Polynucleotide encoding human netrin-1

Details Polynucleotide encoding human netrin-1 Polynucleotide encoding human netrin-1

1998-08-20

2001-04-17

Chan, Christina Y. (Department: 1644)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C536S024310

Reexamination Certificate

active

06218526

ABSTRACT:

INTRODUCTION
1. Field of the Invention
The field of this invention is human netrin proteins and genes.
2. Background
In the developing nervous system, axons project considerable distances along stereotyped pathways to reach their targets. Axon growth and guidance depends partly on the recognition of cell-surface and extracellular matrix cues along these pathways. The identification of such nerve cell growth and guidance cues is the holy grail of neurobiology. These are the compounds that tell neurons when to grow, where to grow, and when to stop growing. The medical applications of such compounds are enormous and include modulating neuronal growth regenerative capacity, treating neurodegenerative disease, and mapping (e.g. diagnosing) genetic neurological defects.
Over decades of concentrated research, various hypotheses involving chemo-attractants and repellents, labeled pathways, cell adhesion molecules, etc. have been invoked to explain guidance. Molecules such as N-CAM and N-cadherin have been reported to provide favorable substrates for axon growth and certain sensory axons may be responsive to NGF and NGF-like factors. Recent reports suggest the existence of diffusible chemotropic molecule(s) which influence the pattern and orientation of commissural axon growth.
Relevant Literature
Ishii et al. (1992) Neuron 9, 873-881 disclose a gene, unc-6, derived from
C. elegans
, which has sequence similarity to the disclosed netrins. Serafini et al (1994) Cell 78, 409-424 and Kennedy et al (1994) Cell 78, 425-435 at page 5, column 1 describe related vertebrate netrins. The work was also reported in
The New York Times,
Section B7, Tuesday, Aug. 16, 1994 and more recently (May 19, 1995) described in Science 268, 971-973 (see also references cited therein).
SUMMARY OF THE INVENTION
The invention provides methods and compositions relating to a human netrin protein and gene. Netrins are a class of proteins which are naturally involved in neural axon guidance. The subject compositions include nucleic acids which encode the specified netrin protein and hybridization probes and primers capable of hybridizing with the specified netrin gene. The netrin proteins finds particular use in modulating neural axon outgrowth. The disclosed compositions also find use variously in screening chemical libraries for regulators of axon outgrowth and orientation, in genetic mapping, as probes for netrin genes, as diagnostic reagents for genetic neurological disease and in the production of specific cellular and animal systems for the development of neurological disease therapy.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides methods and compositions relating to a human netrin-1 protein and gene; including methods and compositions for identifying, purifying, characterizing, and producing the subject proteins and for identifying, characterizing, cloning, expressing, inhibiting the expression of and amplifying the subject nucleic acids. The subject proteins may be incomplete translates of the disclosed netrin cDNA sequence or deletion mutants of the corresponding conceptual translates, which translates or deletion mutants have the human netrin-1 binding activity and specificity described herein. The netrins are isolated, partially pure or pure and are typically recombinantly produced. An “isolated” protein for example, is unaccompanied by at least some of the material with which it is associated in its natural state; generally constituting at least about 0.5%, preferably at least about 2%, and more preferably at least about 10% by weight of the total protein in a given sample; and a pure protein constitutes at least about 50%, preferably at least about 90%, and more preferably at least about 99% by weight of the total protein in a given sample. A wide variety of molecular and biochemical methods are available for generating and expressing the subject compositions, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, New York) or that are otherwise known in the art.
The disclosed netrin compositions may be used to modulate axon outgrowth or guidance in situ or in vivo. For in vivo applications, the compositions are added to a retained physiological fluid such as blood or synovial fluid. For CNS administration, a variety of techniques are available for promoting transfer of the therapeutic across the blood brain barrier including disruption by surgery or injection, drugs which transiently open adhesion contact between CNS vasculature endothelial cells, and compounds which facilitate translocation through such cells. Netrins may also be amenable to direct injection or infusion, topical, intratrachealinasal administration e.g. through aerosol, intraocularly, or within/on implants e.g. fibers e.g. collagen, osmotic pumps, grafts comprising appropriately transformed cells, etc. A particular method of administration involves coating, embedding or derivatizing fibers, such as collagen fibers, protein polymers, etc. with therapeutic proteins. Other useful approaches are described in Otto et al. (1989) J Neuroscience Research 22, 83-91 and Otto and Unsicker (1990) J Neuroscience 10, 1912-1921. Generally, the amount administered will be empirically determined, typically in the range of about 10 to 1000 &mgr;g/kg of the recipient and the concentration will generally be in the range of about 50 to 500 &mgr;g/ml in the dose administered. Other additives may be included, such as stabilizers, bactericides, etc. will be present in conventional amounts.
The invention provides netrin-specific binding agents including isolated binding targets such as membrane-bound netrin receptors and netrin-specific antibodies and binding agents identified in screens of natural and synthetic chemical libraries, and methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. Generally, netrin-specificity of the binding agent is shown by binding equilibrium constants. Such agents are capable of selectively binding the specified netrin, i.e. with an equilibrium constant at least about 10
7
M
−1
, preferably at least about 10
8
M
−1
, more preferably at least about 10
9
M
−1
. A wide variety of cell-based and cell-free assays may be used to demonstrate netrin-specific binding; preferred are rapid in vitro, cell-free assays such as mediating or inhibiting netrin-cell/protein binding, immunoassays, etc.
The invention also provides nucleic acids encoding the subject proteins, which nucleic acids may be part of netrin-expression vectors and may be incorporated into recombinant cells for expression and screening, transgenic animals for functional studies (e.g. the efficacy of candidate drugs for neural disease or injury), etc. and nucleic acid hybridization probes and replication/amplification primers having a disclosed netrin cDNA specific sequence. The hybridization probes contain a sequence common or complementary to the corresponding netrin gene sufficient to make the probe capable of specifically hybridizing to the corresponding netrin gene, and only to the corresponding netrin gene, in the presence of other netrin genes. Hence, the subject probes and primers are uniquely specific to the disclosed cDNA. Hybridization probes having in excess of 100 continuous bases of netrin gene sequence are generally capable of hybridizing to the corresponding netrin cDNA and remaining bound at a reduced final wash stringency of 0.2×SSC (0.9 M saline/0.09 M sodium citrate) and 0.1% SDS buffer at a temperature of 65° C.
The subject nucleic acids are isolated, meaning they comprise a sequence joined to a nucleotide other than that which it is joined to on a natural chromosome, and usually constitute at least about 0.5%, preferably at least about 2%, and more preferably at least about 5% by weight of total nucleic acid present in a given fraction. A pure nucleic acid constitutes at least about 50%, preferably at least abou

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