Note: Descriptions are shown in the official language in which they were submitted.
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VERTEBRATE SLIT DNA SEQUENCE, PROTEIN AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from provisional application 60/124,767,
filed March
17, 1999, which is hereby incorporated by reference in its entirety for all
purposes.
BACKGROUND
This invention relates to newly identified and isolated polynucleotides,
proteins
encoded by the polynucleotides, methods for producing the proteins and uses
for the
polynucleotides and proteins. More particularly the invention relates to the
vertebrate slit
proteins, polynucleotides encoding vertebrate slit proteins, methods for
producing vertebrate
slit proteins and the uses of vertebrate slit proteins and polynucleotides.
More particularly
still, the invention relates to Xenopus slit proteins and polynucleotides,
along with their
production and uses.
Cell migration is essential in species ranging from bacteria to humans (for
recent
reviews, see Mitchison and Cramer, Cell, 84:371-379 (1996); Lauffenburger and
Horwitz,
Cell, 84:359-369 (1996); Montell, Development, 126:3035-3046 (1999)). In the
amoebae
Dictyostelium discoideum, cell migration is involved in chemotaxis towards
food sources and
in cell aggregation and differentiation (reviewed in Devreotes and Zigmond,
Ann. Rev. Cell
Biol., 4:469-686 (1988); Parent and Devreotes, Ann. Rev. Biochem., 65:411-440
(1996); Chen
et al., Trends Genet., 12:52-57 (1996); Parent and Devreotes, Science, 284:765-
770 (1999)).
2 0 In higher vertebrates, cell migration plays important roles in multiple
physiological and
pathological processes. During embryonic and neonatal development, cell
migration is crucial
for morphogenetic movement such as gastrulation, cardiogenesis, formation of
the internal
organs such as the lung and kidney, formation of hematopoietic organs, and the
formation of
the central and peripheral nervous systems (CNS and PNS). In adult animals,
cell migration is
2 5 required for leukocyte trafficking and inflammatory responses. In tumor
growth and
development, tumor-induced angiogenesis and tumor metastasis both involve cell
migration.
Despite its importance, our understanding of mechanisms underlying cell
migration in
mammals is still limited (Lauffenburger and Horwitz, Cell, 84:359-369 (1996);
Mitchison
and Cramer, Cell, 84:371-379 (1996)).
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2
Neural development is the process by which the cells of an embryo organize
themselves
into a complete and functioning nervous system. A striking feature of neural
development is that
the majority of neurons in the developing nervous system have to migrate to
reach their final
positions. Studies of neuronal migration are important for our understanding
of the formation of
the normal nervous system and for understanding the etiology of human diseases
caused by
abnormal migration and to provide a basis for designing therapeutic approaches
to neurological
diseases.
Neuronal migration in the developing central nervous system (CNS) was
initially inferred
from histological observations by classical neuroembryologists. The question
of whether cells
truly migrated or whether cells formed earlier were simply displaced by cells
formed later was
clarified by observations of changes of cell position in a direction opposite
to that expected from
cell displacement through histological examinations in the spinal cord of
chick embryos and
more convincingly by autoradiographic tracing in the cerebrum of rodent
embryos. The fact that
only nuclei were traced in autoradiographic studies raised the possibility
that nuclei, but not
entire cells, moved in the highly structured nervous system. Electron
microscopic (EM)
examination and reconstruction uncovered strong evidence for the migration of
neuronal cell
bodies. Observations of primary neurons and glia cultured in vitro
demonstrated directly that
neurons indeed migrate. Through a considerable amount of work with
histological,
autoradiographic, retroviral tracing, dye labeling and modern imaging
techniques, it is now well
2 0 established that the majority of neurons migrate throughout the developing
nervous system.
In humans, proper migration of neurons is essential for the formation and
normal
functioning of the nervous system. Defects in neuronal migration can cause
multiple
clinically relevant diseases including epilepsy. Migration may also be
important for invasion
of tumors including neuroblastoma and glioblastoma and other tumors of the
nervous
2 5 system. Although it has been known for some time that cell migration is
essential for
postnatal behavior changes in birds, only recently have studies revealed that
neurogenesis and
neuronal migration continue in the brains of postnatal mammals, including
humans. These
findings also highlight that, for successful applications of cell-based
therapies of
neurodegenerative diseases, it is essential to direct correct migration of
neurons or cells
3 0 expressing therapeutic products to the target regions.
Our understanding of the molecular mechanisms guiding neuronal migration is
still
limited. Genetic studies in humans and in mice have led to the identification
of multiple
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3
molecules whose deficiencies cause defects in neuronal migration. Because most
of these are
intracellular molecules, it is unlikely that they can serve as guidance cues
for migrating
neurons. An interesting molecule identified from genetic studies is the
secreted protein
Reelin (D'Arcangelo et al., Nature, 374:719-723 (1995); Hirotsune et al., Nat.
Genet., 10:77-
83 (1995)). Loss of function mutations in the reeler gene cause defects in
central nervous
system (CNS) lamination, and Reelin has been thought to control cell-cell
interactions critical
for cell positioning. It is not clear, however, whether Reelin acts as a stop
signal or an
adhesive molecule for migrating neurons. Because molecules can regulate
neuronal
migration indirectly, it is also not clear whether Reelin affects neuronal
migration directly or
indirectly. Similarly, the precise roles played by the other genetically
identified molecules in
neuronal migration remain to be understood.
An axon is a long projection of a nerve cell that generally carnes nerve
impulses away
from the nerve cell body. Correct projection of axons to their targets is
essential for the
formation and function of the nervous system. Identification of axon guidance
molecules and
determination of their expression patterns and functional properties are
therefore critical for
understanding neural development. In addition, a thorough understanding of the
molecular
mechanisms involved in guiding developing axons to the proper location is
crucial to the
development of new technologies to regenerate damaged nerve tracts following
traumatic
injuries, such as spinal cord injuries, and in treatment of genetic conditions
resulting in
2 0 central nervous system malformation.
Previously, two families of secreted long-range chemoattractants and
chemorepellents, the Netrins and Semaphorins (Sema) were known to function in
directing
axon projections (Tessier-Lavigne and Goodman, Science 274:1123-1133 (1996);
Culotti and
Kolodkin, Curr. Opin. Neurobiol. 6:81-88 (1996); Nieto, Neuron, 17:1039-1048
(1996);
2 5 Puschel, Eur. J. Neurosci. 8:1317-1321 (1996); Varela-Echavarria and
Guthrie, Genes &
Dev. 11:545-557 (1997)).
Recently, a new family of proteins associated with the development of the
nervous
system, termed slit proteins, have been discovered. Slit mutations were
originally observed
in the fruit fly Drosophila in saturation mutagenesis experiments for
mutations affecting
3 0 larval cuticular patterning (Nusslein-Volhard et al., Roux's Arch. Dev.
Biol., 193:267-282
(1984)). Recently, vertebrate slit genes have been found in rats and humans
(Nakayama et
al., Genomics 51:27-34 (1998); Itoh et al., Mol. Brain Res. 62:175-186 (1998);
Holmes et al.,
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4
Mech. Devel. 79:57-72 (1998)). Drosophila slit cDNA was isolated by screening
for genes
encoding epidermal growth factor (EGF) repeats which hybridized to a probe
made from
Notch, a gene involved in cell fate determination (Rothberg et al., Cell
55:1047-1049 (1988)).
Drosophila slit mRNA was found to be expressed in midline glial cells, whereas
the secreted
protein product of the slit mRNA was found in the midline cells and on axons
traversing the
midline cells (Rothberg et al., Cell 55:1047-1049 (1988); Rothberg et al.,
Genes Dev. 4:2169-
2187 (1990)). Loss of slit function is thought to cause defects in the
differentiation of
midline cells and the separation of longitudinal axonal tracts (Rothberg et
al., Cell 55:1047-
1049 (1988); Rothberg et al., Genes Dev. 4:2169-2187 (1990)).
Molecular mechanisms underlying axon guidance at the midline appear to be
conserved between vertebrates and invertebrates. The Netrin protein, which is
secreted by the
floor plate of the neural tube, is attractive to commissural axons in
vertebrates, Drosophila
and the roundworm C. elegans (Ishii et al., Neuron 9:873-881 (1992); Kennedy
et al., Cell
78:425-435 (1994); Serafmi et al., Cell 78:409-424 (1994); Harris et al.,
Neuron 17:217-228
(1996); Mitchell et al., Neuron 17:203-215 (1996); Kolodziej et al., Cell
87:197-204 1996)).
Netrin can also act as a repellent for a specific subset of axons in C.
elegans and vertebrates
(Hedgecock et al., Neuron 4:61-84 (1990); Colamarino and Tessier-Lavigne, Cell
81:621-629
(1995)). Recent studies in Drosophila have revealed that a transmembrane
receptor encoded
by the roundabout (robo) gene plays an important role in ensuring that
commissural axons
2 0 which have already crossed the midline do not re-cross the midline and
that other axons
which stay ipsilateral do not cross the midline (Seeger et al., Neuron 10:409-
426 (1993);
Kidd et al., Cell 92:205-215 (1998); Kidd et al., Nueron 20:25-33 (1998);
Zallen et al., Cell
92:217-227 (1998)). The phenotype of robo mutants and the predicted molecular
features of
the robo protein suggest the existence of a ligand for robo at the midline.
Thus, although the
2 5 robo protein was known and thought to function as a transmembrane
receptor, the ligand for
the robo protein remained unknown. The slit protein of the present invention
is the first
known ligand for the robo protein.
The olfactory system provides a useful model for studying neuronal migration
and
axon guidance. The olfactory bulb (OB) is a structure relaying olfactory
information from
3 0 the olfactory epithelium to the primary olfactory cortex. The major types
of interneurons in
the OB, including the granule cells and the periglomerular cells, are produced
postnatally
from the anterior part of the subventricular zone (SVZa) of the telencephalon
in rodents.
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Neuronal progenitors thus have to migrate in the rostral migratory stream
(RMS) from the
SVZa to the OB.
The importance of cell migration in normal development and homeostasis extends
beyond the nervous system. One area in which cell migration plays an important
role is in
5 the immune system and especially in the area of cell-mediated immunity. The
term cell
mediated immunity originally described localized immune reactions to organisms
mediated
by leukocytes. The term, however, has also been used in a more general sense
for any
immune response in which antibodies play a subordinate role.
Leukocyte chemotaxis was first described by Leber in 1888 (reviewed in
l0 McCutcheon, Physiol. Rev., 26:319-336 (1946); Hams, Physiol. Rev., 34:529-
562 (1954))
and is one of the best-studied phenomena of cell migration in adult mammals
(Boyden, ,l.
Exp. Med., 115:453-466 (1962); Ramsey, Exp. Cell Res., 70:129-139 (1972);
Zigmond,
Nature, 249:450-452 (1974); Devreotes and Zigmond, Ann. Rev. Cell Biol., 4:649-
686
(1988); Downey, Immunol., 6:113-124 (1994); Sanchez-Madrid and Angel del Pozo,
EMBO
J., 18:501-511 (1999)). Work in the past decade has shown that the chemokines
family are
attractive guidance cues for leukocytes (reviewed in Murphy, Ann. Rev.
Immunol, 12:593-
633 (1994); Springer, Cell, 76:301-314 (1994); Rollins, Blood, 90:909-928
(1997);
Baggiolini et al., Ann. Rev. Immunol., 15:675-705 (1997); Luster, N. Eng. J.
Med., 338:436-
445 (1998); Locati and Murphy, Ann. Rev. Med., 50:425-440 (1999)). The first
chemokine
2 0 was isolated and sequenced in 1977 without knowledge of its biological
activity (Duel et al.,
Proc. Natl. Acad. Sci. USA, 24:2256-2258 (1977), and functional studies of the
chemokines
began in 1987 with the identification of IL-8 (Yoshimura et al., Proc. Natl.
Acad. Sci.,
84:9233-9237 (1987). There are more than 40 chemokines known now and they are
structurally related small proteins with 70 to 100 amino acid residues
(Murphy, Ann. Rev.
Immunol, 12:593-633 (1994); Rollins, Blood, 90:909-928 (1997); Baggiolini et
al., Ann. Rev.
Immunol., 15:675-705 (1997); Luster, N. Eng. J. Med., 338:436-445 (1998);
Locati and
Murphy, Ann. Rev. Med., 50:425-440 (1999)). Although chemokines have been
implicated in
a number of other biological processes, their best-characterized roles are
those in leukocyte
chemotaxis. There are four families of chemokines: the CXC (or oc) family with
the first two
3 0 N terminal cysteines separated by one non-conserved amino acid residue,
the CC (or (3)
family with the first two cysteines unseparated, the C (or y) family with one
N terminal
cysteine, and the CX3C (or b) family with the N terminal cysteines separated
by three
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6
residues. Among the best characterized chemokines are CC chemokine RANTES and
CXC
chemokine, stromal cell derived factor 1 (SDF-1) (Tashiro et al., Science
261:600-603
(1993)). Receptors for RANTES have been detected on monocyte, eosinophil,
basophil, T
lymphocytes, natural killer cells and dendritic cells. Receptors for SDF-1
have been found on
monocyte, T lymphocytes and dendritic cells. Both chemokines are implicated in
a number of
inflammatory diseases.
During inflammation, it is well known that leukocyte chemotaxis induced by
chemokines is essential for extravasation and trafficking to target tissues
(e.g., Mohle et al.,
Blood, 91:4523-4530 (1998); Campbell et al., Nature, 400:776-780 (1999)).
Allergic
inflammation including those in the airways such as asthma affects a large
number of people
(Holtzman et al., "Asthma" in Principles of Molecular Medicine, Jameson, ed.,
Humana
Press, 1998, pp. 319-327). Migration to, and accumulation of, leukocytes in
inflammatory
sites are crucially involved in allergic inflammation (reviewed in Seminario
and Gleich, Curr.
Opin. Immunol., 6a;860-864 (1994); Baggiolini and Dahinder, Immunology Today,
15:127-
133 (1994); Holtzman et al., "Asthma" in Principles ofMolecular Medicine,
Jameson, ed.,
Humana Press, 1998, pp. 319-327)). Expression of chemokines is up-regulated in
allergic
inflammation including asthma (Sousa et al., Am. J. Respir. Cell. Mol. Biol.
10:142-147
(1994); MacLean et al., J. Exp. Med. 184:1461-1469 (1996); Ganzalo et al.,
Immunity, 4:1-14
(1996); Teran et al., J. Immunol., 157:1806-1812 (1996)). Chemokines can
attract leukocytes
2 0 and activate them, causing mast cell degranulation and histamine release
(Dahinder et al., J.
Exp. Med., 179:751-756 (1994); Luster and Rothenberg, J. Leukoc. Biol., 62:620-
633
(1997)). Inhibition of chemokine signaling by function-blocking antibodies or
gene knock-
out is effective against airway inflammation including asthma (Harada et al.,
J. Leukoc. Biol.,
56:559-564 (1994); Teran et al., J. Immunol., 157:1806-1812 (1996); Lukacs et
al., J.
Immunol., 158:4398-4404 (1997); Rothenberg et al., J. Exp. Med., 185:785-790
(1997)).
Increased production of chemokines has been implicated in arthritis (for
example, Koch et al.,
J. Clin. Invest., 90:772-779 (1992)). Inhibition of the chemokines IL-8, MCP-1
or RANTES
has been demonstrated to ameliorate arthritis in rabbit (Harada et al., J.
Leukoc. Biol., 56:559-
564 (1994)), mouse (Gong et al., J. Exp. Med., 186:131-137 (1997)) and rat
models (Barnes
3 0 et al., J. Clin. Invest., 101:2910-2919 (1998)). Chemokines are also
implicated in
glomerulonephritis (Harada et al., J. Leukoc. Biol., 56:559-564 (1994); Brown
et al., J.
Leukoc. Biol., 59:75-80 (1996)), inflammatory responses to viruses (Cook et
al., Science,
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7
269:1583-1585 (1995)), PNS demyelination (Fujika et al., J. Neurovirol., 5:27-
31 (1999);
Glabinski and Ransohoff, J. Neurovirol., 5:3-12 (1999)), and chronic
inflammatory responses
such as those in cystic fibrosis (reviewed in Rosenbluth and Brody, "Cystic
Fibrosis" in
Principles ofMolecular Medicine, Jameson, ed., Humana Press, 1998, pp. 329-
338), or in
Ulcerative colitis and Crohn's diseases (Reinecker, et al., Gastroenterology,
108:40-50
(1995); Grimm et al., J. Gastroenterol. Hepatol., 10:387-395 (1995); Garcia-
Zepeda et al.,
Nat. Med., 2:449-456 (1996)).
In the nervous system, chemokines have been implicated in a number of diseases
including encephalitis, encephalomyelitis, meningitis, CNS ischemia, CNS
reperfusion
injury, CNS trauma, CNS tumor, multiple sclerosis, Alzheimer's diseases and
HIV dementia
(reviewed in Karpus, J. Neurovirol., 5:1-2 (1999); Glabinski and Ransohoff, J.
Neurovirol.,
5:3-12 (1999); Fujika et al., J. Neurovirol., 5:27-31 (1999); Hesselgesser and
Horuk, J.
Neurovirol., 5:13-26 (1999); Xia and Hyman, J. Neurovirol., 5:32-41 (1999);
Mennicken et
al., Trends Pharmacol Sci., 20:73-78 (1999)). Multiple sclerosis is an
autoimmune disease
mediated by leukocyte infiltration. Chemokine expression is up-regulated in
multiple
sclerosis or its animal models (Ransohoff et al., FASEB J., 7:592-600 (1993);
Miyagishi et
al., J. Neurol. Sci., 129:223-227 (1995); Godiska et al., J. Neuroimmunol.,
58:167-176
(1995); Ransohoff et al., Cytokine Growth Factor Rev. 7:35-46 (1996);
Ransohoff, J. Leukoc.
Biol., 62:645-652 (1997)); Hvas et al., Scand. J. Immunol., 46:195-203 (1997);
Glabinski and
2 0 Ransohoff, J. Neurovirol., 5:3-12 (1999); Hesselgesser and Horuk, J.
Neurovirol., 5:13-26
(1999)), and inhibition of chemokine receptors is protective against the
induction of
experimental allergic encephalomyelitis, a mouse model of multiple sclerosis
(Karpus et al.,
J. Immunol., 155:5003-5010 (1995)).
Chemokine receptors are essential for the infection of intracellular pathogens
including HIV-1 (Cocchi et al., Science, 270: 1811-5 (1995); Feng et al.,
Science, 272: 872-7
(1996); Oberlin et al., Nature, 382: 833-5 (1996); Bleul et al., Proc. Natl.
Acad. Sci, 94:
1925-30 (1996, 1997); Dragic et al., Nature, 381: 667-73 (1996); Deng et al.,
Nature, 381:
661-6 (1996); Choe et al., Cell, 85: 1135-48 (1996); Doranz et al., Cell, 85:
1149-58 (1996);
Arenzana-Seisdedos et al., Nature, 383: 400 (1996); Schmidtmayerova et al,
Nature, 382: 767
3 0 (1996); Baggiolini, Dewald and Moser, Annu. Rev. Immunol., 15: 675-705
(1997); Hori et
al., J. Immunol., 160: 180-8 (1998); Luster, N. Engl. J. Med., 338: 436-
45(1998); Locati and
Murphy, Annu. Rev. Med. 50: 425-40 (1999); Horuk, Immunol. Today, 20: 89-94
(1999);
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8
Berger et al., Annu. Rev. Immunol., 17: 657-700 (1999)) and the malaria-
causing protozoan
Plasmodium vivax (Horuk et al., Science, 261: 1182-4 (1993)). Several
chemokine receptors
including CXCR4 and CCRS have been shown to be co-receptors for HIV (Cocchi et
al.,
Science, 270: 1811-5 (1995); Feng et al., Science, 272: 872-7 0996); Oberlin
et al., Nature,
382: 833-5 (1996); Bleul et al., Proc. Natl. Acad. Sci, 94: 1925-30 (1996,
1997); Dragic et al.,
Nature, 381: 667-73 (1996); Deng et al., Nature, 381: 661-6 (1996); Choe et
al., Cell, 85:
1135-48 (1996); Doranz et al., Cell, 85: 1149-58 (1996); Simmons et al.,
Science, 276: 276-9
(1997)) and inhibition of receptor signaling has been shown to block HIV
infection (Alfano et
al., J. Exp. Med., 190: 597-605 (1999); Wang and Oppenheim, J. Exp. Med., 190:
591-5
(1999)).
Although it is clear that cell migration plays an important role in both the
nervous and
immune systems, it is much less clear if common mechanisms controlling and/or
guiding cell
migration exist between cell types. Molecularly, all known neuronal guidance
cues function
through single transmembrane receptors including UNC-5, DCC, Eph, neuropilin,
Robo and
plexin (Leung-Hagesteijn et al., Cell, 71: 289-299 (1992); Cheng and Flanagan,
Cell, 79:
157-168 (1994); Keino-Masu et al., Cell, 87: 175-185 (1996); Chan et al.,
Cell, 87: 187-195
(1996); Leonardo et al., Nature, 386: 833-838 (1997); Ackerman et al., Nature,
386: 838-842
(1997); He and Tessier-Lavigne, Cell 90: 739-751 (1997); Kolodkin et al.,
Cell, 90: 753-762
(1997); Feiner et al., Neuron 19: 539-545 (1997); Chen et al., Neuron 21: 1283-
1290 (1998);
2 0 Giger et al., Neuron, 21: 1079-1092 (1998); Winberg et al., Cell, 95: 903-
916 (1998); Kidd et
al., Cell, 92:205-215 (1998); Kidd, et al., Cell, 96:785-794., (1999); Brose
et al., Cell,
96:795-806 (1999); Li et al., Cell 96: 807-818 (1999); Yuan et al., Dev.
Biol., 212:290-306
(1999); Takahashi et al., Cell 99:59-69 (1999); Tamagnone et al., Cell 99:71-
80 (1999);
Bashaw and Goodman, Cell, 97:917-926 (1999)). In contrast, seven transmembrane
2 5 receptors coupled to G proteins (GPCR) are required for all chemokines and
other
chemotactic factors for leukocytes (Hwang, J. Lipid Mediat. 2:123-58 (1990);
Murphy, Ann.
Rev. Immunol., 12:593-633 (1994); Rollin, Blood, 90:909-928 (1997); Baggiolini
et al., Annu.
Rev. Immunol. 15:675-705 (1997); Luster, N. Engl. J. Med., 338:436-45 (1998);
Locati and
Murphy, Annu. Rev. Med., 50:425-40 (1999)) and Dictyostelium (Devreotes and
Zigmond,
3 0 Ann. Rev. Cell Biol. 4:649-686 (1988); Parent and Devreotes, Ann. Rev.
Biochem., 65:411-
440 (1996); Chen et al., Trends Genet., 12:52-57 (1996); Parent and Devreotes,
Science
CA 02365214 2001-09-13
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9
84:765-70 (1999)). Thus, there is no clear connection between the mechanisms
controlling
migration in the nervous and immune systems.
SUMMARY
Accordingly, the present invention provides a polynucleotide encoding the
sequence
for the vertebrate form of the slit protein as well as recombinant
polynucleotides containing
the nucleic acid sequence for the slit protein, recombinant vectors and
expression cassettes
containing the nucleic acid sequence for the slit protein. Also provided are
the protein
encoded by the polynucleotides of the present invention, host cells that have
been genetically
transformed with the polynucleotides of the present invention, and methods for
producing the
slit protein. Additionally the invention provides for pharmacological
compounds containing
the slit protein whose uses include, but are not limited to, the guidance of
axon projection and
neuronal migration, leukocyte migration, and the study of central nervous
system
development. Also, the present invention provides for the therapeutic use of
slit proteins in
directing axon and dendrite growth and projections during nerve regeneration,
in inhibiting
the migration of malignant cells, in modulating the migration of leukocytes to
inhibit
inflammation and graft rejection, and in inhibiting of infection of cell by
HIV. Additionally
the invention provides for the use of slit for increasing cell proliferation,
the use of slit as a
serum replacement in cell culture medium and cell culture media containing
slit proteins.
This invention also provides for the use of slit in wound-healing and organ
repair such as
2 0 regeneration or repair of lung and kidney.
Accordingly one aspect of the invention is to provide an isolated
polynucleotide
comprising a member selected from the group consisting of (a) a polynucleotide
of SEQ ID
NO: 1, fragments of SEQ ID NO: 1, or the complements thereof; (b)a
polynucleotide that has
at least 90% sequence identity with the polynucleotide of (a); (c) a
polynucleotide that
2 5 hybridizes to the polynucleotide of (a) under conditions of SX SSC, 50%
formamide and
42°C, and which encodes a protein having the same biological function;
(d) a polynucleotide
encoding the same amino acid sequence as (a), but which exhibits regular
degeneracy in
accordance with the degeneracy of the genetic code; (e) a polynucleotide
encoding the same
amino acid sequence as (b), but which exhibits regular degeneracy in
accordance with the
3 0 degeneracy of the genetic code; and (f) a polynucleotide encoding the same
amino acid
CA 02365214 2001-09-13
WO 00/55321 PCT/US00/07040
sequence as (c), but which exhibits regular degeneracy in accordance with the
degeneracy of
the genetic code.
Another aspect of the present invention is to provide a recombinant
polynucleotide
comprising a member selected from the group consisting of (a) a polynucleotide
of SEQ ID
5 NO: 1, fragments of SEQ ID NO: l, or the complements thereof; (b)a
polynucleotide that has
at least 90% sequence identity with the polynucleotide of (a); (c) a
polynucleotide that
hybridizes to the polynucleotide of (a) under conditions of 5X SSC, 50%
formamide and
42°C, and which encodes a protein having the same biological function;
(d) a polynucleotide
encoding the same amino acid sequence as (a), but which exhibits regular
degeneracy in
10 accordance with the degeneracy of the genetic code; (e) a polynucleotide
encoding the same
amino acid sequence as (b), but which exhibits regular degeneracy in
accordance with the
degeneracy of the genetic code; and (f) a polynucleotide encoding the same
amino acid
sequence as (c), but which exhibits regular degeneracy in accordance with the
degeneracy of
the genetic code.
In another aspect of the present invention is provided a recombinant vector
comprising a member selected from the group consisting of (a) a polynucleotide
of SEQ ID
NO: 1, fragments of SEQ ID NO: l, or the complement thereof; (b)a
polynucleotide that has
at least 90% sequence identity with the polynucleotide of (a); (c) a
polynucleotide that
hybridizes to the polynucleotide of (a) under conditions of 5X SSC, 50%
formamide and
2 0 42°C, and which encodes a protein having the same biological
function; (d) a polynucleotide
encoding the same amino acid sequence as (a), but which exhibits regular
degeneracy in
accordance with the degeneracy of the genetic code; (e) a polynucleotide
encoding the same
amino acid sequence as (b), but which exhibits regular degeneracy in
accordance with the
degeneracy of the genetic code; and (f) a polynucleotide encoding the same
amino acid
2 5 sequence as (c), but which exhibits regular degeneracy in accordance with
the degeneracy of
the genetic code.
A further aspect of the invention is to provide host cells comprising the
recombinant
vector described above.
Yet another aspect is a protein or polypeptide encoded by a polynucleotide
selected
3 0 from the group comprising a member selected from the group consisting of
(a) a
polynucleotide of SEQ ID NO: l; fragments of SEQ ID NO: l, or the complements
thereof;
(b)a polynucleotide that has at least 90% sequence identity with the
polynucleotide of (a); (c)
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11
a polynucleotide that hybridizes to the polynucleotide of (a) under conditions
of SX SSC,
50% formamide and 42°C, and which encodes a protein having the same
biological function;
(d) a polynucleotide encoding the same amino acid sequence as (a), but which
exhibits
regular degeneracy in accordance with the degeneracy of the genetic code; (e)
a
polynucleotide encoding the same amino acid sequence as (b), but which
exhibits regular
degeneracy in accordance with the degeneracy of the genetic code; and (f) a
polynucleotide
encoding the same amino acid sequence as (c), but which exhibits regular
degeneracy in
accordance with the degeneracy of the genetic code.
Still another aspect of the invention is a protein comprising the amino acid
sequence
of SEQ ID NO: 2 or a fragment of said protein.
Another aspect provides a method for the production of isolated slit protein
comprising growing transformed host cells of the present invention under
conditions where
the cells express slit protein and then isolating the expressed protein or its
fragments.
In a further aspect is provided, a pharmaceutical composition comprising the
protein
or protein fragment of the present invention or a pharmaceutically acceptable
salt thereof, and
a pharmaceutically acceptable Garner, diluent, or excipient.
In an additional aspect is provided a method for altering cell migration
comprising
administering a migration-altering amount of a slit protein or a
pharmaceutically acceptable
salt thereof.
2 0 Yet another aspect provides a method for guiding cell migration comprising
administering a cell migration guiding amount of slit protein or a
pharmaceutically acceptable
salt thereof.
Yet a further aspect provides a method for guiding nerve axons or dendrites
comprising administering a axon- or dendrite-guiding amount of a slit protein
or a slit protein
2 5 derivative.
Another aspect provides a method for treating graft rejection comprising
administering a leukocyte migration inhibiting amount of a slit protein or a
slit protein
derivative.
A further aspect provides a method for increasing cell proliferation by
transforming
3 0 the cells with a vector containing the polynucleotide of the present
invention.
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Additional aspects include a method for culturing cells comprising the
addition of a
slit protein or a slit protein derivative to the culture medium as well as a
culture medium
comprising a slit protein or a slit protein derivative.
Still another aspect is a composition comprising the slit protein of the
present
invention.
Also provided is an expression cassette comprising the polynucleotide of the
present
invention.
Additionally is provided a method for inhibiting the infection of cells by the
HIV
virus comprising administering an infection inhibiting amount of slit protein
or a slit protein
derivative.
Further provided is a method for aiding wound repair, organ repair or organ
regeneration comprising administering a cell proliferating amount of a slit
protein or a slit
protein derivative.
In addition, is provided a method for treating inflammation comprising
administering
a leukocyte migration inhibiting amount of a slit protein.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features, aspects, and advantages of the present invention
will
become better understood with regard to the following description, appended
claims and
accompanying figures where:
2 0 Figure 1 shows nucleotide sequence of the slit protein of the present
invention (SEQ
ID NO: 1 ).
Figure 2 shows the amino acid sequence deduced from the nucleotide sequence of
the
present invention (SEQ ID NO: 2).
Figure 3 shows the cDNA sequence of human slit-2 (SEQ ID N0:3) as contained in
2 5 GenBank (accession number AF055585).
Figure 4 shows the repulsive effect of Slit on neurons migrating from SVZa
explants.
a-c. Distribution of chains of cells from the SVZa explants, co-cultured with
an aggregate of
control HEK cells (a), with cells expressing xSlit (b) or mSlit-1 (c) in the
matrigel for one (a,
b) or two days (c). d-f. Distribution of cells migrating out of the SVZa
explants after being
3 0 co-cultured with control HEK cells (d), with cells expressing xSlit (e) or
mSlit-1 (f) in the
collagen gel.
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Figure 5 shows the effect of slit on neuronal migration in the RMS. DiI
crystals were
placed into SVZa in sagittal sections of postnatal rat brains to label cells
(red in b, c, a and f)
migrating into the RMS. Control or Slit HEK cells were labeled with Di0 (green
in a, c, d
and f). a-c are different views of the same slice in which an aggregate of
control HEK cells
were placed on top of the RMS. d-f are different views of the same slice in
which an
aggregate of Slit cells were placed on top of the RMS. In all panels, the
upper right corner is
towards the SVZa, the origin of the RMS, whereas the lower left corner is
towards to the OB,
the end of the RMS.
Figure 6 shows the effect of Slit on Cells Migrating from LGE Explants. (A)
Symmetric distribution of cells around the LGE explant cocultured with an
aggregate of cells
transfected with the vector (n= 87). (B) Symmetric distribution of migrating
LGE cells when
cocultured with an aggregate of human Sema III-expressing cells (n= 25). (C)
Asymmetric
distribution of migrating LGE cells when cocultured with an aggregate of
mSlitl-expressing
cells (n = 74). (D) Asymmetric distribution of migrating LGE cells when
cocultured with an
aggregate of xSlit-expressing cells (n = 89). (E) Symmetric migration of
endothelial cells
from an aorta explant in the presence of control HEK cells. (F) Symmetric
migration of
endothelial cells from an aorta explant in the presence of HEK cells
expressing xSlit. (G). A
diagram of regions in the rat telencephalon, showing the subventricular zone
in the LGE from
which explants were isolated and cultured, the ventricular zone, and the
mantle layer of LGE.
2 0 Figure 7 shows the effect of slit on migration of cells from the LGE to
the Neocortex.
Results from E16.5 slices are shown here. Similar results have been obtained
with E15.5 and
E17.5 slices. (A-D) Different views of the same explant on which a strip of
Slit cells (green)
has been placed on the junction between the neocortex and the LGE. (E-H)
Different views
of the same explant on which a strip of control cells (green) has been placed
on the junction
2 5 between the neocortex and the LGE. (A) and (E) are Hoechst dye staining to
show outlines
of the brain slices. (B) and (F) show Di0-labeled aggregates of Slit (B) or
control (F) cells;
the green bands are the aggregates. (C) and (G) show DiI-labeled LGE neurons;
note that, in
the neocortex, there were migrating cells in (G) but not in (C). (D) is the
superimposition of
(A), (B), and (C), whereas (H) is the superimposition of (E), (F), and (G);
note the
3 0 relationship of HEK cells (green), the LGE (red) and migrating neurons in
the neocortex
(red), and the outlines of the slices (blue).
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Figure 8 shows the inhibition of RANTES-induced lymph node cell migration by
slit
protein.
Figure 9 shows the inhibition of SDF-1-induced lymph node cell migration by
slit
protein.
Figure 10 shows cell migration in control or robo-overexpressing HEK cells
that were
transfected with CCRS.
Figure 11 shows cell migration in control or robo-overexpressing HEK cells
that were
transfected with CXCR4.
Figure 12 shows the effect of recombinant human slit 2 protein on inhibiting
1 o RANTES-induced lymph-node migration.
Figure 13 shows the effect of recombinant human slit-2 protein on inhibition
of SDF-
1 induced lymph-node cell migration
ABBREVIATIONS AND DEFINITIONS
AP = alkaline phosphatase
HA hemagglutinin
=
PEG = polyethylene
glycol
FCS = fetal calf
serum
HEK = human embryonic kidney
HIV = human immunodeficiency virus
2 0 SVZa = subventricular zone
RMS = rostral migratory stream
OB = olfactory bulb
LGE = lateral ganglionic eminence
RANTES = regulated on activation, normal T cell expressed and secreted protein
2 5 SDF-1 = stromal derived factor 1
DMEM = Dulbecco's modified Eagle's medium
PBS = phosphate buffered saline
PCR = polymerise chain reaction
As used herein in reference to the slit protein of the present invention,
"biological
3 0 function" means the ability of a slit protein to guide cell migration as
in Example 10; or the
ability to repulse axon projection as in Example 8; or the ability of a slit
protein to
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specifically bind to the robo protein as characterized in Example 7; or by the
ability of a slit
protein to inhibit cytokine induced migration of leukocytes as in Example 11
or the ability to
affect cell proliferation as in Example 12.
As used herein, the terms "complementary" or "complementarity" refer to the
pairing
5 of bases, purines and pyrimidines, that associate through hydrogen bonding
in double
stranded nucleic acid. The following base pairs are complementary: guanine and
cytosine;
adenine and thymine; and adenine and uracil. The terms as used herein the
terms include
complete and partial complementarity.
As used herein, the term "hybridization" refers to a process in which a strand
of
10 nucleic acid joins with a complementary strand through base pairing. The
conditions
employed in the hybridization of two non-identical, but very similar,
complementary nucleic
acids varies with the degree of complementarity of the two strands and the
length of the
strands. Thus the term contemplates partial as well as complete hybridization.
Such
techniques and conditions are well known to practitioners in this field.
15 As used herein, the term "amino acid" is used in its broadest sense, and
includes
naturally occurring amino acids as well as non-naturally occurnng amino acids,
including
amino acid analogs and derivatives. The latter includes molecules containing
an amino acid
moiety. One skilled in the art will recognize, in view of this broad
definition, that reference
herein to an amino acid includes, for example, naturally occurring proteogenic
L-amino acids;
2 0 D-amino acids; chemically modified amino acids such as amino acid analogs
and derivatives;
naturally occurring non-proteogenic amino acids such as norleucine, ~3-
alanine, ornithine,
etc.; and chemically synthesized compounds having properties known in the art
to be
characteristic of amino acids.
As used herein, the term "proteogenic" indicates that the amino acid can be
2 5 incorporated into a peptide, polypeptide, or protein in a cell through a
metabolic pathway.
As used herein, the term "pharmaceutically acceptable salts" embraces salts
commonly used to form alkali metal salts and addition salts of free acids or
free bases. The
nature of the salt is not critical, provided that it is pharmaceutically
acceptable.
As used herein, "expression cassette" means a genetic module comprising a gene
and
3 0 the regulatory regions necessary for its expression, which may be
incorporated into a vector.
As used herein, "secretion sequence" or "signal peptide" or signal sequence"
means a
sequence that directs newly synthesized secretory or membrane proteins to and
through
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16
membranes of the endoplasmic reticulum, or from the cytoplasm to the periplasm
across the
inner membrane of bacteria, or from the matrix of mitochondria into the inner
space, or from
the stroma of chloroplasts into the thylakoid. Fusion of such a sequence to a
gene that is to
be expressed in a heterologous host ensures secretion of the recombinant
protein from the
host cell.
As used herein, a "recombinant nucleic acid" is defined either by its method
of
production or its structure. In reference to its method of production, e.g., a
product made by a
process, the process is use of recombinant nucleic acid techniques, e.g.,
involving human
intervention in the nucleotide sequence, typically selection or production.
Alternatively, it can
be a nucleic acid made by generating a sequence comprising fusion of two
fragments which
are not naturally contiguous to each other, but is meant to exclude products
of nature, e.g.,
naturally occurring mutants. Thus, for example, products made by transforming
cells with
any unnaturally occurnng vector is encompassed, as are nucleic acids
comprising sequences
derived using any synthetic oligonucleotide process. Such is often done to
replace a codon
with a redundant codon encoding the same or a conservative amino acid, while
typically
introducing or removing a sequence recognition site. Alternatively, it is
performed to join
together nucleic acid segments of desired functions to generate a single
genetic entity
comprising a desired combination of functions not found in the commonly
available natural
forms. Restriction enzyme recognition sites are often the target of such
artificial
2 0 manipulations, but other site specific targets, e.g., promoters, DNA
replication sites,
regulation sequences, control sequences, or other useful features may be
incorporated by
design.
As used herein, "polynucleotide" and "oligonucleotide" are used
interchangeably and
mean a polymer of at least 2 nucleotides joined together by phosphodiester
bonds and may
2 5 consist of either ribonucleotides or deoxyribonucleotides.
As used herein, "sequence" means the linear order in which monomers occur in a
polymer, for example, the order of amino acids in a polypeptide or the order
of nucleotides in
a polynucleotide.
As used herein, "peptide" and "protein" are used interchangeably and mean a
3 0 compound that consists of two or more amino acids that are linked by means
of peptide
bonds.
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As used herein "recombinant protein" means that the protein, whether
comprising a
native or mutant primary amino acid sequence, is obtained by expression of a
gene carried by
a recombinant DNA molecule in a cell other than the cell in which that gene
and/or protein is
naturally found. In other words, the gene is heterologous to the host in which
it is expressed.
It should be noted that any alteration of a gene, including the addition of a
polynucleotide
encoding an affinity purification moiety to the gene, makes that gene
unnatural for the
purposes of this definition, and thus that gene cannot be "naturally' found in
any cell.
As used herein, "cytokines" means a group of substances from by an animal in
response to infection. They are similar to hormones in their function in that
they are
produced in one cell and stimulate a response in another cell or stimulate in
response in the
cell in which they are produced in an autocrine fashion.
As used herein, the term "chemokines" refers to a large family of small
structurally
related 8- to 10- kDa cytokines that have two or more conserved cysteines
forming disulfied
bonds.
As used herein, the term "animal" includes human beings.
As used herein, "targeting sequence" means in the context of gene or
polynucleotide
insertion, a sequence which results in the gene or polynucleotide being
inserted at a particular
location by homologous recombination. In the context of proteins or peptides,
"targeting
sequence" refers to a nucleotide sequence encoding an amino acid sequence the
presence of
2 0 which results in a protein being directed to a particular destination
within a cell.
DETAILED DESCRIPTION
The following detailed description is provided to aid those skilled in the art
in
practicing the present invention. Even so, this detailed description should
not be construed to
unduly limit the present invention as modifications and variation in the
embodiments
2 5 discussed herein can be made by those of ordinary skill in the art without
departing from the
spirit or scope of the present inventive discovery.
All publications, patents, patent applications and other references cited in
this
application are herein incorporated by reference in their entirety as if each
individual
publication, patent, patent application or other reference were specifically
and individually
3 0 indicated to be incorporated by reference.
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A polynucleotide encoding the slit protein of the frog genus Xenopus has been
isolated from a Xenopus embryonic cDNA library. The sequence of the
polynucleotide has
been determined and is given in SEQ ID NO: 1. The cDNA sequence consists of
5513 bases
and exhibits 59% sequence identity with the marine slit-2 sequence. Although a
particular
embodiment of the nucleotide sequence disclosed herein is given in SEQ ID NO:
1, it should
be understood that other biologically functional equivalent forms of the
nucleic acid sequence
of the present invention can be readily isolated using conventional DNA-DNA
and DNA-
RNA hybridization techniques. Thus the present invention also includes
nucleotide
sequences that hybridize to SEQ ID NO: 1 or its complement under moderate to
high
stringency conditions and encode proteins exhibiting the same or similar
biological activity as
that of protein of SEQ ID NO: 2 disclosed herein. Also included in the
invention are
polynucleotides that exhibit 90%, preferrably 92%, more preferrably 95% ,and
more 98%
sequence identity with SEQ ID NO: 1, its complement or SEQ ID NO: 2. Such
nucleotide
sequences preferably hybridize to the nucleic acid of SEQ ID NO: 1 or its
complement under
high stringency conditions. Exemplary conditions include initial hybridzation
in SX SSPE,l-
SX Denhardt's solution, 10-200 ~g/ml denatured heterologous DNA, 0.5% SDS, at
50- 68°C
for a time sufficient to permit hybridization, e.g. several hours to
overnight, followed two
washes in 2X SSPE, 0.1% SDS at room temperature and two additional 15 minute
washes in
O.1X SSPE, 0.1% SDS at 42°C followed by detection of the hybridization
products. Higher
2 0 stringency washing can accomplished by at least one additional wash in 0.1
% S SPE, 0.1
SDS at 55°C, more preferably at 60°C and more preferably still
at 65°C. High stringency
hybridizations can also be earned out in SX SSPE and 50% formamide at
42°C followed by
washing as described above (Meinkoth and Wahl, Anal. Biochern, 138:267-284
(1984)). As
is well known by those of ordinary skill in the art, SSC can be substituted
for SSPE in the
2 5 above examples so that, for instance, hybridization can be accomplished in
SX SSC in place
of SX SSPE.
It is well known to those of ordinary skill in the art that different
compositions can
result in equal stringency conditions for hybridization depending on well
known factors such
as the concentration of Na+, the % formamide, the temperature, the T", of the
hybrid to be
3 0 formed, and the composition of the hybrid, e.g. DNA-DNA, DNA-RNA, or RNA-
RNA.
Thus the invention also encompasses nucleotide sequence that hybridize under
conditions
equivalent to those described above.
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The amino acid sequence of the protein encoded by the isolated polynucleotide
sequence has been deduced and is given in Figure 2. The protein exhibits 87%
amino acid
identity with the mouse slit-2 protein 85% identity with the human slit-2
protein, 63%
identity with the mouse slit-1 and mouse slit-3 proteins, 83% identity with a
partial chicken
slit protein, 67% with the rat slit protein rMEGFS, 63% with the rat slit
protein rMEGF4
(Nakayama et al., Genomics 51:27-34 (1998)), and 40% identity with the
Drosophila slit
protein. At the amino (N) terminus, there is a putative signal peptide
characteristic of
secreted proteins. There are four leucine rich repeats (LRR), each surrounded
by an N
terminal and a carboxyl (C) terminal flanking region. The leucine rich repeats
are followed
by nine epidermal growth factor (EGF) repeats. Near the C terminus, there is a
laminin G
domain with similarities to agrin, laminin, and perlecan (also known as the
ALPS domain),
followed by a cysteine rich carboxyl terminal region.
The slit protein encoded by the polynucleotide sequence of the present
invention has
been shown to bind to the transmembrane robo protein with a binding affinity
comparable to
that observed in other receptor-ligand interactions. The protein has also been
shown to be
capable of guiding nerve axon projection and neuron migration by repulsion. In
addition, the
slit protein encoded by SEQ ID NO: 1 has been shown to inhibit the cytokine-
induced
migration of leukocytes. The ability to inhibit cytokine induced migration of
lymphocytes
makes the slit protein of the present invention useful in treatment of
inflammation caused by
2 0 the invasion of leukocytes. Therefore, the present invention encompasses
methods and
compositions for the treatment of inflammation resulting from the infiltration
of leukocytes.
Leukocytes also play a major role in delayed graft rejection, especially in
xenograft rejection
(French et al., Reprod. Fertil. Dev., 10:683-696 (1998). Thus, the slit
protein of the present
invention can be used to prevent graft rejection by preventing the migration
of leukocytes
2 5 into the graft. The inhibition or prevention of graft rejection can be
accomplished by the
local or systemic administration of the slit protein. Alternatively, when
cells are transplanted,
either alone or in conjunction with a transplanted organ or tissue, the cells
can be transformed
so as to secrete the slit protein of the present invention and so prevent
damage to the
transplant related to leukocyte invasion.
3 0 The ability of the slit protein of the present invention to influence cell
movement in
systems as diverse as the neural and immune systems demonstrates the
widespread
applicability of the present invention. Thus, the protein encoded by
nucleotide sequences of
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the present invention can be used to affect the movement or migration of any
cell type
expressing the robo protein or any other functional receptor for the slit
protein of the present
invention.
Based on the Drosophila slit sequence, degenerate primers were designed to
amplify
5 possible vertebrate slit homologs by the polymerase chain reaction (PCR).
One pair of
primers allowed isolation of slit homologs from Xenopus and chick embryos. A
probe made
from the PCR fragment of Xenopus slit was used to screen a Xenopus embryonic
cDNA
library, and cDNA clones encoding a full-length Xenopus slit protein were
isolated. By low
stringency hybridization, cDNAs for slit genes were isolated from the mouse.
Sequence
10 comparison showed that the Xenopus slit gene is an ortholog of the slit-2
genes of mice
(mslit-2) and humans (hslit-2).
The predicted full-length Xenopus, mouse and human slit proteins were found to
share features of the Drosophila slit protein. At the amino (N) terminus,
there is a putative
signal peptide characteristic of secreted proteins. There are four leucine
rich repeats (LRR),
15 each surrounded by an N terminal and a carboxyl (C) terminal flanking
region. In Xenopus
slit as well as human and mouse slit-2, there are nine epidermal growth factor
(EGF) repeats,
whereas there are seven EGF repeats in Drosophila slit (Rothberg et al., Genes
Dev. 4:2169-
2187 (1990), Rothberg and Artavanis-Tsakonas, J. Mol. Biol. 227:367-370
(1992)). Near the
C terminus, there is a laminin G domain with similarities to agrin, laminin,
and perlecan (also
2 0 known as the ALPS domain), followed by a cysteine rich carboxyl terminal
region (Rothberg
et al., Genes Dev. 4:2169-2187 (1990); Rothberg and Artavanis-Tsakonas, J.
Mol. Biol.
227:367-370 (1992)).
The present invention also involves recombinant polynucleotides comprising the
isolated sequence for the vertebrate slit protein along with other sequences.
Such
2 5 recombinant polynucleotides are commonly used as cloning or expression
vectors although
other uses are possible. A recombinant polynucleotide is one in which
polynucleotide
sequences of different organisms have been joined together to form a single
unit. A cloning
vector is a self replicating DNA molecule that serves to transfer a DNA
segment into a host
cell. The three most common types of cloning vectors are bacterial plasmids,
phages, and
3 0 other viruses. An expression vector is a cloning vector designed so that a
coding sequence
inserted at a particular site will be transcribed and translated into a
protein.
Both cloning and expression vectors contain nucleotide sequences that allow
the
vectors to replicate in one or more suitable host cells. In cloning vectors,
this sequence is
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21
generally one that enables the vector to replicate independently of the host
cell chromosomes,
and also includes either origins of replication or autonomously replicating
sequences.
Various bacterial and viral origins of replication are well known to those
skilled in the art and
include, but are not limited to the pBR322 plasmid origin, the 2~ plasmid
origin, and the
SV40, polyoma, adenovirus, VSV and BPV viral origins.
The polynucleotide sequence of the present invention may be used to produce
proteins
by the use of recombinant expression vectors containing the sequence. Suitable
expression
vectors include chromosomal, non-chromosomal and synthetic DNA sequences, for
example,
SV 40 derivatives; bacterial plasmids; phage DNA; baculovirus; yeast plasmids;
vectors
derived from combinations of plasmids and phage DNA; and viral DNA such as
vaccinia,
adenovirus, fowl pox virus, retroviruses, and pseudorabies virus. In addition,
any other
vector that is replicable and viable in the host may be used.
The nucleotide sequence of interest may be inserted into the vector by a
variety of
methods. In the most common method the sequence is inserted into an
appropriate restriction
endonuclease sites) using procedures commonly known to those skilled in the
art and
detailed in, for example, Sambrook et al., Molecular Cloning, A Laboratory
Manual, 2nd ed.,
Cold Spring Harbor Laboratory Press, (1989) and Ausubel et al., Short
Protocols in
Molecular Biology, 3rd ed., John Wiley & Sons (1995).
In an expression vector, the sequence of interest is operably linked to a
suitable
2 0 expression control sequence or promoter recognized by the host cell to
direct mRNA
synthesis. Promoters are untranslated sequences located generally 100 to 1000
base pairs
(bp) upstream from the start codon of a structural gene that regulate the
transcription and
translation of nucleic acid sequences under their control. Promoters are
generally classified
as either inducible or constitutive. Inducible promoters are promoters that
initiate increased
2 5 levels of transcription from DNA under their control in response to some
change in the
environment, e.g. the presence or absence of a nutrient or a change in
temperature.
Constitutive promoters, in contrast, maintain a relatively constant level of
transcription. In
addition, useful promoters can also confer appropriate cellular and temporal
specificity. Such
promoters include those that are developmentally-regulated or organelle-,
tissue- or cell-
3 o specific.
A nucleic acid sequence is operably linked when it is placed into a functional
relationship with another nucleic acid sequence. For example, DNA for a
presequence or
secretory leader is operatively linked to DNA for a polypeptide if it is
expressed as a
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22
preprotein which participates in the secretion of the polypeptide;_a promoter
is operably
linked to a coding sequence if it affects the transcription of the sequence;
or a ribosome
binding site is operably linked to a coding sequence if it is positioned so as
to facilitate
translation. Generally, operably linked sequences are contiguous and, in the
case of a
secretory leader, contiguous and in reading frame. Linking is achieved by
blunt end ligation
or ligation at restriction enzyme sites. If suitable restriction sites are not
available, then
synthetic oligonucleotide adapters or linkers can be used as is known to those
skilled in the
art (Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold
Spring Harbor
Laboratory Press, (1989) and Ausubel et al., Short Protocols in Molecular
Biology, 3rd ed.,
John Wiley & Sons (1995)).
Common promoters used in expression vectors include, but are not limited to,
CMV
promoter, LTR or SV40 promoter, the E. coli lac or trp promoters, and the
phage lambda PL
promoter. Other promoters known to control the expression of genes in
prokaryotic or
eukaryotic cells can be used and are known to those skilled in the art.
Expression vectors
may also contain a ribosome binding site for translation initiation, and a
transcription
terminator. The vector may also contain sequences useful for the amplification
of gene
expression.
Expression and cloning vectors can and usually do contain a selection gene or
selection marker. Typically, this gene encodes a protein necessary for the
survival or growth
2 0 of the host cell transformed with the vector. Examples of suitable markers
include
dihydrofolate reductase (DHFR) or neomycin or hygromycin B resistance for
eukaryotic cells
and tetracycline, ampicillin, or kanamycin resistance for E. coli.
In addition, expression vectors can also contain marker sequences operatively
linked
to a nucleotide sequence for a protein that encode an additional protein used
as a marker. The
2 5 result is a hybrid or fusion protein comprising two linked and different
proteins. The marker
protein can provide, for example, an immunological or enzymatic marker for the
recombinant
protein produced by the expression vector. In a preferred embodiment of the
present
invention, alkaline phosphatase (AP), green fluorescence protein (GFP), myc,
histidine tag
(His) and hemagglutinin (HA) are used as markers.
3 0 Additionally, the end of the polynucleotide can be modified by the
addition of a
sequence encoding an amino acid sequence useful for purification of the
protein produced by
affinity chromatography. Various methods have been devised for the addition of
such affinity
purification moieties to proteins. Representative examples can be found in
U.S. Patent Nos.
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23
4,703,004, 4,782,137, 4,845,341, 5,935,824, and 5,594,115. Any method known in
the art for
the addition of nucleotide sequences encoding purification moieties can be
used for example
those contained in Innis et al., PCR Protocols, Academic Press (1990) and
Sambrook et al.,
Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press (1989).
More particularly, the present invention includes recombinant constructs
comprising
the isolated polynucleotide sequence of the present invention. The constructs
can include a
vector, such as a plasmid or viral vector, into which the sequence of the
present invention has
been inserted, either in the forward or reverse orientation. The recombinant
construct further
comprises regulatory sequences, including for example, a promoter operatively
linked to the
sequence. Large numbers of suitable vectors and promoters are known to those
skilled in the
art and are commercially available. In one preferred embodiment, the pCS2+,
the pCEP4
(Invitrogen) and the pIRESneo (Clontech) vectors are used. It will be
understood by those
skilled in the art, however, that other plasmids or vectors may be used as
long as they are
replicable and viable or expressing the encoded protein in the host.
The polynucleotide sequences of the present invention can also be part of an
expression cassette that at a minimum comprises, operably linked in the 5' to
3' direction, a
promoter, a polynucleotide of the present invention, and a transcriptional
termination signal
sequence functional in a host cell. The promoter can be of any of the types
discussed herein,
for example, a tissue specific promoter, a developmentally regulated promoter,
an organelle
2 0 specific promoter, etc. The expression cassette can further comprise an
operably linked
targeting sequence, transit or secretion peptide coding region capable of
directing transport
of the protein produced. The expression cassette can also further comprise a
nucleotide
sequence encoding a selectable marker and a purification moiety.
A further embodiment of the present invention relates to transformed host
cells
2 5 containing the constructs comprising the polynucleotide sequence of the
present invention.
The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a
lower eukaryotic
cell such as an insect cell or a yeast cell, or the host can be a prokaryotic
cell such as a
bacterial cell. Introduction of the construct into the host cell can be
accomplished by a
variety of methods including calcium phosphate transfection, DEAF-dextran
mediated
3 0 transfection, Polybrene mediated transfection, protoplast fusion, liposome
mediated
transfection, direct microinjection into the nuclei, biolistic (gene gun)
devices, scrape loading,
and electroporation.
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24
The present invention also relates to proteins encoded by the isolated
polynucleotide.
As used herein the term protein includes fragments, analogs and derivatives of
the slit
protein. The terms "fragment," "derivative" and "analog" as used herein mean a
polypeptide
that retains essentially the same biological function or activity as the slit
protein encoded by
the sequence of the present invention. For example, an analog includes a
proprotein which
can be cleaved to produce an active mature protein. The protein of the present
invention can
be a natural protein, a recombinant protein or a synthetic protein or a
polypeptide.
Those of ordinary skill in the art are aware that modifications in the amino
acid
sequence of a peptide, polypeptide, or protein can result in equivalent, or
possibly improved,
second generation peptides, etc., that display equivalent or superior
functional characteristics
when compared to the original amino acid sequence. The present invention
accordingly
encompasses such modified amino acid sequences. Alterations can include amino
acid
insertions, deletions, substitutions, truncations, fusions, shuffling of
subunit sequences, and
the like, provided that the peptide sequences produced by such modifications
have
substantially the same functional properties as the naturally occurring
counterpart sequences
disclosed herein. Biological activity or function can be determined by, for
example, the
ability of the protein to guide cell migration (Example 10), or by the ability
to repulse axon
projection (Example 8); by the ability of the protein to specifically bind to
the robo protein
(Example 7) or by the ability to alter cytokine induced migration of
leukocytes (Example 11).
2 0 One factor that can be considered in making such changes is the
hydropathic index of
amino acids. The importance of the hydropathic amino acid index in conferring
interactive
biological function on a protein has been discussed by Kyte and Doolittle ( J.
Mol. Biol., 157:
105-132, 1982). It is accepted that the relative hydropathic character of
amino acids
contributes to the secondary structure of the resultant protein. This, in
turn, affects the
2 5 interaction of the protein with molecules such as enzymes, substrates,
receptors, DNA,
antibodies, antigens, etc.
Based on its hydrophobicity and charge characteristics, each amino acid has
been
assigned a hydropathic index as follows: isoleucine (+4.5); valine (+4.2);
leucine (+3.8);
phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine
(+1.8); glycine (-
3 0 0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3);
proline (-1.6); histidine
(-3.2); glutamate/glutamine/aspartate/asparagine (-3.5); lysine (-3.9); and
arginine (-4.5).
As is known in the art, certain amino acids in a peptide or protein can be
substituted
for other amino acids having a similar hydropathic index or score and produce
a resultant
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peptide or protein having similar biological activity, i.e., which still
retains biological
functionality. In making such changes, it is preferable that amino acids
having hydropathic
indices within ~2 are substituted for one another. More preferred
substitutions are those
wherein the amino acids have hydropathic indices within tl. Most preferred
substitutions are
5 those wherein the amino acids have hydropathic indices within X0.5.
Like amino acids can also be substituted on the basis of hydrophilicity. U.S.
Patent
No. 4,554,101 discloses that the greatest local average hydrophilicity of a
protein, as
governed by the hydrophilicity of its adjacent amino acids, correlates with a
biological
property of the protein. The following hydrophilicity values have been
assigned to amino
10 acids: arginine/lysine (+3.0); aspartate/glutamate (+3.0 ~1); serine
(+0.3);
asparagine/glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ~ 1
); alanine/histidine
(-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine/isoleucine
(-1.8); tyrosine (-
2.3); phenylalanine (-2.5); and tryptophan (-3.4). Thus, one amino acid in a
peptide,
polypeptide, or protein can be substituted by another amino acid having a
similar
15 hydrophilicity score and still produce a resultant protein having similar
biological activity,
i.e., still retaining correct biological function. In making such changes,
amino acids having
hydropathic indices within ~2 are preferably substituted for one another,
those within ~1 are
more preferred, and those within X0.5 are most preferred.
As outlined above, amino acid substitutions in the peptides of the present
invention
2 0 can be based on the relative similarity of the amino acid side-chain
substituents, for example,
their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary
substitutions that take
various of the foregoing characteristics into consideration in order to
produce conservative
amino acid changes resulting in silent changes within the present peptides,
etc., can be
selected from other members of the class to which the naturally occurring
amino acid
2 5 belongs. Amino acids can be divided into the following four groups: (1)
acidic amino acids;
(2) basic amino acids; (3) neutral polar amino acids; and (4) neutral non-
polar amino acids.
Representative amino acids within these various groups include, but are not
limited to: (1)
acidic (negatively charged) amino acids such as aspartic acid and glutamic
acid; (2) basic
(positively charged) amino acids such as arginine, histidine, and lysine; (3)
neutral polar
3 0 amino acids such as glycine, serine, threonine, cysteine, cystine,
tyrosine, asparagine, and
glutamine; and (4) neutral non-polar amino acids such as alanine, leucine,
isoleucine, valine,
proline, phenylalanine, tryptophan, and methionine. It should be noted that
changes which
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26
are not expected to be advantageous can also be useful if these result in the
production of
functional sequences.
The fragment, derivative or analog of the proteins encoded by the
polynucleotide
sequence of the present invention may be, for example and without limitation,
(i) one in
which one or more amino acid residues are substituted with a conserved or non-
conserved
amino acid residue, and such substituted amino acid residue may or may not be
one encoded
by the genetic code; (ii) one in which one or more of the amino acid residues
includes a
substituent group; (iii) one in which the mature protein is fused to another
compound such as
a compound to increase the half life of the protein; (iv) one in which
additional amino acids
are fused to the protein to aid in purification or in detection and
identification; or (v) one in
which additional amino acid residues are fused to the protein to aid in
modifying tissue
distribution or localization of the protein to certain locations such as the
cell membrane or
extracellular compartments.
The term protein also includes forms of the slit protein to which one or more
substituent groups have been added. A substituent is an atom or group of atoms
that is
introduced into a molecule by replacement of another atom or group of atoms.
Such groups
include, but are not limited to lipids, phosphate groups, sugars and
carbohydrates. Thus, the
term protein includes, for example, lipoproteins, glycoproteins,
phosphoproteins and
phospholipoproteins.
2 0 The present invention also includes methods for the production of the slit
protein from
cells transformed with the polynucleotide sequence of the present invention.
Proteins can be
expressed in mammalian cells, plant cells, insect cells, yeast, bacteria,
bacteriophage, or other
appropriate host cells. Host cells are genetically transformed to produce the
protein of
interest by introduction of an expression vector containing the nucleic acid
sequence of
2 5 interest. The characteristics of suitable cloning vectors and the methods
for their introduction
into host cells have been previously discussed. Alternatively, cell-free
translation systems
can also be employed using RNA derived from the DNA of interest. Methods for
cell free
translation are known to those skilled in the art. (Davis et al., Basic
Methods in Moleculaf-
Biology, Elsevier Science Publishing (1986); Ausubel et al., Short Protocols
in Molecular
3 0 Biology, 2"d Ed., John Wiley & Sons (1992)). In the preferred embodiment,
host cells are
HEK 293 cells or 293T cells (American Type Culture Collection).
Host cells are grown under appropriate conditions to a suitable cell density.
If the
sequence of interest is operably linked to an inducible promoter, the
appropriate
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27
environmental alteration is made to induce expression. If the protein
accumulates in the host
cell, the cells are harvested by, for example, centrifugation or filtration.
The cells are then
disrupted by physical or chemical means to release the protein into the cell
extract from
which the protein can be purified. If the host cells secrete the protein into
the medium, the
cells and medium are separated and the medium retained for purification of the
protein.
Larger quantities of protein can be obtained from cells carrying amplified
copies of
the sequence of interest. In this method, the sequence is contained in a
vector that carries a
selectable marker and transfected into the host cell or the selectable marker
is co-transfected
into the host cell along with the sequence of interest. Lines of host cells
are then selected in
which the number of copies of the sequence have been amplified. A number of
suitable
selectable markers will be readily apparent to those skilled in the art. For
example, the
dihydrofolate reductase (DHFR) marker is widely used for co-amplification.
Exerting
selection pressure on host cells by increasing concentrations of methotrexate
can result in
cells that carry up to 1000 copies of the DHFR gene.
Proteins recovered can be purified by a variety of commonly used methods,
including,
but not limited to, ammonium sulfate precipitation, inununo precipitation,
ethanol or acetone
precipitation, acid extraction, ion exchange chromatography, size exclusion
chromatography,
affinity chromatography, high performance liquid chromatography,
electrophoresis, and ultra
filtration. If required, protein refolding systems can be used to complete the
configuration of
2 0 the protein.
Practice of this invention also includes the therapeutic use of the protein
encoded by
the polynucleotide sequence and its inclusion in pharmaceutical compounds. A
greater
understanding of the mechanisms directing nerve axon growth is critical to the
application of
methods for restoring nerve tracts after, for example, spinal cord injury.
Applicants have
2 5 shown that one of the biological functions of the protein encoded by the
polynucleotide
sequence of the present invention is direction of axon growth by directing
axons away from
the source of the protein (repulsive guidance). Another biological function of
the protein is
the guidance of migrating cells by repulsing the migrating cells from the
source of the slit
protein. As used herein the terms repulsing, repulsive and repulsive guidance
mean directing
3 0 the projection (growth) of axons or the migration of cells away from the
source of the slit
protein. A repulsive amount means an amount of slit protein sufficient to
direct growing
axons or migrating cells away from the source of the slit protein. In
addition, by alteration of
conditions, slit protein may act as an attractive guidance cue. Changes in
condition that may
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28
alter slit protein function include, but are not limited to, changes in the
cell type or
developmental stage of the cell or changes in the concentration of cAMP, cGMP
and other
cofactors.
In vitro, transformed cells secreting the slit protein can be used to
determine which
nerve tracts are guided to their target tissue by repulsive forces. For
example, transformed
cells can be enclosed within a collagen matrix along with explants of neural
tissue. The
effect on axon growth from the explant on the sides proximal and distal to the
transformed
cells can then be determined. Alternatively, transformed cells can be enclosed
in
microspheres constructed of material which allows diffusion of the secreted
products of the
cells. In another alternative, the protein can be delivered to explants by the
use of mini
pumps to supply a steady source of the protein in the vicinity of the explant.
Repair of spinal cord injuries requires not only that the nervous tissue be
stimulated to
grow new axons, but in addition, the new axons must be directed to the proper
site within the
nervous system to reestablish the previous nerve tracts. Recently, Xu et al.,
Eur. J. Neurosci.
11:1723-1740 (1999), have reported the regrowth of spinal neurons by the use
of a mini
chamber implanted within the spinal cord. Key to axon regrowth is the presence
of Schwann
cells within the mini-chamber. Although axons can be induced to regenerate
through the use
of the chamber, the growth is not directed. It is believed that by seeding
cells that have been
transformed with the nucleotide sequence of the present invention and that
secrete the protein
2 o encoded by the sequence, the regenerating axons can be directed to a
specific location in the
distal spinal cord.
The slit protein of the present invention is also useful for the treatment of
conditions
involving the migration of leukocytes. As reviewed above, leukocytes are
thought to be
involved in a number of conditions. Thus, the ability to inhibit leukocyte
migration into
2 5 particular organs or tissues would be beneficial in the treatment of
numerous diseases,
conditions or disorders. Representative conditions in which inhibition of
leukocyte migration
by administration of the slit proteins encoded by the nucleotide sequence of
the present
invention include, but are not limited to, asthma, arthritis,
glomerulonephritis, cystic fibrosis,
ulcerative colitis, Crohn's disease, multiple sclerosis, allergic
encephalomyelitis, Alzheimer's
3 0 disease, coronary artery restenosis, and any other condition which may be
alleviated by an
inhibition of migration of leukocytes into the affected tissue or organ.
In addition, the slit protein of the present invention is useful for the
prevention or
inhibition of graft rejection associated with leukocytes, especially delayed
rejection and more
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29
particularly delayed rejection of xenografts. The slit protein acts to lessen
or prevent graft
rejection by inhibiting the migration of leukocytes into the graft. This can
be accomplished
by the systemic or local administration of the protein. For example, local
administration can
be accomplished by transplantation, along with the tissue or organ, of a mini-
pump to slowly
release the slit protein over time. Alternatively, cells can be transformed
with the
polynucleotides of the present invention such that they secrete slit protein.
When the
objective is to transplant the cells themselves, such as the insulin producing
cells of the
pancreas, the secretion of the slit protein can serve to protect the cells.
When organs are
being transplanted, transformed endothelial cells of the blood vessels can be
used to limit
leukocyte invasion. Other methods of using slit proteins to inhibit graft
rejection will be
apparent to those skilled in the art and are within the scope of the present
invention.
Without being limited by theory, the protein of the present invention is
thought to act
by binding to the robo protein. Thus, the present invention can be applied to
direct the
growth or migration of any cell type expressing the robo protein or other
functional slit
protein receptor on its surface, for example to direct the growth of
regenerating tissues or
organs. In addition, the ability of proteins encoded by the nucleotide
sequence to inhibit
andlor redirect cell movement, has use in preventing migration of malignant
cells which
express the robo protein or other functional slit protein receptors.
Use of the slit protein is not limited to cells that naturally express the
robo gene or
2 0 genes for other functional slit protein receptors, but includes cells into
which the robo gene or
genes for other functional slit protein receptors have been introduced through
recombinant
DNA technology. For example, cells could be designed that produce both a
therapeutic agent
and also express the robo gene or other functional slit protein receptor gene.
Such cells could
then be transplanted into the body to a location near where the therapeutic
agent was needed.
2 5 By using the slit protein, the cells producing the agent could be directed
to a location where
the therapeutic agent was needed. For example, cells producing a factor
necessary to
stimulate nerve regeneration could be directed to a specific site where nerve
damage has
occurred. In another example, the slit protein could be used to prevent the
migration of cells.
In this use, cells transplanted to a location in the body, for example, could
be prevented from
3 0 migrating by local administration of the slit protein by, for example, a
transplantable mini
pump or by co-transplantation of cells secreting the slit protein. This method
can also be
applied to inhibiting the migration of malignant cells expressing the robo
protein. By local
administration of the slit protein, the metastasis of the malignant cells can
be prevented or
CA 02365214 2001-09-13
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inhibited. Examples of malignant cells that express the robo protein include,
but are not
limited to, neuroblastoma cells and glioblastoma cells.
The slit protein encoded by the polynucleotide of the present invention has
been
shown to be effective in blocking the action of RANTES and SDF-1. Receptors
for
5 chemokines, including RANTES and SDF-1, have been reported to play a role in
as
recognition sites for HIV infection (See, Baggiolini and Moser, J. Exp. Med.,
186:1189-1191,
1997). Antagonists of chemokines have also been reported as blocking HIV
infection
(Simmons et al., Science, 276:276-279, 1997; Arenzana-Seisdedos et al.,
Nature, 383:400,
1996). Thus, the slit protein of the present invention can be used to limit
HIV infection of
10 cells in vitro or in vivo. When administered in vivo, the amount
administered will vary with
such factors as the size, age, sex, health and virus load of the patient, as
well other factors
well known in the art.
The slit proteins of the present invention can be formulated as pharmaceutical
compositions. Such compositions can be administered orally, parenterally, by
inhalation
15 spray, rectally, intradermally, transdermally, or topically in dosage unit
formulations
containing conventional nontoxic pharmaceutically acceptable carriers,
adjuvants, and
vehicles as desired. Topical administration may also involve the use of
transdermal
administration such as transdermal patches or iontophoresis devices. The term
parenteral as
used herein includes subcutaneous, intravenous, intramuscular, or intrasternal
injection, or
2 0 infusion techniques. Formulation of drugs is discussed in, for example,
Hoover, John E.,
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania
(1975),
and Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel
Decker,
New York, N.Y. (1980).
Injectable preparations, for example, sterile injectable aqueous or oleaginous
2 5 suspensions, can be formulated according to the known art using suitable
dispersing or
wetting agents and suspending agents. The sterile injectable preparation may
also be a sterile
injectable solution or suspension in a nontoxic parenterally acceptable
diluent or solvent, for
example, as a solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that
may be employed are water, Ringer's solution, and isotonic sodium chloride
solution. In
3 0 addition, sterile, fixed oils are conventionally employed as a solvent or
suspending medium.
For this purpose, any bland fixed oil may be employed, including synthetic
mono- or
diglycerides. In addition, fatty acids such as oleic acid are useful in the
preparation of
injectables. Dimethyl acetamide, surfactants including ionic and non-ionic
detergents, and
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31
polyethylene glycols can be used. Mixtures of solvents and wetting agents such
as those
discussed above are also useful.
Suppositories for rectal administration of the compounds discussed herein can
be
prepared by mixing the active agent with a suitable non-irritating excipient
such as cocoa
butter, synthetic mono-, di-, or triglycerides, fatty acids, or polyethylene
glycols which are
solid at ordinary temperatures but liquid at the rectal temperature, and which
will therefore
melt in the rectum and release the drug.
Solid dosage forms for oral administration may include capsules, tablets,
pills,
powders, and granules. In such solid dosage forms, the compounds of this
invention are
ordinarily combined with one or more adjuvants appropriate to the indicated
route of
administration. If administered per os, the compounds can be admixed with
lactose, sucrose,
starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters,
talc, stearic acid,
magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric
and sulfuric
acids, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and/or
polyvinyl alcohol,
and then tableted or encapsulated for convenient administration. Such capsules
or tablets can
contain a controlled-release formulation as can be provided in a dispersion of
active
compound in hydroxypropylmethyl cellulose. In the case of capsules, tablets,
and pills, the
dosage forms can also comprise buffering agents such as sodium citrate, or
magnesium or
calcium carbonate or bicarbonate. Tablets and pills can additionally be
prepared with enteric
2 o coatings.
For therapeutic purposes, formulations for parenteral administration can be in
the
form of aqueous or non-aqueous isotonic sterile injection solutions or
suspensions. These
solutions and suspensions can be prepared from sterile powders or granules
having one or
more of the carriers or diluents mentioned for use in the formulations for
oral administration.
2 5 The compounds can be dissolved in water, polyethylene glycol, propylene
glycol, ethanol,
corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium
chloride, and/or
various buffers. Other adjuvants and modes of administration are well and
widely known in
the pharmaceutical art.
Liquid dosage forms for oral administration can include pharmaceutically
acceptable
3 0 emulsions, solutions, suspensions, syrups, and elixirs containing inert
diluents commonly
used in the art, such as water. Such compositions can also comprise adjuvants,
such as
wetting agents, emulsifying and suspending agents, and sweetening, flavoring,
and perfuming
agents.
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32
The amount of protein that can be combined with the carrier materials to
produce a
single dosage form will vary depending upon the patient and the particular
mode of
administration.
Slit protein or slit protein derivatives can also be use to stimulate cell
proliferation.
Rates of cell proliferation can be increased, for example, by transforming
cells with vectors
containing the polynucleotide encoding the slit protein. Alternatively, cell
proliferation can
be increased by treating cells with slit protein, either by addition of slit
protein to the culture
medium or by conditioning the medium with cells secreting the slit protein.
Conditioned
medium is obtained by culturing cells secreting the slit protein in the
medium, harvesting the
medium, and using the medium to culture additional cells. Ih vivo, slit can be
administered
locally or systemically to stimulate cell proliferation by any of the methods
or routes
described previously, by transplanting transformed cells that secrete the slit
protein, or by the
use of a transplantable mini pump that delivers slit protein to the desired
location. In vivo the
ability of slit to stimulate cell proliferation can be used, for example, to
aid in wound healing
or repair, or in organ regeneration such as lung, liver and kidney
regeneration following .
injury or disease. The exact amount of slit administered will vary based on
the size, age, and
sex of the animal, the route and method of administration, the cell type to be
stimulated, the
amount of cell proliferation desired, and other factors well known in the art.
The slit protein can also be used to reduce or eliminate serum in cell culture
medium.
2 0 Serum, for example fetal bovine serum, is commonly added to cell culture
media to promote
cell growth. The addition of serum to cell culture media is undesirable
because the
composition of serum varies from batch to batch. Thus, the use of serum adds
unknown
variables to experiments using cell culture which can complicate analysis and
cause
inconsistent results. The need for serum in culture medium can be reduced or
eliminated by
2 5 transforming cells with polynucleotides encoding the slit protein. Without
being bound by
theory, it is believed that the need for serum is reduced or eliminated
because the transformed
cells secrete slit protein into the culture medium. Thus, it is also believed
that the need for
serum in culture medium can be eliminated or reduced by the addition of slit
protein to the
culture medium. As used herein, reduced serum culture medium means culture
medium in
3 0 which the amount of serum normally used is decreased by at least 25% due
to the addition of
slit protein or slit protein derivatives.
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33
Example 1
Isolation of Vertebrate Slit cDNA
Degenerate primers corresponding to NPFNCNC (SEQ ID NO: 4) and CETNIDDC
(SEQ ID NO: 5) of Drosophila slit protein (amino acid residues 652-658 and 981-
988
respectively) were used to clone fragments of Xenopus slit cDNA by PCR. A cDNA
library
was constructed using the BKCMV vector (Strategene) with mRNAs extracted from
stage 17
Xenopus embryos. The PCR fragment of Xenopus slit was used to screen the cDNA
library
to obtain full-length Xenopus slit cDNA clones. Human slit-2 cDNA was obtained
using PCR
with cDNA prepared from human fetal brain mRNA (Clontech) and primers
corresponding to
l0 MRGVGW (amino acid residues 1-6) and CTRCVS (the last six amino acid
residues in the
coding region). The full-length Xenopus slit and human slit-2 cDNAs were
sequenced using
an ABI 373A automatic sequencer (Applied Biosystems) in accordance with the
manufacturer's protocol and the amino acid sequence deduced from the
nucleotide sequence.
The human slit-2 cDNA isolated differs from the published huamn slit-2
sequence (Genbank
accession # AF055585) (SEQ ID NO: 3) by an insertion of eight amino acid
residues
(AKEQYFIP) (SEQ ID NO: 6) between residue 479 and 480. This is possibly a
result of
alternative splicing as predicted from sequence analysis. Methods for the
construction and
screening of cDNA libraries are well known to those skilled in the art and can
be found, for
example in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2°a
Ed., Cold Spring
2 0 Harbor Press, (1989) and Ausubel et al., Short Protocols in Molecular
Biology, 2°d Ed., John
Wiley & Sons (1992).
The nucleotide sequence for the Xenopus slit cDNA is given in Figure 1. The
cDNA
sequence consists of 5513 bases and exhibits 59% sequence identity with the
murine slit-2
sequence. The deduced amino acid sequence encoded by the cDNA is given in
Figure 2.
The protein exhibits 87% amino acid identity with the mouse slit-2 protein,
85% identity with
human slit-2 protein, 63% identity with the mouse slit-1 and mouse slit-3
proteins, 83%
identity with chicken slit protein, 67% with the rat slit protein rMEGFS, 63%
with the rat slit
protein rMEGF4 (Nakayama et al., Genomics 51:27-34 (1998)), and 40% identity
with the
Drosophila slit protein. At the amino (N) terminus, there is a putative signal
peptide
3 0 characteristic of secreted proteins. There are four leucine rich repeats
(LRR), each
surrounded by an N terminal and a carboxyl (C) terminal flanking region. In
Xenopus slit, as
with mouse and human slit-2, there are nine epidermal growth factor (EGF)
repeats, whereas
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34
there are seven EGF repeats in Drosophila slit (Rothberg et al., Genes Dev.
4:2169-2187
(1990), Rothberg and Artavanis-Tsakonas, J. Mol. Biol. 227:367-370 (1992)).
Near the C
terminus, there is a laminin G domain with similarities to agrin, laminin, and
perlecan (also
known as the ALPS domain), followed by a cysteine rich carboxyl terminal
region (Rothberg
et al., Genes Dev. 4:2169-2187 (1990), Rothberg and Artavanis-Tsakonas, J.
Mol. Biol.
227:367-370 (1992)).
Example 2
Plasmid Construction
The coding region for full-length Xenopus slit was inserted in-frame into the
pCS2+
vector containing a six-myc epitope tag or containing the secreted form of
alkaline
phosphatase to obtain slit-myc or slit-AP, respectively. The coding region for
full-length
human slit-2 was inserted into the pCS2+ vector containing the myc tag to
obtain hslit-myc
expressing plasmid. In both cases, slit was at the N terminal portion of the
fusion proteins.
To express robo as an epitope tagged protein, the rat robol coding region was
obtained by
PCR using rat spinal cord cDNA and was then inserted into pCS2+ vector
containing a
hemagglutinin (HA) epitope. The HA epitope was at the C terminus of robo-1.
Plasmids
were also constructed to express different fragments of Xenopus slit, human
slit-2 or robo.
The full-length or fragments of coding regions of slit and robo-1 in the
plasmids were verified
by sequencing. Before being used in transfection experiments, individual
plasmids were also
2 0 tested for expression of the corresponding proteins by coupled in vitro
transcription-
translation (Promega). The plasmid construction was carried out following
standard
protocols described in Sambrook et al., Molecular Cloning, A Laboratory
Manual, 2°d Ed.,
Cold Spring Harbor Press, (1989) and Ausubel et al., Short Protocols in
Molecular Biology,
2°d Ed., John Wiley & Sons (1992).
2 5 Example 3
Cell Culture, Transfection and Immunoprecipitation
To directly test the possible ligand-receptor relationship between slit and
robo, cells
were transformed to express the slit and robo fusion proteins and the
interactions of the
proteins examined by immunoprecipitation and Western blotting.
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HEK 293 cells or 293T cells (both from American Type Culture Collection) were
maintained in 10% fetal bovine serum (FBS) in Dulbecco's modified Eagle's
medium
(DMEM) (Gibco). Cells were grown to 70% confluence on 2-10 cm tissue culture
dishes and
transfected with 5-25 ~g of plasmid DNA per plate using lipofectin or
lipofectamine (Life
5 Technology), commercially available liposome preparations or by
electroporation according
to the manufacturer's instructions. Alternatively, cells were transfected
using calcium
phosphate for 6 - 24 hours. GFP-pGL, a plasmid expressing the green
fluorescent protein
(GFP) (Life Technology), was used in most of the transfection experiments to
monitor
transfection efficiency.
10 For co-immunoprecipitation, plasmids encoding slit-myc, slit-AP, or robo-HA
or
control vector plasmids were transfected into HEK or 293T cells as described
above.
Conditioned media containing slit-myc or slit-AP proteins from the transfected
cells were
collected 72 to 96 hrs after transfection and concentrated using a Biomax-100K
ultrafree-15
filter (Millipore). Robo-HA containing cell lysates or control lysates were
prepared with lysis
15 buffer (0.5% NP-40, SO mM Tris pH 7.5, 150 mM NaCI, 1 mM EDTA, SOmM NaF,
1mM
Na3V04, 1mM DTT, 1mM phenylmethylsulfonyl fluoride, 25wg/ml leupeptin, 25~g/ml
aprotinin, 150~g/ml benzamidine). Conditioned media containing slit-myc or
slit-AP were
mixed with lysates from robo-HA or control cells. Immunoprecipitation was
carried out as
described in Kopan et al., Proc. Natl. Acad. Sci. USA 93:1683-1688 (1996)
using anti-myc
2 0 (Babco) or anti-AP (Sigma) antibodies. Precipitated proteins were then
detected after
Western blotting by anti-HA (Babco) with enhanced chemiluminescence according
to the
manufacturer's instructions (Amersham).
Following incubation of slit-myc medium with robo-HA cell lysate, slit-myc
could be
immunoprecipitated with anti-HA antibody. Conversely, robo-HA could be
precipitated from
2 5 the slit containing medium mixed with robo containing cell-lysate by anti-
myc antibodies.
These results provide evidence that the slit protein of the present invention
binds to robo.
Example 4
Cell Surface Binding and Immunocytochemistry
To confirm that the binding of the slit and robo proteins was not confined to
cell-free
3 0 systems, but also occurred at the cell surface, the binding of slit-AP to
cells expressing the
robo-HA protein was determined.
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HEK 293 cells grown in 10 cm dishes were transfected with robo-HA or vector
plasmids as previously described. Approximately 30-48 hours after
transfection, cells were
suspended by pipetting up and down several times and then seeded onto 6- well
or 24-well
dishes to 50% confluence. Cells were grown for another 12 to 18 hrs before
incubation with
either slit-AP fusion protein or lunatic fringe alkaline phosphatase (lFng-AP)
fusion protein
conditioned media containing similar amounts of AP activity (approximately 530
OD/ml/hr).
Lunatic fringe is a vertebrate signaling protein found in Xenopus used here as
a negative
control. The lFng-AP protein was made by the method of Wu et al., Science
273:355-358
( 1996).
Cells were incubated for 1 hr with the slit-AP containing media or the control
media
followed by three to four washes in HBHA buffer (Hank's balanced salt
solution, O.Smg/ml
BSA, 20mM HEPES, pH 7.0), and then fixed for 30 seconds in acetone-
formaldehyde
fixative (60% acetone, 3% formaldehyde, 20mM HEPES pH 7.0). Fixed cells were
washed
three times in HBS (150mM NaCI, 20mM HEPES, pH7.0) and incubated at
65°C for 10 min
to inactivate the endogenous cellular phosphatase activity. AP staining buffer
(100mM Tris,
pH9.5, SOmM MgCl2, 100mM NaCI, 0.1% Tween 20, 0.17mg/ml 5-bromo-1-chloro-3-
indoxyl phosphate (BCIP), 0.33mg/ml nitroblue tetrazolium) was used to detect
slit-AP or
lFng-AP bound at the cell surface. Following three washes, robo-HA expression
on AP
positive cells was confirmed by the double antibody staining method by
treatment with
2 0 mouse anti-HA and anti-mouse immunoglobulin conjugated to cyanine dye 3
(Cy3, Jackson
Immunoresearch). GFP expression indicated similar transfection efficiencies in
vector and
robo-HA transfected cells.
Slit-AP binding was found to correlate with robo-HA expression providing
additional
evidence that slit binds to robo on the cell surface. The lunatic fringe-AP
fusion protein did
2 5 not bind to the robo transfected cells. These results indicate that slit
specifically binds to cell
surface robo proteins.
Example 5
Production of Cell Lines Exhibiting Stable
Expression of Slit or Robo Proteins
3 0 Several stable cell lines were made using a human embryonic kidney (HEK)
cell line
(American Tissue Culture Center Accession Number CRL-1573). Vector control
lines were
made by transfection with the vector plasmid pIRESneo (Clontech) or pCEP4
(Invitrogen).
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Xslit-myc line and hslit-myc line express full-length Xenopus Slit and human
slit2 proteins,
respectively. Stable cell lines were also prepared to express different
regions of human slit-2
protein including N890 (amino-terminal 890 amino acid residues), N1076 (amino-
terminal
1076 amino acid residues) and C 1 (nucleotide sequence encoding carboxyl-
terminal 892-
1529 amino acid residues fused to the sequence encoding the predicted signal
peptide).
Stable cell lines were prepared to express different regions of Xenopus slit
protein including
N882 (amino-terminal 882 amino acid residues), and Cl ( nucleotide sequence
encoding
carboxyl-terminal 883-1530 amino acid residues fused to the sequence encoding
the predicted
signal peptide). Cells expressing full-length Xenopus Slit and human slit2
proteins also
express proteolytic fragments, similar to that reported by Wang, K-H et al.
(Cell 96:771-784,
1999). In addition, human slit2 stable cell lines expressing individual
mutations at amino
acid residue L538S and R172G/C1358Y were made. These two mutant slit-2 stable
lines
have altered pattern in proteolytic cleavage compared to the wild-type slit-2
cells.
Stable cell lines were also established expressing the full-length Robo and
Robo-N
(amino-terminal 730 amino acid residues) as proteins tagged with a
hemagglutinin (HA) tag
at their carboxyl termini. The full-length Robo and Robo-N plasmids for making
stable cell
lines were constructed using the vector modified from pIRESneo (Clontech).
Another Robo-
HA stable line, Robo-HA-hygB, was also constructed using the pCEP4 vector
(Invitrogen).
Linearized or circular plasmids encoding Slit-myc and Robo-HA (full-length or
2 0 derivatives) and their corresponding vector controls were transfected into
HEK cells.
Antibiotics were added 24 to 48 hours after transfection and selection was
carned out for
three to five weeks with the media changed every two to three days. 6418 (200-
500 ~g/ml)
(Life Technology) was used to select for Slit-myc and Robo-HA (full-length or
derivatives)
stable lines. Hygromycin B (100-300 ~.g/ml) (Sigma) was used to select for
Robo-HA-hygB
2 5 stable lines. Stable cell lines expressing Slit-myc and Robo-HA were
obtained after isolating
individual colonies and testing for protein expression by both Western blots
and
immunocytochemical staining (with anti-myc or anti-HA antibodies,
respectively).
Example 6
Purification of Slit Proteins and Their Derivatives
3 0 To purify 220Kd full-length Slit protein, cells stably expressing Slit
were grown to 90-
95% confluence, treated with trypsin and plated at 30-40% confluence. Cells
were then
grown for 20-36 hours before collecting the media. To purify slit fragments,
Slit stable cells
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expressing corresponding slit fragments [N890, N1076, Cl fragments] or
carboxyl-terminal
fragment cleavage product from the full-length slit were grow to confluence
and media were
collected 2-5 days after plating cells.
The Slit protein (full-length or derivatives) containing media was adjusted to
pH 5.5-6.5
with acetic acid and then loaded onto a SP-sepharose ion exchange
chromatography column
(Pharmacia) that had been equilibrated with 10 bed volumes of PBS, pH S.5-6.5.
The column
was then washed with 10 bed volumes of PBS, pH 5.5-6.5. Slit protein was
eluted with a 0-
1.0 M NaCI gradient. The full-length Slit protein was usually eluted out at
0.4 to 0.6 M NaCI
and the N890, N1076, and C1 fragments usually eluted at 0.2 to 0.5 M NaCI.
Fractions were
collected and examined by Western blotting and silver staining to determine
which fractions
contained Slit protein of the desired purity. The desired fractions were
combined for further
purification or concentration. Further purification was achieved with immuno-
affinity
chromatography by 9E10-agarose (Babco) or Superose (Pharmacia) gel filtration.
For
immuno-affinity chromatography, the slit containing fractions were adjusted to
pH 7.5 and
loaded onto a 9E10-agarose column (Babco). The media were passed through the
column
three times and the column was then washed with 10 bed volumes of PBS. Slit
protein was
eluted with 0.1 M Glycine pH 2.9 and the pH of the protein eluted was adjusted
to 7.5 with
Tris-HCl by adding 1/10 volume of 1 M Tris HCI, pH 7.5. Gel filtration was
earned out
using a Superose column (Pharmacia) according to the manufacturer's
instructions. If
2 0 necessary, Slit protein preparations were further concentrated by using
polyethylene glycol
(PEG) (Sigma) or a filter device (Millipore). The slit protein and its
derivatives can be
purified with other variations of ion exchange chromatography with gel
filtration and/or
affinity chromatography. The purity of proteins was determined by silver
staining.
Example 7
2 5 Quantitative Assay of Slit-AP Binding to Robo Expressing Cells
In order to determine if the interaction of the slit and robo proteins
exhibited a binding
affinity similar to that observed between other soluble and cell surface
proteins, the binding
affinity was determined. The method used has been published previously
(Flanagan and
Leder, Cell 63:185-194 (1990); Cheng and Flanagan, Cell 79:157-168 (1994);
Keino-Masu et
3 0 al., Cell 87:175-185 (1996); Leonardo et al., Nature 386:833-838 (1997))
and so will only be
briefly described herein.
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Stable cell lines expressing robo-HA or the vector were seeded in 24 well-
culture
dishes pre-coated with 100mg/ml polylysine. Cells were grown to 95% confluence
and then
incubated at 37°C for lhr in 200~1/well of concentrated, conditioned
media containing
different concentrations of slit-AP with the highest level at approximately
3000 OD/ml/hr of
AP activity. Media were collected to assay for the AP activity as free slit-
AP. Cells were
washed 4 times with the culture medium without slit-AP. Cells were then lysed
using a lysis
buffer containing SOmM Tris, pH 8 and 1% Triton-X 100. Cell lysates were
cleared by
centrifugation for 10 min. Samples were heated at 65°C for 10 min to
inactivate the
endogenous cellular phosphatase activity. AP activity was assayed by adding
equal volumes
l0 of 2X AP buffer (2M diethanolamine, pH9.8, 1mM MgCl2, 20mM homoarginine, 12
mM p-
nitro-phenyl phosphate) to cell lysates in 96-well flat-bottom microtiter
plates. Incubation
was carned out at room temperature and AP activity was determined at 405nm.
The
concentration of slit-AP fusion protein in the conditioned media was estimated
by
comparison with the purified human placenta alkaline phosphatase (HuPAP)
(Sigma) both in
AP activity assays and in Western blots using anti-AP antibody (Genzyme).
An apparent dissociation constant (KD) of 2.75 nM was estimated from a binding
curve and is comparable to other ligand-receptor interactions (Flanagan and
Leder, Cell
63:185-194 (1990); Cheng and Flanagan, Cell 79:157-168 (1994); Keino-Masu et
al., Cell
87:175-185 (1996); Leonardo et al., Nature 386:833-838 (1997)). These results
indicated that
2 0 soluble slit proteins bind with high affinity to robo cell surface
proteins.
Example 8
Effect of Slit protein on Axon Outgrowth from
Olfactory Bulb Explants.
To determine if slit plays a role in axon guidance, olfactory bulb explants
were
2 5 isolated from chick embryos and co-cultured with either HEK cells stably
transfected with
either slit-myc or vector plasmid.
Preparation of rat tail collagen and explant culture in collagen gel matrices
were
carried out according to the protocol described in Guthrie and Lumsden,
Neuroprotocols
4:116-120 (1994). Cell aggregates were prepared by the hanging-drop method
(Fan and
3 o Tessier-Lavigne, Cell 79:1175-1186 (1994)).
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The olfactory bulb explant assay was performed according to Pini, Science
261:95-98
(1993); Keynes et al., Neuron 18:889-897 (1997). Briefly, stage 33 or 34 chick
embryos
were dissected out in Tyrode's solution. Olfactory bulbs were removed and
stored in L15
medium with 5% horse serum (GIBCO) on ice for at least 30 min. Olfactory bulb
explants
5 were trimmed to 200-300 ~.m. Olfactory bulb explants and cell aggregates
were transferred
onto a collagen pad and covered with collagen. The distance between the
olfactory bulb
explants and the cell aggregates ranged from 100 to 400 Vim. After collagen
gel matrices
solidified, DMEM with 10% FCS and 100 ~g/ml of penicillin and streptomycin was
added.
Explants and cells were co-cultured at 37°C with 5% COZ and the effects
of slit on olfactory
10 bulb axons were visible 10-24 hours after culturing. Explants were fixed
after about 12 or 24
hours of co-culture. TuJl antibody was used in immunocytochemistry to
visualize neuronal
processes. TuJl is a monoclonal antibody that recognizes an epitope unique to
neuronal-
associated class III beta-tubulin isotypes (Caccamo et al., Lab. Invest.
60:390-398 (1989)).
Quantification of the axon projections from the olfactory bulb explants was
carried
15 out according to the method of Keynes et al. Neuron 18:889-897 (1997). A
score of 0 was
given if there was no or very few axons growing in the proximal quadrant; a
score of 2 was
given if there were few axons in the proximal quadrant with strong asymmetry
when
compared with the distal quadrant; a score of 4 was given if there was greater
outgrowth in
the proximal quadrant, with axons in the proximal quadrant still more than 50
~,m from the
2 0 cell aggregates, and strong asymmetry between the distal and proximal
quadrants; a score of
6 was given if axons in the proximal quadrant were growing within less than 50
~m from the
cell aggregates, still with asymmetry between the distal and proximal
quadrants; a score of 8
was given if axons were contacting the cell aggregates, but still with
detectable asymmetry;
and a score of 10 was given if axons had grown over the cell aggregates and
there was no
2 5 asymmetry between the proximal and distal quadrants.
When olfactory bulb explants were co-cultured with control cells transfected
with
vector plasmid, axons grew symmetrically from the explant. In contrast, when
slit-myc
transfected cells were used, axon growth was asymmetrical with more axons
growing on the
side of the explant distal to the slit expressing cells than on the side of
the explant proximal to
3 0 the slit expressing cells.
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Example 9
Effect of Slit Protein on the Projection of
Olfactory Bulb Axons into the Telencephalon.
To confirm the effect of slit on axon guidance, the ability of slit to guide
axon growth
was assessed in whole-mount preparations of telencephalon, the natural
environment for
olfactory bulb axon growth.
Whole mount preparations of olfactory bulb-telencephalon co-culture were
earned out
with a protocol similar to that described in Sugisaki et al., J. Neurobiol.
29:127-137 (1996).
Briefly, the telencephalic hemisphere together with the olfactory bulb were
dissected out
from mice at day 12.5 of gestation, freed from the pia mater, and placed on a
collagen gel.
Cells transfected with either vector alone or with slit-myc cDNA were labeled
with Di0
(3,3'dioctadecyloxacarbocyanine, Molecular Probes). Aggregates of these cells
were put on
top of the telencephalon, but not the olfactory bulb. Whole mount preparations
were cultured
with DMEM containing 10% fetal bovine serum at 37°C in 5% COZ. Forty
hours later, small
crystals of the lipophilic dye 1,1'-dioetadecyl-3,3,3',3'-tetramethylindo-
carbocyanine per-
chlorate (DiI; Molecular Probes) were inserted into the olfactory bulbs. Eight
to twelve hours
later, the specimens were fixed with 4% paraformaldehyde in PBS and kept at 4
°C before
examination under a fluorescent microscope.
The results obtained showed that while axons could grow into the part of a
2 0 telencephalon covered with control, vector transfected cells, axons turned
away from the part
of a telencephalon covered with slit-myc transfected cells.
Example 10
Effect of Slit Protein on Neuronal Migration
Co-culture of subventricular zone (SVZa) explants from rats and slit
expressing HEK
2 5 cells was carried out in collagen gel matrices as previously described.
Brains from newborn
Sprague-Dawley (SD) rats (postnatal days 3-7) were embedded in 7% low melting
point
agarose prepared in phosphate buffered saline. Coronal and sagittal sections
of 400 ~,m were
cut with a vibratome. Tissue within the borders of the SVZ in coronal sections
was dissected
out to make SVZa explants of 200-400 ~m in diameter. Septal explants were
isolated form
3 o sagittal sections. Explants were placed into the collagen gel or matrigel
as described
previously (Hu and Rutishauser, Neuron 16:933-940 (1996); Wichterle et al.,
Neuron,
18:779-791 (1997)). The explants were cultured for 16 to 24 hours under 5% COZ
in F-12
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42
medium (Life Technologies) supplemented with 10% fetal bovine serum,
penicillin and
streptomycin. Aggregates of control or slit-expressing cells were made by the
hanging drop
method and placed into collagen gels or the matrigel together with SVZa
explants and
cultured as described above. For quantification, immunofluorescence images of
sample
stained with TuJI were obtained with a Zeiss microscope. Cell distribution was
quantified by
analyzing the distance between the cell body and the nearest edge of the
explant.
As shown in Figure 4, when SVZa explants were co-cultured with HEK cells
stably
transfected with control plasmid, migrating cells symmetrically distributed
around the
circumference of the explants. When SVZa explants were co-cultured with cells
stably
transfected with a plasmid expressing the slit protein of the present
invention, cell migration
was highly asymmetric around each explant with more cells in the quadrant
distal to the slit
secreting cells than in the quadrant proximal to the slit cells. When the
experiments were
repeated using matrigel which allows migration of chains of cells, similar
results were
obtained with symmetrical chain migration around explants when co-cultured
with control
plasmid transfected HEK cells and asymmetrical chain migration when co-
cultured with cell
aggregates expressing the slit protein. The neuronal nature of the migrating
cells was
confirmed by staining with the TuJl antibody. When explants were cultured on
top of slit
expressing HEK cells, neurons still migrated out of the SVZa. These results
show that slit is
repulsive to migrating neurons.
2 0 To test whether slit can repulse neurons migrating in their natural
pathway, brains
were isolated from postnatal rats and sagittal sections cut with a vibratome.
Slices of sagittal
sections containing the SVZa, the rostral migratory stream (RMS) and the
olfactory bulb
(OB) were cultured in collagen gel matrices. Crystals of the lipophilic dye
1,1'-dioctadecyl-3,
3, 3', 3'-tetramethylindo-carbocyanine per chlorate (DiI) were inserted into
the SVZa to label
2 5 neuronal precursors and the slices were cultured as described for the SVZa
explants. After 24
hours of culture, migrating neurons were found in the RMS. To test the effect
of slit, control
HEK cells or cells stably expressing slit were pre-labeled with 3,3'
dioctacedyloxacarbocyanine (Di0), thus allowing visualization of SVZa neurons
and HEK
cells. DiI and Di0 signals were visualized under a microscope using
appropriate filter sets.
3 0 Images were collected by photography using a Spot-camera (Zeiss) and
analyzed.
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When aggregates of control cells were placed on top of the RMS, cells from the
SVZa
migrated into the RMS. In contrast, when slit expressing cells were placed on
the RMS, few
cells migrated into the RMS (Figure 5). These results show that slit can
regulate the
migration of neuronal cells along their natural pathways.
The effect of slit on neuronal migration was also tested in GABAergic neurons
of
lateral ganglionic eminence (LGE) explants. As shown in Figure 6, when LGE
explants were
cocultured with control HEK cells transfected with the vector plasmid or with
a plasmid
expressing another axon repellent protein Semaphorin III, migrating neurons
were
symmetrically distributed around the circumference of the explants (Figure
6A). When LGE
explants were co-cultured with HEK cells stably transfected with a plasmid
expressing the slit
protein of the present invention, cell migration was highly asymmetric around
LGE explants
with more cells in the quadrant distal to the slit expressing cell aggregates
than in the
quadrant proximal to the slit expressing cells (Figure 6C and Figure 6D).
Human slit-2 and
mouse slit-1 expressing cells have similar activity to the cells expressing
Xenopus slit gene in
these assays. These results indicated that slit proteins were chemorepellents
to cells
migrating out of LGE explants.
To test whether slit can act on LGE neurons in their natural migratory
pathway, the
striatal-cortical pathway, to direct neuronal migration from the LGE to the
neocortex, a slice
assay was used in which a coronal section of the rat embryonic brain
containing the entire
2 0 migratory pathway of LGE neurons was preserved in culture. Previous
studies have
established that DiI labeling in this system can trace GABAergic neurons
migrating from the
LGE to the neocortex (Anderson et al., Science 278, 474-476 (1997) ; Tamamaki
et al., . J.
Neurosci. 17, 8313-8323, (1997)). In this assay, slit-expressing cells or
control cells were
placed at the juncture of the LGE and the neocortex and examined their effects
on cells
2 5 migrating from the LGE to the neocortex (Figure 7). Slices of coronal
sections of rat brains
were labeled with Hoechst dye to reveal the outlines of the sections (Figure
7A and Figure
7E). Aggregates of Slit-expressing cells or control cells were labeled with 3,
3'dioctadecyloxacarbocyanine (Di0). In some experiments, slit or control cells
were placed
at the junction of the LGE and the neocortex (Figure 7B and Figure 7F, green
cells). The
3 o migrating LGE neurons were traced by inserting a crystal of DiI in the
subventricular zone of
the LGE (Figure 7C and Figure 7G, red cells). Superimposition of three color
images of
Hoechst dye, DiO, and DiI revealed the positions of LGE cells migrating into
the neocortex
relative to the aggregates of Slit or control cells (Figure 7D and Figure 7H).
In one
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44
experiment with 53 explants, Slit-expressing cell aggregates were placed on
the cortical-
striatal junction on the left side of the brain and control cell aggregates
were placed on the
right junction. Among these explants, neurons migrated from the LGE past the
region
overlaid with an aggregate of control cells into the neocortex in 52 out of 53
slices. In 50 out
of the same 53 slices but on the side with a Slit aggregate, no LGE neurons
migrated past the
region overlaid with Slit aggregates into the neocortex. In the three explants
in which LGE
neurons migrated past Slit cells, the migration of LGE neurons into the
neocortex was
significantly reduced by Slit aggregates when compared to control aggregates.
These results
indicate that Slit is a repellent for LGE neurons migrating in their normal
pathway to the
neocortex.
To test whether endogenous Slit contributes to the repulsive activity in the
ventricular
zone of the LGE, cells expressing RoboN, the extracellular fragment of the
slit receptor
roundabout (Robo), were used. RoboN can bind slit (Wu et al., Nature 400:331-
336, (1999) )
but cannot transduce the signal to intracellular compartments and is thus a
competitive
blocker of Slit. Aggregates of HEK 293T cells transfected with the control
vector or with a
plasmid expressing RoboN were made and placed at the bottom layer of the
collagen gel
matrix. Explants from the ventricular zone and those from the subventricular
zone of the LGE
were cocultured at the top layer. LGE neurons were repelled by the ventricular
zone in the
presence of control HEK 297T cells, whereas the presence of RoboN-expressing
cells
2 0 inhibited the repulsive activity of the ventricular zone. These results
indicate that endogenous
Slit present in the ventricular zone of the LGE is repulsive to LGE neurons.
The slit expressing cells were also tested in migration of cerebellar neurons.
Rat
cerebellar explants of 100-300mM were prepared with a vibratome and co-
cultured either
with the control HEK cells or cells expressing the slit protein. Migrating
neurons were
2 5 symmetrically distributed around the circumference of the explants when
cultured with the
control cells. When the cerebellar explants were co-cultured with HEK cells
stably
transfected with a plasmid expressing the slit protein of the present
invention, cell migration
was highly asymmetric around the cerebellar explants with more cells in the
quadrant distal
to the slit expressing cell aggregates than in the quadrant proximal to the
slit expressing cells.
3 0 In several systems tested so far, including SVZa, LGE and cerebellar
neurons, the
human slit-2 protein has similar effect to the Xenopus slit protein. In
addition, different
fragments of slit protein were tested. Cells expressing amino-terminal
fragments of Slit,
including N890 (amino-terminal 890 amino acid residues) and N1076 (amino-
terminal 1076
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amino acid residues) behave similar to cells expressing full-length slit in
repelling olfactory
bulb axon. Slit fragment N890, however, did not have the repulsive activity in
neuronal
migration assay. Both full-length and slit fragment N1076 were active in
repelling neuron in
migration assay. The cells expressing the carboxyl terminal fragment C 1
(nucleotide
5 sequence encoding carboxyl-terminal 892-1529 amino acid residues fused to
the sequence
encoding the predicted signal peptide) did not show detectable activity in
either axon
guidance or neuronal migration assays.
Example 11
Effect of Slit on Cytokine Stimulated Cell Migration
10 The effect of slit on cell migration in response to cytokines was measured
using the
trans-well assay and the micro-chemotaxis chamber assay using either primary
leukocytes or
transfected HEK cells. Primary leukocytes were isolated from rat lymph node or
peripheral
blood using standard methods (Coligan et al., eds., Current Protocols in
Immunology, Wiley
& Sons, (1991)). HEK cells were co-transfected by the calcium phosphate method
or by
15 electroporation with plasmids encoding the slit protein receptor roundabout
and the
chemokine receptor CXCR4 ( Robo + CXCR4) or co-transfected with plasmids
encoding
roundabout and the chemokine receptor CCRS (Robo+CCRS). CXCR4 receptors bind
SDF-1
and CCRS receptors bind RANTES (Baggiolini et al., Annu. Rev. Immunol., 15:675-
705,
1997). Leukocytes or transfected HEK cells were kept on ice in DMEM
supplemented with
2 0 S-10% heat inactivated fetal bovine serum. Immediately before being
transferred into the
chemotaxis chamber, cells were resuspended at 1-5 x 106 cells/ml in either
DMEM, RPMI or
50% RPMI + 50% M199 medium (Life Technologies) without serum, but containing
0.25-
0.5% BSA.
The chemotaxis assays were preformed as previously described (Taub et al., J.
25 Immunol. Meth., 184:1870198 (1995); Ganju et al., J. Biol. Chem., 273:23169-
23175 (1998)).
Brielfy, media were put in the bottom wells containing different
concentrations of
commercially available recombinant RANTES or SDF 1 (R&D Systems) or test
proteins (slit
arid its derivatives) or combinations of cytokine and purified slit proteins.
Approximately 2-5
x 104 cells were placed in the top wells of a 48 well micro-chemotaxis chamber
(NeuroProbe)
3 0 or 1-5 x 105 cells in the inner inserts of a trans-well chamber (Costar).
The upper and lower
wells were separated by either 5 qm polycarbonate filters for leukocytes or 8
~m
polycarbonate filters for transfected HEK cells. Cells were incubated at
37°C for 80-240
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46
minutes. Cells that migrated into the bottom chamber were counted under a
microscope with
a hemacytometer. Cells that migrated into the pores of the filter were counted
after fixation
in methanol and staining in Diff Quick solution (Baxter).
In one experiment, the effect of differing concentrations of the cytokines
RANTES and
SDF on leukocyte migration in the presence of 0, 100 pM or 200 pM purified
slit protein was
determined. Concentrations of RANTES ranged from 10-6 M to 10-'2 M with
control wells
containing no RANTES. Concentrations of SDF ranged from 10-6 M to 10-'z M. The
results
of the experiment using RANTES are shown in Figure 8A. When no slit protein
was present,
increasing concentrations of RANTES resulted in increased migration of
leukocytes up to 10-
9 M at which point a plateau was reached. Addition of slit protein markedly
decreased the
migration, with migration completely inhibited at 200 pM slit. Similar results
were seen with
SDF (Figure 9A) except that leukocyte migration was completely inhibited with
slit at either
100 or 200 pM. The reciprocal experiment was also conducted in which the
concentration of
purified slit varied between 0 and 1100 pM while the concentration of
cytokine, RANTES or
SDF, was held constant at 10-9 M. The results obtained were similar for either
RANTES
(Figure 8B) or SDF (Figure 9B). In both cases, addition of purified slit
protein at a
concentration of 200 pM completely abolished chemokine stimulated leukocyte
migration.
In another set of experiments, the effect of purified slit on cytokine
stimulated cell
migration was examined using transfected HEK cells expressing robo, the CCRS
receptor, the
2 0 CXCR4 receptor, robo+ CCRS, or robo+CXCR4. In one experiment the
concentration of
RANTES varied from 10-6 to 10-'Z M, while the concentration of purified slit
was either 0 or
100 pM. The results of this experiment are shown in Figure 10. Increasing
concentration of
RANTES up to 10-9 M resulted in increased cell migration in cells expressing
CCRS alone
regardless of the presence of slit at 100 pM. Slit protein at 100 pM, however,
completely
2 5 inhibited migration of cells transfected with both Robo and CCRS. Similar
results were
observed when the experiment was repeated with SDF as the chemokine (Figure 11
). As with
RANTES, inhibition of cell migration by 100 pM slit was only observed in cells
transfected
with both Robo and CXCR4 receptor.
Purified human slit 2 protein has the activity similar to Xenopus slit in
inhibiting
3 0 leukocyte migration induced by RANTES and SDF 1, as shown in Figure 12 and
Figure 13. In
addition, carboxyl terminal fragment C 1 of slit showed similar activity in
inhibiting leukocyte
migration induced by RANTES and SDFl.
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Example 12
Effect of Slit on Cell Proliferation
HEK cells were stably transformed as previously described with either slit or
control
vector. Cells were cultured in DMEM as previously described. After 2 and 5
days of culture
the number of cells present was determined either by direct counting or by
counting on a
hemacytometer after staining with trypan blue. Cells that stably expressed
slit were found to
proliferate at 2-3 times the rate of vector transformed cells.
HEK cells stably transformed with either slit or control vector were cultured
in DMEM
as previously described except that the concentration of fetal bovine serum
was reduced over
1 o a 2-3 week period from 10% to 2% in 2% increments. The reduction in serum
concentration
had no effect on proliferation of slit transformed cells. In contrast, vector
transformed cells
showed significant reductions in rate of proliferation as the serum
concentration decreased.
Mesencephalic neuronal cells were cultured in medium that had been conditioned
with
either cells secreting the slit protein or cells secreting the lunatic fringe
protein. When
compared to cells cultured in medium conditioned with lunatic fringe, neuronal
cells cultured
in slit conditioned medium were more granular and had thicker neuritic masses
indicating
neurotrophic activity. These results indicate that slit has an affect on cell
proliferation.
Conclusion
In light of the detailed description of the invention and the examples
presented above, it
2 o can be appreciated that the several aspects of the invention are achieved.
It is to be understood that the present invention has been described in detail
by way of
illustration and example in order to acquaint others skilled in the art with
the invention, its
principles, and its practical application. Particular formulations and
processes of the present
invention are not limited to the descriptions of the specific embodiments
presented, but rather
2 5 the descriptions and examples should be viewed in terms of the claims that
follow and their
equivalents. While some of the examples and descriptions above include some
conclusions
about the way the invention may function, the inventors do not intend to be
bound by those
conclusions and functions, but put them forth only as possible explanations.
It is to be further understood that the specific embodiments of the present
invention as
3 0 set forth are not intended as being exhaustive or limiting of the
invention, and that many
alternatives, modifications, and variations will be apparent to those of
ordinary skill in the art
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48
in light of the foregoing examples and detailed description. Accordingly, this
invention is
intended to embrace all such alternatives, modifications, and variations that
fall within the
spirit and scope of the following claims.
