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Title:  Composition and method for improving function of embryonic kidney transplants
United States Patent: 
7,074,762
Issued: 
July 11, 2006

Inventors: 
Hammerman; Marc R. (St. Louis, MO); Rogers; Sharon A. (Edwardsville, IL)
Assignee: 
Washington University (St. Louis, MO)
Appl. No.: 
222460
Filed: 
December 29, 1998


 

Covidien Pharmaceuticals Outsourcing


Abstract

A method and composition for enhancing the development of metanephric tissue upon transplantation into an allogenic or xenogenic host are disclosed. Metanephric tissue is removed from an embryonic donor and is contacted with a composition comprising one or more growth factors for metanephric development. The composition can be administered to the metanephric tissue in vitro prior to implantation of the donor tissue in the transplant recipient, or can be administered in vivo either during or subsequent to the transplantation procedure.

SUMMARY OF THE INVENTION

A method and composition for enhancing the development of metanephric tissue upon transplantation into an allogenic or xenogenic host are disclosed. Metanephric tissue is removed from an embryonic donor and is contacted with a composition comprising one or more growth factors for metanephric development. The composition can be administered to the metanephric tissue in vitro prior to implantation of the donor tissue in the transplant recipient, or can be administered in vivo either during or subsequent to the transplantation procedure.

DETAILED DESCRIPTION OF THE INVENTION

The methods described in published European application no. 0 853 942 A2 are applicable to the present invention. The entire contents of that application are incorporated herein by reference. To summarize, that application describes the transplantation of metanephric tissue from an embryonic mammalian donor to an allogenic or xenogenic mammalian recipient. The recipient can be at any developmental stage, including juvenile and adult. A preferred recipient is a human with reduced renal function. The metanephric tissue is taken from the donor at a suitable stage of development, typically within 1 to 5 days after the metanephros begins formation, and is implanted into the recipient either within the omentum, preferably adjacent a host kidney, or under the renal capsule of a host kidney. The metanephric tissue grows and becomes vascularized, in large part by the recipient to form a chimeric kidney. The chimeric kidney develops to form mature structures, including a ureter, which can externalize urine formed by the chimeric kidney after connection to the host's excretory system.

In experiments where rat metanephric allografts were transplanted into the abdominal cavity of non-inbred adult rat hosts, the metanephric tissue developed into functioning chimeric kidneys which were not rejected by the host for as long as 32 weeks after transplantation. It is believed that the vascularization of the metanephric tissue by the recipient facilitates the acceptance of the transplant. In contrast, when developed kidneys from adult rat donors are transplanted into non-inbred adult rat hosts, they are rejected within 7 days.

The present invention is directed to the further discovery that growth factor treatment of the metanephric tissue before, during, and/or after transplantation, enhances the development and functioning of the chimeric kidney. In addition, the removal of host renal tissue prior to transplantation of the donor metanephric tissue further enhances development of the chimeric kidney.

As used herein, the phrase "growth factor for metanephric development" refers to any molecule that promotes the growth, proliferation, and/or differentiation of metanephric tissue. Thus, the phrase encompasses growth factors, ligands that bind to growth factor receptors, vitamins, and other molecules that assist in the development of metanephric tissue. The phrase also encompasses molecules that stimulate endogenous production of growth factors for metanephric development either by the donor metanephric tissue or by the transplant recipient. For example, somatotropin is known to stimulate IGF-I production. Whether a particular growth factor assists in metanephric development can be readily determined by routine experimentation using the procedures described herein.

Presently preferred growth factors include transferrin, prostaglandin E.sub.1 (PGE.sub.1), sodium selenite, ligands of the EGF-receptor such as transforming growth factor alpha, epidermal growth factor (EGF), and amphiregulin; insulin-like growth factors (IGFs), particularly IGF-I and IGF-II vitamin A and derivatives thereof such as retinoic acid; vascular endothelial growth factor (VEGF); hepatocyte growth factor (HGF), nerve growth factor (NGF), cytokines such as TGF-.beta. and other members of the TGF-.beta. family (see Atrisano et al., J. Biochemica et Biophysica Acta (1994) 1222:71 80), and growth hormone (GH) (see Hammerman, M. R., Seminars in Nephrology (1995)). Using known procedures, it can readily be determined whether a particular factor serves as growth factor for the developing metanephroi. For example, in cultures of metanephroi, antibodies can be used to block the action, if any, of a certain factor. An inhibition of development compared to controls indicates whether the factor acts as a growth factor for metanephroi development. As another method, comparisons of development of metanephroi in culture with and without supplements can be used to determine whether a certain factor acts as a growth factor (see Hammerman et al., Pediatr Nephrol (1993) 7(5):616 620); Hammerman M R, Seminars in Nephrology (1995) 15:291 299; Schofield P N and Boulter C A, Exp'l Nephrology (1996) 4:97 104; Pugh et al. Kidney Int (1995) 47(3):774 781); Humes et al., Lab Invest (1991) 64(4):538 545); and Vilar et al. Kidney Int. (1996) 49(5): 1478 1487).

The metanephros can be treated with a single growth factor that enhances development, or combinations of growth factors can be administered either sequentially, or as a growth factor cocktail. Preferred growth factor cocktails comprise any combination of two or more of the following growth factors: transferrin, PGE.sub.1 sodium selenite, nerve growth factor, fibroblast growth factor (FGF), platelet derived growth factor (PDGF), IGF-I, IGF-II, TGF-.alpha., TGF-.beta., HGF, and/or VEGF. The growth factors are dissolved in any physiologically-acceptable solution in which the metanephroi can be immersed. Various cell culture media can be used, such as a 50:50 mixture of Dulbecco's modified Eagles medium and Hams F12 (DMEM:HF120). Physiological saline is another suitable solution, particularly if the growth factors are administered to the transplant recipient during or after the transplantation procedure. The growth factors are usually used at concentrations ranging from about 1 fg/ml to 1 mg/ml. Concentrations between about 1 to 100 ng/ml are usually sufficient for most growth factors. Simple titration experiments can be performed to readily determine the optimal concentration of a particular growth factor.

Prior to the transplantation procedure, metanephric tissue is harvested from one or more suitable mammalian donors at an appropriate stage of fetal development. Preferably, the metanephric tissue is harvested soon after the metanephric kidney begins formation and prior to the presence of blood vessels that either originate within the metanephros or from inside or outside the metanephros. If the embryonic renal tissue is harvested too early in development, it may, once implanted into the recipient, differentiate into non-renal tissues such as hair and gut. Tissue harvested too late in the development of the metanephric kidney, for example, tissue having visible blood vessels, may contain more antigen-presenting cells and cell-surface antigens and thus present more of threat of rejection by the recipient. Preferably, the harvested metanephroi contains metanephric blastema, segments of ureteric bud, and nephron precursors, and does not contain glomeruli.

The preferred developmental stage for harvesting the metanephros will vary depending upon the species of donor. Generally, the metanephros is preferably harvested 1 to 5 days after the metanephros forms. Preferably, the metanephros is harvested from 1 to 4 days after the metanephros forms, and more preferably from about 2 to 4 days after metanephros formation. In rats, the metanephros forms on day 12.5 of a 22-day gestation period, and on day 11 of a 19 day gestation period in mice. In these species, a suitable time frame in which to harvest the donor metanephros of mice or rats is typically between the second and fourth day after the metanephros begins formation. Preferably the metanephros is harvested within 3 days after formation of the metanephros begins.

In species having a longer gestation period, the time-frame during which the metanephros is preferably harvested following its formation, can be longer. Generally, the time frame in which the metanephros is harvested will be less than about one fifth of the total gestation period of the donor, preferably less than about one seventh of the total gestation period of the donor, and more preferably, less than about one tenth of the total gestation period of the donor. Table 1 (see Original Patent) shows the time-course (in days) of metanephros development and gestational period in some vertebrates.

Pigs are preferred xenogeneic donors for humans because of their comparable organ size, and availability. Additionally, the digestive, circulatory, respiratory and renal physiologies of pigs are very similar to those of humans. In the case of renal function, the maximal renal concentrating ability (1080 mOsm I.sup.-1), total renal blood flow (3.0 4.4 ml min.sup.-1 g.sup.-1) and glomerular filtration rates (126 175 ml min.sup.-1 70 kg) of the miniature pig are virtually identical to those of humans (see Sachs D H, Veterinary Immunology and Immunopathology (1994) 43:185 191). The use of metanephroi from transgenic pigs that have been "humanized" to reduce the potential for transplant rejection may provide further advantages (e.g. Pierson et al., J. Heart Lung Transplant (1997) 16:231 239). Pig metanephroi are harvested at about the 10 mm stage. This occurs between approximately embryonic day 20 and embryonic day 30. Human tissue could be used as an allogeneic source for transplantation.

Metanephroi are removed surgically under a dissecting scope and suspended in a suitable holding medium, such as a 1:1 mixture of Dulbecco's modified Eagles Medium and Hams F12 medium (Rogers et al. J. Cell Biol. (1991) 113:1447 1453), and placed under sterile conditions, until they are transplanted. It is preferred to use the whole metanephros, with renal capsule intact, for transplantation. One or more metanephroi may be used per recipient, depending upon the increase in nephron mass that the recipient needs.

If the metanephroi are to be treated with growth factors prior to transplantation, the growth factors can be added directly to the holding medium. When used as a pretreatment, the growth factor composition exerts a positive effect in a surprisingly short period of time. Significant improvement in the development of the implant can be achieved when the metanephric tissue is contacted with the growth factor composition in vitro for less than 24 hours. Preferably, the metanephric tissue is contacted with the growth factor composition in vitro for less than 8 hours, and preferably less than 2 hours. Optimal results can usually be achieved when the metanephric tissue is contacted with the growth factor composition for as little as about 20 to 60 minutes prior to implantation into the transplant recipient.

To transplant the metanephric tissue, surgery is performed on the recipient to expose one or both kidneys. Surgical procedures for renal transplantation are well known in the art (e.g. Cohn et al., Am. J. Physiol (1982) 24:F293 F299). The donor metanephroi can be implanted directly under the renal capsule of the recipient's kidney, or into a fold of the omentum where it forms a chimeric kidney that functions independently of the recipient's kidney. The omentum is a membranous structure which connects the bowels. It is a preferred site for the implant, particularly if the implanted metanephric tissue is intended to replace a malfunctioning or non-functioning kidney which may be removed, either at the time of transplantation or after the donor metanephric tissue develops sufficiently to form a functioning chimeric kidney. Implantation of the metanephric tissue into the omentum is also preferred if it is desired to treat the tissue with growth factors after implantation. The omentum is a more accessible site for the growth factor treatment compared to underneath the renal capsule of the recipient's kidney. An osmotic pump that provides a steady supply of growth factors could be placed in the omentum next to the implanted tissue. Alternatively, the recipient could receive periodic injections of the growth factors in the vicinity of the transplant or the growth factors could be delivered in a manner such that they are present in the recipient's blood that circulates through the transplant.

While a donor metanephros can be placed adjacent to any portion of the omentum, it is preferable to implant it in an omental fold which will retain the developing kidney at the site of implantation. It is most preferable to implant the metanephros at an omental fold located near one of the recipient's kidneys, particularly near the ureter, so that the developing ureter of the metanephros can be readily connected to the recipient's excretory system.

When implanted into the recipient's kidney, an incision, large enough to receive the donor tissue is made in the fibrous renal capsule that surrounds the recipient kidney. The location of the incision can be anywhere in a viable portion of the recipient kidney, but most conveniently will be at an external border of the kidney that is easily accessible during surgery. The donor tissue is placed between the capsule and the cortex of the recipient kidney.

The implanted metanephroi are allowed to grow and differentiate within the recipient under conditions that allow the metanephric tissue to vascularize and develop to form mature, functioning nephrons. Suitable conditions may include the use of pre or post-operative procedures to prevent rejection of the implant in addition to the use of growth factors that facilitate the development and functioning of the metanephric tissue. In some cases of allogeneic transplantation, there may be no host rejection of the transplanted metanephros. However, in the case of xenogeneic transplantation, rejection prevention measures are typically taken. This is usually done by immunosuppressing the recipient after the transplantation. Cyclosporine A (CSA) treatments can provide sufficient immunosuppression to prevent rejection of the donor tissue. CSA treatment protocols to prevent transplant rejection are known in the medical field. Local immunosuppression techniques are described by Gruber, Transplantation (1992)54:1 11. In U.S. Pat. No. 5,560,911, antibodies directed against idiotypes on naturally occurring human anti-animal antibodies are disclosed for use in inhibiting xenograft rejection. The anti-idiotypic antibodies are injected into the xenograft recipient in order to bind to the idiotypes expressed on anti-xenograft antibodies. Anti-idiotypic antibodies that bind human anti-pig antibodies, to prevent rejection of transplanted pig tissues by a human patient are exemplified. Anti-lymphocyte globulins are also known for prevention of transplant rejection (Lacy et al. Diabetes, (1981) 30:285 291). As an alternative to immunosuppression, the implanted metanephros can be treated prior to transplantation to reduce its antigenicity. Exemplary approaches to the reduction of immunogenicity of transplants by surface modification are disclosed by Faustman WO 92/04033 (1992). For xenografts into human transplant recipients, transgenic animals that have been humanized to reduce organ transplant rejection may be used. Finally, agents thought to induce tolerance to transplanted tissue can be administered to recipients such as CTLA4-lg (Lin et al., J. Exp. Med. (1993) 178:1801 1806).

Metanephric kidneys transplanted using the techniques described herein grow, and become vascularized in large part by the recipient, to form chimeric kidneys. It is believed that the vascularization by the recipient may facilitate the acceptance of transplanted xenogeneic tissue. When implanted into the recipient's kidney, the metanephroi become imbedded into the parenchyma of the recipient kidney. The donor metanephroi begin to form various mature structures that are distinguishable from the structures in the adjacent recipient renal tissue, including mature glomeruli and tubules, renal papilla, and ureter. After a sufficient period of development, it is evident that the glomeruli are capable of filtering plasma. Hence, implantation of the metanephric tissue contributes to an increase in the nephron mass of the recipient.

Filtering glomeruli are evidenced by the detection of urine within the donor metanephroi. This can be done by measuring the levels of urea nitrogen and/or creatinine in fluid aspirated from the donor tissue. Such fluid may be contained within one or more cysts associated with the donor tissue (see Example 1). Urine is defined herein as fluid having a concentration of creatinine and/or urea nitrogen that is higher than the concentration of the corresponding components found in the recipient's plasma. The concentration differential varies, and will be reduced with increased hydration of the recipient. However, generally, the concentration of creatinine in the donor metanephroi will be at least twice the concentration found in the recipient's plasma. The concentration of urea nitrogen in the donor metanephroi will generally be at least fifty percent greater than the concentration of urea nitrogen in the recipient's plasma.

In order to facilitate the externalization of the urine that forms within chimeric kidney, a standard ureter to ureter anastomosis procedure can be used to hook up the ureter that forms from the implanted metanephros with the ureter of a kidney of the recipient. During this procedure, the chimeric kidney can be further treated with growth factors by direct administration of a growth factor-containing composition to the chimeric kidney as it is exposed during the procedure. When the metanephroi are implanted into the omentum of the recipient, externalization of urine can be achieved by linking the ureter directly to the recipient's ureter or bladder. These procedures, and other procedures known in the art for the externalization of urine are summarized in Adult and Pediatric Urology, 3rd Ed., Gillenwater, et al., Eds. . 987 994 and 2369 2375 (1996). In some cases, post-transplantation surgery may be unnecessary as the intrarenal transplanted donor kidneys may incorporate into the collecting system of the host.

 

Claim 1 of 15 Claims

1. A method for the treatment of metanephric tissue for transplanted into a recipient comprising: a. contacting said metanephric tissue, in vitro, with a growth factor-containing composition comprising vascular endothelial growth factor; and b. transplanting said metanephric tissue into said recipient.

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