Neuronal factor

Details Neuronal factor Neuronal factor

1995-05-31

2001-01-16

Allen, Marianne P. (Department: 1631)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C536S023500, C435S320100, C435S325000, C435S352000, C435S354000, C435S357000, C435S358000, C435S364000, C435S366000, C435S367000, C435S252300, C435S252330, C435S069700, C435S069800

Reexamination Certificate

active

06174701

ABSTRACT:

FIELD OF THE INVENTION
This application relates to proteins which are involved in the growth, regulation or maintenance of nervous tissue. In particular, it relates to nerve-derived factors having homology to NGF.
BACKGROUND OF THE INVENTION
Nerve growth factor (NGF) is a protein which has prominent effects on developing sensory and sympathetic neurons of the peripheral nervous systems. NGF acts via specific cells surface receptors on responsive neurons to support neuronal survival, promote neurite outgrowth, and enhance neurochemcial differentiation. NGF actions are accompanied by alterations in neuronal membranes (Connolly et al., J. Cell. Biol. 90:176-180 [1981]; Skaper and Varon, Brain Res. 197:379-389 [1980]), in the state of phosphorylation of neuronal proteins (Yu, et al., J. Biol. Chem. 255:10481-10492 [1980]; Haleqoua and Patrick, Cell 22:571-581 [1980]), and in the abundance of certain mRNAs and proteins likely to play a role in neuronal differentiation of function (see, for example, Tiercy and Shooter, J. Cell. Biol. 103:2367-2378 [1986]).
Forebrain cholinergic neurons also respond to NGF and may require NGF for trophic support. (Hefti,
J. Neurosci.,
6:2155 [1986]). Indeed, the distribution and ontogenesis of NGF and its receptor in the central nervous system (GNS) suggest that NGF acts as a target-derived neurotrophic factor for basal forebrain cholinergic neurons (Kosching, TINS, pp 570-573 (Nov/Dec [1986]).
While a number of animal homologues to NGF have become known, it was not until recently that an apparently distinct nerve growth factor was identified that nonetheless bears some homology to NGF (Leibrock et al., Nature 341:149 [1989]). This factor, called brain-derived neurotrophic factor (BDNF), was purified from pig brain, and a partial amino acid sequence determined both from the N-terminal end and from fragments purified after cleavages. The longest sequence, compiled from several overlapping fragments, was used to synthesize two sets of oligonucleotides that were used to prime the amplification of a pig genomic template using the polymerase chain reaction (PCR). The nucleotide sequence between the two primers was determined and used to synthesize specific primers for further PCRs on a complementary DNA template obtained by reverse transcription of total RNA isolated from the superior colliculus of the pig brain. The nucleotide sequence so obtained contained an open reading frame coding for a protein of 252 amino acids, starting with the first methionine codon found after four in-frame stop codons. Leibrock et al. speculate that there is no reason to think that BDNF and NGF should be the only members of a family of neurothrophic proteins having in common structural and functional characteristics, and the authors hope that these common structural features could be sued to aid the discovery of other members.
It is an object to identify other neurotrophic factors which bear homology to NGF and to obtain nucleic acid encoding such factors.
It is another object to synthesize such new factors in recombinant cell culture.
It is yet another object to provide derivatives and modified forms of such new factors.
It is an additional object to prepare immunogens for raising antibodies against such new factors, as well as to obtain antibodies capable of binding them.
Another object is to provide diagnostic and therapeutic compositions comprising such new factors or derivatives thereof and methods of therapeutic treatment.
SUMMARY OF THE INVENTION
These and other objects of the invention apparent to the ordinary artisan are accomplished by first providing a nucleic acid sequence comprising at least a portion of the coding sequence for a new nerve-derived factor related to NGF and BDNF, hereafter termed neuronal factor (NF). An alternative name for NF is neuronotrophin-3, or NT-3. The nucleic acid sequence is inserted into an expression vector and expressed in recombinant host cell culture in order to synthesize NF, or is used in hybridization assays for NF nucleic acid.
NF or fragments thereof (which also may by synthesized by in vitro methods) are fused (by recombinant expression or in vitro covalent methods) to an immunogenic polypeptide and this, in turn, is used to immunize an animal in order to raise antibodies against an NF epitope. Anti-NF is recovered from the serum of immunized animals. Alternatively, monoclonal antibodies are prepared from cells of the immunized animal in coventional fashion. Antibodies identified by routine screening will bind to NF but will not substantially cross-react with BDNF or NGF. Immobilized anti-NF antibodies are useful particularly in the diagnosis (in vitro or in vivo) or purification of NF.
Substitutional, delectional or insertional mutants of NF are prepared by in vitro or recombinant methods are screened for immuno-crossreactivity with NF and for NF antagonist or agonist activity.
NF also is derivatized in vitro in order to prepare immobilized NF and labelled NF particularly for purposes of diagnosis of NF or its antibodies, or for affinity purification of NF antibodies.
NF, tis derivatives or antibodies are formulated into physiologically acceptable vehicles, especially for therapeutic use. Such vehicles include sustained release formulations of NF.
In another aspect, the invention provides a method for producing NF, comprising culturing the transformed host cell and recovering NF from the culture.
NF has been found to be neurothrophic for primary sympathetic neurons, sensory neurons derived from nodose ganglion (placode), and subpopulations of spinal sensory neurons derived from dorsal root ganglion (neural crest). In addition, it has a broad tissue distribution and is structurally related to both NGF and BDNF. NF has a unique range of trophic activities that complement those of NGF and BDNF, and is a differentiation-inducing factor for the phaeochromacytoma cell line, PC-12. Thus, it is likely to play a wide role in defining the fate and function of nerve cells during development. In addition, its presence in the CNS indicates that it can be useful as a therapeutic agent for neurodegenerative diseases and damaged nerves, e.g., nerves damaged as a result of trauma.


REFERENCES:
patent: 5180820 (1993-01-01), Barde et al.
patent: 5266474 (1993-11-01), Miller

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