays Standard curves were generated using the synthetic N-terminal 22-amino acid peptide of Fas L (SC9567). The data generated from the standard curve are indicated below.

Conc. Of SC9567 Absorbance Deviation ng / 200 μl (490 nm) Average Standard 0 0.044, 0.046 0.045 0.001 2.5 0.072, 0.076 0.074 0.002 5.0 0.161, 0.121 0.143 0.029 10.0 0.151, 0.197 0.174 0.033 60.0 0.517, 0.629 0.573 0.079 Using the values for the previous standard curve, the values of the cross-reacting material with Fas L in the medium conditioned with RPE cells (RPE CM) (values per aliquot of 100 μl) were calculated, using the anti-Fas Santa Cruz antibody. L and they are listed right away.

Sample Analyzed. Absorbance Deviation ng of Fas L Medium (490 Standard (per 100 μl) nm) (Absorption) Medium Control 0.063 0.001 2.5 RPE CM 0.091 0.004 5.2 (DMEM / F12, P0) RPE CM 0.114 0.017 6.5 (DMEM / F12, Pl) RPE CM 0.115 0.02 6.6 (DMEM / F12, PE) RPE CM 0.139 0.033 8.0 (RPMI 1640, PO) RPE CM 0.292 0.044 17.0 (RPMI 1640, Pl) RPE CM 0.228 0.031 13.0 (RPMI 1640, P2) RPE CM 0.157 0.011 9.0 (RPMI 1640, PO) RPE CM 0.130 0.013 7.5 RPE CM 0.202 0.004 12.0 (RPMI 1640, Pl) RPE CM 0.167 0.006 9.6 (DMEM / F12, PO) ELISA assays of late pass RPE cells developed in RPMI 1640 + 2% or + 10% fetal calf serum or DMEM / F12 + 10% fetal calf serum are shown below. In the first case, the cells were seeded in plaque on flasks coated with Pronectin F, whereas in the latter case, the cells were seeded in plaque on mouse laminin. The mass calculations of Fas L are normalized to the absorbance at 490 nm for the value of the peptide SC9567 Fas L at 5 ng / assay. A control is also included for the medium not exposed to the RPE cells. The results are as follows: Cells developed in DMEM / F12 + 10% FCS Sample Absorbance Mean Deviation ng of Fas L (490 nm) Standard (Per 100 μl) (Absorption) SC9567, 5 ng 0.461 0.001 5.0 Medium Control 0.063 0.007 0.7 RPE CM, P4 0.870 0.124 9.4 RPE CM, P5 0.544 0.101 6.0 RPE CM, P6 0.442 0.120 4.7 RPE CM, P7 0.136 0.025 1.5 RPE CM, P8 0.529 0.160 5.7 RPE CM, P9 0.793 0.191 8.6 Cells developed in RPMI 1640 + 2% FCS Sample Absorbance Mean Deviation ng of Fas L (490 nm) Standard (Per 100 μl) (Absorption) SC9567, 5 ng 0.461 0.001 5.0 RPMI Medium 0.085 0.012 0.9 Control RPE CM, P4 0.628 0.087 7.0 RPE CM, P5 0.395 0.039 4.3 RPE CM, P6 0.427 0.066 4.6 RPE CM, P7 0.379 0.086 4.1 RPE CM, P8 0.524 0.026 5.7 Cells developed in RPMI 1640 + 10% FCS Sample Absorbance Mean Deviation ng of Fas L (490 nm) Standard (Po r 100 μl) (Absorption) SC9567, 5 ng 0.461 0.001 5.0 RPMI Medium 0.049 0.000 0.5 RPE Control CM, P4 0.653 0.070 7.0 RPE CM, P5 0.418 0.120 4.5 RPE CM, P6 0.452 0.039 5.8 RPE CM, P7 0.425 0.018 4.6 RPE CM, P8 0.359 0.073 4.0 Evaluation of the Conditioned Condition with RPE for the Induction Activity of Apoptosis Against Timocitos The results described above indicate that the RPE cells release material into the culture medium, which is immunologically related to the N-terminal peptide of the Fas ligand in the assays with the Santa Cruz BioTech antibody preparation. Similar experiments were performed using the anti-Fas L antibody obtained from Pharmingen, which confirmed the presence of the cross-reacting material with Fas L. The negative control or positive control cells are treated with FITC-dUTP in the presence of the TdT enzyme. This leads to the incorporation of FITC-dUTP into the DNA fragments found in apoptotic cells. The cells are then stained with propidium iodide and analyzed on u? FACSCANMR by Beckton Dickinson. The presence of apoptotic cells is demonstrated by the increased fluorescence intensity since apoptotic cells are clearly marked with FITC (yellow-green cells), whereas non-apoptotic cells show only red staining of propidium iodide. The results of the FACS analysis are presented in Figure 1 and are summarized in the inserts of the attached table in Figure 2. To briefly summarize, apoptosis in thymocytes incubated in fresh medium (not exposed to RPE cells) was approximately 12%. No indication of apoptosis was observed until the Fas L concentration of the RPE CM had reached its highest value, for example 13 ng / 100 μl of conditioned medium. At this point, the apoptotic value had risen to 24% or approximately twice that of the control medium. Failure to observe apoptosis generated at lower concentrations of Fas L may indicate that Fas L is significantly degraded or may indicate that apoptosis-inducing activity is marginal as free ligand is achieved up to high concentrations.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (29)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for creating an immunologically privileged site in a mammal, characterized in that the method comprises administering retinal pigmented epithelial cells (RPE) at a site in the mammal, in an amount effective to create an immunologically privileged site at said site.

2. A method of treating a disease in a mammal, characterized in that the method comprises administering retinal pigmented epithelial cells (RPE) that deliver a therapeutic protein or a biologically active molecule to a mammal in need of such treatment, wherein the RPE cells are administered in a site in the mammal in an effective amount to create an immunologically privileged site in said site, and sustain a therapeutic effect.

3. A method of treating a disease in a mammal, characterized in that the method comprises co-administering retinal pigmented epithelial cells (RPE) with cells that deliver a therapeutic protein or other biologically active molecule, wherein said RPE cells are administered at a site in the mammal in an effective amount to create an immunologically privileged site at said site, and wherein the cells that deliver a therapeutic protein or other biologically active molecule are administered at the site in an amount effective to sustain a therapeutic effect.

4. A method of treating a disease in a mammal, characterized in that the method comprises administering retinal pigmented epithelial cells (RPE) at a site in the mammal and subsequently administering the cells that deliver a therapeutic protein or other biologically active molecule, wherein said RPE cells are administered in an amount effective to create an immunologically privileged site at said site, and the cells that deliver a therapeutic protein or other biologically active molecule are administered at the site in an amount effective to sustain a therapeutic effect.

5. The method according to claim 2, 3 or 4, characterized in that the therapeutic protein or another biologically active molecule consists of a growth factor, cytokine, hormone, peptide fragment of a hormone, cytokine inhibitor, growth factor or peptide factor. differentiation, interleukin, chemokine, interferon, neurotransmitter, colony stimulating factor or angiogenic factor.

6. The method according to claim 2, characterized in that the RPE cells are transformed by a nucleic acid encoding the therapeutic protein or another biologically active molecule.

7. The method according to claim 3 or 4, characterized in that the cells that produce the therapeutic molecule are cells transformed by a nucleic acid encoding said therapeutic protein or another biologically active molecule.

8. The method according to claim 1, 2, 3 or 4, characterized in that the RPE cells or the cells that supply a therapeutic protein or another biologically active molecule are coupled to a matrix.

9. The method according to claim 3, characterized in that the RPE cells and the cells that deliver a therapeutic protein or another biologically active molecule are coupled to a matrix.

10. The method according to claim 1, 2, 3 or 4, characterized in that the administration is by transplantation.

11. The method according to claim 1, 2, 3 or 4, characterized in that the RPE cells are administered in a dose of 103 to 107 cells.

12. The method according to claim 3 or 4, characterized in that the cells that produce said biological factor are administered in a dose of 103 to 107 cells.

13. The method according to claim 10, characterized in that the transplant is by xenograft.

14. The method according to claim 10, characterized in that the transplant is by allograft.

15. The method according to claim 2, 3 6 4, characterized in that the disease consists of a neurological, cardiac, endocrine, hepatic, pulmonary, metabolic or immunological disease.

16. The method according to claim 1, 2, 3, or 4, characterized in that it further comprises the re-administration of the RPE cells at said site, in an amount effective to sustain an immunologically privileged site at the site.

17. The method according to claim 2, 3 or 4, characterized in that it comprises the readminis of the RPE cells at said site or the cells that deliver the therapeutic protein or another biologically active molecule at the site, in an amount effective to sustain an effect therapeutic.

18. The method according to claim 2, 3 6 4, characterized in that the method further comprises administering a systemic immunosuppressive agent to the mammal.

19. A pharmaceutical composition, characterized in that it comprises retinal pigmented epithelial cells (RPE) and cells that produce a therapeutic protein or other biologically active molecule and a pharmaceutically acceptable carrier.

20. A pharmaceutical composition, characterized in that it comprises retinal pigmented epithelial cells (RPE) and a pharmaceutically acceptable carrier.

21. A pharmaceutical composition, characterized in that it comprises retinal pigmented epithelial cells (RPE) coupled to a matrix.

22. A pharmaceutical composition, characterized in that it comprises retinal pigmented epithelial cells (RPE) and cells that produce a therapeutic protein or another biologically active molecule coupled to a matrix.

23. The composition according to claim 19 or 22, characterized in that the therapeutic protein or another biologically active molecule consists of a growth factor, cytokine, hormone, peptide fragment of a hormone, inhibitor of cytokines, peptide growth or differentiation factor, interleukin, chemokine, interferon, colony-stimulating factor or angiogenic factor.

24. A kit for use in the treatment of a disease in a mammal, characterized in that it comprises a first container adapted to contain retinal pigmented epithelial cells (RPE), and the RPE cells contained within said first container, and a second container adapted to contain cells that produce a therapeutic molecule that is absent or defective from the disease, and cells that produce the therapeutic molecule contained within the second container .

25. A kit according to claim 24, characterized in that the cells that produce a therapeutic molecule are cells of the pancreatic islets of Langerhans.

26. An equipment according to claim 24, characterized in that the RPE cells are coupled to a matrix.

27. An equipment according to claim 24, characterized in that the cells that produce a therapeutic molecule are coupled to a matrix.

28. An article of manufacture, characterized in that it comprises a packaging material and retinal pigmented epithelial cells (RPE) within a packaging material, wherein the RPE cells are effective to create an immunologically privileged site in a mammal, and wherein the material of The container contains a label indicating that said cells can be used to create a univalogically privileged site in a mammal.

29. A method to produce Fas Ligand (Fas L), characterized the method because it comprises (i) the culture of retinal pigmented epithelial cells (RPE) expressing Fas L; and (ii) recovery of Fas L from cell culture.