WO2001093681A1 - Modulating angiogenesis and neurogenesis with goliath proteins - Google Patents

Abstract

The invention provides methods and compositions for modulating angiogenesis or neurogenesis, including pharmaceutical compositions comprising an effective amount of a goliath polypeptide in dosage form. Therapeutic methods include administering to an animal determined to have pathogenic angiogenesis or neurogenesis, a pharmaceutical composition comprising an effective amount of a goliath polypeptide or a goliath polynucleotide and detecting a change in angiogenesis or neurogenesis in the animal an screening methods include contacting an endothelial or nerve cell with an effective amount of a goliath polypeptide or polynucleotide, and detecting a change in function of the cell.

Description

Modulating Angiogenesis and Neurogenesis with Goliath Proteins

INTRODUCTION Field of the Invention

The field of the invention is modulating angiogenesis and neurogenesis with goliath polypeptides and polynucleotides.

Background

The ability to understand the genetic pathways that regulate migration, growth, differentiation and sprouting of vessels has overwhelming implications for treating many human diseases, most certainly cancers. The growth of solid tumors is dependent on the continued development of a vasculature supply from the host. Inhibition of vasculature development in tumors by using antibodies or gene therapy against molecules that regulate vascular growth is an emerging and promising method for blocking tumorigenesis. We disclose a family of "goliath" proteins, which when overexpressed causes the inhibition of vasculature and hematopoietic development in vertebrate animals. In the vast majority of human tumors, including breast, lung, kidney, ovary, endometrium and uterus and in other neovascular diseases, including blindness, endometriosis, and rheumatoid arthritis, VEGF-A is grossly overexpressed or misregulated. Therefore, antibodies that inhibit VEGF signaling are being used to block angiogenesis from forming inappropriately. Several of these VEGF-A antibodies have been shown to block tumorigenesis in vitro and in animal models. Goliath molecules provide similar anti-angiogenic properties without many of the therapeutic limitations of antibodies.

Relevant Art

A number of sequences having similarity with the subject goliath sequences are present in genetic databases, including:

Osada et al. (April 29, 2000), Accession No. BAA95033 (mouse goliath);

Nagase et al. (Nov 11, 1999), Accession No. AB033040 (SEQ ID NOS:2, 12; human goliath);

Peng et al. (May 2, 2000), Accession No. AAF67007 (SEQ ID NOS:3, 13; human goliath-2);

Baker et al. (May 20, 2000), Accession No. AAF05310 (SEQ ID NOS:4, 14; mouse gi);

Bouchard et al. (Feb 1, 1995), Accession No. Q06003 (Drosophila goliath); Adams et al. (Mar 22, 2000), Accession No. AAF47316 (Drosophila goliath-2) and Davidson et al. (Oct 29, 1999), Accession No. T33407 (C. elegans goliath-like).

Prajal is also reported to bear sequence similarity to Drosophila goliath (Mishra et al., 1997,

Oncogene 15, 2361-2368).

SUMMARY OF THE INVENTION

The invention provides methods and compositions for modulating angiogenesis or neurogenesis. In one embodiment, the invention provides pharmaceutical compositions comprising an effective amount of a goliath polypeptide in dosage form. In one embodiment, the subject methods comprise the steps of administering to a vertebrate animal determined to have pathogenic angiogenesis or neurogenesis, a pharmaceutical composition comprising an effective amount of a goliath polypeptide or a goliath polynucleotide and detecting a change in angiogenesis or neurogenesis in the animal. Preferred target animals are mammals, particularly humans and mice. The pathogenicity may derive from excess angiogenesis (e.g. undesirable vascularization of a tumor) or insufficient angiogenesis (e.g. ischemia); from excess neurogenesis (e.g. neuroblastoma) or insufficient neurogenesis (e.g. neurodegenerative disease, unrecovered nerve trauma), etc. In the case of polynucleotide administration, the polynucleotide may be expressed in the animal as a goliath polypeptide, as an antisense complement of an endogenous goliath transcript, etc. The invention also provides methods for modulating endothelial or nerve cell function, comprising the steps of contacting an endothelial or nerve cell with an effective amount of a goliath polypeptide or polynucleotide, and detecting a change in function of the cell. For example, where the cell is an endothelial cell the change in function may comprise vascularization of the cell, and where the cell is a neuron or neuronal stem cell, the change may be in the growth, including proliferation and differentiation, of the cell. This embodiment is particularly suited to high-throughput in vitro screening for modulators, including inhibitors and activators, of goliath activity.

The invention provides a variety of methods and compositions relating to goliath polypeptides having goliath-specific structure and activity, related polynucleotides and modulators of goliath function. The goliath polypeptides may be recombinantly produced from transformed host cells, from the subject goliath polypeptide-encoding nucleic acids or purified from natural sources such as mammalian cells. The invention provides isolated goliath hybridization probes and primers capable of specifically hybridizing with natural goliath genes, goliath-specific binding agents such as specific antibodies, agonists and antagonists, and methods of making and using the subject compositions in diagnosis (e.g. genetic hybridization screens for goliath transcripts), therapy (e.g. goliath inhibitors and activators to modulate angiogenesis) and in the biopharmaceutical industry (e.g. as immunogens, reagents for isolating natural goliath genes and transcripts, reagents for screening chemical libraries for lead pharmacological agents, etc.).

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation. Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms "a" and "an" mean one or more, the term "or" means and/or and polynucleotide sequences are understood to encompass opposite strands as well as alternative backbones described herein.

The subject polypeptide sequences find a wide variety of applications. In one embodiment, the subject sequences are used to synthesize polypeptides which in turn provide a number of applications, including use in proteomic microarrays (e.g. Silzel JW, et al. Clin Chem 1998 Sep;44(9):2036-43), models for rational drug design, immunogens for antibody elicitation, reagents for enzyme assays, etc. The polypeptide sequences are also used to specifically detect sequences having similarity to the disclosed parental SEQ ID NOS: 1-4 or polypeptides comprising such sequences or fragments thereof. Any convenient sequence detection method may be used, including computational methods for direct sequence detection (e.g. BLAST-type algorithms, alignments, etc.) and physical methods for inferential sequence detection of polymers (e.g. mass spectroscopy, etc.).

In addition to direct synthesis, the subject polypeptides can also be expressed in cell and cell-free systems (e.g. Jermutus L, et al., Curr Opin Biotechnol. 1998 Oct;9(5):534-48) from encoding polynucleotides, such as the corresponding parent polynucleotides (SEQ ID NOS: 11-14) or naturally-encoding polynucleotides isolated with degenerate oligonucleotide primers and probes generated from the subject polypeptide sequences ("GCG" software, Genetics Computer Group, Inc, Madison WI) or polynucleotides optimized for selected expression systems made by back-translating the subject polypeptides according to computer algorithms (e.g. Holler et al. (1993) Gene 136, 323-328; Martin et al. (1995) Gene 154, 150- 166).

The subject goliath polypeptides generically encompass polypeptides which comprise a disclosed parental vertebrate goliath sequence (SEQ ID NOS: 1-4), comprise functional deletion mutants thereof, or have sequence similarity to a disclosed parental sequence. The subject fragments or deletion mutants of the recited sequences enhance or inhibit either basal or wild-type goliath induced angiogenesis or neurogenesis, and preferably substantially retain the activity of the full-length sequence polypeptide in one or more of the goliath bio-activity assays described below,^" articularly the HUVEC/Matrigel assay. Preferred fragments include intact distinct functional/structural domains, such as signal sequences (e.g. residues 20-32 and 12-24 of SEQ ID NO:l and 4, respectively), extracellular N-terminal domains (e.g. residues 33-205 and 25-192 of SEQ ID NO: 1 and 4, respectively), transmembrane domains (e.g. residues 206-225 and 193-213 of SEQ ID NO:l and 4, respectively), intracellular C-terminal domains (e.g. residues 226-428 and 214-419 of SEQ ID NO:l and 4, respectively), particularly the C3H2C3 ring finger motif (e;g. residues 277-317, 302-342 and 264-304 of SEQ ID NO:l, 2, and 4, respectively), etc. Exemplary active deletion mutants are shown in Table 1.

Table 1. Goliath deletion mutants retain angiogenic activity (HUVEC/Matrigel assay, below; activity includes enhancement or inhibition of either basal or wild-type goliath induced angiogenesis)

Polypeptide Sequence Angiogenic Activity

SEQ ID NO: 1 , residues 2-428 +++

SEQ ID NO: 1 , residues 18-428 +++

SEQ ID NO:l, residues 32-428

SEQ ID NO: 1 , residues 32-225 +++

SEQ ID NO: 1 , residues 32-206 +++

SEQ ID NO: 1 , residues 206-428 +++

SEQ ID NO: 1, residues 225-428 +++

SEQ ID NO: 1, residues 1-225 +++ SEQ ID NO:l, residues 1-206 +++ SEQ ID NO: 1, residues 2-259 +++ SEQ ID NO:l, residues 32-259

Subject parental sequence homologous polypeptides have goliath activity or goliath inhibitory activity. For such homologs, the requisite sequence similarity is at least 75%, preferably at least 85%, more preferably at least 95%, more preferably at least 98%, more preferably at least 99%, most preferably 100%. These polypeptides comprise, and the similarity or identity extends over at least 60, preferably at least 120, more preferably at least 240, more preferably at least 500 contiguous residues, preferably over a distinct functional/structural domain (supra) and most preferably over the entire polypeptide and/or parental goliath sequence.

Generally, for disclosed polymeric genuses, "percent (%) sequence identity over a specified window size W" with respect to parental sequences is defined as the percentage of residues in any window of W residues in the candidate sequence that are identical with the residues in the parent sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. The % identity values are generated by WU-BLAST-2 .0 al9 obtained from Altschul et al., J. Mol. Biol, 215: 403-410(1990); http://blast.wustl.edu/blast/README.html. WU-BLAST-2.0al9 uses several search parameters, all of which are set to the default values. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. Hence, a % sequence identity value is determined by the number of matching identical residues divided by the window size W for which the percent identity is reported. Exemplary species are readily generated by mutating the corresponding parental sequences and confirming goliath activity. For example, goliath polypeptides defined by SEQ ID NOS:5-10 exemplify an active 98% genus over the full length of parental sequence SEQ ID NO: 1.

The subject polypeptides and fragments thereof are isolated or pure: an "isolated" polypeptide is unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, and more preferably at least about 5% by weight of the total polypeptide in a given sample and a pure polypeptide constitutes at least about 90%, and preferably at least about 99% by weight of the total polypeptide in a given sample. The polypeptides may be synthesized, produced by recombinant technology, or purified from cells. A wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al, Greene Publ. Assoc, Wiley- erscience, NY) or that are otherwise known in the art.

The invention provides binding agents specific to the subject polypeptides, methods of identifying and making such agents, and their use. For example, specific binding agents are useful in a variety of diagnostic and industrial applications and include somatically recombined polypeptide receptors like specific antibodies or T-cell antigen receptors (see, e.g Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory), intracellular binding agents identified with assays such as one-, two- and three-hybrid screens, non-natural intracellular binding agents identified in screens of chemical libraries such as described below, including catalytic substrates, cofactors, agonists, antagonists, etc. In a particular embodiment, the invention provides goliath ligands and assays for goliath ligands, such as adapted from Davis et al., 1996, Cell 87, 1161-1169. In another embodiment, goliath polypeptides provide specific antigens and/or immunogens, especially when coupled to carrier proteins, for generating goliath specific antibodies. For example, peptides are covalently coupled to keyhole limpet antigen (KLH) and the conjugate is emulsified in Freunds complete adjuvant. Laboratory rabbits are immunized according to conventional protocol and bled. The presence of specific antibodies is assayed by solid phase immunosorbant assays using immobilized corresponding polypeptide.

Accordingly, the invention provides complementarity determining region (CDR) sequences and libraries of such sequences. The subject CDR sequences find a wide variety of applications. In one embodiment, the subject CDR sequences are used to synthesize polypeptides which in turn provide a number of applications, including immuno-microarrays, affinity reagents, etc. In addition to direct synthesis, the subject CDR polypeptides can also be expressed in cell and cell-free systems (e.g. Jermutus L, et al., Curr Opin Biotechnol. 1998 Oct;9(5): 534-48) from encoding polynucleotides, such as the corresponding parent polynucleotides or naturally-encoding polynucleotides isolated with degenerate oligonucleotide primers and probes generated from the subject polypeptide sequences ("GCG" software, Genetics Computer Group, Inc, Madison WI) or polynucleotides optimized for selected expression systems made by back-translating the subject polypeptides according to computer algorithms (e.g. Holler et al. (1993) Gene 136, 323-328; Martin et al. (1995) Gene 154, 150-166). Generally, the CDR polypeptides are expressed and used as the binding domain of an immunoglobulin or fragment thereof.

The invention provides efficient methods of identifying agents, compounds or lead compounds for agents which modulate goliath activity. A wide variety of assays is provided including the goliath activity assays detailed below are used to screen candidate agents for their ability to modulate angiogenesis or neurogenesis. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead therapeutic compounds. Candidate agents encompass numerous chemical classes, though typically they are organic compounds; preferably small organic compounds and are obtained from a wide variety of sources including libraries of synthetic or natural compounds.

The subject polynucleotide sequences find a wide variety of applications. For example, the polynucleotide sequences are also used to specifically detect the disclosed parent sequences, related natural vertebrate, preferably mammalian, more preferably mouse or human homologs, or polynucleotides comprising such sequences. Any convenient sequence detection method may be used. In one embodiment, candidate or unknown sequences are determined and compared with one or more parent and/or homolog sequence (SEQ ID NOs: 11-14 and NOS: 15-20, respectively) to classify the candidate or unknown sequences. For example, an algorithm such as BLAST (e.g. Build sol2.5-x86 01:40:37 05-Feb-1998, Copyright (C)1997 Warren R. Gish, using default parameters, Altschul et al., Methods in Enzymology, 215: 403-410 (1997)) may be used to define relatedness to one or more subject sequence diagnostic of goliath expression in computer-based methods. In another embodiment, the parent and/or homolog sequences are used to synthesize and/or are embodied in polynucleotides which in turn provide a number of applications, including microarray-based methodologies, see e.g. Nat Genet 1999 Jan;21(l Suppl), entire issue incl. Debouck C, et al. at 48-50; gene expression analysis, see e.g. Carulli JP, et al., J Cell Biochem Suppl 1998;30-31:286-96; drug target discovery and design, see, e.g. Jones DA, et al., Curr Opin Chem Biol 1999 Feb;3(l):71-6; combinatorial chemistry, see, e.g. Lukas TJ, et al., J Med Chem. 1999 Mar 11;42(5):910-919; ribozymes and therapeutics, see e.g. Rossi JJ, Chem Biol 1999 Feb;6(2):R33-7; mapping; etc. In one embodiment, candidate and/or unknown polynucleotides may be isolated, compared and/or classified (e.g. by relatedness) by hybridization to one or more parent and or homolog polynucleotide, e.g using microarrayed libraries of parent and/or homolog polynucleotides. Such polynucleotides may also be used as probes and/or primers to localize, isolate, amplify, etc., natural genes and transcripts. In another embodiment, the parent and/or homolog polynucleotides or fragments or libraries of such polynucleotides, including reverse complements and antisense counterparts, are transfected into cells for a wide variety of cloning, display, expression, goliath inhibition, etc. applications, including 'n' -hybrid systems, see, e.g. Vidal M, et al., 1999, Nucleic Acids Res. 27(4):919-929 & Proc Natl Acad Sci U S A. 93(19):10315-20 & 10321-6; mapping protein- ligand interactions using whole genome phage display libraries, see e.g. Palzkill T, et al., Gene 1998 Oct 9;221(l):79-83; DNA-based selection and screening of peptide ligands, see e.g. Bartoli F, et al, Nat Biotechnol 1998 Nov;16(l 1): 1068-73, etc.

In a preferred embodiment, the invention provides microarrays of the disclosed polynucleotides and their uses as described or cited herein. A wide variety of materials and methods are known in the art for arraying polynucleotides at discrete elements of substrates such as glass, silicon, plastics, nylon membranes, etc., including contact deposition, e.g. US Pat Nos. 5,807,522; 5,770,151, DeRisi JL, et al. Curr Opin Oncol 1999 Jan;l l(l):76-9, etc.; photolithography-based methods, e.g. US Pat Nos. 5,861,242; 5,858,659; 5,856,174; 5,856,101; 5,837,832, Lipshutz RJ, et al. Nat Genet 1999 Jan;21(l Suρpl):20-4, etc.; inkjet dispensing technologies, e.g. Lemmo AV, et al., Curr Opin Biotechnol 1998 Dec;9(6):615-7; flow path-based methods, e.g. US Pat No. 5,384,261; dip-pen nanolithography-based methods, e.g. Piner, et al., Science Jan 29 1999: 661-663, etc.; etc.

The invention also provides polynucleotides which hybridize to a polynucleotide having a sequence as set forth in any one of parental vertebrate goliath-encoding polynucleotides (SEQ ID NOS: 11-14), or to its reverse complement, under hybridization condition #1, preferably #2, more preferably #3 and so on to #10, as identified and described in Tables A-C. Thus, for example, if hybridization condition #7 is preferred, then the conditions used for identifying and classifying related or homologous polynucleotides employ hybridization buffer M at a hybridization temperature of 40°C, and wash buffer E at a wash temperature of 55°C. Condition #1 identifies polynucleotides having at least about 50% sequence identity with the target polynucleotide (with % identity calculated as described herein). With each subsequent condition, the stringency is such that the isolated polynucleotide has a sequence identity of at least 5% greater than what would be isolated by using the next lower condition number. Thus, for example, condition #2 identifies polynucleotides having at least about 55% sequence identity with the target polynucleotide, and conditions #9 and #10 identify polynucleotides having at least about 90% and 95% sequence identity, respectively, to the target polynucleotide.

Exemplary higher stringency hybridizing polynucleotides of each parent (having parent sequence identities of about 95%) are designated SEQ ID NOS: 15-17, respectively and exemplary lower stringency hybridizing polynucleotides of each parent (having parent sequence identities of about 90%) are designated SEQ ID NOS: 18-20, respectively. In situations where it is desired to classify more closely related polynucleotides, the hybridization condition is increased by increments of one, until the desired specificity is obtained. Preferably, each hybridizing polynucleotide has a length that is at least 30%, preferably at least 50%, more preferably at least 70% and more preferably at least 90% and most preferably 100% of the length of the corresponding parent polynucleotide sequence described herein, or the reverse complement thereof, to which it hybridizes.

In Tables A and B, formamide is expressed as percent (v/v) in a buffered diluent comprising IX to 6X SSC (IX SSC is 150 mM NaCl and 15mM sodium citrate; SSPE may be substituted for SSC, IX SSPE is 150mM NaCl, 10 mM Na H₂PO₄, and 1.25 mM EDTA, pH7.4). Procedures for polynucleotide hybridizations are well-known in the art (see Ausubel et al, Current Protocols in Molecular Biology. Wiley Literscience Publishers, (1995); Sambrook et al., Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Press, 1989; Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 6789-6792; and PCT publication WO 99/01466).

TABLE A TABLE B

TABLE C

The invention also provides fragments of the parent and/or homolog polynucleotides which may be used in the foregoing methods, especially as nucleic acid hybridization probes and replication / amplification primers. These fragments are of length sufficient to specifically hybridize with the corresponding SEQ ID NO or complement thereof, generally comprising at least 12, preferably at least 24, more preferably at least 36 and most preferably at least 96 contiguous nucleotides of the corresponding SEQ ID NO (see, e.g. Table 2).

Table 2. Exemplary polynucleotide fragments which hybridize with a strand of SEQ ID NO:N under hybridization condition #5; N is integers 11-14.

Polynucleotide fragment Hybridization

SEQ ID NO:N, nucleotides 1-36 +

SEQ ID NO:N, nucleotides 18-43 + SEQ ID NO:N, nucleotides 25-51 +

SEQ ID NO:N, nucleotides 35-62 +

SEQ ID NO:N, nucleotides 36-81 +

SEQ ID NO:N, nucleotides 99-138 +

SEQ ID NO:N, nucleotides 168-195 +

SEQ ID NO:N, nucleotides 255-291 +

SEQ ID NO:N, nucleotides 325-351 +

SEQ ID NO:N, nucleotides 709-737 +

SEQ ID NO:N, nucleotides 753-777 +

SEQ ID NO:N, nucleotides 800-828 +

The subject polynucleotides and fragments thereof may be joined to other components such as labels or other polynucleotide/polypeptide sequences (i.e. they may be part of larger sequences) and are of synthetic/non-natural sequences and/or are isolated, i.e. unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, preferably at least about 5% by weight of total nucleic acid present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome. Recombinant polynucleotides comprising the subject SEQ ID NOs, or fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by (i.e. contiguous with) a sequence other than that which it is joined to on a natural chromosome, or flanked by a native flanking region fewer than 2 kb, preferably fewer than 500 bases, more preferably fewer than 100 bases, most preferably fewer than 20 bases, which is at a terminus or is immediately flanked by a sequence other than that which it is joined to on a natural chromosome. While the nucleic acids are usually RNA or DNA, it is often advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide modified stability, etc.

Administration of Goliath Molecules. In numerous embodiments of the present invention, a goliath molecule will be administered to an animal. Such compounds can be administered by a variety of methods including, but not limited to, parenteral, topical, oral, or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment. The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include, but are not limited to, powder, tablets, pills, capsules and lozenges. It is recognized that goliath modulators (e.g., antibodies, antisense constructs, ribozymes, small organic molecules, etc.) when administered orally, must be protected from digestion. This is typically accomplished either by complexing the molecule(s) with a composition to render it resistant to acidic and enzymatic hydrolysis, or by packaging the molecule(s) in an appropriately resistant carrier, such as a liposome. Means of protecting agents from digestion are well known in the art.

In a particular embodiment, the compositions are delivered locally and distribution is restricted. For example, a particular method of administration involves coating, embedding or derivatizing fibers, such as collagen fibers, protein polymers, etc. with therapeutic agents, see also Otto et al. (1989) J Neuroscience Research 22, 83-91 and Otto and Unsicker (1990) J Neuroscience 10, 1912-1921. Another particular embodiment is adapted from treatment of spinal cord injuries, e.g. Schulz MK, et al., Exp Neurol. 1998 Feb; 149(2): 390-397; Guest JD, et al., J Neurosci Res. 1997 Dec 1; 50(5): 888-905; Schwab ME, et al, Spinal Cord. 1997 Jul; 35(7): 469-473; Tatagiba M, et al., Neurosurgery. 1997 Mar; 40(3): 541-546. See Example 3, below. The amount administered depends on the goliath molecule, formulation, route of administration, etc. and is generally empirically determined and variations will necessarily occur depending on the target, the host, and the route of administration, etc.

The compositions for administration will commonly comprise a goliath molecule or modulator dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.

Thus, a typical pharmaceutical composition for intravenous administration would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100 mg per patient per day may be used, particularly when the drug is administered to a secluded site and not into the blood stream, such as into a body cavity or into a lumen of an organ. Substantially higher dosages are possible in topical administration. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania (1980). In addition, the compounds may be advantageously used in conjunction with other neurogenic agents, neurotrophic factors, growth factors, anti-inflammatories, antibiotics etc.; and mixtures thereof, see e.g. Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9^th Ed., 1996, McGraw- Hill, esp. Chabner et al., Antineoplastic Agents at pp .1233.

The compositions for administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. The term "unit dosage forms" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions. In such compositions, the hydroxylamine compound is usually a minor component (from about 0.1 to about 50% by weight or preferably from about 1 to about 40% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.

The compositions containing modulators of goliath can be administered for therapeutic or prophylactic treatments. In therapeutic applications, compositions are administered to a patient suffering from a disease (e.g., a cancer or neuropathy) in an amount sufficient to reduce or arrest pathology. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the agents of this invention to effectively treat the patient.

Introducing Nucleic Acids into Cells. In numerous embodiments, one or more nucleic acids, e.g., goliath polynucleotides, such as antisense polynucleotides or ribozymes, will be introduced into cells, in vitro or in vivo. The present invention provides methods, reagents, vectors, and cells useful for expression of goliath and other polypeptides and nucleic acids using in vitro (cell-free), ex vivo or in vivo (cell or organism-based) recombinant expression systems.

The particular procedure used to introduce the nucleic acids into a host cell for expression of a protein or nucleic acid is application specific. Any of the well known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, spheroplasts, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger), F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1999), and Sambrook et al, Molecular Cloning - A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989.

Vectors. In numerous embodiments of this invention, nucleic acids encoding Goliath polypeptides, or inhibitors thereof, will be inserted into vectors using standard molecular biological techniques. Vectors may be used at multiple stages of the practice of the invention, e.g., for subcloning nucleic acids encoding goliath polypeptides or goliath inhibitors, e.g., goliath ribozymes or antisense sequences, or for subcloning additional elements used to control protein or mRNA expression, vector selectability, etc. Vectors may also be used to maintain or amplify the nucleic acids, for example, by inserting the vector into prokaryotic or eukaryotic cells and growing the cells in culture. In addition, vectors may be used to introduce and express goliath nucleic acids, or goliath-inhibiting nucleic acids, e.g., goliath ribozymes or antisense sequences, into cells for therapeutic or experimental purposes.

A variety of commercially or commonly available vectors and vector nucleic acids can be converted into goliath vectors by cloning a goliath polynucleotide into the commercially or commonly available vector. A variety of suitable vectors are well known in the art: for cloning in bacteria, common vectors include pBR322-derived vectors such as pBLUESCRIPT (Strategene), and bacteriophage derived vectors. In yeast, vectors include Yeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids (the YRp series plasmids) and pGPD-2. Expression in mammalian cells can be achieved using a variety of commonly available plasmids, including pSV2, pBC12BI, and p91023, as well as lytic virus vectors (e.g., vaccinia virus, adeno virus, and baculovirus), episomal virus vectors (e.g., bovine papillomavirus), and retro viral vectors (e.g., murine retroviruses).

Typically, a nucleic acid subsequence encoding a goliath polypeptide is placed under the control of a promoter. A nucleic acid is "operably linked" to a promoter when it is placed into a functional relationship with the promoter. For instance, a promoter or enhancer is operably linked to a coding sequence if it increases or otherwise regulates the transcription of the coding sequence. Similarly, a "recombinant expression cassette" or simply an "expression cassette" is a nucleic acid construct, generated recombinantly or synthetically, with nucleic acid elements that are capable of effecting expression of a structural gene in hosts compatible with such sequences. Expression cassettes include promoters and, optionally, introns, polyadenylation signals, and transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used as described herein. For example, an expression cassette can also include nucleotide sequences that encode a signal sequence that directs secretion of an expressed protein from the host cell. Transcription termination signals, enhancers, and other nucleic acid sequences that influence gene expression, can also be included in an expression cassette.

An extremely wide variety of promoters are well known, and can be used in the vectors of the invention, depending on the particular application. Ordinarily, the promoter selected depends upon the cell in which the promoter is to be active. Other expression control sequences such as ribosome binding sites, transcription termination sites and the like are also optionally included. For E. coli, example control sequences include the T7, trp, or lambda promoters, a ribosome binding site and preferably a transcription termination signal. For eukaryotic cells, the control sequences typically include a promoter which optionally includes an enhancer derived from immunoglobulin genes, SV40, cytomegalo virus, a retrovirus (e.g., an LTR based promoter) etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.

EXAMPLES Functional cloning of Goliath. During gastrulation, two of the three primary germ layers mesoderm and endoderm are formed, and diverse numbers of specialized cell types, including angioblasts and neural tissue, emerge. In mammals, gastrulation leads to the induction of extraembryonic mesoderm, which is essential for establishing vascular connections to the mother. We devised a functional assay to identify the genes required for these pivotal cell fate decisions. We generated two independent cDNA libraries from early (6.5 d.p.c.) and late (7.5 d.p.c.) staged mouse gastrula. These cDNA libraries were arrayed onto 384 well plates, divided into pools of 96 individual cDNAs and made into synthetic mRNA. Each mRNA pool was then injected into the animal pole or marginal zone of one-cell-staged Xenopus embryos. At neurula stages these embryos were analyzed by in situ hybridization for a panel of neural patterning markers. Xenopus embryos injected with pool #88 exhibited ectopic neuronal development. Isolation of the cDNA responsible for this phenotype yielded a novel mouse protein (mouse goliath, SEQ ID NOS: 1, 11 - translate and cDNA, respectively) with homology over a C3H2C3 ring finger motif at the c-terminus to the Drosophila protein, Goliath. In addition to the c-terminal ring finger, mouse Goliath contains a strong signal sequence and a single transmembrane domain. Further Blast searches revealed other family members, including a mouse Goliath polymorphism (Osada et al. (April 29, 2000), Accession No. BAA95033, which has an A to G substitution at +394, resulting in a conservative I to V point mutation at residue 132 - both polymorphism are encompassed by the term mouse goliath); mouse protein (gl, SEQ ID NOS:4, 14), which in addition to strong sequence similarity to mouse Goliath has both the predicted signal sequence and single transmembrane domain, and human homologs (SEQ ID NOS:2, 12 and 3, 13). However, neither the human protein nor Drosophila Goliath contain the signal sequence or transmembrane domain.

Goliath expression influences mesodermal patterning. Xenopus embryos expressing Goliath in the ectodermal animal hemisphere display mesodermal patterning defects during gastrulation. Although a normal mesodermal ring appears to form throughout the equitorial region, some mesodermal cells appear ectopically in the vegetal or animal regions giving the mesoderm a "spotty" appearance. Later in development, at early tadpole stages, muscle is not affected by Goliath expression, but other mesoderm derivatives, including blood and endothelial cells are absent. These data indicate that Goliath influences a select population of mesodermal cell fates during and after gastrulation.

Goliath forms ectopic neurons. Goliath mRNA injections into one blastomere of the two cell Xenopus embryo cause ectopic neural tissue to form throughout the epidermis in a spotty pattern as judged by insitu hybridization for the general neural marker, nrpl . Since this spotty neural pattern is characteristic of neurons, we analyzed Goliath injected embryos by in situ hybridization for the neuron specific marker n-tubulin to determine whether Goliath causes ectopic formation of neurons. Indeed, the misplaced neural tissue is composed of neurons. Close observation of this phenotype reveals that the formation of ectopic neural tissue coincides with a decrease in the neural cohesion and thickness of the spinal cord, indicating that Goliath functions as a neural guidance molecule and not as a direct inducer of neural tissue.

Goliath is co-expressed with VEGF and VEGFR2 at different times during vascular development. Expression analysis during mouse embryogenesis revealed that Goliath is not localized to neural tissues, but is localized to developing endothelial cells. As early as 5.5 d.p.c and throughout gastrulation, Goliath is expressed in the extraembryonic ectoderm and endoderm. By 7.5 d.p.c. Goliath is found primarily in the extraembryonic endoderm. This pattern of Goliath expression parallels that of VEGF-A, indicating that they can regulate one another during gastrulation. During later stages, Goliath continues to be expressed in developing endothelial cells. At 8.5 d.p.c.Goliath is expressed in the developing dorsal aorta and umbilical vein. By 10.5 d.p.c. we detect Goliath in the intersomitic veins, the heart, the limb buds, the bracheal arches, and in regions of the brain.

Goliath inhibits both endothelial and blood development in Xenopus embryos. Since Goliath is co-expressed with VEGF during mouse gastrulation, we tested whether Goliath has an effect on vascular development in Xenopus Laevis. Goliath mRNA was injected into the prospective ectoderm of the one-cell Xenopus embryo. These embryos were harvested in two separate experiments at tadpole stage and examined by insitu hybridization for the endothelial marker, Xhex, the blood marker, b-globin and the general muscle marker, c-actin. The Goliath overexpressing embryos demonstrate a remarkable reduction in the endothelial cells of the intersomic veins, the posterior cardinal veins, and blood islands . However, expression of Xhex in the liver is unaffected by Goliath. Furthermore, embryos overexpressing goliath show a distinct absence of blood, but have normal muscle development. These results indicate Goliath is a negative regulator of endothelial and blood development.

Expression of goliath in CHO cells. To express the mature factor in CHO cells, the goliath cDNA with a segment coding for the FLAG octapeptide (IBI Kodak) at C terminal is amplified by PCR and inserted into the mammalian expression vector pcDNA3 (Invitrogen) under the control of the cytomegalovirus promoter (construct LM357). CHO cells are transfected with LM357 by using calcium phosphate precipitation. Stable clones are selected in DMEM containing 10% FCS and 800 μg/ml G418. To assay the presence of goliath in CHO supernatants, isolated clones are grown in DMEM containing 2% FCS and 800 μg/ml G418 and analyzed by ELISA using anti-goliath rabbit polyclonal antiserum (Orlandini, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93, 11675-11680). These methods, the yeast expression, cornea angiogenic assay, and exogenous administration assay (below) were adapted from Marconcini et al., 1999, Proc Natl Acad Sci USA 96, 9671-9676.

Expression of goliath in yeast. To express goliath in yeast a cDNA fragment encoding the goliath polypeptide with six histidine residues at N terminus is amplified by PCR and inserted into the expression vector Yepsecl immediately downstream from DNA sequence encoding the Kluyveromyces lactis toxin leader peptide (LM375) (Baldari, C. et al. (1987) EMBO J. 6, 229-234). The protein is expressed in Saccharomyces cerevisiae yeast strain by adding galactose to the yeast culture medium because Yepsecl construct contains a galactose upstream activation sequence and the 5' nontranslated leader of the yeast CYC1 gene, up to position 4 from the ATG translation initiation codon (Baldari, C. et al. (1987) EMBO J. 6, 229-234).

In Vivo Cornea Angiogenic Assay. The angiogenic activity of goliath is assayed in vivo by using the rabbit cornea assay previously described (Ziche, M. et al. (1989) Lab. Invest. 61, 629-634). Corneal assays are performed in male New Zealand albino rabbits (Charles River, Calco, Lecco, Italy) Briefly, after being anaesthetized with sodium pentothal (30 mg/kg), a micro pocket (1.5 x 3 mm) is surgically produced by using a pliable iris spatula 1.5 mm wide in the lower half of the cornea. Into the micro-pocket are implanted (a) the cell suspension (from 2.5 to 4 x 10⁵ cells/5ml) with and without slow-release pellets of Elvax-40 (DuPont) containing purified VEGF (human recombinant VEGF, expressed in Sβl insect cells; CAS 127464-60-2, Sigma-Aldrich) or (b) slow-release pellets containing goliath (recombinant, purified from yeast, supra) with and without the VEGF pellets. Subsequently daily observation of the implants is made with a slit lamp stereomicroscope without anesthesia. An angiogenic response is scored positive when budding of vessels from the limbal plexus occurs after 4 days and capillaries progress to reach the implanted pellet according to the scheme of (Ziche, M. et al. (1997) J. Clin. Invest. 99, 2625-2634). The potency of angiogenic activity is evaluated on the basis of the number and growth rate of newly formed capillaries, and an angiogensis score is calculated as described (Ziche, M. et al. (1997) J. Clin. Invest. 99, 2625-2634). Corneas are removed at the end of the experiment as well as at defined intervals after surgery and/or treatment and fixed in formalin for histological examination. Both CHO clones expressing goliath and goliath pellets inhibit basal and VEGF-induced corneal vascularization.

HUVEC Exogenous Administration Assay.

Cell Cultures. Human endothelial cells are isolated from umbilical cord vein by collagenase treatment as described (Bussolino. F. et al. (1989) Nature (London) 337, 471- 473) and used at passage 1-4. Cells are grown in gelatin-coated plastic, in medium M 199 supplemented with 20% heat-inactivated FCS, penicillin (100 units/ml) streptomycin (50 μg/ml), heparin (50 μg/ml), and bovine brain extract (100 μg/ml) (Life Technologies, Milan, Italy).

In Vitro Angiogenesis. Fifty microliters of Matrigel (Collaborative Research, lot 901448) (Kleinman, H.K., et al. (1986) Biochemistry 25, 312-318) is added per well of 96- well tissue culture plates and allowed to gel at 37°C for 10 min. HUVECs are starved for 24 h in Ml 99 with 1% FCS before being harvested in PBS-EDTA. Cells (10⁴) are gently added to each of triplicate wells and allowed to adhere to the gel coating for 30 min at 37°C. Then, medium is replaced with indicated concentrations of goliath.

Treatment with VEGF induces in vitro morphological changes resembling capillarylike structure formation; cells become elongated, forming thin cords of interconnecting cells. Treatment of VEGF treated cells with goliath demonstrates a dose-dependent reversal of the in vitro angiogenesis.

Goliath and VEGF Adeno virus Vectors. The replication-deficient recombinant adenovirus (Ad) vector containing the cDNA for goliath or VEGF is engineered according to (Maeda H. et al. (1994) Gastroenterology 106, 1638-1644). Briefly, the cDNA including the signal sequence for secretion (Leung DW. et al. (1989) Science 246, 1306-1309), is inserted into an expression plasmid (Maeda H. et al.) and is under the control of the constitutive CMV immediate-early promoter/enhancer. The expression plasmid also contains the Ad 5 sequence from nucleotide 5778 (9.24 to 16.05 map units), which serves as the homologous recombination sequence. The plasmid carrying the cDNA for goliath or VEGF is cotransfected with the plasmid pJM17 into 293 cells (American Type Culture Collection, CRL1573). The plasmid pJM17 contains the full-length Ad5 DNA (36 kb) and pBRX, a 4.3- kb insert placed in the El region, thus exceeding by ~2 kb the maximum packaging limit of DNA into the Ad capsid (McGrory, WJ. et al. (1998) Virology 163, 614-617). Homologous recombination between the expression plasmid and pJM17 in 293 cells replaced the El region and pBRX insert with the expression cassette from the expression plasmid. The growth of these El -deleted Ads is limited to 293 cells, a human embryonic kidney cell line that has been transformed by Ad 5 and expresses the El region in trans. Culture medium for the 293 cells is improved minimal essential medium with 10% heat-inactivated fetal bovine serum, 2mmol/L glutamine, 50 U/mL penicillin, and 50 μg/ml streptomycin (all from Biofluids). After cotransfection, individual viral plaques are isolated and amplified in 293 cells. The control vector is AdCMV.βgal, which carries the cDNA for the Escherichia coli lacZ gene and codes for the enzyme β-galactosidase (Hersh, J. et al. (1995) Gene Ther. 2, 124-131). AdCMB.Gol, AdCMV.VEGF and AdCMV.βgal are propagated in 293 cells and are purified by CsCl density purification. Subsequently, the preparations are dialyzed and stored in the dialysis buffer (10 mmol/L Tris-HCl and 1 mmol/L MgCl₂, pH 7.4) with 10% glycerol at -70°C. The titer of each viral stock is determined by plaque assay in 293 cells as previously described (Rosenfeld, MA. et al. (1992) Cell 68, 143-155), and the titers consistently range between 5xl0⁹ and 2xlO^u pfu/mL. These methods and the HUCEV transfection assays (below) were adapted from Muhlhauser et al., 1995, Circ Res 77, 1077- 1086.

HUVEC Transfection Assay. In Vitro. HUVECs (supra) in serum-free MCDB131 medium (Clonetics) and without growth supplements are infected with AdCMV.Gol or with ADCMV.βgal (20 pfu per cell) 48 hours before trypsinization and replating. Exposure to the Ad vector lasts 24 hours. Another group of uninfected cells is used as a second control. HUVECs are harvested 48 hrs after the infection with trypsin/EDTA and plated in 16-mm wells (8 x 10⁴ cells/well) previously coated with reconstituted basement membrane (Matrigel, 0.3 mL per well, 10 mg/mL) for 1 hr at 37°C as described in Kubota Y. et al. (1988) J Cell Biol. 107, 1589-1598, except the culture medium is supplemented with VEGF (10 ng/mL). After 24 hrs. the cells are fixed in PBS-buffered 10% formalin containing 2.5% glutaraldehyde. Capillary-like structures formed by HUVECs are visualized with an inverted microscope (Diaphot), photographed with a Polaroid camera, and quantified by optical imaging (IMAGE- 1 analysis system, Universal Imaging Corp). AdmCMV.Gol transfection inhibits basal in vitro angiogenesis.

HUVEC Co-transfection Assay. In Vivo. In order to assess the effects of Ad- mediated gene transfer in vivo, AdCMV.Gol, AdCMVNEGF, (AdCMV.Gol + AdCMVNEGF) or AdCMV.βgal (2xl0¹⁰ pfu) is resuspended in 0.5 mL Matrigel. Subsequently, C57BL mice (Jackson Laboratories, Bar Harbor, Me) are injected subcutaneously, near the abdominal midline, with 0.5 mL Matrigel containing a vector or cocktail. Additional animals are injected with uninfected Matrigel. Mice are studied according to four different protocols: (1) To establish whether Ad vectors resuspended in Matrigel infect the surrounding tissues, mice are injected either with Matrigel containing AdCMV.βgal or Matirgel alone. The animals are killed 6 days after injection, and the Matrigel plugs are removed and fixed as described above for endothelial cells. Subsequently, the Matrigel plugs are sectioned, stained with X-gal as previously described (Hersh, J. et al.), and examined for evidence of blue staining. (2) To establish the duration of transgene expression in vivo, mice are injected either with Matrigel containing AdCMV.NEGF, AdCMV.Gol, (AdCMV.Gol + AdCMV.VEGF), AdCMV.βgal, or Matrigel alone. Animals are killed and the Matrigel plugs are removed 3, 7, and 21 days after injection. Tissue blocks are immersed in OCT compound (Miles h e) and rapidly frozen in liquid nitrogen. Tissue blocks are stored at -70°C for <1 month. For immunohistochemical evaluation, 10-μm frozen sections (Microm cryotome) were mounted on salinated slides (Digene Diagnostics). Sections are air-dried for 15 minutes, and either stored at -70°C for up to 48 hours or fixed immediately in lx Histochoice (Amresco) containing 0.1% Triton X-100 (Sigma Chemical Co) for 12 minutes. After they are washed with PBS (pH 7.4), slides are incubated in 0.5% hydrogen peroxide in methanol to inhibit endogenous peroxidase activity. Anti-goliath or anti-VEGF primary rabbit antibodies (see below) are detected by using biotinylated goat anti- rabbit IgG secondary antibody and the avidin-biotin complex and visualized by diaminobenzidine (all detection reagents are from Vector Laboratories). Procedures are performed according to package directions, except sections are kept in blocking solution for at least 45 minutes before the addition of the primary antibody, and incubations with anti- goliath, anti-VEGF or control serum (1:6000 dilution) are performed overnight at 4°C. Sections are counterstained in hematoxylin. Anti-goliath and anti-VEGF antibodies are produced in rabbits as previously described (Berkman, RA. et al. (1993) J Clin Invest. 91, 153-159), except the peptide is conjugated to a carrier protein, KLH, by 0.2% glutaraldehyde. Antibodies to KLH alone are also raised and used as a negative control. Antibody specificity is determined by recognizing human goliath or VEGF on Western blots, and both anti-KLH and prebleed serum are used as negative controls to determine background staining. (3) The presence of newly formed blood vessels is evaluated as previously described (Passaniti, A. et al. (1992) Lab Invest. 67, 519-528) in mice killed 14 days after the injection of the Matrigel (n=8 mice for each Ad vector; 4 mice are used in each of two separate experiments). The gels are recovered by dissection and fixed. Histological sections are stained with Masson's trichrome stain and evaluated for the presence of neovascularization. The thickness of the stroma surrounding the Matrigel is assessed by measuring the distance between the surface of the Matrigel and the abdominal muscle in two different histological sections from each plug. Ten measurements are obtained at 50- to 100-μm intervals from each histological section, and the 20 measurements from the two sections are averaged to express stromal thickness for each individual plug. (4) The angiogenic response is quantified by the hemoglobin content of the Matrigel plugs (Passaniti et al.). Transfection with AdCMV.VEGF and AdCMV.Gol promotes and inhibits, respectively, basal in vivo angiogenesis. Cotransfection with AdCMV.Gol demonstrates a dose-dependent reversal of VEGF-induced in vivo angiogenesis.

Corticospinal Tract (CST) Regeneration Assay. Goliath molecules can improve corticospinal tract (CST) regeneration following thoracic spinal cord injury by promoting CST regeneration into human Schwann cell grafts in the methods of Guest et al. (supra). For these data, the human grafts are placed to span a midthoracic spinal cord transection in the adult nude rat, a xenograft tolerant strain. Goliath molecules incorporated into a fibrin glue are placed in the same region. Anterograde tracing from the motor cortex using the dextran amine tracers, Fluororuby (FR) and biotinylated dextran amine (BDA), are performed. Thirty-five days after grafting, the CST response is evaluated qualitatively by looking for regenerated CST fibers in or beyond grafts and quantitatively by constructing camera lucida composites to determine the sprouting index (SI), the position of the maximum termination density (MTD) rostral to the GFAP-defined host/graft interface, and the longitudinal spread (LS) of bulbous end terminals. The latter two measures provide information about axonal die-back. In control animals (graft only), the CST do not enter the SC graft and undergo axonal die-back. Goliath molecules reduce axonal die-back and cause sprouting.

Peripheral Nerve Regeneration Assay. Goliath molecules are incorporated in the implantable devices described in US Pat No. 5,656,605 and tested for the promotion of in vivo regeneration of peripheral nerves. Prior to surgery, 18 mm surgical-grade silicon rubber tubes (ID. 1.5 mm) are prepared with or without guiding filaments (four 10-0 monofilament nylon) and filled with goliath molecules. Experimental groups consist of: 1. Guiding tubes plus Biomatrix 1™ (Biomedical Technologies, Inc., Stoughton, Mass) ; 2. Guiding tubes plus Biomatrix plus filaments; 3-23. Guiding tubes plus Biomatrix 1™ plus goliath molecules.

The sciatic nerves of rats are sharply transected at mid-thigh and guide tubes containing the test substances with and without guiding filaments sutured over distances of approximately 2 mm to the end of the nerves. In each experiment, the other end of the guide tube is left open. This model simulates a severe nerve injury in which no contact with the distal end of the nerve is present. After four weeks, the distance of regeneration of axons within the guide tube is tested in the surviving animals using a functional pinch test. In this test, the guide tube is pinched with fine forceps to mechanically stimulate sensory axons. Testing is initiated at the distal end of the guide tube and advanced proximally until muscular contractions are noted in the lightly anesthetized animal. The distance from the proximal nerve transection point is the parameter measured. For histological analysis, the guide tube containing the regenerated nerve is preserved with a fixative. Cross sections are prepared at a point approximately 7 mm from the transection site. The diameter of the regenerated nerve and the number of myelinated axons observable at this point are used as parameters for comparison.

Measurements of the distance of nerve regeneration document the therapeutic effect of goliath molecules. Similarly, plots of the diameter of the regenerated nerve measured at a distance of 7 mm into the guide tube as a function of the presence or absence of one or more antagonists of the device demonstrate a similar therapeutic effect. No detectable nerve growth is measured at the point sampled in the guide tube with the matrix-forming material alone. The presence of guiding filaments plus the matrix-forming material (no antagonist) induces only very minimal regeneration at the 7 mm measurement point, whereas dramatic results, as assessed by the diameter of the regenerating nerve, are produced by the device which consisted of the guide tube, guiding filaments and antagonist compositions. Finally, treatments using guide tubes comprising either a matrix-forming material alone, or a matrix- forming material in the presence of guiding filaments, result in no measured growth of myelinated axons. In contrast, treatments using a device comprising guide tubes, guiding filaments, and matrix containing goliath compositions result in axon regeneration, with the measured number of axons being increased by the presence of guiding filaments.

All publications and patent applications cited in this specification and all references cited therein are herein incorporated by reference as if each individual publication or patent application or reference were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising an effective amount of a goliath polypeptide in dosage form, wherein the goliath polypeptide has sequence identity to a sequence selected from the group consisting of SEQ JD NO: 1, 2, 3 and 4, wherein the sequence identity is at least 75% over the entire polypeptide, and wherein the goliath polypeptide (a) inhibits angiogenesis in an assay selected from the group consisting of a Xenopus embryo transfection assay, a rabbit cornea assay, a HUVEC/Matrigel exogenous administration assay, a HUVEC/Matrigel transfection in vitro assay, and a HUVEC/Matrigel cotransfection in vivo assay; or (b) promotes neurogenesis in an assay selected from the group consisting of a Xenopus embryo transfection assay, a human/rat xenograft corticospinal tract regeneration assay and a rat peripheral nerve regeneration assay.

2. A composition according to claim 1, wherein the sequence identity is at least 85%.

3. A composition according to claim 1, wherein the sequence identity is at least 95%.

4. A composition according to claim 1, wherein the sequence identity is 100%.

5. A method for inhibiting angiogenesis or promoting neurogenesis, comprising the steps of administering to an animal determined to have pathogenic angiogenesis or neurogenesis, a composition comprising an effective amount of a goliath polypeptide or a goliath polynucleotide, and detecting an inhibition of said angiogenesis or promotion of said neurogenesis in the animal, wherein the goliath polypeptide has sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, 2, 3 and 4, wherein the sequence identity is at least 75% over the entire polypeptide, and wherein the goliath polypeptide (a) inhibits angiogenesis in an assay selected from the group consisting of a Xenopus embryo transfection assay, a rabbit cornea assay, a HUVEC/Matrigel exogenous administration assay, a HUVEC/Matrigel transfection in vitro assay, and a HUVEC/Matrigel cotransfection in vivo assay; or (b) promotes neurogenesis in an assay selected from the group consisting of a Xenopus embryo transfection assay, a human/rat xenograft corticospinal tract regeneration assay and a rat peripheral nerve regeneration assay, and the goliath polynucleotide encodes or inhibits the function of the goliath polypeptide.

6. A method according to claim 5, wherein the sequence identity is at least 85%.

7. A method according to claim 5, wherein the sequence identity is at least 95%.

8. A method according to claim 5, wherein the sequence identity is 100%.

9. A method according to claim 5, wherein the pathogenic angiogenesis comprises ischemia.

10. A method according to claim 5, wherein the pathogenic angiogenesis comprises undesirable vascularization of a tumor.

11. A method according to claim 5, wherein the composition comprises the polynucleotide and the polynucleotide is expressed in the animal as a goliath polypeptide.

12. A method according to claim 5, wherein the composition comprises the polynucleotide and the polynucleotide is expressed in the animal as an antisense complement of an endogenous goliath transcript.

13. A method according to claim 5, comprising the steps of administering to an animal determined to have pathogenic angiogenesis or neurogenesis, a composition comprising an effective amount of a goliath polypeptide, and detecting an inhibition of said angiogenesis or promotion of said neurogenesis in the animal, wherein the goliath polypeptide has sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, 2, 3 and 4, wherein the sequence identity is at least 85% over the entire polypeptide, and wherein the goliath polypeptide (a) inhibits angiogenesis in an assay selected from the group consisting of a Xenopus embryo transfection assay, a rabbit cornea assay, a HUVEC/Matrigel exogenous administration assay, a HUVEC/Matrigel transfection in vitro assay, and a HUVEC/Matrigel cotransfection in vivo assay; or (b) promotes neurogenesis in an assay selected from the group consisting of a Xenopus embryo transfection assay, a human/rat xenograft corticospinal tract regeneration assay and a rat peripheral nerve regeneration assay.

14. A method for inhibiting angiogenesis or promoting neurogenesis, comprising the steps of contacting an endothelial or nerve cell with an effective amount of a goliath polypeptide or polynucleotide, and detecting an inhibition of angiogenesis or promotion of neurogenesis of the cell, wherein the goliath polypeptide has sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, 2, 3 and 4, wherein the sequence identity is at least 75% over the entire polypeptide, and wherein the goliath polypeptide (a) inhibits angiogenesis in an assay selected from the group consisting of a Xenopus embryo transfection assay, a rabbit cornea assay, a HUVEC/Matrigel exogenous administration assay, a HUVEC/Matrigel transfection in vitro assay, and a HUVEC/Matrigel cotransfection in vivo assay; or (b) promotes neurogenesis in an assay selected from the group consisting of a Xenopus embryo transfection assay, a human/rat xenograft corticospinal tract regeneration assay and a rat peripheral nerve regeneration assay, and the goliath polynucleotide encodes or inhibits the function of the goliath polypeptide.

15. A method according to claim 14, wherein the sequence identity is at least 85%.

16. A method according to claim 14, wherein the sequence identity is at least 95%.

17. A method according to claim 14, wherein the sequence identity is 100%.

18. A method according to claim 14, wherein the cell is an endothelial cell and the function comprises vascularization of the cell.

19. A method according to claim 14, wherein the cell is a neuron and the function comprises growth of the cell.

20. A method according to claim 14, comprising the steps of contacting an endothelial or nerve cell with an effective amount of a goliath polypeptide, and detecting an inhibition of angiogenesis or promotion of neurogenesis of the cell, wherein the goliath polypeptide has sequence identity to a sequence selected from the group consisting of SEQ JD NO: 1, 2, 3 and 4, wherein the sequence identity is at least 85% over the entire polypeptide, and wherein the goliath polypeptide (a) inhibits angiogenesis in an assay selected from the group consisting of a Xenopus embryo transfection assay, a rabbit cornea assay, a HUVEC/Matrigel exogenous administration assay, a HUVEC/Matrigel transfection in vitro assay, and a HUVEC/Matrigel cotransfection in vivo assay; or (b) promotes neurogenesis in an assay selected from the group consisting of a Xenopus embryo transfection assay, a human/rat xenograft corticospinal tract regeneration assay and a rat peripheral nerve regeneration assay.