Method for inducing growth and enhancing survival of nervous tissue

[0002] This is a continuation-in-part of co-pending application Ser. No. 9/302,094, filed Apr. 29, 1999 now abandoned, the content of which is hereby incorporated by reference in its entirety.

[0004] Death or destruction of nervous tissue such as neurons or glial cells is associated with a variety of degenerative disorders of the nervous system. These disorders include, for example, degenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's chorea, and amyotrophic lateral sclerosis. Necrosis or loss of neurons is also associated with neuropathies of the central, peripheral or motor neurons as a result of disease states such as diabetes. In addition, damage to neurons may result from ischemia due to stroke, trauma (e.g. burns and wounds), kidney dysfunction as well as the toxic effects of drugs used to treat cancer and AIDS. Further, the loss of neurons may result in dementia associated with aging.

[0005] Currently, there are no known effective treatments for damaged or degenerated nervous tissue. Certain growth factors such as ciliary neurotrophic factor (CNTF) have been proposed for the treatment of neurodegenerative diseases such as Alzheimer's disease. However, the use of CNTF has not been successful in clinical trials. Thus, there is a present need for the identification of new agents for treating damaged and degenerated nervous tissue.

[0006] Kothapalli, et al. described the identification of a novel human gene encoding endometrial bleeding associated factor protein (“ebaf”) that is associated with abnormal endometrial bleeding (J. Clin. Invest., 99(10):2342-50, 1997). The ebaf gene is located on human chromosome 1 at band q42.1, and the nucleotide and deduced amino acid sequences are known (Kothapalli, et al., 1997). The sequence of the ebaf protein also shows homology and structural features of the members of the TGF-β superfamily (Kothapalli, et al., 1997). The ebaf gene is expressed in human endometrium in the late secretory and menstrual phases and absent in the early and mid-secretory endometria (Tabibzadeh, et aL, Mol. Hum. Reprod, 4(6):595-602, 1998), and also in certain adenocarcinomas that exhibited mucinous differentiation including colonic, duodenal, and ovarian carcinomas (Tabibzadeh, et al., Front. Biosci., 15(2):18-25, 1997).

[0007] Meno, et al. have described two mouse members of the TGF-β superfamily, designated Lefty-1 and Lefty-2. These are expressed in a left-right asymmetric fashion in mouse embryos, and are separated by about 30 kb on mouse chromosome 1H2 (Meno, et al., Genes Cells, 2(8):513-24, 1997). Meno, et al. also reported that Lefty-1 induces NCAM-1 (a neural marker) in the absence of mesoderm induction, and hypothesized that the direct neutralizing activities of Lefty-1, similar to BMP antagonists such as noggin and chordin, may antagonize BMP (bone morphogenic protein)-mediated signals in tissues positioned on the left side of mouse embryos.

[0008] Recently, Kosaki, et al. described the identification of two human lefty genes, designated Lefty-A and Lefty-B, that are separated by approximately 50 kb on chromosome 1q42 (Am. J. Hum. Genet. 64(3):712-21, 1999). Lefty-A was described as being identical to ebaf. Although human Lefty-A and Lefty-B and mouse Lefty-1 and Lefty-2 are similar, Kosaki, et al. described that human Lefty-A and Lefty-B are more similar to each other than to mouse Lefty-1 and Lefty-2. In this regard, Kosaki, et al. described that Lefty-A and Lefty-B are 96% homologous and mouse Lefty-1 and Lefty-2 are 90% homologous, while the cross-species homology is only 81-82%. Thus, Kosaki, et al. suggested that sequence analysis alone precludes determination of specific orthologous relationships (i.e. whether Lefty-A is the functional equivalent of Lefty-1).

[0009] The present invention is based on the discovery that ebaf is associated with the development and growth of nervous tissue. Based on this finding, the present invention provides a method for inducing the growth or enhancing survival of nervous tissue comprising contacting the nervous tissue with an amount of ebaf effective to induce the growth or enhance the survival of the nervous tissue. The present invention also provides a method for treating a subject having damaged or degenerated nervous tissue comprising administering to the subject an amount of ebaf effective to treat the damaged or degenerated nervous tissue.

[0010] The present invention further provides a method for treating a neurodegenerative disease in a subject comprising administering an amount of ebaf effective to treat the neurodegenerative disease. The present invention still further provides a method for preventing the onset or reducing the severity of damaged or degenerated nervous tissue in a subject comprising administering an amount of ebaf effective to prevent the onset or reduce the severity of the damaged or degenerated nervous tissue.

[0011] Lastly, the present invention provides a method for inducing growth or enhancing survival of nervous tissue comprising contacting the nervous tissue with a modulator of ebaf expression in an amount effective to induce or enhance expression of ebaf and induce the growth or enhance the survival of the nervous tissue. Additional objects of the present invention will be apparent in view of the description which follows.

[0012] FIGS. 1A-1K demonstrate the effect of ebaf on the gene transcription in the development of the various tissue markers in the blastocyst of the Xenopus laevis. The ebaf RNA was prepared by in vitro transcription and was injected into the two cell stage Xenopus laevis blastocyst as described in the example. The animal caps were removed at 24 hours and the RNA was reverse transcribed and then cDNA was subjected to PCR using primers to the Xenopus laevis mRNA as shown. FIG. 1A=actin (mesoderm marker); FIG. 1B=XAG1 (cement gland marker); FIG. 1C=H8 (endodermal marker); FIG. 1D=ctokeratin (epithelial marker); FIG. 1E=globin (ventral mesoderm); FIG. 1F=X1hbox9 (spinal cord marker); FIG. 1G=NRP-1 (general marker); FIG. 1H=EN2 (mid-hind brain marker); FIG. 1I=OTXA (forebrain marker); FIG. 1J=KROX20(hindbrain marker); FIG. 1K=EF1 α (housekeeping gene). FIGS. 1A-1D: Lane 1: whole embryo RNA used as control. Lane 2: uninjected with ebaf RNA. Lane 3: injected with 20 pg of ebaf RNA. Lane 4: injected with 200 pg of ebaf RNA. Lane 5: injected with injected with 2 ng of ebaf RNA. FIGS. 1E-1K: Lane 1: whole embryo RNA used as control. Lane 2: RT-PCR control in which the reverse transcriptase was omitted from the RT reaction. Lane 3: uninjected with the ebaf RNA. Lane 4: injected with 20 pg of ebaf RNA; Lane 5: injected with 200 pg of ebaf RNA. Lane 6: injected with 2 ng of ebaf RNA.

[0013] FIG. 2 depicts the nucleotide sequence (SEQ ID NO: 1) and corresponding amino acid sequence (SEQ ID NO: 2) for ebaf.

[0014] FIG. 3 depicts the effect of ebaf on proliferation of rat embryo forebrain cells. Cultures of 16 day old (E16) rat embryonic forebrain cells were incubated with supernatants of cells producing ebaf and the control cultures received DMEM medium alone in presence of BrdU for one day, followed by staining for BrdU. Bars represent mean BrdU+ cell counts±standard errors of the mean (SEM).

[0015] FIG. 4 depicts the effect of ebaf on number of TG-2 positive rat embryo forebrain cells. Cultures of 16 day (E16) old rat embryonic forebrain cells were incubated with supernatants of cells producing ebaf, recombinant ebaf (5 ng/ml) and the DMEM medium alone. After 9 days in culture, these were stained with TG-2 antibody. Bars represent mean TG2+ cells±standard errors of the mean (SEM) on repeated measurements.

[0016] FIG. 5 depicts the effect of ebaf on neuronal number measured by ELISA. The forebrain cells were cultured as described in the text and the OD was determined for cells stained for TG-2 and NeuN2. Results shown are from the means of duplicate samples.

[0017] The present invention provides a method for inducing growth or enhancing survival of nervous tissue that comprises contacting the nervous tissue with an amount of ebaf effective to induce the growth or enhance the survival of the nervous tissue. The method of the present invention may be used in the culturing of nervous tissue in vitro and also for the inducing growth and enhancing survival of nervous tissue in vivo.

[0018] As used herein, “nervous tissue” includes the neurons and neuroglia. “Neurons” are any of the conducting or nerve cells of the nervous system that typically consist of a cell body containing the nucleus and surrounding cytoplasm (perikaryon), several short radiating processes (dendrites), and one long process (the axon), which terminates in twiglike branches (telodendrons) and may have branches (collaterals) projecting along its course. “Neuroglia” are the neuroglial cells or glial cells which form the supporting structure of the nervous tissue. “Nervous tissue” includes the nervous tissue present in both the central nervous system and the peripheral nervous system.

[0019] As used herein, “growth” is an increase in thickness, diameter, length, mass and/or number of one or more of the components of the nervous tissue including but not limited to the perikaryon, the neurofibril, the nissl bodies, the axon, the dentrites, the telodenria, the myelin sheath, the neurilemma, the schwann cells, and/or the neuroglial or glial cells, and includes the generation or regeneration of one or more of the components of the nervous tissue. “Enhance the survival” of the nervous tissue is the full or partial protection of the nervous tissue from further death, degeneration, damage or injury.

[0020] With respect to the culturing of nervous tissue in vitro, it is believed that the ability of ebaf to induce the growth or enhance the survival of nervous tissue renders ebaf particularly useful for culturing nervous tissue in vitro. In this connection, ebaf may be introduced into the culture media by adding the ebaf protein directly to the culture media or by introducing nucleic acid encoding ebaf to the nervous tissue or other cells in a manner permitting expression of ebaf in amounts sufficient to induce the growth or enhance the survival of the nervous tissue. The culturing of nervous tissue in vitro may be desirable for preparing nervous tissue for transplantation, diagnostics, drug screening, and the like.

[0021] Concerning in vivo treatment, the ability of ebaf to induce the growth or enhance the survival of nervous tissue renders ebaf particularly useful for treating damaged or degenerated nervous tissue in a subject. The subject is preferably a mammal (e.g. humans, domestic animals, commercial animals), and most preferably a human. The damaged or degenerated nervous tissue may be associated with a neurodegenerative disease such as Alzheimer's disease, Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis, dementia, or Pick's disease, congenital hydrocephalus, and the like. It is also within the confines of the present invention that the damaged or degenerated nervous tissue may result from an injury associated with trauma, cerebral hemorrhage, aneurysms, hypertensive encephalopathy, subarachanoid hemorrhage, diabetes, kidney dysfunction, ischemia, the treatment of therapeutic agents such as chemotherapy agents and antiviral agents, and other diseases or conditions prone to result in damaged or degenerated nervous tissue.

[0022] Thus, by treating the damaged or degenerated nervous tissue, it is believed that ebaf is useful for the treatment of neurodegenerative diseases. Similarly, by treating damaged or degenerated nervous tissue resulting from injury associated with trauma, cerebral hemorrhage, aneurysms, hypertensive encephalopathy, subarachanoid hemorrhage, diabetes, kidney dysfunction, ischemia, the treatment of therapeutic agents such as chemotherapy agents and antiviral agents, and other diseases or conditions prone to result in damaged or degenerated nervous tissue, it is believed that ebaf would be effective either alone or in combination with therapeutic agents used in the treatment of these diseases, conditions or disorders.

[0023] Furthermore, the ability of ebaf to induce the growth or enhance the survival of nervous tissue renders ebaf useful for preventing the onset or reducing the severity of damaged or degenerated nervous tissue. For example, a subject recently diagnosed with a neurodegenerative disease or predisposed to having a neurodegenerative disease based on family history may be considered a candidate for ebaf treatment. Similarly, it is envisioned that ebaf may be used in treating patients with diabetes, cancer or AIDS to prevent the onset or reduce the severity of damage or degenerative nervous tissue resulting from the disease or drugs used to treat these diseases or conditions.

[0024] In addition, since ebaf induces the growth or enhance the survival of nervous tissue, ebaf may be useful for enhancing wound healing, organ regeneration, organ transplantation (e.g., heart, kidney, lung, and liver), the transplantation of artificial organs, and in the acceptance of grafts (e.g skin, appendages, etc.).

[0025] In accordance with the methods of the present invention, the contacting or administration of ebaf may be effected by introduction or administration of the ebaf protein itself, or by the introduction or administration of a nucleic acid encoding ebaf in a manner permitting expression of the ebaf protein. The ebaf protein may be produced synthetically or recombinantly, or may be isolated from native cells, but is preferably recombinantly produced using the cDNA encoding ebaf (as set forth in FIG. 2) and conventional techniques. As used herein, the ebaf protein has the amino acid sequence set forth in FIG. 2. However, it is within the confines of the present invention that the protein includes functional variants thereof (i.e. proteins having ebaf protein activity) that are preferably 90% or greater in homology. In addition, the present invention also includes fragments of the ebaf protein with biological activity (i.e. peptide fragments that induce growth and/or enhance survival of neurons) and related peptide analogues thereof that exert similar biological activity. The ebaf protein may be administered to a tissue or subject by known techniques for the administration of proteins such as, for example, by injection or transfusion. When the damaged or degenerated nervous tissue is localized to a particular portion of the body, such as the brain, it may be desirable to administer the protein directly to the nervous tissue by injection or some other means (such as introducing ebaf into the cerebrospinal fluid or the blood). The amount of ebaf protein is an amount effective to promote the growth or enhance the survival of the nervous tissue, and is readily determinable to the skilled artisan.

[0026] ebaf also may be administered by introducing the nucleic acid encoding ebaf into a sufficient number of the cells of the nervous tissue (such as neurons, glial cells or Schwann cells, for example) in a manner permitting expression of ebaf in sufficient quantities to treat the damaged or degenerated nervous tissue. The nucleic acid may be introduced using conventional procedures known in the art including but not limited to electroporation, DEAE Dextran, monocationic liposome fusion, polycationic liposome fusion, protoplast fusion, DNA coated microprojectile bombardment, by creation of an in vivo electrical field, injection with recombinant replication-defective viruses, homologous recombination, naked DNA transfer, gene therapy, viral vectors, expression vectors, or a combination thereof. Recombinant viral vectors suitable for gene therapy include but are not limited to vectors derived from the genomes of viruses such as HSV, adenovirus, adeno-associated virus, Semiliki Forest virus, cytomegalovirus and vaccinia virus. It also is within the confines of the present invention that the nucleic acid encoding ebaf may be introduced into suitable cells in vitro (e.g. Schwann cell or glial cells) using conventional procedures. The cells expressing ebaf may then be administered to the subject to treat the damaged or degenerated nervous tissue. To reduce rejection, the cells are preferably removed from the patient, subjected to the DNA techniques to incorporate the nucleic acid encoding ebaf, and then reintroduced into the patient.

[0027] Depending upon the desired use of ebaf, it is within the confines of the invention that ebaf may be used alone or in combination with one or more therapeutic agents such as growth factors to treat the damaged or degenerated nervous tissue. In addition, ebaf may be used in combination with other therapeutic agents such as chemotherapeutic agents or antiviral agents.

[0028] Finally, the present invention provides a method for inducing growth or enhancing survival of nervous tissue comprising contacting the nervous tissue with a modulator of ebaf expression in an amount effective to induce or enhance expression of ebaf and induce the growth or enhance the survival of the nervous tissue. Examples of modulators of ebaf expression include but are not limited to retinoic acid, estrogen or progesterone. It is also within the confines of the present invention that the modulators of ebaf expression may be used for both in vitro and in vivo applications as discussed above, including, for example, the treatment of damaged or degenerated nervous tissue, the treatment of neurodegenerative diseases, for preventing the onset or reducing the severity of damaged or degenerated nervous tissue, and the like.

[0029] The present invention is described in the following examples which are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter.

[0030] ebaf RNA was injected into two cell stage blastocysts of the Xenopus laevis. The RNA was injected in various doses to show a dose response effect. The blastocysts were allowed to grow in vitro for several hours and then the animal caps were dissected away and further maintained in vitro until removed. Blastocysts that did not receive the ebaf RNA and the whole embryo served as controls. The RNAs were extracted from the injected, uninjected animal caps and the whole embryos which served as a positive control. These RNAs were reverse transcribed into cDNA and then amplified by PCR using various primer sets against markers of different tissues. The findings demonstrate that while ebaf has no effect on the development of endoderm and the mesoderm layers, ebaf inhibited the epidermization and enhanced the development of neuronal markers (see FIG. 1). ebaf also induced the neural marker NCAM (not shown). These tests also demonstrate that ebaf does not enhance the development of the spinal cord or hindbrain, rather its effect is precisely confined to the development of the forebrain (FIG. 1). In addition, morphologically, the brains of the frogs with ebaf induction were larger than the controls (not shown).

[0031] The activity of the ebaf is remarkably similar to those elicited by BMP antagonists such as noggin, chordin and follistatin (Weinstein D, et aL, Neural inducation in the frog Xenopus laevis. In: Inhibin, activin and follistatin. Serono Symposia USA, Norwell, Mass. Eds: Aono T, Sugino H, and Vale WW. A Serono Symposia SA Publication. Springer-Verlag, N.Y., 214-219, 1997). These proteins bind directly to ligands and block their binding to the receptors. In view of the lack of the cysteine residue required for dimerization of the members of the TGF-beta family (Kothapalli, et al., 1997), ebaf is likely not to form dimers and may bind to the receptor of the TGF-beta family members rather than to the ligand. By virtue of this binding, ebaf may inhibit the activity of one or several members of the TGF-beta superfamily.

[0032] The biological effects of the members of the TGF-beta family are signaled through two classes of molecules designated as type I and type II receptors. These are transmembrane serine-threonine kinases that share homology with each other but have distinctive features. The dimerized ligand first binds the type II receptor and the type I receptor is subsequently recruited leading to the formation of a heteromeric complex. Within this complex, the type II receptor which is constitutionally active, phosphorylates the type I receptor in the GS (glycine-serine rich) domain. In the case of BMP and activin, the ligand first binds to the type I receptor (Padgett, et al, Bioessays, 20(5):382-90, 1998). The ebaf monomers may bind to the receptor of the TGF-beta family members and may prevent their activity.

[0033] The following experiments were done in vitro to assess the neurogenic potential of ebaf. First, the mitogenic effect of ebaf on the cells isolated from rat embryo forebrain was determined. Then, whether ebaf increases the number of neurons in such cultures was determined. Based on the findings presented below, it is concluded that ebaf increases proliferation of forebrain cells and increases their maturation to neuronal cells.

[0034] Effect of Ebaf on Proliferation of Rat Embryo Forebrain Cells in Culture.

[0035] Cultures of dissociated, embryonic, day 16 (E16), rat forebrain were established in 24-well tissue culture plates with equal number (250×10³/well) of cells. These were maintained overnight in a chemically defined medium. Next morning, the experimental culture wells received overnight culture supernatant of confluent ebaf transfected cells collected in DMEM medium. The control culture wells received DMEM medium alone. Bromodeoxyuridine (BrdU) was added to all culture vessels (1 μl of a 1 mM solution per ml). The next day, these cultures were stained for BrdU. The experiment was done in triplicate. Results are shown in FIG. 3.

[0036] Effect of Ebaf on Neuronal Number in Rat Embryo Forebrain Cell Cultures.

[0037] Cultures of dissociated, embryonic, day 16 (E16), rat forebrain were established in 24-well tissue culture plates with equal number (250×10³/well) of cells. These were maintained overnight in a chemically defined medium. Next morning, the experimental culture wells received overnight culture supernatant of confluent ebaf transfected cells collected in DMEM medium. Other cultures received a recombinant E coli ebaf (26 kD) at 5 ng/ml. The control culture wells received DMEM medium alone. These were incubated for nine days without changing the medium. After nine days of culturing, cells stained with TG-2 antibody which marks the neurons (TG-2: Honer, et al., Psychol. Med 26: 1919-195, 1996). TG-2 positive cells were counted under a microscope at 200× magnification in each dish. The experiment was done in duplicate. The results are shown in FIG. 4.

[0038] Effect of Ebaf on Neuronal Markers in Rat Embryo Forebrain Cell Cultures.

[0039] Cultures of dissociated, embryonic, day 16 (E16), rat forebrain were established in 24-well tissue culture plates with equal number (250×10³/well) of cells. These were maintained overnight in a chemically defined medium. The next morning, culture supernatants from confluent ebaf-transfected cells (diluted 25 fold) and DMEM medium alone were added respectively to the experimental and control wells. These were incubated for nine days in culture. Medium was replaced on day five. On day 9, the well was stained with TG-2 and NeuN antibody which both mark neuronal cells (TG-2: Honer, et al., Psychol. Med. 26: 1919-195, 1996; NeuN: Mullen, et al., Development 116: 201-211, 1992). The reaction product was produced in solution in presence of o-phenylenediamine dihydrochloride (OPD). The solutions were the transferred to a 96 well plate and the absorbance was read at 490 nm. The experiment was done in duplicate. The results are shown in FIG. 5.

[0040] All publications mentioned herein above are hereby incorporated in their entirety. While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of the disclosure that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.