WO1998029547A1

WO1998029547A1 - Modulators of radial glia-astrocyte differentiation and transformation, and diagnostic and therapeutic uses thereof

Info

Publication number: WO1998029547A1
Application number: PCT/US1997/023818
Authority: WO
Inventors: Mary E. Hatten; Kim E. Hunter
Original assignee: The Rockefeller University
Priority date: 1996-12-31
Filing date: 1997-12-31
Publication date: 1998-07-09

Abstract

Modulators of radial glia-astrocyte differentiation and transformation comprising a secreted radial glial differentiation factor have been identified, isolated, purified and characterized. This radializing factor, herein called RF60, possesses an apparent molecular weight of approximately 60kD and functions as a radial glial maintenance signal, with the availability of the protein regulating the phenotype of cortical glia. RF60 can be utilized to reverse the glial scarring occurring during CNS trauma, and thus reverse the glia to their pre-event stage. RF60 can thus be utilized to induce cells to return to their embryonic form, and regenerate normally so as to provide treatment of brain or spinal cord injury or surgery, treatment of neurological disorders or diseases where there has been a diminution of the normal activity of the brain or central nervous system, such as dementia, Alzheimer's Disease, multiple sclerosis, and situations where regeneration of brain or central nervous system tissue is therapeutic, such as surgery, trauma, stroke due to ischemic or hemorrhagic occurrences, bacterial and viral infections, neoplasms, and the like.

Description

MODULATORS OF RADIAL GLIA-ASTROCYTE DIFFERENTIATION AND TRANSFORMATION, AND DIAGNOSTIC AND THERAPEUTIC USES THEREOF

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to modulators of radial glia-astrocyte differentiation and transformation, and to various diagnostic and therapeutic uses therefor.

BACKGROUND OF THE INVENTION

Radial glial fibers provide the scaffolding that sets forth the pattern of the mammalian forebrain. Postmitotic neurons leaving the ventricular zone use radial glia as a migratory substrate; cortical laminae form in a temporally regulated manner, the oldest neurons being positioned in deep layers, and the youngest, superficially (Angevine and Sidman, 1961; Caviness and Sidman, 1973; Rakic, 1972, 1974; Schmechel and Rakic, 1979a). Though young neurons can migrate tangentially (Fishell et al., 1993; O'Rourke et al. 1992, 1995) and sibling cells can ultimately become dispersed over widespread areas of cortex (Luskin et al. , 1988; Austin and Cepko, 1990; Walsh and Cepko, 1992, 1993) it is recognized that the majority of neuronal migration is radial and glial-guided (O'Rourke et al. , 1995).

The functions of radial glia are exclusively developmental; correspondingly, in the early postnatal cortex they undergo a dramatic change of phenotype and function by transforming into mature astrocytes (Schmechel and Rakic, 1979; Voigt, 1989; Culican et al. , 1990). Though some of the molecules involved in neuronal migration along the glial fibers have recently been identified, such as astrotactin (Zheng et al. 1996), factors regulating radial glial cell development remain to be elucidated. A radial glial differentiation signal was previously described which is present in embryonic cortex but absent postnatally; this signal is a protein which will induce astrocytes to express a radial glial phenotype (Hunter and Hatten, 1995), suggesting the radial glia-astrocyte developmental pathway is bidirectional. The differentiation signal is therefore a 'maintenance' cue for the radial glia scaffold, and its perinatal downregulation precipitates the transformation of the elongated glial forms that support migration into the astrocytes of the mature cortex. The question remains as to how this signal functions to control the temporal program of glial development in vivo.

The response of astroglial cells to injury is based upon several basic assumptions about such cells. The "reactive astroglia" seen in injured brain are considered to be generic cells, rather than a diverse family of glial cells descended from a variety of progenitors in different brain regions. There is the assumption that injury induces supportive astrocytes to become inhibitory cells which impede axon growth, [see, Reier et al. , in Spinal Cord Reconstruction, Kao et al, ed. , Raven Press, New York, pp 163-196 (1983) and Reier et al , in Astrocytes, vol. 3, Federoff et al , eds, Academic Press, New York, 263-324 (1986)]. Injury is thus assumed to induce glial proliferation, resulting in the formation of a scar. However, in recent years, experimental analyses of the role of glial fibrillary acidic protein (GFAP) expression in glial differentiation, the role of glia in the support of axon growth, the role of neuronal regulation of glial proliferation, and of astroglial cell diversity among brain regions have called into question all of these assumptions.

Analyses of neuron-glia relationships in the developing brain have revealed an interdependence between neurons and glial cells, with cell-cell contacts between neurons and glia regulating both neuronal and glial differentiation. One of the first responses of the adult central nervous system (CNS) to traumatic invasive injury such as a stab wound is reactive gliosis, i.e. , the rapid proliferation of astrocytes to form a glial "scar". While this scar tissue does provide structural repair at the site of the CNS wound, the glial scar is well-recognized as being a limiting factor in the capacity of the CNS to regenerate in that it presents a physical barrier which prevents axonal regrowth. Although a number of therapies are currently being developed which attempt to facilitate axonal regeneration in the mammalian, and particularly the human, CNS, for instance local administration of neurotrophic factors to damaged neurons, it is recognized that even though axonal regrowth may be induced, a major impeding factor is the glial scar. Other therapeutic approaches involve transplanting embryonic neural cells or neural cell lines into the injured CNS to "replace" the damaged neurons, but again, the glial scar presents a non-supportive environment for either migration of these cell in vivo or for new axonal growth. Thus, there remains a need for useful methods of treating such trauma in the central nervous system.

SUMMARY QF THE INVENTION

In accordance with the present invention, modulators of radial glia-astrocyte differentiation and transformation comprising a secreted radial glial differentiation factor have been identified, isolated, purified and characterized. This radializing factor, herein called RF60, possesses an apparent molecular weight of approximately 60kD and functions as a radial glial maintenance signal, with the availability of the protein regulating the phenotype of cortical glia. RF60 is present embryonically, but absent postnatally, so that its use in the mature mammal can reverse the glia to an embryonic-like state.

In a further embodiment, RF60 has been utilized to reverse the glial scarring occurring during CNS trauma, and thus reverse the glia to an embryonic-like state.

In its broadest aspect, the present invention extends to a secreted radializing factor, its variants, functionally active fragments thereof, analogs thereof, and alleles thereof, the foregoing having the following characteristics: an apparent molecular weight of approximately 60kD; and the capacity to induce expression of a radial glial phenotype by cultured astrocytes.

In a still further aspect, the present invention extends to a radializing factor (RF60) which induces adult astrocytes to assume properties normally associated with their embryonic precursors. Thus, astrocytes, normally epithelioid in shape, become radial and elongated in the presence of RF60, express the embryonic glial marker RC2 and have dramatically reduced levels of GFAP.

In a particular embodiment, the present invention relates to all members of the herein disclosed family of radializing factors (RF60).

The present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a radializing factor (RF60); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the radializing factor (RF60). In another embodiment, the human and murine DNA sequences of the radializing factor (RF60) of the present invention or portions thereof, may be prepared as probes to screen for complementary sequences and genomic clones in the same or alternate species. The present invention extends to probes so prepared that may be provided for screening cDNA and genomic libraries for the radializing factor (RF60). For example, the probes may be prepared with a variety of known vectors, such as the phage λ vector.

The present invention also includes radializing factor (RF60) proteins having the activities noted herein, and that display the characteristics set forth and described above.

In a further embodiment of the invention, the full DNA sequence of the recombinant DNA molecule or cloned gene so determined may be operatively linked to an expression control sequence which may be introduced into an appropriate host. The invention accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding the present radializing factor (RF60). According to other preferred features of certain preferred embodiments of the present invention, a recombinant expression system is provided to produce biologically active animal or human radializing factor (RF60).

The concept of the radializing factor (RF60) contemplates that specific factors exist for correspondingly specific ligands. Accordingly, the exact structure of each will understandably vary so as to achieve this ligand and activity specificity. It is this specificity and the direct involvement of the radializing factor (RF60) in the chain of events leading to glial scar formation, that offers the promise of a broad spectrum of diagnostic and therapeutic utilities.

The present invention naturally contemplates several means for preparation of the radializing factor (RF60), including as illustrated herein known recombinant techniques, and the invention is accordingly intended to cover such synthetic preparations within its scope. The isolation of the cDNA and amino acid sequences disclosed herein facilitates the reproduction of the radializing factor (RF60) by such recombinant techniques, and accordingly, the invention extends to expression vectors prepared from the disclosed DNA sequences for expression of radializing factors such as RF60 in host systems by recombinant DNA techniques, and to the resulting transformed hosts.

The invention further extends to a class of cells, and particularly, to astrocytes, including adult astrocytes, that exhibit a radial glia phenotype, and more particularly, to astrocytes that exhibit such phenotype as a result of the presence of the radializing factor (RF60). Specifically, such astrocytes possess a radial adn elongated shape, express the glial cell marker RC2 and have dramatically reduced levels of GFAP.

The invention includes an assay system for screening of potential drugs effective to modulate radializing factor (RF60) activity in target mammalian cells by modulating or potentiating the activity of the radializing factor (RF60). In one instance, the test drug could be administered to a cellular sample with radializing factor (RF60), or an extract containing the activated radializing factor (RF60), to determine its effect upon the activity of the radializing factor (RF60) to any chemical sample (including DNA), or to the test drug, by comparison with a control.

The assay system could more importantly be adapted to identify drugs or other entities that are capable of stimulating the production of radializing factor (RF60), either in the cytoplasm or in the nucleus, thereby increasing or potentiating radializing factor (RF60) activity. Such assay would be useful in the development of drugs that would be specific against particular cellular activity, or that would potentiate such activity, in time or in level of activity.

One of the characteristics of the present radializing factor (RF60) is its capacity to induce expression of a radial glial phenotype by cultured astrocytes.

The diagnostic utility of the present invention extends to the use of the present radializing factor (RF60) in assays to screen for the presence of radializing factor (RF60) agonists.

The present invention likewise extends to the development of antibodies against the radializing factor (RF60), including naturally raised and recombinantly prepared antibodies. For example, the antibodies could be used to screen expression libraries to obtain the gene or genes that encode the radializing factor (RF60). Such antibodies could include both polyclonal and monoclonal antibodies prepared by known genetic techniques, as well as bi-specific (chimeric) antibodies, and antibodies including other functionalities suiting them for additional diagnostic use conjunctive with their capability of modulating radializing factor (RF60) activity.

Thus, the radializing factors (RF60), its variants and fragments, their analogs, and any antagonists or antibodies that may be raised thereto, are capable of use in connection with various diagnostic techniques, including immunoassay s, such as a radioirnmunoassay, using for example, an antibody to the radializing factor (RF60) that has been labeled by either radioactive addition, or radioiodination.

In an immunoassay, a control quantity of the antagonists or antibodies thereto, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a cellular sample. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached.

In the instance where a radioactive label, such as the isotopes ³H, ¹⁴C, ³²P, ³⁵S, ³⁶C1, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re are used, known currently available counting procedures may be utilized. In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.

The present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of the radializing factor (RF60), or to identify drugs or other agents that may mimic or block their activity. The system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the radializing factor (RF60), their agonists and/or antagonists, and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand, capable either of binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s).

In a further embodiment, the present invention relates to certain therapeutic methods which would be based upon the activity of the radializing factor, its (or their) variants, or active fragments thereof, or upon agents or other drugs determined to possess the same activity. A first therapeutic method is associated with the prevention or inhibition of the formation of radial glial scar formation related to brain or central nervous system trauma or injury, and comprises administering a radializing factor, its variants, active fragments, or other agent or drug determined to possess the same activity, either individually or in mixture with other agents in an amount effective to prevent the development of such conditions in the host.

More specifically, the therapeutic method generally referred to herein could include the method for the treatment of various pathologies or other cellular dysfunctions and derangements which occur in brain or central nervous system or trauma by the administration of pharmaceutical compositions that may comprise the radializing factor (RF60),its variants or fragments, or other equally effective drugs developed, for instance, by a drug screening assay prepared and used in accordance with a further aspect of the present invention. For example, drugs or other binding partners to the radializing factor (RF60) or proteins may be administered to increase or potentiate the glial scar inhibitory activity. Thus, the agents of the present invention find broad utility in the treatment of brain or other central nervous system trauma, including use in the treatment of brain or spinal cord injury or surgery, treatment of neurological disorders or diseases where there has been a diminution of the normal activity of the brain or central nervous system, such as dementia, Alzheimer's Disease, multiple sclerosis, and situations where regeneration of brain or central nervous system tissue is therapeutic, such as surgery, trauma, stroke due to ischemic or hemorrhagic occurrences, bacterial and viral infections, neoplasms, and the like.

In particular, the proteins of radializing factor (RF60), their antibodies, agonists, antagonists, or active fragments thereof, could be prepared in pharmaceutical formulations for administration in instances wherein therapy is appropriate, such as to treat brain or spinal cord injury. The specificity of the radializing factor (RF60) proteins hereof would make it possible to better manage the aftereffects of brain or central nervous system trauma or injury.

Accordingly, it is a principal object of the present invention to provide a radializing factor (RF60), its variants and fragments, in purified form that exhibits certain characteristics and activities associated with the prevention, inhibition or reversal of the formation of glial scar formation, especially in connection with brain or central nervous system trauma or injury.

It is a further object of the present invention to provide antibodies to the radializing factor (RF60) as aforesaid, and methods for their preparation, including recombinant means.

It is a further object of the present invention to provide a method and associated assay system for screening substances such as drugs, agents and the like, potentially effective in either mimicking the activity of the radializing factor (RF60) and/or its variants and fragments, in mammals.

It is a still further object of the present invention to provide a method for the treatment of mammals to prevent, inhibit or reverse the formation of glial scar formation, especially in the instance of brain or central nervous system trauma or injury by administration of a therapeutic amount of the radializing factor (RF60), its variants and fragments, or subunits thereof, so as to alter the adverse consequences of brain or central nervous system trauma or injury.

It is a still further object of the present invention to provide pharmaceutical compositions for use in therapeutic methods which comprise or are based upon the radializing factor (RF60), its variants and fragments, their binding partner(s), or upon agents or drugs that potentiate or enhance the production, or that mimic or antagonize the activities of the radializing factor (RF60). Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing description which proceeds with reference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1 is photograph of acrylamide gel utilized to show that a single gel band region defines the radial glial differentiation protein as RF60. A sample of the monoQ sepharose final eluent pool loaded onto a 10% denaturing acrylamide gel then silver stained to reveal site and abundance of protein bands present. The sample contains 25 abundant bands (a). 20 of these bands were excised from a parallel unfixed lane. The protein was eluted from each gel slice, renatured, and screened for bioactivity on astrocytes which were scored for conversion to a radial glia phenotype; this is expressed as units of bioactivity per gel slice (1 unit of activity induces 25% of astrocytes to express a radial phenotype. The bioactivity was found to be present only in one gel band in the molecular weight region of approximately 60kD (b).

FIGURES 2A-2C show comparisons of the results of administration of radializing factor (RF60) on normal and reeler astrocytes. A cell-autonomous defect prevents reeler astrocytes from responding to RF60. Bioactivity profiles of fractionated embryonic cell conditioned medium to yield enriched samples of the glial differentiation signal, when screened in parallel on normal and reeler astrocytes for its ability to effect expression of a radial phenotype by astrocytes. Size fractionation and screening on normal astrocytes shows the bioactivity to elute in a sharp peak, estimated to be in the range 50-60kD, as previously described (Hunter and Hatten, 1995); however when screened on reeler astrocytes these cells show very little ability to respond to the activity, resulting in only a very flattened peak (FIGURE 2A). Further enriched samples were obtained following FPLC purification on heparin agarose, wheat germ agglutinin and monoQ sepharose affinity matrices and screened in serial dilutions on normal and reeler astrocytes. Normal astrocytes showed increased response to the progressively enriched fractions (FIGURE 2B). In contrast, reeler astrocytes showed almost no increase in differentiation in response to greater or enriched amounts of bioactivity, rather, simply a flat, baseline response (FIGURE 2B). However in a comparison screening of soluble factors secreted by reeler and normal E14 cortical cells, on reeler and normal astrocytes, both cortical sources were found to secrete comparable amounts of activity although reeler astrocytes were unresponsive to both (FIGURE 2C).

FIGURES 3A-3F are photographs illustrating the pattern of RC2 and GFAP protein distribution in developing normal and reeler cortex. Confocal images showing coronal sections of normal (FIGURES 3 A, 3C, 3E) and reeler (FIGURES 3B, 3D, 3F) cerebral cortex immunostained with RC2 (FIGURES 3A, 3B) and anti-GFAP (FIGURES 3C-3F) to illustrate the changing form of astroglia. In E14 normal cortex radial glia are well differentiated and form a dense, organized fiber scaffold (FIGURE 3A); in E14 reeler cortex, however, radial glia are poorly differentiated and present a looser, more disrupted scaffold (FIGURE 3B). In P0 normal cortex, although a few stellar astrocytes have begun to accumulate beneath the pial surface, the radial glial scaffold is still substantially present (FIGURE 4C) while in P0 reeler cortex the scaffold is more fibrous and a large number of astrocytes are already present (FIGURE 3D). In P4 normal cortex the predominant cell form recognized is the transitional astroglial phenotype, bearing features of both radial glia and astrocytes (FIGURE 3E), while in P0 reeler cortex all evidence of the radial scaffold has disappeared and only fully mature astrocytes are seen (FIGURE 3F). Scale bar = 100mm

FIGURE 4 is a comparison of GFAP protein levels in developing normal and reeler cortex using a Western blot of cortex harvested from normal and reeler animals aged E14-P21, and probed with anti-GFAP. In normal cortex, only negligible amounts of GFAP are present through embryonic stages, until the time of birth when the protein is detectable. In contrast, GFAP is present in reeler cortex at the earliest developmental stage probed (El 4); levels continue to be greater in reeler than in normal cortex, even in mature cortex (P21).

FIGURES 5A and 5B are photographs depicting the results of stab wound experiments in cortex, presenting a comparison of GFAP protein levels in injured, untreated cortex (Figure 5A), and injured but treated with RF60 (Figure 5B), 5 days after injury. The stab wound is to the left side of each figure. Note the dense, strongly GFAP+ reactive astrocytes in the untreated cortex (see arrow, Figure 5A). In contrast, in the RF60 infused cortex, GFAP levels are much lower and the astrocytes assume a radial morphology (see arrow, Figure 5B).

DETAILED DESCRIPTION

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g. , Sambrook et al, "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology" Volumes I-III [Ausubel, R. M., ed. (1994)]; "Cell Biology: A Laboratory Handbook" Volumes I-III [J. E. Celis, ed. (1994))]; "Current Protocols in Immunology" Volumes I-III [Coligan, J. E. , ed. (1994)]; "Oligonucleotide Synthesis" (M.J. Gait ed. 1984); "Nucleic Acid Hybridization" [B.D. Hames & S.J. Higgins eds. (1985)]; "Transcription And Translation" [B.D. Hames & S.J. Higgins, eds. (1984)]; "Animal Cell Culture" [R.I. Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

Therefore, if appearing herein, the following terms shall have the definitions set out below.

The terms "radializing factor, " "RF60," "radial glial differentiation factor, " and any variants not specifically listed, may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous material including single or multiple proteins, and extends to those proteins having the profile of activities set forth herein and in the Claims. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits. Also, the terms "radializing factor," "RF60," and "radial glial differentiation factor(s)" are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.

The amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L- amino acid residue, as long as the desired functional property of immunoglobulin- binding is retained by the polypeptide. NH₂ refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature, J. Biol. Chem. , 243:3552-59 (1969), abbreviations for amino acid residues are shown in the following Table of Correspondence:

TABLE OF CORRESPONDENCE

SYMBOL AMINO ACID

1 -Letter 3-Letter

Y Tyr tyrosine

G Gly glycine

F Phe phenylalanine

M Met methionine

A Ala alanine

S Ser serine

I He isoleucine L Leu leucine

T Thr threonine

V Val valine

P Pro proline K Lys lysine

H His histidine

Q Gin glutamine

E Glu glutamic acid

W Trp tryptophan R Arg arg ine

D Asp aspartic acid

N Asn asparagine

C Cys cysteine

It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino- terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the begmning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues. The above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.

A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.

A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double- stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

An "origin of replication" refers to those DNA sequences that participate in DNA synthesis.

A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy 1) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the mmimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.

An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.

A "signal sequence" can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that cornmunicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

The term "oligonucleotide," as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.

The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.

As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.

A cell has been "transformed" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., DNA Cloning, Volumes. I & II, supra; Nucleic Acid Hybridization, supra.

It should be appreciated that also within the scope of the present invention are DNA sequences encoding radializing factor (RF60) which code for a radializing factor (RF60). Also, included within the scope of the invention are other DNA sequences which are degenerate to such sequences. By "degenerate to" is meant that a different three-letter codon is used to specify a particular amino acid. It is well known in the art that the following codons can be used interchangeably to code for each specific amino acid:

Phenylalanine (Phe or F) UUU or UUC

Leucine (Leu or L) UUA or UUG or CUU or CUC or CUA or CUG Isoleucine (He or I) AUU or AUC or AUA

Methionine (Met or M) AUG Valine (Val or V) GUU or GUC of GUA or GUG Serine (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro or P) CCU or CCC or CCA or CCG Threonine (Thr or T) ACU or ACC or ACA or ACG Alanine (Ala or A) GCU or GCG or GCA or GCG Tyrosine (Tyr or Y) UAU or UAC Histidine (His or H) CAU or CAC Glutamine (Gin or Q) CAA or CAG Asparagine (Asn or N) AAU or AAC Lysine (Lys or K) AAA or AAG

Aspartic Acid (Asp or D) GAU or GAC Glutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU or UGC Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG Glycine (Gly or G) GGU or GGC or GGA or GGG Tryptophan (Trp or W) UGG Termination codon UAA (ochre) or UAG (amber) or UGA (opal)

It should be understood that the codons specified above are for RNA sequences. The corresponding codons for DNA have a T substituted for U.

Mutations can be made such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e. , by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. A non-conservative change is more likely to alter the structure, activity or function of the resulting protein. The present invention should be considered to include seguences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein.

The following is one example of various groupings of amino acids:

Amino acids with nonpolar R groups Alanine

Valine

Leucine

Isoleucine

Proline Phenylalanine

Tryptophan

Methionine

Amino acids with uncharged polar R groups Glycine

Serine

Threonine

Cysteine

Tyrosine Asparagine

Glutamine

Amino acids with charged polar R groups (negatively charged at Ph 6.0) Aspartic acid Glutamic acid Basic amino acids (positively charged at pH 6.0)

Lysine

Arginine

Histidine (at pH 6.0)

Another grouping may be those amino acids with phenyl groups:

Phenylalanine Tryptophan Tyrosine

Another grouping may be according to molecular weight (i.e., size of R groups):

Glycine 75

Alanine 89

Serine 105

Proline 115

Valine 117

Threonine 119

Cysteine 121

Leucine 131

Isoleucine 131

Asparagine 132

Aspartic acid 133

Glutamine 146

Lysine 146

Glutamic acid 147

Methionine 149

Histidine (at pH 6.0) 155

Phenylalanine 165

Arginine 174 Tyrosine 181

Tryptophan 204

Particularly preferred substitutions are: - Lys for Arg and vice versa such that a positive charge may be maintained;

- Glu for Asp and vice versa such that a negative charge may be maintained;

- Ser for Thr such that a free -OH can be maintained; and

- Gin for Asn such that a free NH₂ can be maintained.

Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced a potential site for disulfide bridges with another Cys. A His may be introduced as a particularly "catalytic" site (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces -turns in the protein's structure.

Two amino acid sequences are "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions.

A "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g. , a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein. An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,816,397 and 4,816,567.

An "antibody combining site" is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.

The phrase "antibody molecule" in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab' , F(ab')₂ and F(v), which portions are preferred for use in the therapeutic methods described herein.

Fab and F(ab')₂ portions of antibody molecules are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See for example, U.S. Patent No. 4,342,566 to Theofilopolous et al. Fab' antibody molecule portions are also well- known and are produced from F(ab')₂ portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide. An antibody containing intact antibody molecules is preferred herein.

The phrase "monoclonal antibody" in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.

The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, the formation of glial scar formation in the situation where brain or central nervous system trauma or injury has occurred.

A DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence. The term "operatively linked" includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.

The term "standard hybridization conditions" refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65 °C for both hybridization and wash. However, one skilled in the art will appreciate that such "standard hybridization conditions" are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide, and the like. Also important in the determination of "standard hybridization conditions" is whether the two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20°C below the predicted or determined T_m with washes of higher stringency, if desired.

In its primary aspect, the present invention concerns the identification, isolation and purification of a radializing factor (RF60).

In a particular embodiment, the present invention relates to all members of the herein disclosed radializing factor (RF60).

As stated above, the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a radializing factor (RF60), or a fragment thereof, that possesses a molecular weight of about 60 kD and that exhibits the structural and mophological characteristics of a radial glial cell; preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the 60 kD radializing factor.

The possibilities both diagnostic and therapeutic that are raised by the existence of the radializing factor (RF60), derive from the fact that the factors appear to participate in direct and causal protein-protein interaction between the radializing factor (RF60), and those factors that thereafter mediate the formation of glial scar formation. As suggested earlier and elaborated further on herein, the present invention contemplates pharmaceutical intervention in the cascade of reactions in which the radializing factor (RF60) is implicated, to utilize the activity initiated by the radializing factor (RF60), and consequently affect the formation of glial scar formation and its deleterious effects. Thus, in instances where it is desired to reduce or inhibit the resultant effects of brain or central nervous system trauma or injury, the introduction of additional quantities of the radializing factor (RF60),its variants and fragments, including its chemical or pharmaceutical cognates, analogs, fragments and the like can be utilized to obviate or minimize the formation of glial scarring.

As discussed earlier, the radializing factor (RF60), its active fragments, or analogs thereof, may be prepared in pharmaceutical compositions, with a suitable carrier and at a strength effective for administration by various means to a patient experiencing an adverse medical condition associated with specific brain or central nervous system trauma or injury. A variety of administrative techniques may be utilized, among them parenteral techniques such as subcutaneous, intravenous and intraperitoneal injections, catheterizations and the like. Average quantities of the radializing factor (RF60), its variants and fragments, may vary and in particular should be based upon the recommendations and prescription of a qualified physician or veterinarian.

Also, antibodies including both polyclonal and monoclonal antibodies, and drugs that modulate the production or activity of the radializing factor (RF60) and/or its variants and fragments, or their subunits may possess certain diagnostic applications and may for example, be utilized for the purpose of detecting and/or measuring the extent of the formation of the glial scar formation. For example, the radializing factor (RF60) or its variants and fragments, or subunits may be used to produce both polyclonal and monoclonal antibodies to themselves in a variety of cellular media, by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells. Likewise, small molecules that mimic or antagonize the activity (ies) of the radializing factor (RF60) of the invention may be discovered or synthesized, and may be used in diagnostic and/or therapeutic protocols. The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al. , "Hybridoma Techniques" (1980); Hammerling et al., "Monoclonal Antibodies And T-cell Hybridomas" (1981); Kennett et al., "Monoclonal Antibodies" (1980); see also U.S. Patent Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; 4,493,890.

Panels of monoclonal antibodies produced against radializing factor (RF60) peptides can be screened for various properties; i.e. , isotype, epitope, affinity, etc. Of particular interest are monoclonal antibodies that neutralize the activity of the radializing factor (RF60), its variants and analogs. Such monoclonals can be readily identified in radializing factor (RF60) activity assays. High affinity antibodies are also useful when immunoaffinity purification of native or recombinant radializing factor (RF60) is possible.

Preferably, the anti-radializing factor (RF60) antibody used in the diagnostic methods of this invention is an affinity purified polyclonal antibody. More preferably, the antibody is a monoclonal antibody (mAb). In addition, it is preferable for the anti-radializing factor (RF60) antibody molecules used herein be in the form of Fab, Fab', F(ab' ₂ or F(v) portions of whole antibody molecules.

As suggested earlier, the diagnostic method of the present invention comprises examining a cellular sample or medium by means of an assay including an effective amount of an antagonist to a radializing factor (RF60)/protein, such as an anti- radializing factor (RF60) antibody, preferably an affinity-purified polyclonal antibody, and more preferably a mAb. In addition, it is preferable for the anti- radializing factor (RF60) antibody molecules used herein be in the form of Fab, Fab', F(ab')₂ or F(v) portions or whole antibody molecules. As previously discussed, patients capable of benefiting from this method include those suffering from various forms of brain or central nervous system trauma or injury. Methods for isolating the radializing factor (RF60) and inducing anti-radializing factor (RF60) antibodies and for determining and optimizing the ability of anti-radializing factor (RF60) antibodies to assist in the examination of the target cells are all well- known in the art.

Methods for producing polyclonal anti-polypeptide antibodies are well-known in the art. See U.S. Patent No. 4,493,795 to Nestor et al. A monoclonal antibody, typically containing Fab and/or F(ab')₂ portions of useful antibody molecules, can be prepared using the hybridoma technology described in Antibodies - A

Laboratory Manual, Harlow and Lane, eds. , Cold Spring Harbor Laboratory, New York (1988), which is incorporated herein by reference. Briefly, to form the hybridoma from which the monoclonal antibody composition is produced, a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with a radializing factor (RF60)- binding portion thereof, or radializing factor (RF60), or an origin-specific DNA- binding portion thereof.

Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000. Fused hybrids are selected by their sensitivity to HAT. Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact with the present radializing factor (RF60) and their ability to inhibit specified radializing factor (RF60) activity in target cells.

A monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity. The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by well-known techniques. Media useful for the preparation of these compositions are both well-known in the art and commercially available and include synthetic culture media, inbred mice and the like. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol. 8:396 (1959)) supplemented with 4.5 gm/1 glucose, 20 mm glutamine, and 20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Methods for producing monoclonal anti-radializing factor (RF60) antibodies are also well-known in the art. See Niman et al., Proc. Natl. Acad. Sci. USA, 80:4949-4953 (1983). Typically, the present radializing factor (RF60) or a peptide analog is used either alone or conjugated to an immunogenic carrier, as the immunogen in the before described procedure for producing anti-radializing factor (RF60) monoclonal antibodies. The hybridomas are screened for the ability to produce an antibody that immunoreacts with the radializing factor (RF60) peptide.

The present invention further contemplates therapeutic compositions useful in practicing the therapeutic methods of this invention. A subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of a radializing factor (RF60), polypeptide analog thereof or fragment thereof, as described herein as an active ingredient. In a preferred embodiment, the composition comprises radializing factor (RF60) together with any necessary pharmaceutical adjuvants.

The preparation of therapeutic compositions which contain polypeptides, analogs or active fragments as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.

A polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The therapeutic polypeptide-, analog- or active fragment-containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e. , carrier, or vehicle.

The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, and the severity of the condition under treatment. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations of ten nanomolar to ten micromolar in the blood are contemplated.

The therapeutic compositions may further include an effective amount of the radializing factor (RF60) or analog thereof, and one or more of the following active ingredients: an antibiotic, a steroid. Exemplary formulations are given below:

Formulations

Intravenous Formulation I

Ingredient mg/ml cefotaxime 250.0 radializing factor (RF60) 10.0 dextrose USP 45.0 sodium bisulfite USP 3.2 edetate disodium USP 0.1 water for injection q.s.a.d. 1.0 ml

Intravenous Formulation II Ingredient mg/ml ampicillin 250.0 radializing factor (RF60) 10.0 sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injection q.s.a.d. 1.0 ml Intravenous Formulation III

Ingredient mg/ml gentamicin (charged as sulfate) 40.0 radializing factor (RF60) 10.0 sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injection q.s.a.d. 1.0 ml

Intravenous Formulation IV Ingredient mg/ml radializing factor (RF60) 10.0 dextrose USP 45.0 sodium bisulfite USP 3.2 edetate disodium USP 0.1 water for injection q.s.a.d. 1.0 ml

As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "μg" mean microgram, "mg" means milligram, "ul" or "μl" mean microliter, "ml" means milliliter, "1" means liter.

Another feature of this invention is the expression of the DNA sequences disclosed herein. As is well known in the art, DNA sequences may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to an expression control sequence, of course, includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence. A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCRl, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage λ, e.g., NM989, and other phage DNA, e.g. , M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2μ plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

Any of a wide variety of expression control sequences — sequences that control the expression of a DNA sequence operatively linked to it — may be used in these vectors to express the DNA sequences of this invention. Such useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the 1-77? system, the major operator and promoter regions of phage λ, the control regions of fd coat protein, the promoter for

3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast o.-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

A wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture.

In a particular aspect of the invention, astrocytes of a host, e.g. for which treatment in accordance with the invention is desirable, may be transformed to incorporate the nucleic acid molecules of the present invention within their genome, to facilitate the expression by such transformed astrocytes of the radializing factor (RF60), and to thereby promote the adoption by such cells of the characteristics of radial glia.

It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this invention. For example, in selecting a vector, the host must be considered because the vector must function in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors will normally be considered. These include, for example, the. relative strength of the system, its controllability, and its compatibility with the particular DNA sequence or gene to be expressed, particularly as regards potential secondary structures. Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products. Considering these and other factors a person skilled in the art will be able to construct a variety of vector/expression control sequence/host combinations that will express the DNA sequences of this invention on fermentation or in large scale animal culture.

It is further intended that radializing factor (RF60) analogs may be prepared from nucleotide sequences of the protein complex/subunit derived within the scope of the present invention. Analogs, such as fragments, may be produced, for example, by pepsin digestion of radializing factor (RF60) material. Other analogs, such as muteins, can be produced by standard site-directed mutagenesis of radializing factor (RF60) coding sequences. Analogs exhibiting "radializing factor (RF60) activity" such as small molecules, may be identified by known in vivo and/or in vitro assays.

As mentioned above, a DNA sequence encoding radializing factor (RF60) can be prepared synthetically rather than cloned. The DNA sequence can be designed with the appropriate codons for the radializing factor (RF60) amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol. Chem. , 259:6311 (1984).

Synthetic DNA sequences allow convenient construction of genes which will express radializing factor (RF60) analogs or "muteins". Alternatively, DNA encoding muteins can be made by site-directed mutagenesis of native radializing factor (RF60) genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.

A general method for site-specific incorporation of unnatural amino acids into proteins is described in Christopher J. Noren, Spencer J. Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science, 244: 182-188 (April 1989). This method may be used to create analogs with unnatural amino acids.

The present invention also relates to a variety of diagnostic applications, including methods for detecting the extent of brain or central nervous system trauma or injury, by reference to the ability to elicit the activities which are mediated by the present radializing factor (RF60). As mentioned earlier, the radializing factor (RF60) can be used to produce antibodies to itself by a variety of known techniques, and such antibodies could then be isolated and utilized as in tests for the presence of particular radializing factor (RF60) activity in suspect target cells.

As described in detail above, antibody (ies) to the radializing factor (RF60) can be produced and isolated by standard methods including the well known hybridoma techniques. For convenience, the antibody (ies) to the radializing factor (RF60) will be referred to herein as Ab, and antibody (ies) raised in another species as Ab₂.

The presence of radializing factor (RF60) in cells can be ascertained by the usual immunological procedures applicable to such determinations. A number of useful procedures are known. Three such procedures which are especially useful utilize either the radializing factor (RF60) labeled with a detectable label, antibody Ab, labeled with a detectable label, or antibody Ab₂ labeled with a detectable label. The procedures may be summarized by the following equations wherein the asterisk indicates that the particle is labeled, and "RF60" stands for the radializing factor:

A. RF60* + Ab, = RF60*Ab,

B. RF60 + Ab* = RF60Ab,*

C. RF60 + Ab, + Ab₂* = RF60Ab,Ab₂*

The procedures and their application are all familiar to those skilled in the art and accordingly may be utilized within the scope of the present invention. The "competitive" procedure, Procedure A, is described in U.S. Patent Nos. 3,654,090 and 3,850,752. Procedure C, the "sandwich" procedure, is described in U.S. Patent Nos. RE 31,006 and 4,016,043. Still other procedures are known such as the "double antibody," or "DASP" procedure.

In each instance, the radializing factor (RF60) forms complexes with one or more antibody(ies) or binding partners and one member of the complex is labeled with a detectable label. The fact that a complex has formed and, if desired, the amount thereof, can be determined by known methods applicable to the detection of labels.

It will be seen from the above, that a characteristic property of Abj is that it will react with Ab,. This is because Ab, raised in one mammalian species has been used in another species as an antigen to raise the antibody Ab₂. For example, Ab₂ may be raised in goats using rabbit antibodies as antigens. Ab₂ therefore would be anti-rabbit antibody raised in goats. For purposes of this description and claims, Ab, will be referred to as a primary or anti-radializing factor (RF60) antibody, and Ab₂ will be referred to as a secondary or anti-Ab, antibody.

The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.

A number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.

The radializing factor (RF60) or its binding partner(s) can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The preferred isotope may be selected from ³H, ¹⁴C, ³²P, ³⁵S, ³⁶C1, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, *^>Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re. Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques. The enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase. U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.

A particular assay system developed and utilized in accordance with the present invention, is known as a receptor assay. In a receptor assay, the material to be assayed is appropriately labeled and then certain cellular test colonies are inoculated with a quantity of both the labeled and unlabeled material after which binding studies are conducted to determine the extent to which the labeled material binds to the cell receptors. In this way, differences in affinity between materials can be ascertained.

Accordingly, a purified quantity of the radializing factor (RF60) may be radiolabeled and combined, for example, with antibodies or other inhibitors thereto, after which binding studies would be carried out. Solutions would then be prepared that contain various quantities of labeled and unlabeled uncombined radializing factor (RF60), and cell samples would then be inoculated and thereafter incubated. The resulting cell monolayers are then washed, solubilized and then counted in a gamma counter for a length of time sufficient to yield a standard error of < 5 % . These data are then subjected to Scatchard analysis after which observations and conclusions regarding material activity can be drawn. While the foregoing is exemplary, it illustrates the manner in which a receptor assay may be performed and utilized, in the instance where the cellular binding ability of the assayed material may serve as a distinguishing characteristic. An assay useful and contemplated in accordance with the present invention is known as a "cis/trans" assay. Briefly, this assay employs two genetic constructs, one of which is typically a plasmid that continually expresses a particular receptor of interest when transfected into an appropriate cell line, and the second of which is a plasmid that expresses a reporter such as lucif erase, under the control of a receptor/ligand complex. Thus, for example, if it is desired to evaluate a compound as a ligand for a particular receptor, one of the plasmids would be a construct that results in expression of the receptor in the chosen cell line, while the second plasmid would possess a promoter linked to the luciferase gene in which the response element to the particular receptor is inserted. If the compound under test is an agonist for the receptor, the ligand will complex with the receptor, and the resulting complex will bind the response element and initiate transcription of the luciferase gene. The resulting chemiluminescence is then measured photometrically, and dose response curves are obtained and compared to those of known ligands. The foregoing protocol is described in detail in U.S. Patent No. 4,981,784 and PCT International Publication No. WO 88/03168, for which purpose the artisan is referred.

In a further embodiment of this invention, commercial test kits suitable for use by a medical specialist may be prepared to determine the presence or absence of predetermined radializing factor (RF60) activity or predetermined radializing factor (RF60) activity capability in suspected target cells. In accordance with the testing techniques discussed above, one class of such kits will contain at least the labeled radializing factor (RF60) or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g., "competitive," "sandwich, " "DASP" and the like. The kits may also contain peripheral reagents such as buffers, stabilizers, etc.

In accordance with the above, an assay system for screening potential drugs effective to modulate the activity of the radializing factor (RF60) may be prepared. The radializing factor (RF60) may be introduced into a test system, and the prospective drug may also be introduced into the resulting cell culture, and the culture thereafter examined to observe any changes in the radializing factor (RF60) activity of the cells, due either to the addition of the prospective drug alone, or due to the effect of added quantities of the known radializing factor (RF60).

The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLE 1

Isolation and purification of radializing factor (RF60)

A protein present in embryonic mouse forebrain which functions as a radial glial differentiation factor was previously described (Hunter, K.E. and Hatten, M.E.

(1995), Radial glial cell transformation is bidirectional: regulation by a diffusible factor in embryonic forebrain. Proc. Natl. Acad. Sci.92:2061-2065).

Partial purification/enrichment of the differentiation protein. 10 liters of GCB6 conditioned medium were loaded onto an Econocolumn™ (BioRad) packed with 40mls heparin agarose affigel matrix (BioRad) through a Pharmacia FPLC system with P-500 pump at a flow rate of 5 ml/minute. Partially purified bioactivity was obtained by elution from this column with 0.4M KC1 and the activity pool loaded onto an Econocolumn™ packed with ml wheat germ agglutinin matrix (Vector Laboratories Inc, Burlingame, CA). Bioactivity was eluted from this using a gradient of N-acetyl-glucosamine and loaded onto an Econocolumn™ packed with 5 ml MonoQ Sepaharose matrix (Pharmacia). A final pool of partially purified glial differentiation protein was obtained by elution with 0.4M KC1. Total protein determinations were taken for whole conditioned medium, and for the heparin agarose and mono Q bioactive pools and these were screened on normal and reeler astrocytes as described above at serial dilutions 10 ' to lO-¹² ng/ml total protein. After 48 hours cells were fixed, immunostained with anti-GFAP (anti-glial fibrillary acidic protein) and scored for cell phenotype as described above.

Elution of individual bands from acrylamide gel. A 5 mg sample of partially purified RF60 (eluted as 'final pool' from monoQ sepharose column as described above) was loaded into one lane of a 10% SDS-PAGE gel adjacent to a lane in which 10 ml of SeeBlue protein standard (Novex, San Diego, CA) was loaded. A duplicate set of these samples were loaded onto the same gel. The gel was run out for a sufficient time to ensure maximum separation of the 50kD and 64kD size markers. The piece of gel containing one pair of lanes was excised and placed at 4°C. The piece of gel containing the other pair of lanes was fixed and silver stained (Plus one silver staining kit, Pharmacia, Uppsala, Sweden) to visualize individual protein bands. The most abundant bands were numbered. By placing this fixed stained gel on a light box next to the unfixed gel it was possible to excise small slices from the unfixed gel which should contain the same individual bands as those visualized in the silver stained gel. 25 bands isolated in this way. Each was placed in a 1.5 ml tube containing 1001 elution-renaturation buffer (Ossipow et al., 1993), incubated at 37°C for 3 hours, then microfuged for 10 minutes to sediment the polyacrylamide residues. The supernatant was removed from each sample and transferred to a fresh tube. Each sample was diluted 1: 100 in DMEMF12 then passed through a 0.22mm filter. Diluted samples were assayed for bioactivity by screening on cultured astrocytes as described above. After 48 hours cells were fixed, immunostained with anti-GFAP (anti-glial fibrillary acidic protein) and scored for cell phenotype as described above.

To isolate, purify and characterize this protein, large quantities of protein were harvested from culture medium conditioned by the previously described cell line GCB6 (Gao and Hatten, 1994). Comparison of culture medium harvested from GCB6 cells and E14 cortical cells, the source of bioactivity for previously reported purifications (Hunter and Hatten, 1995) revealed that these two cell populations produce similar levels of the differentiation factor (K.E. Hunter and M.E. Hatten, unpublished data). Therefore, for the purposes of ongoing purification, the GCB6 cell line provides an accessible large scale source of protein.

GCB6 conditioned medium was fractionated using three affinity matrices in sequence: heparin agarose, wheat germ agglutinin and monoQ sepharose, each of which had been found separately in preliminary assays to bind, and permit enrichment of, the glial differentiation protein (data not shown). As shown in FIGURE 2, and discussed below in more detail, following each purification step the eluted bioactive pool became progressively more potent in its capacity to induce radial differentiation of astrocytes harvested from normal cortex. The number of different proteins in the final pool, i.e. pooled eluent from the monoQ sepharose column, were determined by SDS-PAGE analysis. A sample of this pool was loaded onto a denaturing acrylamide gel and silver staining was used to permit visualization of the distribution, number and separation of prominent protein bands present.

The final pool was found to contain a number of abundant protein bands but these were well separated visually on the gel (FIGURE la). By running a second lane of the protein on the same gel and leaving this unfixed/unstained, then comparing this gel piece to its silver stained equivalent, it was possible to excise, from the unfixed gel piece, slices of gel representing the approximate position of each of 20 abundant bands. The protein band in each gel slice was eluted and renatured and each sample was screened for bioactivity (defined as the capacity to induce expression of a radial glial phenotype by cultured astrocytes (Hunter and Hatten, 1995)). Bioactivity was found to be restricted to a region of three bands on the gel located around the 60kD molecular weight range (FIGURE lb). This correlates with the previous report that the bioactivity elutes from a size exclusion column in the 50-60kD molecular weight range (Hunter and Hatten, 1995). Allowing for the possibility that bioactivity may have diffused into neighboring bands, and considering the fact that the gel pieces were essentially excised 'blind' from an unmarked gel, it seems therefore that the radial glial differentiation signal is represented by a single gel band in the approximate molecular weight region of 60kD. According to its function and approximate molecular weight, this was termed radializing factor 60 (RF60), which occurs as a single protein band on SDS-PAGE analysis.

EXAMPLE 2 To elucidate the molecular events which regulate radial glial cell development the following experiments were conducted with neurological mutant reeler mice.

In reeler cortex, radial glia differentiate poorly, as evidenced by the extension of only short, truncated processes; the radial scaffold is maintained for a shorter time than normal, and radial glia transform prematurely into astrocytes. In these experiments, although astroglial cells from normal animals are induced to elaborate a radial glia phenotype by RF60, reeler astroglia show only impaired differentiation in response to the protein. This suggests that an intrinsic defect in glial differentiation contributes to the phenotype of abnormal cortical lamination seen in reeler mouse.

The murine autosomal recessive mutation reeler broadly affects cellular organization throughout the central nervous system, including in the cerebral and cerebellar cortices. The reeler phenotype is evident at E14 as a failure in the formation of the cortical plate. Subsequently, patterning of neurons during migration and laminar formation are disturbed, thus leading to an inversion of the normal cortical layers (Falconer, 1951; Goffmet, 1979; Caviness, 1982; Pinto-Lord et al., 1982; Caviness et al., 1988). Molecular cloning of the reeler gene has revealed that the gene encodes a 388kD extracellular protein, reelin. In wild-type cerebral cortex, reelin is expressed in Cajal Retzius neurons, cells which are localized to the outermost layer, layer 1, and form a postmitotic, fully differentiated neuronal population well before the bulk of cortical neurogenesis occurs. The early differentiation of Cajal Retzius cells, together with their expression of the reelin protein, indicates a critical role for these early neurons in cortical patterning (Miao et al., 1994; D'Arcangelo et al., 1995). The reeler mouse provides therefore a genetic model for the study of cortical patterning, including the development of the glial scaffold and its role in corticogenesis.

The reeler system can be used to elucidate the molecular events which regulate radial glial cell development and the role of the radial glial differentiation signal, radializing factor (RF60) in this. The progression of radial glia and astrocyte phenotype during the perinatal period in reeler and normal cortex can be examined and the effect of the radial glial differentiation factor on reeler and normal astroglia can thus be compared. These analyses aim to determine whether or not the developmental program of radial glial cell development in reeler cortex is normal, whether these cells might directly cause the reeler cortical phenotype, and ultimately, to reveal how the glial differentiation factor functions in the developing forebrain.

Experimental Procedures

Preparing tissue sections, rl/rl and 4-/rl mice were acquired from matings of rl/rl or +/rl females with +/rl males (Jackson Laboratories, Bar Harbor, ME). Day of plug detection was determined as embryonic day 0 (E0) and day of birth as postnatal day 0 (P0). Pups of stage E14, P0 and P4 were sacrificed and the forebrains removed in ice cold CMF-PBS (calcium-magnesium free buffered saline; GIBCO BRL, Grand Island, NY), fixed overnight in 2% paraformaldehyde in PBS (phosphate buffered saline; reconstituted from tablets (Sigma Chemical Co., St. Louis, MO)), washed in PBS for 3 hours then embedded in agarose (3% low EEO agarose (Sigma) in dH₂0). Brains were cut into lOOμm coronal

Vibratome sections. Two sections from each brain were counterstained with cresyl violet then examined wet and unmounted under light microscopy and phenotyped as normal or reeler according to presence or absence of a cortical plate, respectively. Immunostaining for RC2 and GFAP (^"glial fibrillary acidic protein) distribution in vivo. Cortical sections were permeablized/preblocked (1 % Tween20 (BioRad, Hercules, CA), 20% normal goat serum (GIBCO) in PBS, 90 minutes) then incubated in either monoclonal mouse-anti-RC2 (tissue culture supernatant, cell line a generous gift of Dr. M. Yamamoto, Tsukuba University, Japan) diluted 1:1 in preblocking solution, or rabbit-anti-cow GFAP (Dako Corporation, Carpinteria, CA) diluted 1:200 in preblocking solution (each overnight at 4°C) then either FITC-conjugated goat-anti-mouse IgM or TRITC-conjugated goat-anti-rabbit IgG (Sigma) diluted in preblocking solution (3 hours at room temperature). Sections were washed in PBS (3x15 minutes) between and after these steps. Sections were then mounted wet on glass slides in a droplet of DABCO mountant (see Hunter et al. , 1993). Sections were imaged by optical sectioning and image reconstruction as previously described using a BioRad confocal microscope with fluorescein filter block (see Gao and Hatten, 1993).

Western blot analysis. Cortices were harvested from litters of reeler strain matings of developmental ages E14, E17, E19, P0, P2 and P21. In each case one hemisphere was fixed for phenotyping as described above. The other hemisphere was homogenized in 500 ml protein sample buffer (Laemmi, 1970) and protein concentrations determined (BCA protein assay kit (Pierce, Rockford, IL)). A sample of each was boiled for 5 minutes, and 5 mg total protein from each sample were loaded onto a 10% acrylamide gel. The gel was run, then blotted onto nitrocellulose paper using a semi-dry blotting system (BioRad). The blot was then probed to determine the relative levels of GFAP (glial fibrillary acidic protein) in each sample by probing with rabbit-anti-GFAP (1:2500 dilution) followed by alkaline phosphatase conjugated anti-rabbit secondary antibody (Promega, Madison, WI) (1 :7500 dilution). Bound secondary antibody was detected by bromochloroindoyl phosphate/nitro blue tetrazolium development as described (Towbin et al., 1979). Screening size fractionated protein on reeler and normal astrocytes. Astrocytes were purified from forebrains of PO +/rl and rl/rl pups. Brains were harvested as described above and the cortex divided into two hemispheres. One hemisphere was placed in fix for phenotyping as described above and the other to prepare primary astrocytes as previously described (Hunter et al., 1993), plating each single cortex into a T25 flask (Falcon).

Cell conditioned medium was harvested from cultures of the GCB6 cerebellar cell line (Gao and Hatten, 1994) as follows: GCB6 cells were plated into Integrid ™ culture plates (Falcon) at 1/10 dilution in DMEMF12 culture medium (GIBCO) supplemented with 10% fetal bovine serum (FBS; GIBCO). On reaching confluence (5-7 days) GCB6 cells were transferred to a minimal volume of serum free DMEMF12. This medium was harvested and replaced daily; harvested medium was filtered through a 0.22mm membrane filter to remove cell debris, then stored at 4°C. 5mls of conditioned medium was fractionated on a HiLoad Superdex 16/60 size exclusion column (Pharmacia) as previously described (Hunter and Hatten, 1995). Fractions eluted from the sizing column were screened on normal and reeler astrocytes; after 48 hours cells were fixed in 2% paraformaldehyde, and immunostained for GFAP (glial fibrillary acidic protein) expression as previously described (Hunter and Hatten, 1995). Approximately 200 cells on each coverslip were scored according to phenotype as 'normal' or 'radial' , and the percentage of radial cells on each coverslip was calculated. 1 unit of bioactivity was defined as the amount of protein pool required in each case to effect 'conversion' (switch from epithelioid to radial phenotype) of 25% of astrocytes.

Preparation of radializing factor (RF60 . Radializing factor (RF60) was prepared and purified as described in Example 1.

Cell ceiling culture assays. Forebrains were harvested from reeler strain litters of E15 as described above. Half of each cortex was used for phenotyping as described above; the remaining halves were dissociated and used to prepare embryonic cell 'ceilings' as previously described (Hunter and Hatten, 1995). Ceilings were assayed for bioactivity by coculture with normal astrocytes. After 48 hours astrocytes were fixed, immunostained with anti-GFAP and scored for cell phenotype as described above.

RESULTS

The radial glial scaffold develops poorly and disappears prematurely in reeler cortex The architecture and organization of the radial glial scaffold was examined in coronal sections of reeler and normal cortex of developmental stages E14, P0 and P4. These stages represent the early, middle and late phases of the radial glia-astrocyte developmental pathway. Radial glia, marked by expression of the RC2 antigen (Misson et al., 1988) are fully elaborated in the cortical wall at E14 and persist throughout the late embryonic period; around the time of birth they begin to transform into astrocytes, as evidenced by expression of glial intermediate filament protein, glial fibrillary acidic protein (GFAP) (Dahl and Bignami, 1973; Levitt and Rakic, 1980). Transformation is complete by the end of the first postnatal week (Misson et al. , 1991) at which time highly elongated, RC2-positive cells have disappeared and stellate GFAP-positive astrocytes are prevalent. The switch from RC2 expression to GFAP expression in the perinatal period occurs concomitantly with the establishment of the cortical layers, suggesting that the transformation of radial glial into astrocytes follows changing roles for astroglial cells in corticogenesis. To visualize the glial scaffold throughout the period of differentiation and transformation in the developing forebrain, E14 cortical sections were immunostained with RC2 antibody while P0 and P4 sections were immunostained with anti-GFAP.

Radial glia in the E14 reeler cortex were organized in an approximately radial manner across the thickness of the cortical anlage. However the radial scaffold which they constituted was sparse and the fibers were loosely packed. Instead of projecting in the radial plane, fibers projected in all directions within the cortical wall. These fibers were poorly differentiated, extending shorter processes that often failed to form endfeet along the pial surface (FIGURE 3B). In contrast, the forms of radial glia in normal E14 cortex recognized by RC2 antibody were as reported in classical studies (reviewed in Misson et al., 1991). These radial glia formed a dense scaffold of remarkably well-aligned fibers arranged perpendicular to the pial surface and spanning the thickness of the cortical anlage. At the glial limitans along the pial surface, bifurcating endfeet were evident; overall glial fibers appeared more abundant in normal cortex than in reeler at E14 (FIGURE 3A). Therefore it seems as though radial glia in the E14 reeler cortex are less well differentiated, more sparse and disorganized than their counterparts in the normal cortex. Thus, the patterning of radial glial fibers appears severely disrupted in reeler cortex.

In the PO reeler cortex, the radial glial scaffold was present but the cell processes were thick and fibrous, the whole scaffold having a coarse appearance. However the most striking feature of the PO reeler cortex was the abundance of mature astrocytic forms present. These cells were located directly beneath the pial surface, being highly abundant and forming a dense network of stellar cell forms (FIGURE 3D).

In contrast, in normal PO cortex, anti-GFAP immunostaining revealed that the overall structure of the glial scaffold is still fully in evidence and composed of well differentiated radial glia (FIGURE 3C). In contrast to reeler cortex, GFAP expression in normal cortex at this time was still relatively low; immunostaining of parallel sections with RC2 antibody (data not shown) revealed that GFAP and RC2 antigen are equally represented in normal PO cortex. Radial glia formed a well organized scaffold and still bear fine cell processes, these extending to the pial surface, retaining contact with the limitans (FIGURE 3C) Though it is clear that the process of transformation of radial glia into astrocytes has begun, only a few astrocytic forms were present in the cortical wall (FIGURE 3C), in comparison to the large number in reeler cortex of the same age. Therefore it seems as though the astroglial cells in normal PO cortex are more immature than in reeler cortex of the same age.

The contrast in distribution of astroglial forms between reeler and normal cortex was even more marked at P4, at which time GFAP immunostaining showed that the radial scaffold had completely disappeared. All of the astroglial forms present in the P4 reeler forebrain were astrocytic, bearing characteristic short stellar processes (FIGURE 3F).

In comparison the predominant astroglial phenotype in normal P4 cortex was reminiscent of that classically described as transitional astroglia, cells which bear features of both radial glia and astrocytes, having long radial glia-like cell processes oriented perpendicular to the pial surface, as well as shorter, astrocytic processes close to the cell body (FIGURE 3E). The abundance of these cell forms is indicative of the fact that the transformation of radial glia into astrocytes is still in progress within the cortical wall. However as seen in PO cortex, the astroglial forms present are of more immature forms than those in P4 reeler animals. Therefore, transformation of radial glia into astrocytes, normally not concluded until the end of the second postnatal week (reviewed in Misson et al., 1991), seems to be completed ahead of schedule in the reeler cortex.

Glial transformation is accelerated in reeler cortex

The ήnmunostaining of PO cortical sections hinted that GFAP (glial fibrillary acidic protein) levels appear higher in reeler than normal cortex at this time, lending support to the idea that progress along the radial glia-astrocyte transformation pathway is accelerated in reeler cortex. In order to confirm at the molecular level whether there is a difference in the developmental expression of GFAP (glial fibrillary acidic protein) between normal and reeler cortex, a Western Blot of protein extracts from normal and reeler cortex ranging in age from E14 to P21 was prepared and probed with anti-GFAP. An intriguing developmental pattern of GFAP (glial fibrillary acidic protein) levels was seen in reeler cortex. GFAP was present in reeler cortex at the earliest developmental stage screened, El 4, a time when the radial glial scaffold is most well differentiated, and transformation has not yet been initiated. GFAP levels then remained constant throughout the embryonic period but there was a marked increase in at the time of birth, and very high levels of GFAP were seen in the P21 reeler cortex. Overall, in reeler cortex levels of GFAP were substantial throughout the embryonic and postnatal periods.

In contrast, GFAP was absent from normal cortex during embryogenesis, being first detectable at PO, a time more in keeping with its observed appearance in immunostained sections, as shown here and previously (reviewed in Misson et al., 1991). Furthermore, even at PO GFAP levels were still low in normal cortex, particularly when compared to levels in reeler cortex at this time. Indeed of all stages of normal cortex screened, only the most mature, that harvested from P21 cortex, contained substantial amounts of GFAP, and again this was markedly lower than levels in P21 reeler cortex (FIGURE 4). Indeed, GFAP levels in PO reeler cortex were comparable to those in the mature P21 normal cortex (FIGURE 4).

Western blot analysis therefore demonstrates clearly that GFAP, the characteristic astrocyte marker, is expressed precociously in reeler cortex from the earliest stages of radial glial differentiation, and continues to be expressed at higher levels in reeler than normal cortex, even in maturity, when transformation of radial glia into astrocytes is completed anyway. This enhanced expression of GFAP confirms and supports the immunohistochemical finding that in reeler cortex there is premature transformation of radial glia into mature astrocytes and further shows that astrocytes in mature reeler cortex express levels of GFAP which are in excess of those seen in normal cortex.

Reeler astroglia have an autonomous defect in their ability to respond to RF60 The comparison of astroglial phenotype in tissue sections of reeler and normal cortex indicated that reeler radial glia differentiate poorly and transform prematurely into astrocytes, suggesting that the radial glia-astrocyte developmental program is defective in reeler cortex. This raised the question as to whether RF60 is either functional or present in reeler cortex. The questions were therefore addressed as to whether reeler astroglia are able to differentiate in response to RF60, and whether absence of reeler gene expression alters cortical levels of this protein. Partially purified RF60 was prepared as previously described by elution from a size exclusion column (Hunter and Hatten, 1995). Serial fractions eluted from the size exclusion column were screened in vitro as before on normal cortical astrocytes for effect on cell phenotype. The activity eluted as previously described within the range of only a few fractions of around 60kD (Hunter and Hatten, 1995; FIGURE 2A). However a very different result was obtained in a parallel screen of these fractions on reeler cortical astrocytes. Although the general bioactivity profile paralleled that of normal astrocytes, the overall response of reeler astrocytes was dramatically reduced (FIGURE 2A). Only a small percentage of reeler astrocytes were induced to express a radial phenotype, the peak response being only 20% compared to almost 70% in the case of normal astrocytes; instead, the majority of cells remained epithelioid in form (FIGURE 2A). This suggested reeler astrocytes may have a reduced ability to respond to RF60. One explanation for this result is that reeler astrocytes have a shifted dose-response for RF60, i.e. require higher levels than normal astrocytes to exhibit the same degree of phenotype change. To assess this, GCB6 conditioned medium was progressively purified by fractionation on three sequential affinity matrices: heparin agarose, wheat germ agglutinin and monoQ sepharose, steps which progressively enriched RF60. The bioactivity pool from each column was screened in serial dilution on reeler and normal astrocytes. After each step the protein pool became more potent in inducing radial differentiation of normal astrocytes (FIGURE 2A, 2B, 2C). However as in the size fractionation screen, overall, very few reeler astrocytes were induced to differentiate. Furthermore, there was no dose-response effect of RF60 on reeler astrocytes; even in the presence of the highly RF60-enriched eluent pool from the monoQ sepharose column, very few astrocytes differentiated (FIGURE 2B). Reeler astrocytes therefore do not have a shifted dose-response to RF60, but rather, possess overall only a restricted ability to respond to the factor.

In order to determine whether RF60 is present in reeler cortex a previously described assay was used (Hunter and Hatten, 1995). Astrocytes harvested from either normal or reeler cortex were cocultured with non-contacting 'ceilings' of E14 normal or reeler cortical cells. This model provides a means of testing the effects of soluble factors, released by the cells plated into the ceilings, on the cells plated on the substrate beneath. Whether in the presence a normal or reeler embryonic cortical cell ceiling, reeler astrocytes failed to differentiate, again remaining epithelioid in form (FIGURE 2C). In contrast, a proportion of normal astrocytes did differentiate and exhibit a radial phenotype approximately to the same extent whether in the presence of a normal or reeler cortical cell ceiling (FIGURE 2C). This functional assay therefore supports the conclusion that RF60 is indeed present in the reeler cortex and that the defect lies autonomously in the astroglial cells of reeler cortex.

DISCUSSION

It was previously proposed that radial glial differentiation and transformation into astrocytes is bidirectional in the mammalian forebrain is bidirectional, with the availability of a diffusible factor present in embryonic but not adult brain, acting to regulate the form and function of astroglial cells (Hunter and Hatten, 1995). Here, this factor was purified by biochemical methods, using a previously described bioassay, and defined as a single bioactivity, termed RF60. The findings presented here reveal that there are intrinsic defects in the program of development of reeler radial glia development and transformation in reeler forebrain which involves the function of RF60. Although RF60 is present in embryonic reeler cortex, reeler astroglia are unresponsive to this; the protein is ineffectual in inducing a radial glia phenotype in cultured reeler astrocytes; even saturating levels of RF60 are ineffective in inducing a radial phenotype in these cells, suggesting there is a glia-autonomous defect in reeler cortex which involves a failure of RF60 function, either in terms of expression of receptors for this protein, or in its signalling pathway.

EXAMPLE 3 To ascertain the effect/ impact of radializing factor (RF60) in vivo, stab wounds in the adult mouse cortex were studied both with and without the administration of radializing factor (RF60), infused into the would using as osmotic micropump system. The form of the astrocytes around the wound were examined at two, five and seven days after the wounding/infusion.

Methods Adult mice were anaesthetized with Nembural according to body weight; during surgery deep anaesthesia was maintained by administering the vaporous anaesthetic Aerrane™. An incision was made through the skin and muscle over the forebrain. Using a stereotactic device a fine needle was driven through the skull and into the wall of the left cortical hemisphere. A catheter was inserted into this hole and then the catheter was connected via fine tubing to an Alzet™ osmotic micropump, which had been pre-filled with a partially purified fraction of RF60 in saline. The pump itself was inserted under the skin of the back of the mouse. As a control, a catheter only was inserted in the brain, or, a catheter connected to a pump carrying only saline. The skull wound area was then sealed and the pump held in place by layering dental acrylic over the skull and allowing this to set. Animals were kept warm until fully recovered. Animals were sacrificed at two, five and seven days after surgery. Animals were perfused and the brains harvested and cut on a Vibratome into lOOum coronal sections. Sections from immediately around the wound site were immunostained to determine protein distribution of GFAP and RC2. Corresponding results are depicted in Figures 5 A and 5B, representing the repetition of the above described stab wound experimental protocol. Results

In the cortical hemispheres of the control animals in which no stab wound had been made (see Figure 5A), anti-GFAP antibody recognized stellate cells with the characteristic appearance of mature astrocytes. GFAP levels appeared to be constant across the cortical mass in these cases, i.e., no regional variations. RC2 antibody did not detect antigen at any time in these animals, indicating that this embryonic marker is not normally expressed in adult cortex.

In the animals in which an "empty" catheter or one carrying only saline had been inserted, the following pattern was seen. At two days, reactive gliosis was recognized around the wound site by high expression of GFAP. Furthermore, astrocytes in these region also had the phenotype of reactive astrocytes with characteristic long, stellar, meshed processes. A similar pattern was seen in these cortices at five and seven days, indicating that reactive gliosis persists during this period. RC2 antigen was not detected in cortex at any time in these animals.

In animals in which a catheter carrying RF60 had been inserted (see Figure 5B), the following pattern was seen. At two days there was an absence of the features of reactive gliosis around the wound site such as observed in the catheter-wounded group above. Instead, GFAP levels around the infusion site were unusually low, being dramatically lower than when a control catheter was inserted, and lower even that levels in the adjacent untreated cortical hemisphere. Further, those GFAP-expressing cells seen exhibited the "radialized" phenotype of astrocytes in vitro when they are treated with RF60. In parallel stained sections, a number of cells of this radial phenotype were found to be expressing RC2. At five days, the appearance and GFAP levels of astrocytes around the wound/infusion site were almost the same as those seen after five days in cortex which had received a control catheter. At five days, there were still some RC2-expressing cells close to the wound site in RF60 treated cortex, but levels were much lower than those seen at two days. At seven days, the appearance of astrocytes was similar to that seen at five days except that RC2 is no longer expressed at this time. Indeed, at this time, the form and antigen expression patterns of astrocytes in RF60 treated cortex are identical to those seen in cortex which had received a control catheter.

Summary In normal adult cortex astrocytes have a characteristic stellar appearance, and express GFAP, but not RC2, the latter being an embryonic glial marker. Following stab injury of the cortex, astrocytes usually assume a reactive phenotype, becoming even more stellar and increasing their expression of GFAP. This reactive cell phenotyped is seen at two days following injury and is still in evidence even seven days after injury. These reactive astrocytes do not express RC2 at any time.

If, however, RF60 protein is infused into the wound site following injury, a very different pattern is observed. Two days after injury, reactive astrocytes are not seen at the wound site. Rather, GFAP levels are reduced, being even lower than in unstabbed cortical areas; however, a number of astrocytes are recognized by RC2 antibody and a number of these cells have a "radialized" phenotype. Five days after wounding/infusion these cortices look very similar to those wounded but not infused with RF60 protein.

These experiments provide evidence that RF60 functions as an anti-gliosis factor in the injured CNS when infused at the wound site following injury. As seen in vitro, the in vivo effects of RF60 are transient and repeated infusion would be expected to maintain astrocytes at the wound site in a "radialized" form. Doing so could limit the impeding effects of the glial scar on ongoing axonal regrowth at the wound site and could therefore present a therapeutic method for preventing or reversing the formation of glial scarring, thus facilitating mammalian, and especially human, CNS regeneration.

While the invention has been described and illustrated herein by references to various specific material, procedures and examples, it is understood that the invention is not restricted to the particular material combinations of material, and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art.

The following is a list of documents related to the above disclosure and particularly to the experimental procedures and discussions. The documents should be considered as incorporated by reference in their entirety.

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Claims

WHAT IS CLAIMED IS:

1. A radializing factor, comprising a material selected from the group consisting of a protein, active fragments thereof, agonists thereof, mimics thereof, and combinations thereof, said factor having the following characteristics: a) an apparent molecular weight of approximately 60kD; and b) the capacity to induce expression of a radial glial phenotype by cultured astrocytes.

2. The radializing factor of Claim 1 which is isolated from GCB6 cells or E14 cortical cells.

3. The radializing factor (RF60) of Claim 1 which is derived from mammalian cells.

4. The radializing factor (RF60) of Claim 1 labeled with a detectable label.

5. The radializing factor (RF60) of Claim 4, wherein the label is selected from enzymes, chemicals which fluoresce and radioactive elements.

6. An antibody to a radializing factor (RF60), the radializing factor (RF60) to which said antibody is raised having the following characteristics: a) an apparent molecular weight of approximately 60kD; and b) the capacity to induce expression of a radial glial phenotype by cultured astrocytes.

7. The antibody of Claim 6 which is a polyclonal antibody.

8. The antibody of Claim 6 which is selected from the group consisting of monoclonal antibodies and chimeric antibodies.

9. An immortal cell line that produces a monoclonal antibody according to Claim 8.

10. The antibody of Claim 6 labeled with a detectable label.

11. The antibody of Claim 10, wherein the label is selected from enzymes, chemicals which fluoresce and radioactive elements.

12. A DNA sequence or degenerate variant thereof, which encodes a radializing factor (RF60), or a fragment thereof.

13. The DNA molecule of Claim 12, wherein said DNA sequence is operatively linked to an expression control sequence.

14. A recombinant DNA molecule comprising a DNA sequence or degenerate variant thereof, which encodes a radializing factor (RF60), or a fragment thereof.

15. The recombinant DNA molecule of Claim 14, wherein said DNA sequence is operatively linked to an expression control sequence.

16. The recombinant DNA molecule of Claim 15, wherein said expression control sequence is selected from the group consisting of the early or late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC system, the 77?C system, the major operator and promoter regions of phage ╬╗, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase and the promoters of the yeast ╬▒-mating factors.

17. A probe capable of screening for the radializing factor (RF60) in alternate species prepared from the DNA sequence of Claim 12.

18. A unicellular host transformed with a recombinant DNA molecule comprising a DNA sequence or degenerate variant thereof, which encodes a radializing factor (RF60), or a fragment thereof; wherein said DNA sequence is operatively linked to an expression control sequence.

19. The unicellular host of Claim 18 wherein the unicellular host is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeasts, CHO, Rl. l, B-W, L-M, COS 1, COS 7, BSCl, BSC40, and BMTIO cells, plant cells, insect cells, and human cells in tissue culture.

20. A method of testing the ability of a drug or other entity to modulate the activity of a radializing factor (RF60) which comprises A. culturing a colony of test cells which has a receptor for the radializing factor (RF60) in a growth medium containing the radializing factor (RF60); B. adding the drug under test; and C. measuring the reactivity of said radializing factor (RF60) with the receptor on said colony of test cells, wherein said radializing factor (RF60) has the following characteristics: a) an apparent molecular weight of approximately 60kD; and b) the capacity to induce expression of a radial glial phenotype by cultured astrocytes.

21. An assay system for screening drugs and other agents for ability to modulate the production of a radializing factor (RF60), comprising: A. culturing an observable cellular test colony inoculated with a drug or agent; B. harvesting a supernatant from said cellular test colony; and C. examining said supernatant for the presence of said radializing factor (RF60) wherein an increase or a decrease in a level of said radializing factor (RF60) indicates the ability of a drug to modulate the activity of said radializing factor (RF60), said radializing factor (RF60) having the following characteristics: a) an apparent molecular weight of approximately 60kD; and b) the capacity to induce expression of a radial glial phenotype by cultured astrocytes.

22. A method of preventing or reversing glial scar formation in the brain or central nervous system of a mammal in need of such therapy, comprising administering to a mammal a therapeutically effective amount of a material selected from the group consisting of a radializing factor (RF60), an agent capable of promoting the production and/or activity of said radializing factor (RF60), an agent capable of mimicking the activity of said radializing factor (RF60), an agent capable of inhibiting the production of said radializing factor (RF60), and mixtures thereof, or a specific binding partner thereto, said radializing factor (RF60) having the following characteristics: a) an apparent molecular weight of approximately 60kD; and b) the capacity to induce expression of a radial glial phenotype by cultured astrocytes.

23. The method of Claim 22 wherein said disease states include treatment of brain or spinal cord injury or surgery, treatment of neurological disorders or diseases where there has been a diminution of the normal activity of the brain or central nervous system, and situations where regeneration of brain or central nervous system tissue is therapeutic.

24. A pharmaceutical composition for the prevention or treatment of glial scar formation in the brain or central nervous system of a mammal in need of such therapy, comprising: A. a therapeutically effective amount of a material selected from the group consisting of a radializing factor (RF60), an agent capable of promoting the production and/or activity of said radializing factor (RF60), an agent capable of mimicking the activity of said radializing factor (RF60), an agent capable of inhibiting the production of said radializing factor (RF60), and mixtures thereof, or a specific binding partner thereto, said radializing factor (RF60) having the following characteristics: a) an apparent molecular weight of approximately 60kD; and b) the capacity to induce expression of a radial glial phenotype by cultured astrocytes; and B. a pharmaceutically acceptable carrier.

25. A radializing factor (RF60) implicated in the prevention or reversal of glial scar formation in brain or central nervous system cells in response to injury or trauma to the brain or central nervous system, said factor having the following properties: a) it has an apparent molecular weight of approximately 60kD; and b) it possesses the capacity to induce expression of a radial glial phenotype by cultured astrocytes.

26. An antibody produced by injecting a substantially immunocompetent host with an antibody-producing effective amount of an radializing factor (RF60) polypeptide, and harvesting said antibody, said radializing factor (RF60) polypeptide having the following properties: a) it has a molecular weight of about 60kD; and b) it has the capacity to induce expression of a radial glial phenotype by cultured astrocytes.

27. The antibody of either of Claims 25 or 26 which is monoclonal.

28. The antibody of either of Claims 25 or 26 which is polyclonal.

29. The antibody of either of Claims 25 or 26 which is chimeric.

30. A recombinant virus transformed with the DNA molecule, or a derivative or fragment thereof, in accordance with Claim 12.

31. A recombinant virus transformed with the DNA molecule, or a derivative or fragment thereof, in accordance with Claim 13.

32. The recombinant DNA molecule of Claim 15 composing plasmid pGEX- 3X, clone E3 or plasmid PGEX-3X, clone E4.

33. Astrocytes that exhibit a radial glia phenotype, such astrocytes having increased levels of the radializing factor of Claim 1.

34. The astrocytes of Claim 33, which possess a radial and elongated shape, express the glial cell marker RC2 and have dramatically reduced levels of GFAP.