PROSTHETIC IMPLANT AND METHODS OF USE FOR THERAPEUTIC GENE EXPRESSION

Abstract: The invention provides a method of systemic delivery of a gene product to an individual. The method consists of implanting within the wall of the digestive tract of an individual a prosthetic chamber containing an effective amount of cells secreting the gene product. The prosthetic chamber can be implanted within or proximal to a connective tissue layer including, for example, between the tunica serosa and the tunica muscularis of the digestive tract wall. The gene product to be delivered can be selected from cytokines, hormones, growth factors and blood clotting factors including, for example, Epo or insulin. Expression of the gene product can be constitutive or inducible. Therefor, the invention provides a method of treating a pathology mediated by the deficiency of a gene product. The method consists of implanting a prosthetic chamber containing an effective amount of cells expressing therapeutic levels of the deficient gene product.

Description: PROSTHETIC IMPLANT AND METHODS OF USE FOR THERAPEUTIC GENE EXPRESSION This invention was made with government support under grant numbers DK 43727, DK 47754 and DK 50686 awarded by the National Institutes of Health. The United States Government has certain rights in this invention. BACKGROUND OF THE INVENTION This invention relates to generally to methods of gene delivery and, more specifically to cell implantation methods for sustained engraftment and long term gene expression. The availability of recombinant cloning and expression me.thods has provided significant advances in the therapeutic treatment of human diseases. For example, the availability of recombinant human erythropoietin (Epo) , a regulator of red blood cell production and maintenance, has provided a significant advance in the treatment of renal failure patients receiving dialysis with the elimination of attendant dangers of transfusion therapy and an increase in the quality of life of these patients. The administration of recombinant Epo is now widely used for long-term treatment of anemia associated with chronic renal failure, cancer chemotherapy, and human immunodeficiency virus infections. Long-term, sustained delivery of this hormone, and others like it, by cell therapy rather than by repeated injections would provide substantial clinical and economic benefits. Unfortunately the DNA regulatory sequences which control the expression of Epo in response to tissue oxygenation are too large to be suitable for insertion in a retro viral vector. Similar problems exist for the expression of other hormones and therapeutic proteins as well . Long-term in vivo gene expression requires both target cells and gene delivery vectors that permit continuous vector encoded activity. Of the three prevalent virus-based methods of gene transfer, retro viral vectors are likely the most useful for ex vivo gene transfer. Adeno-associated virus (AAV) vectors have some attractive features, such as safety and ability to transduce non- proliferating cells but do not possess advantages over retro viruses for ex-vivo gene transduction . Replication defective retro viral vectors can be made with high titers, will infect a wide variety of cell types and infection results in stable proviral integration into the host chromosome providing gene expression for the lifetime of the cell and its progeny. Therapeutic genes can be expressed at high level from the viral long terminal repeat (LTR) promoter/enhancer or strong internal promoters and the incorporation of internal ribosome entry sites from picornaviruses into retro viral vectors has allowed the generation of bicistronic vectors conferring linked-gene selection. However, there is always great concern when using replication defective vectors because contamination with a single replication compentant vector can result in infection of the patient with a disease producing virus. Non-hematopoietic cells studied as alternative vehicles for gene therapy include skin fibroblasts, myoblasts and vascular smooth muscle cells. Skin fibroblasts are easily obtained, cultured and transduced but have a major disadvantage of inactivating vector sequences after transplantation. Myoblasts represent one target cell type for gene therapy. Transduced skeletal myoblasts have been used to deliver Epo in mice and to treat alpha-L-iduronidase deficiency in dogs following transplantation. Additionally, intramuscular injection of plasmid DNA has produced systemic expression of Epo and other therapeutic proteins in mice. Smooth muscle cells are present within the vasculature as a multilayered mass of long-lived cells in proximity to the circulation and also have been investigated as targets for gene therapy. Moreover, transduced vascular smooth muscle cells seeded into carotid arteries in a rat model has provide sustained expression of both marker and therapeutic genes, including for example Epo and granulocyte-colony stimulating factor (G-CSF) . However, this procedure is significantly hindered for human therapeutic applications because it requires arterial injury to achieve cell engraftment. Thus, there exists a need for a reliable and reproducible method which provides long-term sustained gene expression of a therapeutic or beneficial protein with minimal adverse side effects. The present invention satisfies this need and provides related advantages as well . SUMMARY OF THE INVENTION The invention provides a method of systemic delivery of a gene product to an individual. The method consists of implanting within the wall of the digestive tract of an individual a prosthetic chamber containing an effective amount of cells secreting the gene product. The prosthetic chamber can be implanted within or proximal to a connective tissue layer including, for example, between the tunica serosa and the tunica muscularis of the digestive tract wall. The gene product to be delivered can be selected from cytokines, hormones, growth factors and blood clotting factors including, for example, Epo or insulin. Expression of the gene product can be constitutive or inducible. Therefore, the invention provides a method of treating a pathology mediated by the deficiency of a gene product. The method consists of implanting a prosthetic chamber containing an effective amount of cells expressing therapeutic levels of the deficient gene product. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a schematic cross section of a implantation site under the tunica serosa of the stomach. Figure 2 shows the hematocrit values of animals implanted with Epo-secreting, LrEpSN-transduced (closed symbols), and ADA-screting, LASN-transduced (open symbols), vascular smooth muscle cells as a function of time . Figure 3 shows histological cross-sections of PTFE chambers containing transduced smooth muscle cells implanted in the stomach. Figure 4 shows expression levels of endogenous Epo mRNA in animals receiving Epo-secreting cell implants compared to ADA-secreting control cell implants. DETAILED DESCRIPTION OF THE INVENTION This invention is directed to cell transplantation methods for the long-term, sustained delivery of a therapeutic or beneficial protein product. The methods of the invention employ a biocompatable prosthetic chamber surgically implanted into an individual and seeded with the cells expressing a therapeutic or beneficial gene product. The implanted cells become engrafted, providing consistent, long-term expression of the therapeutic product. The method is safe, enables easy engraftment and removal of implanted smooth muscle cells, and can be applied generally to the systemic delivery of a variety of therapeutic proteins including, for example, cytokines, hormones, enzymes and clotting factors. In one embodiment, the prosthetic chamber is implanted at the tissue plane between the tunica serosa and the tunica muscularis which provides a rich growth environment for retention of transduced cells. Therefore, one advantage of the method is that this tissue is composed of smooth muscle cells, is well vascularized and able to provide nutrition for implanted cells with concommitant secretion of the gene product into the circulatory system. In another embodiment, the methods of the invention implant under the tunica serosa genetically modified vascular smooth muscle cells for stable long-term cell engraftment expression of Epo. Smooth muscle cells are present at this location and advantageously potentiate the engraftment efficiency and long-term survival of the transplanted cells because they are a normal constituent of the implanted area. In this embodiment, autologous transduced cells are injected into a chamber created from a polytetrafluoroethylene (PTFE) ring placed between the serosa and muscularis of the stomach. The engrafted PTFE ring becomes vascularized and permits both the long-term survival of transduced smooth muscle cells and an area protected from ingrowth of non-transduced cells. As used herein, the term "systemic" when used in reference to expression of a gene product is intended to mean delivery of the gene product to the circulatory system of an individual. The circulatory system includes those parts of the body receiving blood through either the aorta or the pulmonary artery. As used herein, the term "delivery" is intended to mean presenting to the hematopoietic system the expressed gene product. Presentation can include, for example, direct secretion into the circulatory system or indirect secretion into the extracellular space such that diffusion into capillary beds occurs. Presentation also can include surface expression on the plasma membrane of the implanted cells where cells of the hematopoietic system can come in contact with the expressed gene product. Specific examples of secreted gene products include direct or indirect secretion of cytokines, hormones, growth factors, extracellular matrix proteins and vaccine antigens. Delivery of cell surface proteins can include, for example, cell vaccines which express tumor antigens. Those skilled in the art will know which mode of delivery is applicable to achieve a particular result for a particular gene product. As used herein, the term "gene product" is intended to mean both polypeptide and nucleic acid products that can be expressed or replicated from a nucleic acid. Specific examples of a polypeptide gene product includes peptides, polypeptides or proteins encoded by structural genes. Specific examples of a nucleic acid gene product includes RNA such as mRNA, antisense RNA or DNA and viral vectors for example. As used herein, the term "implanting" is intended to mean the introduction or transplantation of cells in a prosthetic chamber into an individual wherein the cells remain viable after implantation and maintain their gene product expression capability. Implanting includes, for example, directly introducing the cell- containing prosthetic chamber into a tissue, introduction of the cell-containing prosthetic chamber with accessory cells which are capable of grafting to the surrounding tissue or which facilate long-term stability of the prosthetic chamber or gene product expression capability of the implanted cells. Such accessory cells can be, for example, located within the prosthetic chamber, attached to, or, associated with the perimeter of the chamber. Implanting additionally includes, for example, introduction of the cell-containing prosthetic chamber into the tissue with other components such as extracellular matrix components, fragments or other molecules which facilitate adhesion of the cells as well as with soluble or substrate bound growth factors, for example. Implanting is generally performed by surgical procedures but other methods can similarly be employed so long as the manipulation is compatible for use with the particular type of prosthetic chamber being used in to implant the cells. Those skilled in the art will know, or can determine, which mode of introduction is appropriate for a particular type of chamber. As used herein, the term "prosthetic chamber" is intended to mean a devise that is capable of compartmentalizing cells. Compartmentalization allows the initial separation of implanted cells from endogenous cells within the implanted area without substantially affecting the exchange of nutrients, cell by-products and factors necessary for cell viability and proliferation. The compartmentalization effects performed by the prosthetic chamber should also allow the cells to maintain their gene product expression capability. Barriers which form the compartment can be temporary or permanent and can effect the complete encasement of the cells, or alternatively, can allow growth of the implanted cells over and around the barriers as the cells become engrafted and enlarged in the area of implantation. Temporary barriers can include, for example, biodegradable material whereas permanent barriers can include, for example, metals such as titanium and stainless steel, synthetic polymers and biopolymer. The sizes of such prothetic chambers can vary depending on the number of cells to be transplanted and can be about 10mm or greater, between about 7-lOmm and preferably between about 3-6mm in outer diameter or width. However, sizes larger or smaller can be used which can accommodate a desired number of cells or which is suitable for a particular location for implantation. Those skilled m the art will know what sizes within the above range of sizes, as well as prosthetic chambers that lager or smaller than these sizes depending on the intended application. Compartmentalization using partial physical barriers that contain the cells during implantation but are open to the surrounding cells and tissue components m one or more places includes, for example, devices having shapes and structures in the form of a ring, cylinder, horseshoe, planer porous structures or any variation thereof which functionally results in containment of the cells at implantation. A specific example of a prosthetic chamber that compartmentalizes cells using partial physical barriers is a ring of polytetrafluoroethylene (PTFE). Compartmentalization can also be accomplished through the use of a three- dimensional biomatrix containing the cells to be implanted, beads or particles coated with cells being attached through receptor ligand interactions as well as any combination of such compartmentalization devices and biomatricies . Examples of biomatrix include extracellular matrix, including for example, collagen gels. An example of a combination of compartmentalization devises includes cells casted in a collagen gel contained within a PTFE ring. Complete encasement of cells includes, for example, the use prosthetic chambers constructed of semipermeable materials, such as membranes, that are barriers to cells but allow passage out of the barrier of nutrients, cell by-products, growth factors and secreted gene products. For example, cells can be placed into an ampule or sack constructed of this material and then physically sealed. The semipermeable barrier also can be coated with a biomatrix such as extracellular matrix, or the cells can be contained in a biogel matrix or attached to particals coated with extracellular matrix components or cell attachment fragments thereof, for example. As used herein, the term "effective amount" when used in reference to the number of cells secreting a gene product is intended to mean the number of cells that can be implanted and become engrafted so as to secrete a gene product at a level which will reduce one or more symptoms of the target disease. For example, an effective amount of cells expressing Epo consists of the number of cells that would cause a stabilization or increase in red blood cell number as well as a decrease in the rate of dispearance of red blood cell number. Moreover, clinical manifestations of a target disease can also be used as a measure of effective amount of cells. For example, an increased quality of life for blood transfusion patients following implantation of Epo expressing cells or, an increased immune tolerance or an increased rate of progenitor and mature cell production for bone marrow transplant patients following implantation of G-CSF expressing cells. Similarly, an effective amount of cells also is intended to mean the number of cells that can be implanted, become engrafted and secrete a gene product at sufficient levels to produce a desired effect on a biological or biochemical component cells or tissues of the individual. Therefore, where a specific effect on a clinical symptom or phenotype is absent due to the complexity of the target disease or condition, for example, a measurable effect on a biological or biochemical component of the pathway due to the secreted gene product is sufficient to render the number of implanted cells within the definition of an effective amount as used herein. Depending on the level of expression and inherent activity of the gene product, cell populations between about 10 ' and 10 " are sufficient number of cells that can be implanted and become engrafted so as to secrete a gene product at a level which will reduce one or more symptoms of the target disease. However, a cell population greater than about 10', preferably greater than about 10% and more preferably greater than about 10 or more cells when implanted in an individual can provide therapeutic effects in vivo . The invention provides a method of systemic delivery of a gene product. The method consists of implanting within the wall of the digestive tract of an individual a prosthetic chamber containing an effective amount of cells secreting said gene product. The methods of the invention employ cell engraftment as a vehicle for the systemic delivery of a gene product. Long-term engraftment of cells expressing a desired gene product is facilitated through the use of a prosthetic chamber. The chamber is surgically implanted at the site of engraftment and cells are seeded into the chamber either during or previous to implantation. The function of the prosthetic chamber is to contain cells at the site of implantation, which provides an initial favorable microenvironment for cell viability with an increase the engraftment efficiency of implanted cells. The location of the prosthetic chamber can be essentially any tissue in an individual that is accessible to surgical procedures and will depend on, for example, the type of prosthetic chamber used, the cell type being implanted and the gene product or expression elements employed for production of the gene product. Those skilled in the art will know, or can determine a suitable location for implantation given the teachings and guidance provided herein. Generally, a suitable location for implantation of the prosthetic chamber is a tissue, or region of a tissue which is adequately vascualrized and capable of supplying nutrients and growth factors necessary for cell survival. Such tissues can include, for example, muscle including skeletal and smooth muscle, connective tissue, epithielia and endothelia cell linings, basement membrane structures, epidermis and dermis. Certain tissues and organs have morphologies that provide additional advantages for the use of prosthetic chambers as described herein because they naturally form a stable compartment which further facilitates the function of the chamber. Implantation into these regions allows for the use of a broad range of prosthetic chamber types. Tissues which exhibit favorable morphologies include essentially any tissue in which there is a plane between two layers of tissue which are vascularized and can stably support the surgical insertion of a prosthetic chamber. For example, the layers of the digestive tract is one such tissue location that exhibits a morphology favorable for implantation. Briefly, the wall of the digestive tract is composed of four layers. The first, outermost layer is a thin layer of mesothelium and connective tissue and is known as the tunica serosa or serosa. The next layer inward is the tunica muscularis, composed of muscle layers, which is followed by a second layer of connective tissue known as the submucosa. The innermost layer also contains connective tissue and is additionally lined with a layer of epithelial cells. This inner layer of the digestive tract is known as the mucosa. All of these layers are well vascularized which will support cell growth and viability. Therefore, a prosthetic chamber can be implantated into, or between any of the above layers and seeded with cells secreting a desired gene product to achieve systemic delivery of the product. A prosthetic chamber can be implantated into any of the above described tissues or morphological tissue layers to form a microcompartment for cells secreting a desired gene product. Insertion at a plane between tissue layers is one location which can be used for the efficient implantation of a prosthetic chamber. For example, the plane between any of the four digestive tract layers described above are easily accessable by routine surgical procedures, can be separated without substantial damage to the morphological structure or biological function of the tissue layer and inherenty provide a natural crevice once segregated for which a prosthetic device can be stably placed and seeded with cells. For example, the muscle cell bed at the plane between the tunica muscularis and the tunica serosa on the stomach can serve as a location to receive a prosthetic chamber occupied by cells expressing a gene product of interest. This tissue site is well vascularized and will therefore provide nutrition for implanted cells. The long-term survival of implanted cells requires access to nutrients such as oxygen from the blood supply. As discussed further below, smooth muscle cells are a normal part of the architecture of this site. Modifying this cell type to secrete the desired gene would therefore provide additional advantages for the survival of transplanted cells because they are a normal constituent of the targeted area. A specific example of lmpimplantmg vascular smooth muscle cells retrovirally transduced to express erythropoietm and G-CSF is described further below in the Examples. Implantation of a prosthetic chamber can be accomplished by surgical placement of the chamber into any of the tissues or morphological tissue layers described above. Briefly, the chamber is implanted by making an incision at the entry site of the appropriate depth and length to expose the target tissue and physically placing the chamber within the tissue incision. Alternatively, the target tissue can be externalized, followed by one or more incisions until an area in the target location has been created for implantation. Methods for implanting a wide variety of artificial devices and prothesis as well as cell, tissue and organ transplantation and engraftment procedures are well known to those skilled in the art. Similarly, a wide variety of surgical methods applicable for the treatment of diseases, diagnostic indications and cosmetic procedures are also well known to those skilled in the art and are all applicable surgical methods which can be used for implanting a prosthetic chamber of the invention. For example, the use of endoscopes is well known to those skilled in the art and affords a minimally invasive procedure to access and perform surgery on the stomach and intestin. Various modification of and combinations of such methods known to those skilled in the art similarly applicable for implanting one or more prosthetic chambers in an individual for the systemic delivery of a gene product. Specific examples of a surgical procedure which can be used for implanting a prosthetic device and seeding with cells modified to express a desired gene product is described further below in the Examples. Therefore, the invention provides a method of systemic delivery of a gene product where a prosthetic chamber containing an effective amount of cells secreting said gene product is implanted within or proximal to a connective tissue layer of the digestive tract of an individual. The location can be, for example, between the tunica serosa and the tunica musclaris of the digestive tract wall. The invention also provides a method of systemic delivery of a gene product by implanting a prosthetic chamber containing an effective amount of cells secreting said gene product where the prosthetic chamber has an outer diameter or width that is greater than about 10mm, preferably between about 7.0-lOmm, more preferably between about 3-6mm. The prosthetic chamber can consist of various shapes and can be open at one or more locations to the surrounding tissue, or it can be completely enclosed. Prosthetic chambers used in the methods of the invention can be of various shapes and sizes so long as they function to restrain cells seeded into the chamber at the time of implantation and can be surgically manipulated into a desired location within the recipient individual. As described previously, the chamber can either partially or completely encase the cells to provide a beneficial microenvironment for cell growth, viability and long-term engraftment. Prosthetic chambers which particially encase seeded cells include chambers open at one or more locations to the surrounding tissue. Such chambers exhibit the advantage in that they allow for expansion of the engrafted cells by proliferation and migration out of the chamber into the surrounding tissue regions . Shapes of such prosthetic chambers include, for example, a ring, horseshoe and cylinder or any geometric shape or motif that contains an inner region which can contain seeded cells and an outer barrier which serves to restrain the cells. For example, a ring having an inner diameter of about 4mm for seeding cells and an outer diameter of about 6mm is one example of a prosthetic chamber that is partially open to surrounding tissue and capable of containing cells at the site of implantation. Cells seeded into the center of the ring are initially restrained by the outer barrier at implantation. As the implanted cells engraft and expand they are able to migrate out of the ring and over the outer barrier forming a patch of stably engrafted cells. Other shapes having similar functional features such as those listed above as well as squares, hexagons, rectangles, triangles and the like also can be used for a prosthetic chamber in the methods of the invention. Therefore, the shape of the chamber is unimportant so long as the chamber functions to contain cells seeded at the site of implantation . Prosthetic chambers having various alternative forms to those described above also can be used in the methods of the invention. For example, prosthetic chambers composed of a planar, porous material will similarly function to contain cells at the site of implantation, allow access to the surrounding tissue and exchange of nutrients, factors and cell by-products. Cells can be seeded into the pores which completely, or partially extend, through the planar material. One advantage with this structure for a prosthetic chamber is that the surface for cell containment is increased portional to the number and pores. Additionally, cells can be seeded onto the outer surfaces of the plane by, for example, coating it with cell adhesion proteins or substrates and allowing the cells to attach through receptor-ligand interactions. Concave disks of various shapes and depths can similarly function as a prosthetic chamber to contain cells at the site of implantation. As with porous chambers, concave disks can be coated with adherent proteins to facilitate cell attachment at the time of implantation or, alternatively, preseeded with cells prior to implantation. Prosthetic chambers that can be used in the methods of the invention also can be of various three- dimensional shapes and sizes. For example, the chambers can be cylinders and cones which have an inner area for containment of the cells and an outer barrier. Additionally, chambers can be particles or spheres which can be coated which adherent cells prior to or during implantation. The particles or spheres can be porous or textured so as to increase the surface area and therefore the number of cells capable of being implanted using such a chamber. Therefore, the chambers which can be used in the methods of the invention are not limited to a particular geometric shape, structure or three- dimensional form. The selection of a particular shape or form for use in the methods of the invention will depend, for example, on the site of implantation, number cells to be seeded and surgical preference by those skilled in the art. For example, if the prosthetic chamber is to be implanted between tissue layers, such as between the tunica serosa and the tunica muscularis of the digestive tract, those skilled in the art can select, for example, a ring, horseshoe, other geometric shape, or other planar chamber that can be inserted between the tissue layers with minimal damage and preturbation of the surrounding tissue. Those skilled m the art will know, or can determine, what shape and structure of the prosthetic chamber is appropriate for a particular tissue location. Prosthetic chambers which completely encase seeded cells include chambers such as spheres, ampules, and sacks constructed of semipermiable barriers . Such chambers can be advantageous when using non-autologous cells because they physically block host immune cell attack and therefore reduce graft rejection by this mechanism. The use of such completely enclosed prosthetic chambers therefore allows for the implantation of non-autologous cells and reduces or eliminates the need for immunosuppressive drugs which compromise the recipient's immune system. The shape of such chambers can additionally be any of those described previously except that the exposed areas of the previously described chamber shapes would instead be enclosed with a semipermeable barrier. For example, a ring can be used where the top and bottom planes are enclosed with, for example, a semipermeable membrane, thus forming an enclosed cylinder or oval-shaped structure having cells seeded into the internal area prior to implantation. The prosthetic chambers used in the methods of the invention can be of various sizes and will depend on the desired number of cells to be implanted, in addition to the site of implantation and associated considerations described above in regard to selection of the shape of the chamber. For example, a ring having an inner diameter of about 4mm will accommodate about 1x10 or greater number of cells. Larger chambers larger will accommodate proportionally more cells and conversely, smaller chambers can be used to implant proportionally less cells. In addition, the total number of cells can be substantially increased by about 2- to 10-fold, or more if porous or textured chambers are used. The chambers therefore can be essentially any size which the tissue at the site of implantation can physically accommodate and which is in acceptable medical parameters for implantation of a prosthetic device. For example, a prosthetic chamber between about 7 and 10mm or greater in outer diameter or width and as small as 3 to 6mm can be accomodiated at the tissue plane between the tunica serosa and the tunica muscularis. The inner diameter or width of a prosthetic chamber used in the methods of the invention similarly can be within these same size ranges and can depend, for example, on the type of material used and the methods of fabrication. The height of the prosthetic chamber is generally about 3mm to allow sufficient diffusion of oxygen into the implanted cell area. However, the height of a chamber can be as small as l-2mm and as large as 4-5mm or greater. Moreover, the height can be of various other sizes depending on the site of implantation and the shape and surface area exposed to the surrounding tissue and vasulature. Those skilled in the art will know how to vary the height of the prosthetic chamber to achieve sufficient diffusion of oxygen and other nutrients to maintain viability and support engraftment of the implanted cells given the teachings and guidance provided herein . The above described prosthetic chamber sizes will accommodate populations of cells from about 1x10 " or smaller. The size of the cell population to be implanted can be, for example, as small as 1x10- or less. However, larger populations such as 1x10 , 1x10 , 1x10" and 1x10 are, in general, the amount of implanted cells that will secrete a sufficient amount of the desired gene product to accomplish the targeted functional effect. Therefore, any of the prosthetic chambers described above, as well as others known to those skilled in the art are applicable for use in the methods of the invention The number of cells required for an effective amount will vary depending on the gene product being secreted, its expression and secretion level into the extracellular space and its inherent biochemical activity. For example, a gene product that has a high affinity for substrate or ligand will inherently require less amounts to be secreted to achieve the same level of activity when compared to a lower affinity homologue. Similarly, a gene product with a longer half life also will require less amounts or rate of secretion to achieve comparable activities or accumulation levels to a shorter lived gene product. Therefore, an effective amount of cells will vary depending on inherent properties of the gene product to be secreted and expression characteristics of the modified cells, all of which are criteria well know to those skilled in the art. Therefore, the invention also provides a method of delivering a gene product systemically by implanting an effective amount of cells secreting the gene product where the number of cells is a population of about 1x10' cells or greater, preferably about 1x10' cells or greater, more preferably about 1x10' cells or greater. Fabrication materials for the prosthetic chambers can be of any solid, porous or degradable material so long as it is biocompatable . For example, the chamber composition can include surgical grade metals, synthetic polymers and biopolymers. Examples of synthetic fabrication material is polytetrafluoroethylene (PTFE), gortex© and carbon fiber. Surgical grade metals include, for example, titanium and stainless steel whereas biopolymers can included, for example, collagen, fibronectin, proteoglycans and active fragments thereof. The prosthetic chambers can additionally include a wide variety of intracellular and systemic biomolecules that augment cell viability and proliferation of implanted cells. Such biomolecules can include, for example, growth factors and hormones. Additionally, prosthetic chambers containing semi-permeable barriers can be composed of, for example, natural or synthetic membranes with a pore size that excludes cell-cell contact. Generally, a pore size of about 0.22 mm is sufficient to allow exchange of macromolecules such as polypeptides and proteins. However, other pore sizes can also be used without affecting the function of the chamber or of the implanted cells. Specific examples of chambers made from some of these materials are described below and in the Examples. Other materials known to those skilled in the art also can be used for the prosthetic chambers used in the methods of the invention. Briefly, prosthetic chambers used in the methods of the invention can be fabricated with PTFE. This material is non-immunogenic, easily fabricated into various shapes, sizes and formats, can be prepared for implantation using methods well known to those skilled in the art for sterilization and handling and can be manipulated by routinely surgical procedures for implantation. Prosthetic chambers also can oe fabricated using various lengths of 18g cerclage wire, or surgical grade stainless steel wire, which is bent into a "U" or horseshoe shape to restrain cells seeded into the chamber. Modification to the prosthetic chambers can additionally be made to help maintain the shape and volume of the chamber. For example, short lengths or disks of about 0.5-lcm in length or diameter and about lmm thick PTFE or other compatible material can be inserted in the chamber after the cells are introduced to achieve this effect. A further modification includes casting the cells that are to be grafted into the chamber m a contracting collagen matrix which serves to both concentrate the cells and to hold the cells place after engraftment. This type of prosthetic chamber is applicable with any biocompatible material that can be manipulated into a shape to contain cells. The inner diameters of any of such chambers can exceed 10 mm and allows for the engraftment of at least about 1x10 or more modified cells. Systemic delivery of a desired gene product utilizes cells as a vehicle for delivery and long-term production of the gene product m the recipient individual. The cells can be modified ex vivo to express in a secreted form the desired gene product, or alternatively, the implanted cells can be natural producers of the gene product. Cell implantation and engraftment is facilitated using the prosthetic chambers as described above. The cell type to be implanted can be any cell type, population of cell types and heterogeneous mixtures, that are compatible with the site of implantation and the recipient's major histocompatibility (MHC) antigens responsible for graft rejection. Compatibility includes modification of the implanted cells or treatment of the recipient so as to allow self- recognition by the recipient's immune system or to down regulate the recipient ' s immune system by immunosuppressive agents. Therefore, and as described further below, selection of the cell type to be implanteα follows criteria for cell, tissue and organ engraftment and/or transplantation which is well known to those skilled in the art. Cell types to be selected for generating the modified cells used in the methods of the invention are those which are capable of polypeptide synthesis and secretion and compatible witn the recipient's immune system and the site of implantation. With the exception of highly specialized cell types, such as red blood cells, the large majority of cells meet these criteria and can be used for constructing the modified cells used in the methods of invention. Alternatively, cells which naturally express the desired gene product can be obtained, for example, from a donor individual or tissue and implanted directly without the need for genetic modification . The cell type chosen for modification is selected according to the biological characteristics of the cell and according to gene expression criteria well known m the art. For example, objective criteria such as the ease of culture efficiency, the ease of genetic modification and other routine cellular and molecular manipulations can be used to evaluate and select the cell type for modification. Those cell types which can be passaged in culture for multiple generations without substantial loss in viability are preferable candidates for modification as systemic delivery vehicles for a desired gene product. As will be described further below, such cell types include, for example, both primary cells as well as cell lines. Additionally, criteria such as the proliferation characteristics can also be evaluated for selection of the cell type to be modified. Cell types are additionally selected according the efficiency with which they can be modified to express and secrete a gene product in the methods of the invention. Cell types that can be readily modified and selected for the expression of the introduced gene or genes by any of a variety of methods known in the art are applicable for constructing the implantable cells of the invention. Availability of promoter and regulatory elements can also be included as a criteria for selecting a particular cell type for modification. Such characteristics and criteria are routine and well know to those skilled in the art. Various combinations of the above exemplary characteristics as well as other characteristics can additionally be used for selecting a cell type to modify. For example, if the objective is to achieve a particular level of gene product secretion using a relatively small number of cells, then a cell type that can be efficiently modified to yield high levels of expression can be selected to achieve the desired result. In contrast, if cell number is not a limiting factor, then it can be desirable to base selection of the cell type on favorable growth or proliferation characteristics. Additionally, various expression elements can be utilized to augment or modulate the level of expression and secretion so as to complement advantageous characteristics or to overcome any deficiencies of the chosen cell type for modification. Such criteria and characteristics are well known or can be determined by those skilled in the art. The cells selected for use in the methods of the invention can therefore originate from essentially any tissue or organ. However, the engraftment efficiency can be inherently augmented by selecting a cell type or composition of cell types that are natural constituants of the tissue where the prosthetic chamber is to be implanted. For example, cellular constituents of the layers of the digestive tract include connective tissue of mesothelial origin, muscle and epithelial cells. Therefore, selecting one of these cell types for implantation at the corresponding tissue layer will yield engraftment into an environment of similar or identical cells and provide a compatible habitat for cell viability and growth. A specific example, is the implantation of modified smooth muscle cells between the layers of the tunica serosa and the tunica muscularis of the digestive tract because the latter layer is composed primarily of smooth muscle cells. Another specific example of a cell type that is useful for engraftment with the methods and prosthetic chambers of the invention is a fibroblast cell. Fibroblast cells can be obtained from a variety of tissue sources including, for example, skm, liver, muscle or arterial tissue. Fibroblast cells are also advantageous m that they are easily obtained and isolated, routinely modified, and can proliferate to higher densities within a graft. For example, a 10 cm area within a prosthetic chamber can contain 10 fibroblast cells, or up to 20 layers of cells, and can secrete essentially any desired gene product at effective amounts to achieve therapeutically beneficial results. Therefore, the invention provides a method of systemic delivery of a gene product where cells secreting the gene product are smooth muscle cells or fibroblasts. The gene product can be constitutively secreted, or alternatively, it can be secteted by mducible regulation . For implanting primary cells, a tissue and therefore a site for implanting a prosthetic chamber, should be selected that is easily accessible and contains cells that exhibit desirable growth and expression characteristics such as those described herein. Additional considerations when selecting a tissue source include choice of a tissue that contains cells that can be isolated, cultured and modified to secrete a desired gene product. In addition to the cellular constituents of the digestive tract layers, other examples of cell types that can be modified to secrete a desired gene product include, for example, muscle (smooth, skeletal or cardiac), fibroblast, liver, fat, hematopoietic, epithelial, endothelial, endocrine, exocr e, kidney, bladder, spleen, stem and germ cells. Other cell types are similarly known in the art that are capable of being modified to secrete gene products and can similarly be obtained or isolated from a tissue source from a recipient or donor individual. Although human tissue sources are advantageous for therapeutic purposes, the species of origin of the cells can be derived from essentially any mammal, so long as the cells exhibit the characteristics that allow for secretion of the gene product of interest. Cells to be used m a prosthetic chamber car be adherent cell types, which can require isolation methods that temporarily digest or release the cells from their surrounding tissue. For example, smooth muscle cells can be obtained from a segment of venous or arterial tissue. The smooth muscle cells can be obtained following enzymatic digestion in trypsin and collagenase and purified by positive selection using muscle cell-specific antigens such as von Willebrand factor (Lejnieks et al . , Blood, 92:1-7 (1998). Alternatively, cells can be isolated following digestion and further purified by centrifugation. The centrifugation can be performed the presence of a gradient such as a sucrose gradient, which would allow for further separation of cell populations based on their density. Methods for the isolation of primary cells from a tissue source are well known in the art (see, for example, Freshney, Animal Cell Culture: A Practical Approach, 2nd ed., IRL Press at Oxford University Press, New York (1992). Maintenance of the cells prior to modification and implantation can be as a cell suspension, adherent cell culture or as organ culture. Conditions for the maintenance and culture of primary and clonal cells are well known in the art. For therapeutic applications, the above cell types are additionally chosen to be implantable in an individual and remain viable in vivo without being substantially rejected by the host immune system. Therefore, the donor origin of the cell type is a further characteristic that should be evaluated when selecting cells for therapeutic application. Those skilled in the art know what characteristics should be exhibited by cells to remain viable following implantation. Moreover, methods well known m the art are available to augment the viability of cells following implantation into a recipient individual . One characteristic for an implanted donor cell type is to exhibit substantial lmmunological compatibility with the recipient individual. A cell is lmmunologically compatible if it is either histocompatible with recipient host antigens or if it exhibits sufficient similarity m cell surface antigens so as not to elicit an effective host anti-graft immune response. Specific examples of lmmunologically compatible cells include autologous cells isolated from the recipient individual and allogeneic cells which have substantially matched major histocompatiDility (MHC) or transplantation antigens with the recipient individual, lmmunological compatibility can be determined by antigen typing using methods well known in the art. Using such methods those skilled m the art will know or can determine what level of antigen similarity is necessary for a cell or cell population to be lmmunologically compatible with a recipient individual. Tolerable differences between a donor cell and a recipient can vary with different tissues and can be readily determined by those skilled in the art. In addition to selecting cells which exhibit characteristics that maintain viability following implantation into a recipient individual, methods well known in the art can be used to reduce the severity of an anti-graft immune response. Such methods can be used to further increase the m vivo viability of lmmunologically compatible cells or to allow the m vivo viability of less than perfectly matched cells or of non- lmmunologically compatible cells. Therefore, for therapeutic applications, it is not necessary to select a cell type from the recipient individual to achieve viability of the modified cell following implantation. Instead, and as described further below, alternative methods can be employed which can be used m conjunction with essentially any donor cell to confer sufficient viability of the modified cells to achieve a particular therapeutic effect. For example, in the case of partially matched or non-matched cells, immunosuppressive agents can be used to render the host immune system tolerable to engraftment of the implanted cells. The regiment and type of immunosuppressive agent to be administered will depend on the degree of MHC similarity between the modified donor cell and the recipient. Those skilled in the art know, or can determine, what level of histocompatibility between donor and recipient antigens is applicable for use with one or more immunosuppressive agents. Following standard clinical protocols, administration and dosing of such immunosuppressive agents can be adjusted to improve efficiency of engraftment and the viability of the cells of the invention. Specific examples of immunosuppressive agents useful for reducing a host anti-graft immune response include, for example, cyclosporm, corticosteroids, and the immunosuppressive antibody known in the art as OKT3. Another method which can be used to confer sufficient viability on partially-matched or non-matched cells is through the masking of the cells or of one or more MHC antigen (s) to protect the cells from host immune surveillance. Such methods allow the use of non- autologous cells in an individual. Methods for masking cells or MHC molecules are well known in the art and include, for example, physically protecting or concealing the cells, as well as disguising them, from host immune surveillance. Physically protecting the cells can be achieved, for example, by encapsulating the cells within a semi-permeable prosthetic chamber as described previously. Such a barrier prevents contact of host immune cells such as T-cells with the cells contained within the chamber but still allows secretion of the desired gene product into the extracellular space and circulation system. Alternatively, antigens can be disguised by treating them with binding molecules such as antibodies that mask surface antigens and prevent recognition by the immune system. lmmunologically naive cells also can be used for constructing the implantable cells of the invention, lmmunologically naive cells are devoid of MHC antigens that are recognized by a host anti-graft immune response. Alternatively, such cells can contain one or more antigens in a non-recognizable form or can contain modified antigens that faithfully mirror a broad spectrum of MHC antigens and are therefore recognized as self-antigens by most MHC molecules. The use of immunologically naive cells therefore has the added advantage of circumventing the use of the above-described immunosuppressive methods for augmenting or conferring immunocompatibility onto partially or non-matched cells. As with autologous or allogeneic cells, such immunosuppressive methods can nevertheless be used in conjunction with immunologically naive cells to facilitate viability of the glucose-regulated insulin producing cells. An immunologically naive cell, or broad spectrum donor cell, can be obtained from a variety of undifferentiated tissue sources, as well as from immunologically privileged tissues. Undifferentiated tissue sources include, for example, cells obtained from embryonic and fetal tissues. An additional source of immunologically naive cells include stem cells and lineage-specific progenitor cells. These cells are capable of further differentiation to give rise to multiple different cell types. Stem cells can be obtained from embryonic, fetal and adult tissues using methods well known to those skilled in the art. Such cells can be used directly or modified further to enhance their donor spectrum of activity. Immunologically privileged tissue sources include those tissues which express, for example, alternative MHC antigens or immunosuppressive molecules. A specific example of alternative MHC antigens are those expressed by placental cells which prevent maternal anti-fetal immune responses. Additionally, placental cells are also known to express local immuno-suppressive molecules which inhibit the activity of maternal immune cells . An immunologically naive cell or other donor cell can be modified to express genes encoding, for example, alternative MHC or immuno-suppressive molecules which confer immune evasive characteristics. Such a broad spectrum donor cell, or similarly, any of the donor cells described previously, can be tested for lmmunological compatibility by determining its lmmunogenicity in the presence of recipient immune cells. Methods for determining lmmunogenicity and criteria for compatibility are well known in the art and include, for example, a mixed lymphocyte reaction, a chromium release assay or a natural killer cell assay. lmmunogenicity can be assessed by culturmg donor cells together with lympohocyte effector cells obtained from a recipient individual and measuring the survival of the donor cell targets. The extent of survival of the donor cells is indicative of, and correlates with, the viability of the cells following implantation. Once a cell type has been selected as described above, cells expressing a desired gene product in a secretable form are generated by introducing a vector expressing the encoding gene into the selected cell type. An expressible nucleic acid encoding a desired gene product can be constructed using methods well known in the art. Such methods are described in, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989)) and Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York (1998)). For example, a nucleic acid sequence encoding the gene product can be obtained using polymerase chain reaction (PCR) to amplify the sequence from an appropriate tissue or cell type. Encoding nucleic acids can be obtained by other methods known to those skilled in the art as well as by cloning or chemical synthesis using sequences available in scientific databases. Once obtained the encoding gene is operationally linked to expression and regulatory elements which direct expression and secretion of the gene product into the extracellular space. Such promoter and regulatory elements are also well know in the art and are selected for compatibility with the cell type to be implanted. For example, retroviral promoter and enhancers achieve constitutively high levels of expression in a wide variety of different cell types, including smooth muscle cells. Whereas a constitutive promoter yielding relatively strong expression levels in fibroblasts can be selected from a variety of extracellar matrix proteins such as fibronectin, collage and laminin, from structural genes such as actin, tubulin and intermediate filaments and from general housekeeping genes such as those in the glycolysis pathway. Selection of compatible expression and regulatory elements for a particular cell type also is well known to those skilled in the art. Promoters can be constitutive such as those described above, or alternatively they can be mducible. A specific examples of mducible promoters is the glucose regulated promoter describe below and in the Examples. For example, glucose-regulated expression of a gene product of interest can be achieved using a variety of promoter elements that control expression of a downstream gene in response to changes in levels of glucose. Elements that are mducible by glucose generally exhibit low activity in the absence of glucose and are up- regulated in the presence of increased glucose or a metabolite within a glucose-associated pathway. For example, it is known that TGF- promoter activity is responsive to glucose (McClain et al., Proc. Natl. Acad. Sci. USA 89:8150-8154 (1992); and Raja et al., Mol . Endocrinol . 5:514-420 (1991)). Also, sequences from other glucose-responsive promoter elements can be used such as promoter elements from wild-type insulin, fibroblast growth factor (FGF), epidermal growth factor (EGF) , PC2 or PC3 genes (Sander et al . , Proc. Natl. Acad. Sci. USA 95:11572-11577 (1998)). Other glucose responsive promoter elements can be found, for example, in genes for acetyl-CoA carboxylase, 6-phosphofructo-2- kmase, and L-type pyruvate kmase (Zhang and Kim, Arch. Biochem. Biophvs . 15:227-232 (1997); Dupriez and Rousseau, DNA Cell Biol. 16:1075-1085 (1997); Antome et al., J. Biol. Chem. 272:17937-17943 (1997); and Kennedy et al., J. Biol. Chem. 272:20636-20640 (1997)). A wide variety of other mducible promoters applicable for use in the methods of the invention are well known in the art. Selection of a particular mducible promoter will depend, for example, on the desired level of expression and as well as the choice of mducer to administer to a recipient individual. Similarly, selection and operational linkage of the encoding nucleic acid to regulatory sequences for processing and secretion into the extracellular space is similarly well known in the art. For delivery of one or more desired gene products to the circulatory system it is necessary for the product to be secreted. Therefore, the encoding nucleic acids should contain a signal sequence for routing to the endoplasmic reticulu and secretion outside of the cell. The signal sequence can be derived from the authentic gene for the product of interest, or alternatively from a heterologous gene and linked in frame for secretion into the extracellular space. Signal sequences and methods of using them are well known in the art. Vectors used in the methods of the invention can be any vector compatable with the host cell or tissue where the desired gene product is to be expressed. For example, the vectors can be plasmid or viral based vectors. The vectors can be known, or constructed from components well known in the art. In the case of a viral vector, the vector can contain, for example, at least one viral long terminal repeat and a promoter sequence upstream and operably linked to a nucleotide sequence encoding the gene product of interest, followed by at least one viral long terminal repeat and polyadenylation signal downstream of the sequence encoding the gene product of interest. Where multiple gene products are to be expressed, a single vector can be used which encodes more than one gene of interest, each gene of interest can be expressed as separate transcription units. Alternatively, multiple genes can be transcribed as a single transcription unit containing internal πbosome entry sites that allow expression of the genes from a polycistronic messenger RNA (Adam et al., J. Virol. 65:4985-4990 (1991)). Additional nucleic acid sequences can be inserted into the vector using methods well known in the art. Representative retroviral vectors suitable for use in the invention and methods for their design are described for example, in Osborne and Miller, Proc. Natl. Acad. Sci. USA, 85:6851-6855 (1988); Osborne et al., Proc. Natl. Acad. Sci. USA. 92:8055-8058 (1995); Ramesh et al., Nuc. Acids. Res. , 24:2697-2700 (1996); and Hock et al., Blood, 74:876-881 (1989). Other vectors may also be used and are well known in the art, such as lentiviral vectors, DNA vectors, adenoviral vectors, pseudotype retroviral vectors, Epstein-Barr viral vectors, adeno- associated viral (AAV) vectors, vesicular stomatitis virus-g (VSV-g) , VL30 vectors, and liposome mediated vectors. AAV vectors provide an advantage in that they integrate into the host cell chromosome and do not substantially cause an immune response from the recipient host individual. Representative plasmid vectors include, pcDNA and various other mammalian cell expression vectors which are commercially available through a variety of sources . Methods for introducing such vectors into a cell are also well known in the art (see for example, Osborne et al., supra (1995)). One method of introducing a vector into a cell is by transfection of plasmid or DNA vectors. Transfection methods are well known in the art and include, for example, calcium phosphate precipitation, electroporation, liposome-mediated transfection, and microinj ection as described, for example, in Sambrook et al. supra (1989) and Ausubel et al., supra (1998). Alternatively, a retroviral or an AAV vector can be transduced into a cell. Methods for transduction of retroviral and adenoviral-type vectors are also well known in the art and are described further below in the Examples. Following transfection or transduction of cells with vectors of the invention, the cells are selected using a selectable marker that is either on the same vector as the gene of interest or is co-transfected on a separate vector. Methods of selecting cells for expression of a selectable marker encoded by a transfected vector are well known to those skilled in the art (see, for example, Ausubel et al. supra (1998)). Following selection, an isolated population of cells expressing the desired gene product or products is obtained. Verification that the population of cells expresses and secretes the desired gene product or products can be determined using methods well known in the art. For example, a modified population of cells can be verified for the ability to secrete the gene product assaying the amount of product secreted into the culture media under expression-promoting conditions. The can be measured by, for example, enzyme-linked lmmunoaff ity assay, radioimmunoassay or by a functional assay for one of the known biological functions of the gene product. Such assays are well known to those skilled in the art. Additional methods of selecting cells expressing the desired gene product include Northern analysis and solution hybridization of mRNA obtained from the cells, m si tu hybridization, lmmunohistology, and immunofluorescence using antibodies specific for the gene product. Further selection of a population of cells suitable for use in the invention can be performed using in vivo models known to those skilled in the art. Once a population of modified cells has been obtained, the cells can be seeded into a prosthetic chamber and implanted into a recipient individual. The invention provides a method of systemic delivery of a gene product wherein the gene product is selected from the group consisting of cytokines, hormones and growth factors. The secreted gene product can be Epo and insulin. The methods of the invention are effective for expressing essentially any gene product which can have an effect on a target disease or pathological condition if it can, for example, effect a change in the phenotypic or biochemical characteristics of the target tissue's cellular constituents by direct contact of the secreted gene product with the target cells, or alternatively, by indirect contact with one or more factors, including other gene products, induced by the secreted gene product with the target cells. Indirect effects of the secreted gene product also include, for example, negatively regulating a factor which plays a detrimental role in the target disease or pathological condition by inducing down regulation of that factor through secretion of a negative regulator using the methods of the invention. Therefore, the methods for treating a pathology mediated by a deficiency of a gene product of the invention are directed to those pathologies having deficiencies in the expression or regulation of a extracellular gene product, including gene products found systemically, or having their effect via the systemic system. Those skilled in the art will know which particular gene product will have therapeutic or beneficial effects on a particular disease and are applicable for use in the methods of the invention given the teachings and guidance provided herein. For example, the methods of the invention can be used to implant an effective amount of cells secreting a wide variety of gene products which reduce the severity of a number of diseases or pathological conditions. Exemplarily gene products include, for example, hormones, cytokines, growth factors, blood clotting factors and enzymes. A specific example of a clotting factor is factor IX which can be used for reducing the severity of individuals with hemophilia. Glucocerebrosidase is a specific example of an enzyme which can be secreted from modified cells and implanted to treat the severity of Gauchers disease. Other gene products and categories of gene products which can be systemically delivered using the modified cells as implantable vehicles are known to those skilled in the art and similarly applicable for use in the methods so the invention. Hormones, cytokines and growth factors are three classes of factors that can be secreted by implanted cells into a recipient to achieve a wiαe range of beneficial effects, and thus, reduce the severity of the condition. For example, Epo is a hormone which stimulates red blood cell production. Secretion of this cytokme from engrafted cells following implantation can be used in conjunction with a variety of diseases and pathological conditions to augment production of red cells. Such diseases and pathological conditions include, for example, organ and tissue transplants, anemia, blood transfusion, renal failure and human immunodeficiency virus infection. Another hormone that can be secreted from implanted cells to treat or reduce the severity of a disease is insulin. In diabetic individuals blood glucose can be high due to the lack of insulin production. Secretion of this hormone from engrafted cells following implantation will facilitate cellular uptake of blood glucose following digestion of a food m a diabetic individual. Moreover, the insulin encoding gene can be placed under the control of a glucose regulated promoter to achieve regulation of insulin production that parallels increases in blood sugar and achieves glucose homeostasis similar to that of normal individuals. Therefore, the long term engraftment of insulin producing cells will alleviate the need for intravenous injection of insulin. Interleukms are a class of immune-related cytokines which can be used to treat a wide variety of diseases and pathological conditions. These molecules function, for example, in immune cell regulation and can be used to augment immune responses for the treatment of immune disorders, including autoimmune diseases, as well as for the treatment of cancer. For example, in immune related disorders, regulatory T cells can be stimulated with interleukms to augment their ability to induce or inhibit an immune response. Similarly, in cancer treatment, immune cells can be stimulated with interleukms to facilitate immune responses against the pathological cells or to increase the effectiveness of other therapies. Other cytokines which function similarly can also be used either by themselves or in conjunction with an mterleukm. Tnose skilled in the art know the physiological and regulatory effects of a cytokme and can therefore select the appropriate molecule or combination of molecules to effectively treat or reduce the severity of an immune disorder or cancer. Therefore, interleukms are described herein as exemplarily cytokines for the treatment of these pathological conditions and can readily be substituted or combined with another cytokines known to those skilled in the art to have an effect on a target disease such as an immune- related disease or cancer. Other diseases and pathological conditions exist which can similarly be treated through the implantation and engraftment of cells expressing a number of different cytokines. The treatment of these conditions by expressing cytokines having known therapeutic effects can similarly be accomplished using the method of the invention. Growth factors such as granulocyte-colony stimulating factor (G-CSF) , granulocyte, macrophage- colony stimulating factor (GM-CSF) , and related factors which stimulate cell proliferation and differentiation within the hematopoietic cell lineages also can be used for treatment or reduction in severity of a variety of diseases and pathological conditions using the methods described herein. For example, the stimulation of leukocyte production following bone marrow transplantations can be increased by implanting into the bone marrow recipient cells expressing G-CSF or GM-CSF. These cells are necessary for immune surveillance and are critical for protection against pathogenic organisms and therefore early survival of a recipient individual following bone marrow ablation and transplantation. Other factors within the hematopoietic lineage similarly induce the differentiation and proliferation of other cell lineages and also can be secreted by implanted cells as described herein to stimulate some or all of the cell lineages of this system. A variety of growth factors other than the specific examples listed above also can be used m the methods of the invention. Such growth factors include, for example, msulm-like growth factor (IGF), nerve growth factor (NGF) , epidermal growth factor (EGF) , mhibms, tumor growth factor (TGF) , tumor necrosis factor (TNF) , hepatocyte growth factor (HGF) , platelet- derived growth factor (PDGF), fibroblast growth factor (FGF), stem cell factor, leukemia inhibitory factor and thrombopoietm . Such factors and their biological effects as well as those of the mterleuk family of cytokines can be found described in, for example, Molecular Biology and Biotechnology. Ed R.A. Myers, VCH Publishers, Inc., New York, N.Y., 1995, which is incorporated herein by reference. Other growth factors, hormones and cytokines known in the art can similarly be used in the methods of the invention to treat or reduce the severity of a disease or pathological condition. The selection of a particular factor or factors to be secreted using the cell implantation methods of the invention can be made given the teachings and guidance provided herein together with that which is known in the art for the treatment of a particular disease. It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also included within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention. EXAMPLE I Lonσ-Term In Vivo Expression of Therapeutic Levels of Erythropoietin This Example describes the use of a prosthetic chamber for systemic, long-term delivery of Epo from implanted cells to achieve elevated levels of red blood cell production. Smooth muscle cells were used as the implantation and delivery vehicle for Epo secretion. The site of implantation was in the digestive tract between the tunica serosa and the tunica muscularis. Briefly, expression and secretion of Epo was accomplished from smooth muscle cells transduced with a retroviral vector expressing the rat Epo cDNA. The retroviral vector, LrEpSN, was made by inserting an EcoRI-BamHI fragment of the rat Epo cDNA into vector LXSN (Osborne et al., 1995, supra ; Miller et al., Biotechmques 7:980 (1989)) A plasmid containing the rat erythropoietin gene was obtained from Drs. JPR Boissel and HF Bunn, and is describe in Wen et al., Bl ood 82:1507 (1993). The control vector, LASN, was made by inserting human ADA cDNA into LPNSN-2, a vector encoding human purine nucleoside phosphorylase, as described by Hock et al., Blood 74:876-881 (1989). Similarly, the amphotropic retroviruses were generated as described by Hock et al. Rat smooth muscle cells were used for implantation following transduction with the above described vectors. Briefly, rat smooth muscle cell cultures were prepared Joy enzymatic digestion of the aorta from a male Fisher 344 rat, and the cells were characterized by positive staining for muscle cell- specific actms with HHF35 antibody and negative stammg for von Willebrand factor, which is an endothelial cell specific marker (see for example, Geary et al., Hum . Gene Ther . 5:1213 (1994)). Primary cultures of rat smooth muscle cells as well as ecotropic PE501 and amphotropic PA317 retrovirus packaging cell lines, and NIH 3T3 thymidme kmase negative cells were grown m Dulbecco/Vogt-modifled Eagle's medium (DMEM) supplemented with 10% fetal bovine serum in humidified 5% CO at 37°C (Miller et al., 1989, supra , and Miller and Buttimore Mol . Cell Biol . 6:2895 (1986)). For studies showing the cell distribution in PTFE implants, LrEpSN transduced cells were first labeled with the fluorescent marker 1, l'dιoctadecyl-3, 3, 3' 3-' tetra ethylindocarbocyanine perchlorate (Dil ) as described by Clowes et al., J. Clm . Inves t . 93: 644 (1994) . To transduce the cells, early passage smooth muscle cells were exposed to 16-hr virus harvests from PA317-LrEpSN or PA317-LASN amphotropic virus-producing cell lines for a period of 24 hr m the presence of polybrene (4 μg/ml) . Infected cells were selected medium containing G418 at 1 mg/ml. Vascular smooth muscle cells infected with LrEpSN and selected in 1 mg/ml G-418 antibiotic secreted 6.7 mιllιunιts/24 h per 10 cells of Epo. To achieve cell implantation, two PTFE rings per animal were positioned under the serosal plane of the rat stomach to create an area above the muscle layer enclosed by the serosa membrane. Figure 1 shows a schematic cross section of a PTFE chamber implanted under the tunica serosa of the stomacn. The cross section shows a view of a pocket which is created by a PTFE ring when positioned on the muscle layer and seeded with transduced vascular smooth muscle cells. Briefly, rats were anesthetized by mtrapeπtoneal ( IP) injection with 44mg/kg ketamme, 5mg/kg xylazme, and 0.5mg/kg acepromazme . All rats received 0.04 mg dexamethasone IP just prior to surgery. An area from the thoracic mlet to the pubis was prepared for surgery and a 3cm midlme abdominal incision was made from the xyphoid to the umbilicus. The stomach was temporarily exteriorized and held in place with a mosquito hemostat. A 0.5 cm superficial incision was made in the capsule on the cranial face of the body of the stomach and a small pocket of approximately 0.6 cm diameter was created under the capsule using blunt dissection. A small PTFE ring (inner diameter 4mm, outer diameter 6 mm) was inserted into the pocket and sutured in place using 5-0 maxon on a taper needle in a simple continuous pattern. The suture material was drawn tightly to constrict the ring to a final inner diameter of 2 to 3mm before finishing the knot. The fibrous tunic directly overlying the ring was cryofrozen using a steel probe, and the ring was mechanically elevated to prevent the freezing of the underlying muscular layex to minimize tissue damage. Prior to cell implantation, the PTFE ring was rinsed with 0.9% saline to remove any blood clots formed during surgery. Cell aliquots of 1 x 10 c cells/50 ml media were then introduced into the center of the ring through a 24-g intravenous (IV) catheter. Animals received two rings each containing 1 x 10 l transduced vascular smooth muscle cells expressing either Epo or human ADA. The effect of Epo-secreting cells on the rats was determined by measuring the hematocrit values for the animals before and at various times after implantation. Hematocrit and other hematological values were made from 300 μl anticoagulated blood samples obtained from the tail vein. Hematocrit, hemoglobin, platelet, and white blood cell (WBC) number were measured using a Coulter counter (Coulter Immunology, Hialeah, FL) following procedures recommended by the manufacturer. Reticulocyte counts were determined by vital stammg with brilliant cresyl blue and cell counting by standard techniques. The hematocrits of animals implanted with Epo- secretmg transduced cells increased steadily over 50 days from a mean of 42.3 ± 1.6% to a maximum of 67.5 ± 3.1% and remained elevated with a mean value of 56.0 ± 4.0% (P<.001) over the 12-month observation period. Greater than 95% of the treated animals showed significant increases in red cell production, indicating that this procedure results in stable secretion of the gene product from the implanted cells that is very reproducible. Extrapolation of the increases in red cell production obtained over 12 months indicates that the cell implant would function to deliver therapeutic levels for greater than 3 years which is longer than the life- time of a rat. The hematocrit values obtained for the observation period for animals implanted with Epo- secreting cells and control animals having cells secreting ADA implanted is shown in Figure 2. Table 1 shows the preimplantation and the 1 year post-implantation hematocrit values for the above animals as well as values for other blood cell components. Consistent with the increase in hematocrit values was a corresponding increase in hemoglobin levels in animals implanted with Epo-secreting cells. Specifically, the hemoglobin levels rose in these animals from a presurgery mean of 15.2 ± 0.4 g/dl to a maximum of 22.6 ± 0.1 g/dl at around 7 weeks and at 12 months were significantly elevated with a mean value of 19.5 ± 1.3 g/dl (P<.001). The hematocrit and hemoglobin levels of control animals implanted with transduced cells secreting human ADA were not significantly different from baseline during the experiment (P>.05; Table 1). The hematological levels for other cells in the implanted animals were in agreement with normal rat blood values (see, for example, Schalm et al., Veterinary Hema tology (Ed 3) Philadelphia, PA, Lee and Febiger, 1975) . Specifically, the white blood cell (WBC) and platelet counts remained within the normal range during the course of the study in both the Epo-secreting and the ADA-secretmg cell implanted animals (Table 1) . This result demonstrates the selective effect of sustained Epo delivery on hematopoiesis without significant changes m other blood cell types including leukocyte or megakaryocyte production. Table 1. Treated and Control Rat Blood Cell Values Presurgery 1 year Values Postsurgery Values LASN Control (n=5) Hct (%) 45.5(11.5) 46 (±2.5) Hb (g/dL) 15.7 (±0.4) 16.4 (±0.5) * WBC (xlO ~ %/ L 9.8 (±2) 7.8 (±1.6) * Pit (xl0 "3 )/ L 807 (±109) 737 (±101) * LrEpSN treated (n=9) Hct (%) 42.3 (±1.6) 56 (±4) Hb (g/dL) 15.2 (±0.4) 19.5 (±1.3) * WBC (xlO ~ ) /μL 10.7 (±1.5) 10.7 (±2.3) * Pit (xlO "3 )/ L 727 (±63) 687 (±62) * Abbreviations: Hct, nematocπt; Pit, platelets; WBC, white blood cells; Hb, hemoglobin. * P > .05 t P < .001. The formation of reticulocyte formation, red blood cell precursor, also were evaluated in animals with Epo-secreting cell implants. The mean preimplantation value presurgery was 1.9%, with a range of 0.5% to 3.5%, and the postimplantation mean was 3.7%, with a range of 1.3% to 10.3%. The reticulocyte counts peaked between 10 and 30 days. In comparison, implantation of transduced smooth muscle cells on the carotoid artery yields peak levels at about 8 to 14 days, Osborne et al., 1995, s upra . The single administration of dexamethasone at the time of surgery increased the red cell production of treated animals to implantation of Epo-screenmg cells. At 2 months animals receiving dexamethasone had a mean hematocrit of 59% compared with a mean hematocrit of 51% recorded from untreated animals. Dexamethasone is a powerful synthetic glucocorticoid. The use of a single administration of this glucocorticoid at the time of surgery was based on preliminary studies showing that dexamethasone enhanced the response of animals to implantation of Epo-secreting cells. The viral LTR promoter, which drives Epo cDNA expression m the vectors described previously, contains a known steroid responsive element but the short serum half-life of dexamethasone (1.8 to 4.7 hours) makes an effect mediated through this promoter element unlikely to be sustained for 12 months. Dexamethasone has been used clinically to decrease surgical inflammation and edema and to decrease immune-mediated tissue destruction, making preservation of the transplanted cells by dexamethasone treatment the most probable explanation for increased cell survival and hematocrit. The use of dexamethasone however, is not necessary to achieve successful implantation, cell engraftment and long-term secretion of a gene product. Histological analysis of the implanted chambers was also assessed to determine the extent of cell engraftment following implantation. Briefly, a rat showing an elevated hematocrit of 63% at 12 months post surgery was sacrificed, and the PTFE chamber was removed, fixed, and stained with Hematoxylm and Eosm (H&E) . A photomicrograph of a cross-section is shown m Figure 3, panels 1 and 2, and reveals tissue within and around the PTFE graft being fully integrated and well vascularized. This result indicates that the PTFE structure is well tolerated between the muscle and serosal layers. To show that transduced vascular smooth muscle cells expressing Epo are contained within the PTFE ring structure, Epo-secretmg cells were labeled with the fluorescent dye Dil before implantation as described previously. Unlabeled Epo-secretmg cells were implanted in a control group of rats. At 2 months after surgery a rat with an hematocrit of 68% was sacrificed and the tissue, frozen, cross-sectioned and photographed using a Nikon Microphot FXA equipped with a rhodamme filter. Figure 3 shows PTFE chamber implants containing Epo- secretmg vascular smooth muscle cells unlabeled (panel 3) or labeled with Dil (panel 4) . PTFE material is denoted as "P" and mucosal tissue as "M." Panels 1, 2 and 4 of Figure 3 are at 40x the original magnification and panel 3 is at lOOx the original magnification. The photographs of Figure 3 show a large mass of dye marked fluorescmg cells evident in the area encompassed by the PTFE ring. In contrast, fluorescent cells were not visible in adjacent areas or in the control sections of unlabeled cell implants. These results demonstrate that transduced vascular smooth muscle cells remain in the PTFE-enclosed area when implanted between the tunica serosa and the tunica muscularis of the digestive tract and provide therapeutic Epo expression for at least 12 months. To determine if vector-encoded Epo expression resulted in downregulation of endogenous Epo, rats implanted with Epo-secretmg cells were sacrificed after elevated hematocrit and hemoglobin levels were established. Rats implanted with ADA-secretmg cells were sacrificed at similar time points. Epo expression was assessed by specific amplification of endogenous Epo message from kidney. Specifically, RNA obtained from kidney was subjected to reverse transcription followed by polymerase chain reaction (RT-PCR) using rat Epo-specific probes as described below. Total RNA was isolated from rat kidney by homogenization in the presence of RNAzol . Reverse transcription was performed using Superscript reverse transcriptase (GIBCO-BRL, Gaithersburg, MD) , incubated together with lμg of total RNA the presence of random hexamer primers. Amplification of the reverse transcribed cDNA was accomplished with 30 cycles of polymerase chain reaction (PCR) using the following rat Epo specific primers: 5' AGG CGC GGA GAT GGG GGT GC 3' and 5' CCC CGG AGG AAG TTG GAG TAG 3' (SEQ ID NOS : 1 and 2, respectively). This procedure results in the specific amplification of a 540 bp Epo encoding cDNA fragment. An aliquot of the amplified reaction mixture was electrophoresed in a 2% agarose gel and, after DNA blot transfer of the amplification reaction to membranes, the membrane was hybridized with a -P-labeled 500bp Epo cDNA probe . As a control for RNA extraction, integrity, reverse transcription and amplification, β-actin-specific primers were additionally used to amplify a 500 bp fragment from the same cDNA preparation. The primer sequences were: 5' GTG GGG CGC CCC AGG CAC CA 3' and 5' CTC CTT AAT GTC ACG CAC GAT TTC 3' (SEQ ID NOS : 3 and 4, respectively) . Equal amplification of the samples was confirmed by both ethidium bromide stammg and subjecting diluted aliquots of each sample to agarose electrophoresis, DNA blot transfer and hybridization of the transferred DNA to a β-actm-specific P-labeled cDNA probe . Expression levels for endogenous Epo message are shown m Figure 4. Endogenous Epo mRNA kidneys of rats seeded with LrEpSN-transduced cells and analyzed at 11 and 12 months was greatly reduced m comparison to kidney obtained from an animal implanted with ADA- secretmg cells (Figure 4). This result demonstrates a significant downregulation of endogenous Epo production m animals implanted with Epo-secretmg cells. Epo mRNA in liver was not detectable by this RT-PCR method. Band intensities from the DNA blots showed that the RT-PCR amplification of actin mRNA isolated from Epo- and ADA- secretmg tissues were similar, indicating that the RNA isolation and amplification procedures were equivalent between the analyzed samples (Figure 4) . These data evidence the long-term, high-level Epo expression from transplanted cells and further corroborate that the elevated red cell production observed was mediated by implanted transduced cells with a minimal contribution from endogenous Epo. Prolonged red cell production in excess of 60% from genetically modified cells is a significant result from this cell transduction and implantation method. In addition to the above-described studies, expression of the bacterial neomycm phosphotransferase from the viral expression vector was also assessed in regard to possible detrimental effects on cell implantation. Specifically, expression of the foreign neomycm gene product from the LrEpSN vector did not appear to cause an immune-mediated loss of transplanted cells. This result is supported by the longevity of cell survival and the sustained red cell overproduction. The elimination of autologous transduced cells by an immune- mediated mechanism to foreign transgenes has been reported m rats receiving glioma cells and human T-cell transplantations. The smooth muscle cells targeted for gene expression and implantation are not usually involved n antigen processing and presentation and the neomycm gene is expressed in the cytosol, which explains an apparent lack of immune-mediated cell loss. Throughout this application various publications have been referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the disclosed embodiments, those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention. It should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims: What is claimed is: 1. A method of systemic delivery of a gene product comprising implanting within the wall of the digestive tract of an individual a prosthetic chamber containing an effective amount of cells secreting said gene product . 2. The method of claim 1, further comprising implanting said prosthetic chamber within or proximal to a connective tissue layer. 3. The method of claim 1, further comprising implanting said prosthetic chamber between the tunica serosa and the tunica muscularis of said digestive tract wall . 4. The method of claim 1, wherein said prosthetic chamber comprises an outer diameter or width of the chamber being greater than about 10 mm, preferably between about 7.0 and 10 mm, more preferably between about 3 and 6 mm. 5. The method of claim 1, wherein said cells secreting said gene product are smooth muscle cells or fibroblasts . 6. The method of claim 1, wherein said effective amount of cells secreting said gene product comprises a population of about 1x10 cells or greater, preferably about 1x10° cells or greater, more preferably about 1x10 cells or greater. 7. The method of claim 1, wherein said gene product is selected from the group consisting of cytokines, hormones, growth factors and clotting factors. 8. The method of claim 7, wherein said gene product is Epo or insulin. 9. A method of systemic delivery of a gene product comprising implanting within the wall of the digestive tract of an individual a prosthetic chamber containing an effective amount of cells modified for mducible expression of said gene product, and inducing expression of said gene product. 10. The method of claim 9, further comprising implanting said prosthetic chamber within or proximal to a connective tissue layer. 11. The method of claim 9, further comprising implanting said prosthetic chamber between the tunica serosa and the tunica muscularis of said digestive tract wall . 12. The method of claim 9, wherein said prosthetic chamber comprises an outer diameter or width of the chamber being greater than about 10 mm, preferably between about 7.0 and 10 mm, more preferably between about 3 and 6 mm. 13. The metnod of claim 9, wherein said cells secreting said gene product are smooth muscle cells or fibroblasts . 14. The method of claim 9, wherein said effective amount of cells secreting said gene product comprises a population of about 1x10 cells or greater, preferably about lxlO c cells or greater, more preferably about 1x10 cells or greater. 15. The method of claim 9, wherein said gene product is selected from the group consisting of cytokines, hormones, growth factors and clotting factors. 16. The method of claim 15, wherein said gene product is, Epo or insulin. 17. The method of claim 9, wherein said cells modified for mducible expression of said gene product comprise a nucleic acid encoding said gene product operable linked to an mducible promoter/regulatory element. 18. The method of claim 9, wherein said induction of said gene product expression comprises administering to said individual an effective amount of a compound which increases the activity of said promoter/regulatory element. 19. A method of treating a pathology mediated by the deficiency of a gene product comprising implanting a prosthetic chamber containing an effective amount of cells expressing therapeutic levels of said deficient gene product. 20. The metnod of claim 19, further comprising implanting said prosthetic chamber within or proximal to a connective tissue layer. 21. The method of claim 19, further comprising implanting said prosthetic chamber between the tunica serosa and the tunica muscularis of said digestive tract wall . 22. The method of claim 19, wherein said prosthetic chamber comprises an outer diameter or width of the chamber being greater than about 10 mm, preferably between about 7.0 and 10 mm, more preferably between about 3 and 6 mm. 23. The method of claim 19, wherein said cells secreting said gene product are smooth muscle cells or fibroblasts . 24. The method of claim 19, wherein said effective amount of cells secreting said gene product comprises a population of about 1x10 cells or greater, preferably about lxlO 6 cells or greater, more preferably about 1x10 cells or greater. 25. The method of claim 19, wherein said gene product is selected from the group consisting of cytokines, hormones, growth factors and clotting factors. 26. The method of claim 25, wherein said gene product is Epo or insulin. 27. A method of treating a pathology mediated by the deficiency of a gene product comprising implanting a prosthetic chamber containing an effective amount of cells modified for mducible expression of therapeutic levels of said gene product, and inducing expression of therapeutic levels of said gene product. 28. The method of claim 27, further comprising implanting said prosthetic chamber within or proximal to a connective tissue layer. 29. The method of claim 27, further comprising implanting said prosthetic chamber between the tunica serosa and the tunica muscularis of said digestive tract wall . 30. The method of claim 27, wherein said prosthetic chamber comprises an outer diameter or width of the chamber being greater than about 10 mm, preferably between about 7.0 and 10 mm, more preferably between about 3 and 6 mm. 31. The method of claim 27, wherein said cells secreting said gene product are smooth muscle cells or fibroblasts. 32. The method of claim 27, wherein said effective amount of cells secreting said gene product comprises a population of about 1x10' cells or greater, preferably about lxlO 6 cells or greater, more preferably about 1x10 cells or greater. 33. The method of claim 27, wherein said gene product is selected from the group consisting of cytokines, hormones, growth factors and clotting factors. 34. The method of claim 33, wherein said gene product is Epo or insulin. 35. The method of claim 27, wherein said cells modified for inducible expression of said gene product comprise a nucleic acid encoding said gene product operable linked to an inducible promoter/regulatory element. 36. The method of claim 27, wherein said induction of said gene product expression comprises administering to said individual an effective amount of a compound which increases the activity of said promoter/regulatory element.

Inventors: LEJNIEKS DANIEL J, OSBORNE WILLIAM R A, LEJNIEKS, DANIEL, J., OSBORNE, WILLIAM, R., A.
Applicants: LEJNIEKS DANIEL J, OSBORNE WILLIAM R A, UNIV WASHINGTON, LEJNIEKS, DANIEL, J., OSBORNE, WILLIAM, R., A., UNIVERSITY OF WASHINGTON
Application year: 1999
Main classification: A61K48
IPC codes: A61K38, A61K48, A61P5